research article rhein elicits in vitro cytotoxicity in...

Research ArticleRhein Elicits In Vitro Cytotoxicity in PrimaryHuman Liver HL-7702 Cells by Inducing Apoptosis throughMitochondria-Mediated Pathway

Guy-Armel Bounda1 Wang Zhou2 Dan-dan Wang2 and Feng Yu123

1Department of Clinical Pharmacy China Pharmaceutical University No 24 Tong Jia Xiang Jiangsu Nanjing 210009 China2Department of Pharmacology China Pharmaceutical University No 24 Tong Jia Xiang Jiangsu Nanjing 210009 China3Key Laboratory of Drug Quality Control and Pharmacovigilance China Pharmaceutical University Ministry of EducationNanjing 210009 China

Correspondence should be addressed to Feng Yu yufengcpu14gmailcom

Received 1 April 2015 Accepted 24 May 2015

Academic Editor Cheorl-Ho Kim

Copyright copy 2015 Guy-Armel Bounda et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary humanhepatic cells Results Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent mannerFollowing rhein treatment (25120583M 50 120583M and 100 120583M) for 12 h the detection of apoptotic cells was significantly analyzed by flowcytometry and nuclear morphological changes by Hoechst 33258 respectively Fatty degeneration studies showed upregulationlevel of the relevant hepatic markers (P lt 001) Caspase activities expressed significant upregulation of caspase-3 caspase-9and caspase-8 Moreover apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK caspase-9 inhibitor and caspase-3 inhibitor respectively Overproduction of reactive oxygen species lipid peroxidation and loss ofmitochondrial membrane potential were detected by fluorometry Additionally NAC a ROS scavenger significantly attenuatedrhein-induced oxidative damage in HL-7702 cells Furthermore real-time qPCR results showed significant upregulation of p53PUMA Apaf-1 and Casp-9 and Casp-3 mRNA with no significant changes of Fas and Cytochrome-c Immunoblotting revealedsignificant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression Conclusion Taken togetherthese observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway withinvolvement of oxidative stress mechanism

1 Introduction

Polygonum multiflorum Thunb (PMT Polygonaceae) alsoknown as Fo-Ti is a traditional Chinese herbal medicinecommon in northeast Asia Its roots have been widelyused as therapeutic agent including antiallergy antitumorantibacterial spasmolytic antialopecia vasorelaxant andanti-aging agent for many centuries in Asian traditionalmedicine [1ndash4] Mounting data of pharmacological effects ofthis herb and its components including anti-inflammationantioxidative and neuroprotective as well as improvedlearning and memory have been recently published [5ndash8] The genus Polygonum is the source of a wide range ofphenolic compounds flavonoids anthraquinones stilbenes

and tannins [9] including a number of anthraquinones inthe stilbene class such as (E)-23410158405-tetrahydroxystilbene-2-120573-D-glucoside rhein emodin aloe-emodin chrysophanolphyscion and their derivatives [10]

In recent years drug-induced liver injury (DILI) has beenone of the interesting topics which have led to conductingseveral researches on herbal medicines Published data havereported the toxicological effect of Polygonummultiflorum onthe liver [11 12] In liver like other organs apoptosis plays akey role during physiological cell renewal [13 14] and in cellu-lar depletion after stimulation with mitogens or hyperplasia-inducing treatments [15] A growing number of publishedevidences suggest that hepatocyte apoptosis can contribute tothe development of many liver diseases including alcoholic

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 329831 19 pageshttpdxdoiorg1011552015329831

2 Evidence-Based Complementary and Alternative Medicine

OH

OHOH O

O

O

Figure 1 Chemical structure of rhein (4 5-dihydroxyanthraquin-one-2-carboxylic acid) Molecular formula C

15H8O6 Molecular

weight 28422 gmol

liver injury chronic viral hepatitis cholestatic liver diseasesand hepatic fibrosis [8 16 17]

With thousands of years of medical practice TraditionalChinese Medicine (TCM) has accumulated rich theories(including yin yang chrsquoi (qi) meridian five-phase (or five-element) and zang-fu theories) and a great deal of valuableexperience in the prevention and treatment of several diseasesor medical condition [18 19] According to the theory ofTCM the liver is regarded as a special organ in the bodyit is related to the eyes via the meridian connections andits condition is reflected in the nails It is involved by virtueof its role in regulating or ensuring the free flow of qiaround the body and its role in regulating blood volume(ldquostoring bloodrdquo) [20] Based on long period of traditionaland clinical practice TCM considered that the rhizome ofPolygonum multiflorum possesses sweet and slightly warmproperties making it especially suitable for treating patientswho have not only blood deficiency but also a mild Yangand Qi deficiency of the body It opens the meridians andcollaterals able to tonify the blood as well the kidney essence(Jing) strengthens the tendons and bones and improves sleep[21] TCM theory said that both graying of hair and hairloss may be due to lack of essential essences explaining whyPolygonummultiflorum has been used to promote the growthof hair and to treat premature greying of hair which is doneby nourishing the kidneyrsquos ldquoyinrdquo energy replenishing andcooling down the blood [22] However the long-term use ofPolygonum multiflorummay lead to liver and kidney toxicityas several clinical cases of hepatotoxicity have been linked toits consumption

Rhein (4 5-dihydroxyanthraquinone-2-carboxylic acidFigure 1) is one of the most important bioactive componentsof PMT Mounting published reports demonstrated thatits pharmacological effects including anti-inflammatory [8]antiallergic [23] antifungal [24] antibacterial [25] antiviral[26] and anticancer ones [27ndash29] Recently the suppressionof Hep-G2 cells proliferation induced by rhein was expressedvia mitochondrial permeability transition but the oxidativestress injury mechanism was not investigated in this study[30] Mostly the mechanism of rhein antitumor activity incancer cells listed previously is commonly due to its abilityto induce apoptosis in corresponding cancer cells [28 29]

Apoptosis or programmed cell death (PCD) a geneticallycontrolled process whereby cells die in response to environ-mental or developmental cues contributes to the pathogene-sis of disease or removal of cells in adult organism [31] It is

characterized by the activation of biochemical pathways thatlead to changes in cellular morphology DNA fragmentationperturbation of mitochondrial membrane function decreaseof mitochondrial membrane potential translocation of phos-phatidylserine (PS) to the external cell surface and changesin the plasma membrane [32] Understanding and regula-tion of apoptosis are critical for normal development andtissue homeostasis and disruption of this process can havesevere consequences [33] Too much cell death may produceneurodegenerative diseases and impaired development whileinsufficient cell death can lead to increased susceptibility tocancer and sustained viral infection [34] Numerous scientificdata have proven that apoptotic signaling within the cellmay occur by two fundamental pathways (1) death receptoror extrinsic pathway and (2) the mitochondria or intrinsicpathway [35] Rhein has been investigated and shown toinduce cytotoxicity and apoptosis in primary cultures ofrat hepatocytes [36] Measurement of apoptosis has becomean essential component of the evaluation of cytotoxicity ofchemicals [31]

Liver cells especially hepatocytes are notable for theirwide variety of metabolic and other functional capacitiesspanning over 500 classes of functions such as energymetabolism bile production and synthetic or detoxificationfunctions [37] Primary human hepatocytes remain differ-entiated and sustain the major drug-metabolizing enzymesactivities they represent a unique in vitro system and serveas a ldquogold standardrdquo for studies of drug metabolism andtoxicity [38] HL-7702 cell expressed a distinct ultrastructurecompared to hepatic carcinoma and is considered an ideal invitromodel of a primary Chinese nonmalignant liver [39] Invitro and in vivo studies have been conducted to assess thebiosynthetic activities of HL-7702 cells in order to explore thepossibility to use this cell line for a liver support system Ina 7-day in vitro study done by Yang et al [40] the authorsfound that HL-7702 cells could keep their function of proteinsynthesis by culturing on thermoresponsive hydrogen Albu-min secretion continuously grew and the function of ureasynthesis was significantly increased with in time delayedfrom (016 plusmn 002) 120583molmL to (041 plusmn 004) 120583molmL [40]Furthermore the proliferation state of cells by cell cycleanalysis was proven to not be damaged In another studyconducted by Hu and colleagues [41] the biosynthetic func-tion of HL-7702 cells in terms of albumin uridine diphos-phate glucuronosyltransferase (UGT) and cytochrome P4503A4 gene and protein was significantly expressed In thesame study in an in vivo acute liver failure (ALF) modelestablished by 90 partial hepatectomy rats transplantedwith HL-7702 cells showed significantly improved survivalof 70 versus 0 in controls (119875 lt 001) Moreover theenzymatic analysis of various enzymes or liver markers suchas albumin alanine transaminase aspartate transaminaseserum ammonia alkaline phosphates and total and directbilirubin revealed a significant improvement compared to thecontrol groups [41] These evidences support that HL-7702cells could proliferate and keep their biosynthetic functionsat the same time suggesting them to be a feasible sourcefor liver support system and ideal for pharmacological andtoxicological studies

Evidence-Based Complementary and Alternative Medicine 3

Despite few toxicological studies done on HL-7702 cells(also known as L-02 cells) and knowing the paradoxicalhepatotoxicity and hepatoprotection of rhein no availablestudy had ever addressed the effects of rhein on apoptosisin primary human liver HL-7702 line cell Here we showsome evidence about the missing information using primaryhuman hepatic cell line HL-7702 cells which is one ofthe commonly used human primary liver cells for in vitroevaluation apoptosis induced by drugs as this primaryhuman liver cell line has been well proven and studied innumerous researchers published data [40ndash43] to analyze thedrug-induced hepatotoxicity Thus the aim of this study wasto investigate the in vitro cytotoxic activity of rhein in HL-7702 cells and to assess the possible relation between liverinjury and cellular uptake of rhein and possible mechanismsinvolved

2 Materials and Methods

21 Reagents and Antibodies Rhein (4 5-dihydroxyanthra-quinone-2-carboxylic acid purity gt 9999) was purchasedfrom the Chinese National Institute for Food and DrugControl and then dissolved in dimethyl sulfoxide (DMSOSigma-Aldrich USA) to a concentration of 20mgmLDMEM high glucose (Dulbeccorsquos modified Eaglersquos mediumhigh glucose containing L-glutamine) FBS (fetal bovineserum) Maxima SYBR Green qPCR Master Mix andtrypsin were obtained fromThermo Fischer Scientific USA3-(45-Dimethylthiazol-2-yl)-25-di-phenyl tetrazolium bro-mide (MTT) and the total RNA extraction reagent werepurchased from Nanjing Sunshine Biotechnology (China)Annexin V-FITCPI apoptosis detection kit alanine amino-transferase (ALT) and aspartate aminotransferase (AST)assay kits were obtained from Nanjing Jiancheng Bioengi-neering Institute The triglyceride (TG) reagent assay andtotal cholesterol (TC) reagent assay kits were purchasedfrom Zhejiang Dongrsquoou Diagnostic Products Co Ltd Thecell lysis buffer for western and PI phenylmethylsulfonylfluoride (PMSF) LDH cytotoxicity assay kit trypan blue JC-1 mitochondrial membrane potential detection kit HoechstStaining Kit enhanced BCA protein kit cell mitochondriaisolation kit reactive oxygen species assay kit N-acetyl-L-cysteine (NAC) lipid peroxidation (MDA) assay kit totalsuperoxide dismutase (SOD) assay kit and caspase-3 -9 and-8 activity assays kits were purchased fromBeyotime Instituteof Biotechnology China The various caspase inhibitors Z-IETD-FMK (caspase-8) Z-LEHD-FMK (caspase-9) and Z-DEVD-FMK (caspase-3) were purchased from Calbiochem-Novabiochem Co (San Diego CA USA) AMV First StrandcDNA Synthesis Kit and all the primers were designed andsynthesized by Shanghai Sangon Biotechnology Co LtdTheprimary antibodies (including anti-Fas anti-Cytochrome-c and anti-120573-actin) and secondary antibody conjugated tohorseradish peroxidase were purchased from Cell SignalingTechnology Inc USA All other reagents were of analyticalgrade

22 Cells Line and Cells Culture Primary human liver HL-7702 cells also known as L-02 cells were purchased from

the Institute of Biochemistry and Cell Biology SciencesChinese Academy of Sciences (Shanghai China) Expressinga distinct ultrastructure compared to hepatic carcinoma cellsHL-7702 cells are considered an ideal in vitro model ofChinese nonmalignant liver cells [39 44 45] These cellswere routinely grown as monolayer in DMEM containing4500mgL glucose supplemented with 10 fetal bovineserum and maintained at 37∘C in a cell culture humidifiedincubator with 95 air and 5 CO

2 The HL-7702 cells were

used in all experiments described below here

23 Determination of Cell Viability MTT assay was used toassess cell viability as a function of redox potential as onlyviable cells have functioning mitochondrial dehydrogenaseenzymes which can reduce MTT to formazan [46] BrieflyHL-7702 cells were seeded in 96-well microplates at a celldensity of 8 times 103 cells per well After pretreatment withdifferent concentrations of rhein for 24 and 48 h respectivelycell viability was assessed by incubating cells with 20 120583L ofMTT (5mgmL) for 4 h at 37∘C The medium was thenremoved and replaced by 150 120583L of DMSO in each cultureand mixed by pipetting and the plates were then vibratedfor 10min to uniformly dissolve the crystals Absorbancereadings were performed at 570 nm using amicroplate reader(Molecular Devices USA) with the optical density (OD)Theresults were calculated with the following formula survivalrate () = (OD treated well minus OD blank)(OD control well minusOD blank) times 100

24 Lactate Dehydrogenase (LDH) Assay After treatmentwith different concentrations of rhein for 24 and 48 hrespectively LDH activity was measured by using a LDHcytotoxic assay kit (Beyotime Institute of BiotechnologyChina) according to the manufacturerrsquos protocol Briefly theHL-7702 cells were seeded on 12-well cultures plates at adensity of 15 times 106 cellswell At the end of the treatmentthe medium was collected to assess the LDH activity Todetermine the intracellular LDH activity the cells werewashed by PBS and then 150120583L PBS were added into eachwell and the cells were lysedwith 200 120583L of 01TritonX-100LDH activities in both the culture supernatants and the celllysates were determined by adding 60 120583L of substrate solutionfrom the kit followed by incubation at 25∘C for 30min Theabsorbance of the samples was recorded at 490 nmThe LDHleakage was expressed as the percentage () of the total LDHactivity (LDH in the medium + LDH in the cell) accordingto the equation LDH released = (LDH activity in themediumtotal LDH activity) times 100

25 Assessment of Liver Marker Enzymes Serum ALT andAST were determined using the commercial kits purchasedfrom Jiancheng Institute of Biotechnology (Nanjing China)Briefly HL-7702 cells were placed and seeded in 96- wellplates The following day medium was refreshed and thecells were treated with rhein (10120583Mndash400 120583M) for 12 hoursof incubation At the end of incubation period the mediumwas discarded wells were gently washed twice with 1mL PBSCells were centrifuged (1000 g 10min) and the supernatant


was discarded Cells were homogenized on ice in the corre-spondent assay buffer ALT and AST levels in the collectedcell lysates were analyzed using the colorimetric assay kitaccording to the manufacturerrsquos instructions

26 Nuclear Staining with Hoechst 33258 Cell death wasassessed based on nuclear morphological changes that weredetermined following nuclei staining with Hoechst StainingKit Briefly HL-7702 cells were seeded on glass cover slips in6-well plates At 12-hour treatmentwith rhein (25 120583M 50 120583Mand 100 120583M) plates were rinsed twice with PBS and thenfixed with stationary liquid for 10min at room temperatureThe cells were then stained with Hoechst 33258 solution inthe dark for 5min at 37∘C After washing twice with PBSthe morphological features of apoptosis (nuclear shrinkagechromatin condensation intense fluorescence and nuclearfragmentation) were monitored by fluorescence microscopywith inverted Leica microscope and a UV filter (Leica 2500Leica Corporation Germany) Apoptotic cells were identifiedas those whose nuclei exhibited brightly staining condensedchromatin nuclear fragmentation or apoptotic bodies whilethosewith uniformly stained nuclei were identified as healthy

27 Annexin VPI Analysis for Cell Apoptosis The AnnexinVPI double staining assay was further used to determinethe apoptotic cells according to the manufacturerrsquos protocolIn brief HL-7702 cells were plated in a 6-well culture plateat a density of 40 times 105 cellswell and treated with rhein(25 120583M 50 120583M and 100 120583M) for 12 hours At the end of thetreatment they were digested with trypsin and harvestedby centrifugation and then resuspended in 500120583L buffersolution Then Annexin V-FITCPI staining solution (PI(5 120583L) and Annexin V (5 120583L)) was added After incubationfor 10min at room temperature in the dark then the stainedcells were analyzed in FACScan flow cytometry analyzer(Becton Dickinson Biosciences CA USA) Excitation wavewas at 488 nm and the emitted green fluorescence of Annexin(FL1) and red fluorescence of PI (FL2) were measured using525 nm and 575 nm band pass filters respectively A total ofat least 10 000 cells were analyzed per sample The amountof healthy cells early apoptosis and late apoptosisnecrosiswere determined as the percentage of Annexin VminusPIminusAnnexin V+PIminus or Annexin V+PI+ cells respectivelyusing CellQuest software

28 Effects of Rhein on Enzymes Involved in Lipid Metabolism

281 Total Cholesterol (TC) and Triglycerides (TG) AssaysBriefly the cells were plated in a 96-well culture plate andtreated with rhein (25120583M 50 120583M and 100 120583M) for 12 hCollect 10 times 106 cells by centrifugation at 1000timesg for 10 min-utes Discard the supernatant and resuspend in 1mL of coldPBS containing 1 Triton X-100 Homogenize or sonicatethe cell suspension 20x at one-second bursts Centrifuge cellsuspension at 10000timesg for 10minutes at 4∘CCarefully collectthe supernatant and should be stored on ice for immediateuse Cell lysates must be further diluted before assaying (1 5or greater) The TC and TG levels in the collected cell lysates

were analyzed using the colorimetric assay kit according tothe manufacturerrsquos instructions

282 Assessment of HGM-CoAR ACoAC and GPAT RelativeTranscripts Hydroxymethylglutaryl coenzyme A reductase(HMG-CoAR) regulates several pathways within animalcells as it is the rate-limiting enzyme in the biosynthesisof cholesterol and represents the sole major drug targetfor contemporary cholesterol-lowering drugs Acetyl-CoAcarboxylase (ACoAC) plays a critical role in the regulation oflong-chain fatty acid synthesis The initial and rate-limitingstep of glycerolipid synthesis is the acylation of glycerol-3-phosphate (G3P) with long-chain fatty acyl-CoA to formlysophosphatidic acid (LPA) This reaction is catalyzed byglycerol-3-phosphate acyltransferase (GPAT) To assess thetoxicological effect of Rhein on the lipid biosynthesis inHL-7702 cells the transcripts of these three enzymes wereanalyzed The assay procedure is described in Section 213

29 Mitochondrial Membrane Potential (Δ120593119898119894119905

) Assay Lossof mitochondrial membrane potential (Δ120593mit) was assessedby fluorescence spectrophotometry (Shimadzu Japan) usingthe mitochondria-specific lipophilic cationic fluorescent dyeJC-1 In healthy cells with high mitochondrial Δ120593mit JC-1spontaneously aggregates and gives intense red fluorescence[47] On the other hand in apoptotic or unhealthy cellswith low Δ120593mit JC-1 remains in the monomeric formwhich shows only green fluorescence Briefly to monitormitochondrial membrane potential Δ120593mit cells grown in 96-well polystyrene culture plates were treated with differentconcentrations of rhein (25120583M 50120583M and 100 120583M) for 12hours Then JC-1 staining working solution (5120583gmL) wasadded to the culture and then incubated in the dark at 37∘Cfor 20min Cells were then washed twice with ice-cold PBSand then qualitatively and quantitatively analyzed by fluores-cence spectrophotometry 5 120583L of 10 120583M of the protonophorecarbonyl cyanide m-chlorophenylhydrazone (CCCP) was apositive control that could induce mitochondria membranedepolarization Laser scanning confocal microscope (Zeiss)with the excitation wavelength of 488 nm emission of greenand red fluorescence intensity wavelength of 545 nm wasused for detection The ratio of redgreen fluorescence wascalculated and presented in arbitrary units A decrease inthis ratio indicates mitochondrial depolarization (ie loss ofΔ120593mit)

210 Measurement of Intracellular Reactive Oxygen Species(ROS) Production The production of intracellular reactiveoxygen species (ROS) was monitored by fluorescence spec-trophotometer (Shimadzu Japan) after staining with 2101584071015840-dichlorofluorescin diacetate (DCFH-DA) Briefly after treat-ment with rhein (25120583M 50120583M and 100 120583M) for 12 hoursor pretreated with 5mM NAC for 1 hour HL-7702 cells(1 times 106 cellsmL) were washed twice with PBS and thenincubated with the 2101584071015840-dichlorofluorescin diacetate (DCFH-DA) working solution (10 120583Mfinal concentration) for 20minat 37∘C in the dark The intensity of the fluorescence signalwas then detected dose dependently at 488 nm excitation and


525 nm emission 90 by using trypan blue The results ofROS production are expressed as increase in fluorescence inrespect of control

211 Determination of Lipid Peroxidation (MDA) and Super-oxide Dismutase (SOD) The cell culture and rhein treatmentwere conducted in the same manner described above Afterrhein exposure the cells were harvested washed twice withPBS and lysed in cell lysis buffer centrifuged at 14000timesgfor 5min at 4∘C The lysates were then collected and storedat minus20∘C until further use The supernatants were used formeasuring cellular MDA and SOD using the commerciallyavailable assay kits (Beyotime Institute of BiotechnologyChina) The MDA level was calculated by evaluating thethiobarbituric acid reacting substances at a wavelength of532 nm The activity of SOD was determined by making useof the hypoxanthine and xanthine oxidase system [43] Oneunit of SOD activity was defined as the amount of enzymerequired to inhibit oxidation by 50 in a 1mL reaction in theabsorbance at 450 nm All the operations process were doneaccording to the manufacturerrsquos instructions and measuredwith a microplate reader (VersaMax USA) The proteinconcentration of each treatment group was determined usingthe BCA protein assay kit (Beyotime China) The results forMDA and SOD were defined as 120583M120583g protein and U120583gprotein respectively

212 Caspase-3 -8 and -9 Activity Assay Caspase-3 -8and -9 activities were measured by colorimetric assay usingthe cleavage of a colorless substrate specific for caspase-3(Ac-DEVD-120588NA) caspase-8 (Ac-IETD-120588NA) or caspase-9(Ac-LEHD-120588NA) releasing the chromophore 120588-nitroaniline(120588NA) Assays were carried out according to the manufac-turerrsquos instructions Briefly cell lysates were prepared aftertheir respective treatment Assays were performed on 96-wellmicrotitre plates by incubating 20120583L cell lysate protein persample in 70 120583L reaction buffer containing 10 120583L caspase-3-8 or -9 substrates Lysates were incubated at 37∘C for 1 hThe release of 120588NA was quantitated spectrophotometricallyby measuring absorbance at 405 nm using a microplatereader (Molecular Devices USA) and enzyme activity wascalculated with reference to standard curve of 120588NA con-centration versus absorbance The data were represented asthe UmgPro Protein content was measured according toBradford method Assays were done in triplicate

To elucidate the antiapoptotic effects of caspase inhibitorson the apoptosis induced by rhein cells were preincu-bated with 25 120583M Z-IETD-FMK (caspase-8) Z-LEHD-FMK(caspase-9) or Z-DEVD-FMK (caspase-3) for 1 h Followingincubation with rhein for 12 h apoptosis was determined byFACS analyses

213 RNA Extraction and Quantitative RT-PCR Assay Totalcellular RNA was extracted from cells using the TRIzolmethod to guarantee an OD260280 ratio in the range of18ndash20 RNA was reverse transcribed into single-strandedcDNA by the Revert Aid First Strand cDNA Synthesis KitThe synthesized cDNA with primer and SYBR Green Master

Mix (Thermo Fischer Scientific USA) was then amplifiedby quantitative real-time PCR qRT-PCR were performedusing forward and reverse primers shown in Table 1 andrun on a Mastercycle ep realplex real-time PCR system(Eppendorf Germany) Glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) was used as an internal control in par-allel for each run The cycle number at which the fluorescentsignal crosses the detection threshold was denoted as thethreshold cycle (Ct) All samples were run in triplicate andunderwent 40 amplification cycles according to themanufac-turer protocol (95∘C for 10min followed by 40 cycles at 95∘Cfor 15 s 60∘C for 30 s and 72∘C for 30 s) Relative amountsof target RNA were quantified by the 2minusΔΔCt method andnormalized to the corresponding GAPDH values Specificityof real-time PCR products was determined by melting curveanalysis

214 Protein Extraction and Western Blotting Analysis ofFas and Cyt-c To determine the expression of associatedproteins western blotting was performed HL-7702 cells wereharvested after rhein treated (25120583M 50 120583M and 100 120583M)for 12 h The total proteins were extracted using Lysis buffer(PMSF was added previously to a total concentration of1mM) Cytosolic (deprived of mitochondria protein) andmitochondrial proteins were extracted using cell mitochon-drial isolation kit according to the manufacturerrsquos protocolProtein concentration was determined using enhanced BCAprotein assay kit The extracted protein samples were mixedwith 5x SDS loading buffer and boiled for 5min They wereseparated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a 045 120583m (forFas) or 022 120583m (for Cyt-c) nitrocellulose (NC) membranes(BOSTER China) The membranes were blocked with TBS-T (Tris 20mM pH 76 NaCl 150mM and 01 Tween-20)containing 5 bovine serum albumin (BSA) for 1 h at roomtemperature and the membranes were incubated with dilutesolution (1 1000 in 5 wv BSA 1x TBS and 01 Tween)of primary antibodies including anti-Fas anti-Cytochrome-c and anti-120573-actin overnight at 4∘C After washing fourtimes with TBST for 8min the membranes were incubatedwith secondary antibody (1 10000 dilution) conjugated tohorseradish peroxidase for 1 h at room temperature Themembranes were then washed four times for 8min withTBST Immune-reactive proteins were detected using ECLwestern blotting detection system (Millipore Germany) andvisualized with the ChemiDoc XRS+ system (Bio-Rad Lab-oratories USA) Densitometric analysis of immunoblots wasperformed by using Quantity One software and 120573-actin wasused for standardization

215 Statistical Analysis All experiments were performed atleast in 3 independent in vitro experiments (unless statedotherwise) all results are presented as the mean plusmn standarddeviation (SD) and were processed with SPSS 180 software(SPSS Chicago IL) Statistical significancewas assessed usinga two-way ANOVA followed by Tukeyrsquos post hoc test Thesignificance level was set at 119875 lt 005 Error bars denote SDunless stated otherwise


Table 1 Sequences of the primers used in real-time PCR

Gene description Primer Sequence (51015840rarr 31015840) Length (bp)

GAPDH GAPDH-F CAGGAGGCATTGCTGATGAT 20GAPDH-R GAAGGCTGGGGCTCATTT 18

HGM-CoAR HGM-CoAR-F AGCCTGAATAGCCCGACAG 19HGM-CoAR-R CATCCTCCACAAGACAATGC 20

GPAT GPAT-F TGAACAACTGGGCAAACCTAA 21GPAT-R AAATCCACTCGGACACAACC 20

ACoAC ACoAC-F CTCTTGACCCTGGCTGTGTA 20ACoAC-R GATGGAGTTTCTCGCCTCTG 20

p53 p53-F GCCATCTACAAGCAGTCACAG 21p53-R ATTTCCTTCCACTCGGATAAGA 22

PUMA PUMA-F GAAGAGCAAATGAGCCAAAC 20PUMA-R CAGAGCACAGGATTCACAGTCT 22

Fas Fas-F ACACTCACCAGCAACACCAAGT 22Fas-R CCTTTCTCTTCACCCAAACAAT 22

Cyt-c Cyt-c-F TACTCTTACACAGCCGCCAATA 22Cyt-c-R AGTCTGCCCTTTCTTCCTTCTT 22

Apaf-1 Apaf-1-F GTGAAGTGTTGTTCGTGGTCTG 22Apaf-1-R CGTGTGGATTTCTCCCAATAG 21

Casp-8 Casp-8-F ATGTTGGAGGAAAGCAATCTGT 22Casp-8-R CCTGCCTGGTGTCTGAAGTT 20

Casp-9 Casp-9-F ACTAACAGGCAAGCAGCAAAGT 22Casp-9-R ACATCACCAAATCCTCCAGAAC 22

Casp-3 Casp-3-F AGCAATAAATGAATGGGCTGAG 22Casp-3-R GTATGGAGAAATGGGCTGTAGG 22

020406080

100120

012 037 12 37 12 37 120 370

Cel

l via

bilit

y (

)

Rhein concentration (120583M)

24h48h

Figure 2 Rhein induces in vitro cytotoxicity of HL-7702 cells ina dose-and time-dependent manner HL-7702 cells were incubatedwith different concentration of rhein for 24 h or 48 h and thenprocessed for MTT assay Cell viability was made relative tountreated control cells (100) Data are expressed as mean plusmn SDfrom three independent experiments

3 Results

31 Rhein-Induced Inhibition of HL-7702 Cells ViabilityCytotoxicity of rhein was assessed with the MTT assay Adose and time-dependent relationship study of HL-7702 cellstreated with a series of concentrations of rhein (012120583Mndash370 120583M) respectively for 24 h and 48 h was conducted As

shown in Figure 2 cell viability expressed both dose andtime-dependent relationships It significantly decreased after24 h of rhein treatment with 37120583M 120120583M and 370 120583MconcentrationsMeanwhile the cell viability greatly decreasedafter 48 h of rhein treatment with the following concentra-tions 12 120583M 37 120583M 120120583M and 370 120583MTo compare the cellviability IC

50values 13179 120583M and 1845 120583Mwere calculated

respectively at 24 h and 48 h

32 Rhein Damaged the Integrity of Cell Membrane Lactatedehydrogenase (LDH) is abundant in the cytoplasm andcannot pass through the normal cell membrane but would bereleased into the extracellular medium if only cells are dam-aged or deadTherefore an elevated level of LDH leakage willreflect cellmembrane damageWe treated cells with a series ofrheinrsquos concentrations (10 120583Mndash400 120583M) respectively for 24 hand 48 h The results are shown in Figure 3(a) These resultsshowed that rhein was able to induce LDH leakage of HL-7702 cell in all groups in a dose- and time-dependentmanner

The cell membrane integrity was also further investigatedusing Hoechst 33258 fluorescent staining technique Themorphological characteristics of the human primary liverHL-7702 cells following treatment with rhein (25120583M 50 120583Mand 100 120583M) for 12 h were observed and then photographedunder a fluorescence microscope The results shown inFigure 3(b) confirmed that rhein ravaged the integrity of


05

101520253035404550

Control

LDH

rele

ase (

o

f con

trol)

Rhein24h48h

50120583M 100120583M 200120583M 400120583M10120583M

lowast lowast lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

(a)

Control

Rhein

50120583M25120583M 100120583M

(b)

020406080

100120140

Karm

en u

nit

ASTALT

Control

Rhein

50120583M 100120583M 200120583M 400120583M10120583M

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(c)

Figure 3 Rhein ravaged the integrity of cell membrane and induced cell morphological changes (a) Lactate dehydrogenase assay was usedto evaluate the extent of rhein cytotoxicity The method was based in measuring LDH leakage in the culture medium after rhein treatmentwith various concentrations (10120583Mndash400120583M) during 24 h and 48 h Values given are the mean plusmn SD from three independent experiments(lowastlowast119875 lt 001 versus control and lowast119875 lt 005 versus control) (b) HL-7702 cells were stained with Hoechst 33258 and examined under fluorescentmicroscope (mag 400x) Nuclei chromatin margination and condensation were shown by arrows (c) HL-7702 cells were incubated with awide range of doses of rhein for 12 h and then processed for AST and ALT colorimetric assay Analysis of AST and ALT elevation was maderelative to untreated control cells (100) Data are expressed as mean plusmn SD from three independent experiments (lowast119875 lt 005 versus controland lowastlowast119875 lt 001 versus control)

cell membrane inducing apoptosis in HL-7702 cells in dose-dependent mannerThe treated cells showed strong morpho-logical alterations (nuclear shrinkage chromatin condensa-tion and intense fluorescence) Condensed chromatin couldalso be found in many treated cells which is one the classiccharacteristics of apoptotic cells

ALT and AST are cytosolic enzymes in the liver whichserve as biomarkers of hepatocyte damage that are involvedin various reactions in the liver A considerable increase in theplasma levels of these enzymes indicates liver injury [44]Theliver markers enzymes assessment showed that ALT and ASTactivities increased considerably after 12 hours of treatment


with rhein (50120583Mndash400 120583M) (Figure 3(c)) indicating livertoxicity

All these observations to a certain extent confirmedrhein exerted liver damage in vitro through ravaged integrityof cell membrane thus rhein-induced apoptosis in HL-7702cells

33 Rhein-Induced Apoptosis in Primary Human HL-7702Cells To further investigate and quantify the extent ofapoptosis in the total cell population the flow cytometricmeasurement was applied Following the incubation of HL-7702 cells with three different concentrations (25120583M 50 120583Mand 100 120583M) of rhein for 12 h the percentage of early apop-totic late apoptoticnecrotic cells was assessed Significantdifferences were observed between the control and the rhein-treated cells (Figure 4(b)) The percentage of early apoptoticcells in 25 120583M rhein-treated did not express a statisticalsignificance when compared to the control group But weobserved a statistically significant increase in 50120583M rhein-treated cells and in 100 120583M rhein-treated cells (Figures 4(a)and 4(b)) Taken together these results demonstrated thatrhein induced a dose-dependent apoptosis in HL-7702 cells

34 Rhein Altered the Regulation of Enzymes Involved in LipidMetabolism Treatment of primary human hepatic HL-7702cells with 100 120583M rhein for 12 hours revealed a significantbiosynthesis of triglycerides (TG) and total cholesterol (TC)(Figure 5(a)) An increase in their serum levels can reflectactive hepatocellular damage which is of value as markersof chronic exposure to rhein as lipid accumulation in theliver is the major hallmark of nonalcoholic fatty liver disease(NAFLD)

HGM-CoAR GPAT and ACoAC are three key enzymesinvolved in lipid regulation metabolism Their activity isconsiderably induced in liver damage After 12 h of rheinexposure the transcripts analysis showed significant statis-tical upregulation of these three enzymes in HL-7702 cells(Figure 5(b)) Thus these results indicated that toxic effectsof rhein could alter lipid regulation metabolism

35 Measurement of Mitochondrial Membrane Potential(Δ120593mit) Given that Δ120593mit is abrogated during apoptosiswe evaluated Δ120593mit dissipation using the cationic lipophilicprobe JC-1 The redgreen ration in HL-7702 cells was sig-nificantly decreased by 100120583M rhein treatment for 12 h (119875 lt001) or the 10 120583MCCCP treatment (119875 lt 005) as the resultsare shown in Figure 6 CCCP was the positive control thatcould induce mitochondria membrane depolarization Theloss of Δ120593mit was especially significantly elicited by exposureto rhein (50 120583M and 100 120583M) with comparison to control

36 Reactive Oxygen Species (ROS) and Lipid PeroxidationMitochondrion is considered to be a major site of ROSproduction that can be involved in cell death and ROS burstin mitochondrion may cause mitochondrial dysfunction asaccumulating evidences support [48] To determine whetherthis event occurs in rhein-induced apoptosis we exam-ined the intracellular production of ROS by fluorescence

spectrophotometry using DCFH-DA assay HL-7702 cellswere exposed to rhein at 25120583M 50120583M and 100 120583M for12 h The intracellular ROS production results are shown inFigure 7(a)The increase of intracellular ROS production wassignificantly elicited by exposure to 50120583M rhein and 100 120583Mrhein compared to control To further confirm that ROSacted as initiators in rhein-induced HL-7702 cells apoptosisthe cells were preincubated with 5mM NAC prior to 100 120583Mrhein for 12 h As expected the ROS scavenger significantlydecreased the level of ROS to 12167 plusmn 223

119875 lt 001

(Figure 7(a))To elucidate the effects of rhein on oxidative damage in

the HL-7702 cells to a certain extent the content ofMDAwasdeterminedMDA a secondary product of lipid peroxidationis frequently used as indictor of tissue damage [49] Theradical formation resulting in lipid peroxidation is measuredas MDA Lipid peroxidation content (MDA) in cells wassignificantly increased (2116 and 2374 resp lowast119875 lt 005 andlowastlowast

119875 lt 001) in response to rhein 50 120583M and 100 120583M for 12 hcompared to the corresponding control (Figure 7(b))

37 Effect of Rhein Treatment on the Antioxidant EnzymeIncreased activities of many antioxidant enzymes in cellreflect a defense on oxidative stress induced by drugs orenvironmental stress Thus maintaining a high antioxidantcapacity to scavenge the toxic ROS is critical for the cell life[50] SOD a scavenger of superoxide is the most importantprotective enzyme that provides the first line of enzymaticantioxidant defense against oxidative stress in the liver [51]The activities of liver antioxidant SOD (153 and 129 resplowastlowast

119875 lt 001) were significantly decreased in a dose-dependentmanner in response to rhein treatment as compared to thecorresponding control (Figure 8)These results suggested thatrhein induces oxidative damage in HL-7702 cells

38 Rhein Activated Caspases inHL-7702 Cells Despite vary-ing conditions that can lead to apoptosis caspase activationremains a universal event because the caspase family of cys-teine proteases plays an important role in apoptosis and hasbeen recognized as hallmarks of apoptosis [52] To determinewhether caspases are attributed to rhein-induced apoptosis inHL-7702 cells caspase-3 -8 and -9 activities were detectedResults showed that rhein significantly increased caspase-3 -8 and -9 activities in dose-dependent manner withmaximum activities at 100 120583M (Figure 9(a)) However thethreshold concentrations for caspase-3 -8 and -9 activationswere 25 120583M 100 120583M and 50 120583M respectively

To confirm that caspase activation is a key step inrhein-induced apoptosis HL-7702 cells were pretreated with25 120583M of Z-DEVD-FMK (caspase-3 inhibitor) Z-IETD-FMK (caspase-8 inhibitor) and Z-LETD-FMK (caspase-9inhibitor) for 1 h and then subsequently exposed to 100 120583Mrhein for 12 h As shown in Figure 9(b) caspase-9 inhibitorand caspase-3 inhibitor significantly inhibited the antiprolif-erative activity of rhein Rhein significantly triggered caspaseprotease activity in HL-7702 cells and pretreating cells withinhibitors of caspase-9 and caspase-3 respectively could leadto significant abolishing of rhein-induced caspase activity


Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)

Figure 4 Apoptosis induced by rhein (a) A representative result of flow cytometry of HL-7702 cells stained with Annexin VPI aftertreatment with rhein at concentration of 25120583M 50120583M and 100120583M for 12 h (Annexin VminusPIminus represents viable cells Annexin V+PIminusrepresents early apoptotic cells and Annexin V+PI+ represents late apoptosis or necrotic cells) (b)The experiment was repeated three timesand the percentage of early apoptotic cells (mean plusmn SD) for each treatment group is shown (lowastlowast119875 lt 001 versus control)


0

50

100

150

200

250

Triglyceride (TG)Total cholesterol (TC)

lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)

Figure 5 (a) Triglyceride (TG) and total cholesterol (TC) studies Rhein induced in vitro triglyceride and total cholesterol upregulation ina dose-dependent manner HL-7702 cells were incubated with different concentration of rhein for 12 h and then processed for TG and TCcolorimetric assay Analysis of TG and TC elevation was made relative to untreated control cells (100) Data are expressed as mean plusmn SDfrom three independent experiments (b) Quantitative analysis of HGM-CoAR ACoAC and GPAT mRNA expression levels in HL-7702cells exposed to different doses of Rhein GAPDH was used as internal positive control standard The relative expression of target genes wascalculated using 2minusΔΔCt method Data are expressed as mean plusmn SD from three different experiments (lowast119875 lt 005 versus control and lowastlowast119875 lt 001versus control)

02468

101214161820

Control Positivecontrol

Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast

Figure 6 Mitochondrial membrane potential (Δ120593mit) detection bymeasurement of JC-1 fluorescence in HL-7702 cells in a 96-wellplate format The fluorescent intensity for both J-aggregates andmonomeric forms of JC-1 was measured with a 96-well plate reader(J-aggregates excitationemission = 525590 nm JC-1 monomersexcitationemission = 490530 nm)The JC-1 redgreen fluorescenceintensity ratio was statistically significant in 100 120583M rhein treatedcells We also observed statistical significance in positive controlgroup (CCCP) Data are expressed as mean plusmn SD from threeindependent experiments (lowast119875 lt 005 lowastlowast119875 lt 001 versus controlgroup)

(119875 lt 001) whereas pretreatment with caspase-8 inhibitorcould not prevent the apoptosis induced by rhein in HL-7702 cells (119875 lt 001) Therefore based on these significantevidences rhein-induced apoptosis inHL-7702 cells could bemediated through mitochondria-dependent pathway

39 Relative mRNA Levels in Rhein-Induced Apoptotic HL-7702 Cells The induction of rhein has been shown to besignificantly involved in apoptosis by caspase-dependent

pathway Apoptosis is induced by p53 via transcription-dependent and transcription-independent processes Apop-tosis can be induced by either the intrinsic mitochondrialpathway or the extrinsic cell death receptor pathway Togain a broader understanding of molecular mechanisms forrheinrsquos biological effects in primary human liver HL-7702cells by trying to elucidate the upstream molecular eventsleading to the activation of caspase-8 and caspase-9 uponrhein stimulation gene expression analysis was carried outWe determined whether regulation of p53 PUMA Fas Cyt-c Apaf-1 and Casp-8 Casp-9 and Casp-3 was mediatedvia modulating the expression of their respective mRNAthrough quantitative real-time PCR Their specific productsgenerated by qRT-PCR were normalized with respect toGAPDH Results are summarized in Figure 10 Rhein at thethree different concentrations (25 120583M 50 120583M and 100 120583Mafter 12 h exposure) significantly increased the expression ofp53 mRNA and PUMA mRNA (Figure 10(a) 119875 lt 001)whereas it also significantly increased the expression of Apaf-1 Casp-8 -9 and -3 mRNA In the same time rhein didnot significantly change the expression of Fas mRNA andCyt-c mRNA (Figure 10(b) 119875 lt 001) Consequentlyquantitative real-time RT-PCR analysis significantly revealedand strengthened the evidences that rhein-induced apoptosisin HL-7702 cells is principally mediated through intrinsic(mitochondria) pathway

310 Effect of Rhein on Expressions of Apoptosis-RelatedProteins To further investigate the mechanism of rhein-induced apoptosis in HL-7702 cells western blot analysiswas performed to examine the protein expression levelsof Fas mitochondrial Cyt-c and Cytosolic Cyt-c in thetreated cells Immunoblots are shown in Figure 11(a) Asindicated in Figure 11(b) levels of cytosolic Cyt-c protein were


020406080

100120140160180

Control 25 50 100 NAC

ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)

Figure 7 Rhein-induced oxidative stress in HL-7702 cells (a) The cells were pretreated without or with 5mM NAC for 1 h and then treatedwith rhein for 12 h The generation of ROS was measured fluorometrically by using the fluorescent dye DFC-DA The intracellular ROSproduction significantly increased after 50 120583M rhein and 100120583M rhein NAC was significantly effective in preventing ROS production inresponse to rhein (100 120583M) Data are expressed as mean plusmn SD from three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 versus control119875 lt 001 compared to rhein (100120583M) (b) Cells exposed for 12 h to rhein were used to analyze colorimetrically the oxidative deterioration

of lipids to certain extend The intracellular lipid peroxidation activity significantly increased after 50 120583M and 100 120583M rhein exposure Datawere expressed as mean plusmn SD from three different experiments lowast119875 lt 005 lowastlowast119875 lt 001 significantly different from control values

0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)

Figure 8 Total superoxide dismutase (SOD) was assessed in HL-7702 cells exposed to rhein for 12 h Cells exposed for 12 h torhein were used to analyze colorimetrically the SOD activity Theintracellular SOD activity significantly decreased after 50 120583M and100120583M rhein exposure Data were expressed as mean plusmn SD fromthree different experiments lowastlowast119875 lt 001 significantly different fromcontrol values

significantly increased in a concentration-dependentmannerafter treatment with rhein Compared to the control theincrease was significant with 50 120583M and 100 120583M for 12 h (119875 lt001) In contrast the levels of mitochondrial Cyt-c proteinwere significantly decreased after incubation with rhein at100 120583M over a period of 12 h (119875 lt 005) Meanwhile thelevels of Fas protein did not show any significant changesThe leakage of Cytochrome-c from mitochondria into thecytosol and the no expression of Fas related protein (Figure 11)significantly strengthened and corroborated the hypothesisthat rhein-induced apoptosis in HL-7702 cells is mediatedthrough mitochondria pathway

4 Discussion

Herbal medicines have been increasingly used worldwideas they often regarded by the public as harmless remedies

for a variety of medical ailments [53] However recentlyresearchers had paid special attention to herbal hepatotoxi-city in order to insure herbal medicines safe dosage and tox-icity profile [12] Several clinical cases of Polygonum multiflo-rum-induced hepatotoxicity have been reported worldwideIn order to understand the toxicological effect and mecha-nism of Polygonummultiflorum and to investigate a potentialclinical significance for ensuring the safety of administrationherbal medicines we conducted series of experimental pro-cedures of rhein (one of the main bioactives of Polygonummultiflorum) on primary human noncarcinogenic HL-7702cells a widely used model cell line for toxicity model Thecytotoxicity studies conducted in the primary human hepaticcells after rhein exposure at different concentration for 24 hand 48 h suggested that rhein could induce inhibiting cellviability in primary human hepatic cells in dose- and time-dependent manner (Figure 2) IC

50values were calculated

as 13179 120583M and 1845 120583M at 24 h and 48 h respectivelyA 48 h acute toxicity study done by Bironaite and Ollinger[54] has found that LD

50of rhein in primary cultures of

rat hepatocytes was 20120583M Toxicity of rhein was furthertested by Mahbub et al [55] in their 24 h acute toxicity studywhere human cancer cells were treated with rhein at variousdoses (2ndash500120583M) It was found that the IC

50of rhein was

equal or more than 135 120583M suggesting that the effectivenessof this polyphenol varies depending on the leukemia celllineage (lymphoid versus myeloid) and in some cases withinthe cell lines from the same lineage [55] Our study showedthat after treating the human primary liver cells with rheinat various concentrations (012ndash370 120583M) the IC

50values

were calculated as 13179120583M and 1845 120583M at 24 h and 48 hrespectively These values were relatively lower than the onesfrom the two previous studies mentioned [54 55] suggestinga comparative corroborationwith the concentrations of rheinused in our study

Our data from the LDH release experiments (Figure 3(a))showed that with a threshold concentration of 50120583M


0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)

Caspase-3Caspase-9Caspase-8

Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()

lowastlowastlowastlowastlowastlowast

minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+

+Z-DEVD-FMKZ-IETD-FMKZ-LETD-FMK

Rhein (100120583M)

(b)

Figure 9 (a) Rhein-induced differential activation of caspase-3caspase-8 and caspase-9 in HL-7702 cells following treatment withrhein (25120583M 50 120583M and 100 120583M) for 12 h Data are expressedas mean plusmn SD from three independent experiments (lowast119875 lt 005lowastlowast

119875 lt 001 versus control group) (b) Effect of caspase inhibitors onapoptosis in HL-7702 cells following treatment with rhein (100 120583M)for 12 h Data are expressed as mean plusmn SD from three independentexperiments lowastlowast119875 lt 001 compared to rhein (100120583M) treated cells

rhein ravaged the integrity of the cytomembrane causingapparent LDH leakage Characteristicmorphological featuresor alterations such as cell shrinkage chromatin condensa-tion and formation of apoptotic bodies are associated withapoptotic cells To explore the apoptosis-inducing effect ofrhein we carried out an observation of the cell outline andnucleus using a light and fluorescence microscopy and cameto the significant results that HL-7702 cells treated withrhein exhibited the typical apoptotic morphological features(Figure 3(b)) ALT and AST are cytosolic enzymes mainlyfound in the liver Their levels are valuable aid primarily inthe diagnosis of liver disease it can be used in combina-tion with other enzymes to monitor the courses of variousliver disorders as they are also biological catalysts Theiroverconcentration in hepatocytes is quite a histopathologicalindicator of hepatocellular injury [56] Figure 3(c) suggesteda hepatocellular injury thus asserting the fact that rheincould induce in vitro toxicity in primary human hepaticcells by destroying the cytomembrane These aforemen-tioned apoptotic morphological features were more evident

as we carried out a flow cytometry assay The results of theexperiment have indicated that rhein has dose-dependenttoxic effects on HL-7702 cells and were consistent with theprevious observations of changes in cellular ultrastructureconfirming 50 120583M as an apparent threshold concentration ofrhein-inducing cytotoxicity in HL-7702 cells (Figure 4(b))

The liver plays a key role in regulation of whole bodylipid thus lipid deposition in the liver is associated withmetabolic disorders including fatty liver disease type IIdiabetes and hepatocellular cancer [57] In order to broadenour understanding to the investigation in the mechanismsinvolved in rhein-induced apoptosis in the primary humanhepatic cells we investigated the behavior of some lipid andrelevant genes as lipid metabolism plays important role inthe life activities Isolated hepatocytes undergo lipoapoptosisa feature of hepatic lipotoxicity on treatment with saturatedfree fatty acids [58] Lipoapoptosis occurring due to an excessof saturated free fatty acids is a key pathogenic event inthe initiation of nonalcoholic fatty liver disease Althoughcholesterol plays a vital role in regulating physical propertiesof membranes [59] its accumulation in cells is toxic andcauses fatal diseases such as Niemann Pick type C diseaseswhich is a fatal neurodegenerative disease and the secondmost common cause of neonatal cholestasis characterizedby lysosomal storage of cholesterol and glycosphingolipids[60] Moreover elevated levels of cholesterol and triglyc-erides (Figure 5(a)) have been linked to liver diseases astriglyceride deposition within the hepatocyte is the hallmarkof both alcoholic and nonalcoholic fatty liver diseases [61]HMG-CoAR transcripts are highly enriched in liver cells(hepatocytes) where cholesterol is converted into bile saltsand where lipoproteins involved in transporting cholesterolare synthesized and exported GPAT is involved in the firststep in glycerolipid synthesis and is localized in both theendoplasmic reticulum andmitochondriaMoreoverACoACcatalyzes the formation of malonyl-CoA which in turn isutilized by the fatty acid synthetase complex for the de novosynthesis of fatty acids HMG-CoAR GPAT and ACoACare commonly overexpressed or overactivated in diseasesstates associated with fatty liver or liver damaged [62ndash65]In the present study the upregulation of genes involved inlipid synthesis in rhein treated HL-7702 hepatic cells suchas HMG-CoAR ACoAC and GPAT (Figure 5(b)) couldlead to increased glycerolipid and cholesterol biosynthesisExperimental studies have shown higher lipid levels afterexposure to Polygonum multiflorum impaired normal cellsignaling and causing cellular dysfunction [66 67] Based onthe aforementioned results the findings suggested that lipidoverload and fatty degeneration could be involved in rhein-induced cell death

Mitochondria as dynamic organelles have a crucial rolein maintaining both cellular bioenergetics and regulatingsignaling pathways to meet the high energy demands inthe cells Thus any alterations to the mitochondrial home-ostasis will lead to loss of integrity or damage resulting toapoptosis [68] While mitochondria consume oxygen andsubstrates to generate ATP they produce reactive oxygenspecies in the process In mitochondria Cytochrome-c isrequired as an electron carrier in oxidative phosphorylation


005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M

lowastlowast lowastlowast


lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control

Cyt-cApaf-1Caspase-8

Caspase-9Caspase-3

Rhein

lowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)

Figure 10 Quantitative analysis of (a) p53 PUMA and Fas and (b) Cyt-c Apaf-1 Casp-8 Casp-9 and Casp-3 mRNA expression levels inHL-7702 cells exposed to different doses of rhein GAPDHwas used as an internal positive control standardThe relative expression of targetgenes was calculated using 2minusΔΔCt method Data were expressed as mean plusmn SD from three different experiments lowast119875 lt 005 lowastlowast119875 lt 001significantly different from control values

and shuttles electrons from one complex (Complex III)to another (Complex IV) however the electron transportbetween these two complexes generates a proton gradientacross the inner mitochondrial membrane which maintainsΔ120593mit [69] Cytochrome-c release from mitochondria is akey step of apoptosis [70] The mitochondrial dysfunctionfeatures including loss of mitochondrial membrane potential(Δ120593mit) and leakage of Cytochrome-c from the mitochon-drion into the cytosol were also investigated in our studyIn this study the significant loss of Δ120593mit (Figure 6) andthe translocation of Cytochrome-c (Figures 11(a) and 11(b))were significantly observed in rhein-treated HL-7702 cellsMoreover excessively production of ROS (Figure 7(a)) mayalter the mitochondria membrane [48] causing a disruptionof Δ120593mit and the release of Cytochrome-c [71] that inturn triggers mitochondrial membrane permeability andapoptosis By immunofluorescence staining we found therelease of Cytochrome-c from mitochondria (Figure 11(a))These findings suggested that the cascade reactions of ROSproduction lipid peroxidation loss of Δ120593mit and releaseof Cytochrome-c from mitochondria may play an involvedrole in rhein-induced apoptosis in HL-7702 cells in a dose-dependent manner

Oxidative stress and liver injury are strongly associatedOxidative stress in the liver can be triggered during differentconditions and by specific etiologies including hepatotoxinssuch as rhein Oxidative stress is a state of imbalancebetween the production of reactive oxygen species (ROS)and the cellular antioxidant defense neutralizing the reactiveintermediates and triggering damage At a physiological levelmitochondria are primary source of ROS Moreover ROS isinvolved in regulation of the intracellular signaling pathwaysas ldquoredox messengerrdquo whereas excessive production of ROScan lead to lipid peroxidation mitochondrial oxidative stressand DNA damage inducing oxidative modification of cellu-lar macromolecules inhibit protein function and promoteapoptotic cell death [48 72ndash74] Meanwhile experimentsshowed that ROS act upstream of mitochondrial membrane

depolarization Cytochrome-c release execution of caspaseactivation and nuclear fragmentation [75] In our currentstudy we observed the significant excess production ofROS in HL-7702 cells especially with rhein at 50120583M and100 120583M (Figure 7(a)) Pretreatment with ROS scavengerNAC could impressively reverse the action triggered by100 120583M rhein (Figure 7(a)) In a comparative study of threeanthraquinones (rhein danthron and chrysophanol) doneon primary cultures of rat hepatocytes [36] only rhein at50 120583M was found to be the most effective in producing freeradicals and was the only tested compound that could induceapoptosis The in vitro doses of rhein used in our study andthe pharmacological outcome (apoptosis) observed here inHL-7702 cells were significantly in accordance with previousstudies [36] Rhein which contains one carboxyl group issuitable for one-electron-reducing enzymes and an effectiveredox cycler which leads to the production of oxygen-derivedfree radicals that eventually induced apoptotic cell death[36] Lipid peroxidation is one of the main manifestations ofoxidative damage initiated by ROS and it has been linked tothe alteredmembrane structure and enzyme inactivation andexcessive damage leading to cell death [76] In our study (asshown in Figure 7(b)) rhein may facilitate these deleteriouseffects by promoting the lipid peroxidation process thusincreasing the formation of MDA The amount of MDA wassignificantly high in cells treated with rhein at 50120583M (lowast119875 lt005) and 100 120583M (lowastlowast119875 lt 001) Cells maintain a variety ofdefenses in response to oxidative stress through the inductionof antioxidant enzymes with SOD being one of the mostimportant endogenous enzymatic antioxidants In our studySOD activity was significantly decreased by treatment withrhein (from 50 120583M) at 12 h (Figure 8) Previous studies doneon HL-7702 cell suggested that oxidative stress is associatedwith apoptosis in this same cell line [77ndash79] Furthermorea study of rheinrsquos metabolism done in primary culturesrat hepatocytes caused production of oxygen-derived freeradicals by redox cycling initiation of lipid peroxidationwhich eventually led to cell death [54] The activities of ROS


Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)

FasCytosolic Cyt-cMitochondrial Cyt-c

50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)

Figure 11 Western blot analysis of Fas and mitochondrialcytosolicCytochrome-c protein expression levels in HL-7702 cells exposedto different doses of rhein (a) Representative results of westernblotting analysis 120573-actin served as a loading control (b) Thequantification of the immunoblots was analyzed by densitometricscanning Band densities were digitized and relative band intensitiesof target proteins were normalized against the 120573-actin levels Datawere expressed as mean plusmn SD from three different experimentslowast

119875 lt 005 lowastlowast119875 lt 001 significantly different from control values

superoxide dismutase and lipid peroxidation serve as reliableindicators of oxidative damage In our study these resultsindicated that treatment with rhein induced increase in ROSand MDA and loss in activity of SOD which resulted inoxidative stress and the concentration-dependent increasesof apoptotic cells NAC treatment was able to ameliorate theoxidative stress Based on the evidences reported above thereis a reason to speculate that oxidative stress could be involvedin rhein-induced apoptosis in HL-7702 cells and these resultsare consistent with the ROS-mediated toxicity of medicinalherbs in the same cell line

The p53 network mediates cellular responses to diverseforms of stress (eg DNA damage oncogene activation andhypoxia) Functioning primarily as a transcription factor p53regulates the expression of genes involved in cell cycle arrestDNA repair senescence and apoptosis [80] It can regulatethe intrinsic mitochondrial-mediated apoptotic pathway andthe extrinsic apoptotic pathway In the intrinsic mitochon-drial pathway p53 induces transcription of several genessuch as PUMA (p53 upregulated modulator of apoptosis)which has an expression pattern consistent with a causativerole in p53-dependent apoptosis [81] Upon p53 activationresulting in functional PUMA mRNA accumulation con-firmed the results of our RT-qPCR analyses (Figure 10(a))These observations in correlation with the results of otherin vitro studies [80] suggested that PUMA a major effectorof p53-mediated cell death may play an important rolein in vitro regulator of apoptosis when it overexpressedMoreover the results of the overexpression of p53 mRNA andPUMA mRNA (Figure 10(a)) taken together corroboratedthe findings expressed by Tsai and Barton [82] suggestingapparently that the branch of apoptosis put in evidence herewas the intrinsic mitochondrial pathway Following cellularinsults that cause DNA damage such as ionizing radiation(IR) ultraviolet (UV) radiation and oxidative stress maylead to p53 upregulation which afterward this cascade ofevents will eventually lead to apoptosis [77]

In the intrinsic mitochondrial pathway p53 inducesPUMA and as a result we assist to the mitochondrial mem-brane depolarization causing the release of Cytochrome-cAfter release from mitochondria Cytochrome-c binds toApaf-1 which additionally is a direct target of p53-regulatedactivation of transcription Afterwards there is execution ofcaspase activation The caspase family of cysteine proteasesplays an important role in apoptosis therefore the caspaseactivation is considered as an apoptotic marker Moreoverthe apoptosis can be either intrinsic pathway involvingmitochondrial injury and caspase-9 activation or extrin-sic pathway due to FasFasL receptor-mediated caspase-8activation both consequently leading to the activation ofcaspase-3 (known as executor of cell death) In our studyto our surprise caspase-3 caspase-8 and caspase-9 wereall activated in rhein-treated HL-7702 cells by activationanalysis (Figure 9(a)) Additionally caspase-9 or caspase-3 inhibitor could stop rhein-inducing apoptotic effect (Fig-ure 9(b)) suggesting that rhein is able to induce apoptosisin HL-7702 cells through mitochondria-mediated pathwayThe activation of caspase-9 resulted from the leakage ofCytochrome-c and then the activated caspase-9 could induceactivation of caspase-3 The RT-qPCR analysis showed a sig-nificant upregulation of Apaf-1 and Casp-9 and -3 transcripts(Figure 10(b)) confirming the intrinsic apoptotic pathwayHowever to our surprise even though the expression ofFas from the RT-qPCR (Figure 10(a)) and the western blotsanalysis (Figure 11(b)) did not reveal any significant changesbut caspase-8 belonging to the death receptor apoptoticpathway was significantly activated (Figure 9(a)) This couldbe explained as scientific evidences suggested that caspase-8 can be activated in the early stage as an initiator caspaseby activated caspase-3 [83 84] The activation of Apaf-1


Table 2 Conversion of animal doses to HED based on BSA

Species Weight (kg) BSA (m2) 119870119898factor

HumanAdult 60 16 37Child 20 08 25

Baboon 12 06 20Dog 10 05 20Monkey 3 024 12Rabbit 18 015 12Guinea pig 04 005 8Rat 015 0025 6Hamster 008 002 5Mouse 002 0007 3Values based on data from FDA Draft Guidelines [84]HED human equivalent dose BSA body surface areaTo convert dose in mgkg to dose in mgm2 multiply by 119870

119898value

resulted from the leakage of Cytochrome-c triggering theupregulation of caspace-9 and then the activated caspase-9could induce activation of caspase-3 (Figure 10(b)) There-fore the present results suggested that the activation ofcaspase-8 might be induced by the activated caspase-3 andstrengthening the confirmation that based on the presentaforementioned results the mechanism of rhein-inducedapoptosis in primary human liver HL-7702 cells is throughintrinsic mitochondria- mediated pathway

Pharmacokinetics analyses have been conducted to inves-tigate the rational clinical dose of rhein from herbal formulasSeveral pharmacokinetics studies done on rats after oraladministration of rhein at 70mgsdotkgminus1 [85] 119mgsdotkgminus1 [86]and 70mgsdotkgminus1 [87] have led to analyze rheinrsquos pharma-cokinetics parameters to a certain extent suggesting thatrhein has a rapid absorption and a slow elimination Forcalculations of determining the starting dose in humansas extrapolated from animals scientists have used normal-ization of body surface area (BSA) method [88] and theconversation of animals dose to human equivalent dosesbased on BSA [89] respectively shown in (1) and Table 2 asfollows

Formula for dose translation based on BSA [83]

HED (mgkg) = Animal dose (mgkg)Animal 119870

119898

Human 119870119898

(1)

where HED is human equivalent dose and BSA is bodysurface area Clinical studies done in human by Tan etal [90] Zhu et al [91] Jiang et al [92] and Hao et al[87] demonstrated that linear pharmacokinetics for rhein inChinese healthy patients after a single oral administration isin the range of 50ndash200mg (equivalent to 17 and 67 times ofthe upper dose of human stipulated in China Pharmacopoeia(05 gsdotkgminus1)) [93] and enlightened that the pharmacokineticsparameters of rhein as a single compound are significantlydifferent from those of rhein as a compound in a herbal plantor formula Further the clearance scaling of in vivo-in vitroin the same species approach has been shown to be successful

for in vivo-in vitro data of rhein using a physiological basedpharmacokinetic (PBPK) model [87] Based on the resultsof these toxicity studies there is a clear understanding ofthe pharmacokinetic behavior of rhein as a single drug andas a component of herbal formulae leading us to furtherinvestigating the extrapolated dosed in various clinical studymodels

5 Conclusion

To date the hepatotoxicity reported fromherbalmedicines orrelated herbal bioactive component still remains an impor-tant issue to address for drug safety in clinical applicationIn this present study we demonstrated that rhein is ableto decrease primary human hepatic HL-7702 cells viabilityRhein exerted toxicological effects in HL-7702 cells thatcould be done viamitochondria-mediated pathway in a dose-dependent manner The apoptosis induced by rhein (50 120583Mand 100120583M for 12 h) in HL-7702 cells is associated withseveral morphological changes and biochemical signals suchas the following (i) oxygen radicals that can affect the perme-ability and potential of the inner mitochondrial membrane(ii) leakage of Lactate dehydrogenase (iii) overproductionof ROS lipid peroxidation loss in activities of SOD (iv)dysregulation of enzymes involved in lipid metabolism (v)loss of Δ120593mit leaking Cytochrome-c from mitochondria intocytosol and (vi) subsequently enhancing PUMA Apaf-1 andcaspase-9 and -3 activities Based on all evidences reportedabove these findings provide a mechanistic explanationfor the hepatotoxicity of rhein in drug-induced oxidativeliver injury from herbal medicine To our knowledge thisis the first report of rhein-induced apoptosis in primaryhuman hepatic HL-7702 cells The findings of our studyare conducive to further conduct both in vitro and in vivopharmacokinetics studies with primary human HL-7702liver cells as hepatic support system in order to investigateand ensure the clinical administration safety of Polygonummultiflorum or associated herbs containing rhein and othermain phytochemical compounds andmoreover to investigatevarious signaling involved in herbal hepatotoxicity

Abbreviations

PMT Polygonum multiflorumThunbHL-7702 cells Primary human liver cellsDILI Drug-induced liver injuryPCD Programmed cell deathPS PhosphatidylserineUGT Uridine diphosphate

glucuronosyltransferaseALF Acute liver failureALT Alanine aminotransferaseAST Aspartate aminotransferaseTG TriglycerideTC Total cholesterolPMSF Phenylmethylsulfonyl fluorideNAC N-Acetyl-L-cysteineMDA Lipid peroxidationSOD Total superoxide dismutase


HMG-CoAR Hydroxymethylglutaryl coenzyme Areductase

ACoAC Acetyl-CoA carboxylaseG3P Glycerol-3-phosphateLPA Lysophosphatidic acidGPAT Glycerol-3-phosphate acyltransferaseΔ120593mit Mitochondrial membrane potentialCCCP Carbonyl cyanide

m-chlorophenylhydrazoneDCFH-DA Dichloro-dihydro-fluorescein diacetateROS Reactive oxygen speciesGAPDH Glyceraldehyde-3-phosphate

dehydrogenase

Conflict of Interests

The authors declare that they have no conflict of interests todisclose

Authorsrsquo Contribution

Guy-Armel Bounda and Wang Zhou contributed to thestudyrsquos design performed the experiments the interpretationof data and wrote the paper Wang Zhou contributed withthe technical handling of cell culture Guy-Armel Boundacontributed to the critical revision of the paper Dan-danWang contributed with technical assistance in ROS andmitochondrial membrane potential experiments Feng Yucontributed to the concept design and supervision of thestudy All the authors read and approved the final version ofthe paper

References

[1] Z Zhang Q Q Zhuang and M Z Mei ldquoEffect of some drugson plasma and liver lipoprotein lipase activities and plasmacholesterol levels in ratsrdquo Yao Xue Xue Bao vol 18 no 6 pp468ndash471 1983

[2] P-G Xiao S-T Xing and L-WWang ldquoImmunological aspectsof Chinese medicinal plants as antiageing drugsrdquo Journal ofEthnopharmacology vol 38 no 2-3 pp 167ndash175 1993

[3] T C Kuo J C Ou J Tsai C L Wu and C M Sun ldquoEvaluationof Chinese herbs that affect the cell-mediated immunity (II)rdquoJournal of Chinese Medicine vol 7 no 2 pp 119ndash131 1996

[4] J K H Kam ldquoMutagenic activity of Ho Shao Wu (Polygonummultiflorum Thunb)rdquo American Journal of Chinese Medicinevol 9 no 3 pp 213ndash215 1981

[5] Z Liu Y Liu Z Chao Z Song C Wang and A Lu ldquoIn vitroantioxidant activities ofMaillard reaction products produced inthe steaming process of Polygonum multiflorum rootrdquo NaturalProduct Communications vol 6 no 1 pp 55ndash58 2011

[6] R Wang Y Tang B Feng et al ldquoChanges in hippocampalsynapses and learning-memory abilities in age-increasing ratsand effects of tetrahydroxystilbene glucoside in aged ratsrdquoNeuroscience vol 149 no 4 pp 739ndash746 2007

[7] L L Zhang L H Huang L W Chen D J Hao and J ZChen ldquoNeuroprotection by tetrahydroxystilbene glucoside inthe MPTP mouse model of Parkinsonrsquos diseaserdquo ToxicologyLetters vol 222 no 2 pp 155ndash163 2013

[8] B-H Lee Y-Y Huang P-D Duh and S-C Wu ldquoHepatopro-tection of emodin and Polygonum multiflorum against CCl4-induced liver injuryrdquo Pharmaceutical Biology vol 50 no 3 pp351ndash359 2012

[9] L-C Lin S M Nalawade V Mulabagal M-S Yeh and H-S Tsay ldquoMicropropagation of Polygonum multiflorum Thunband quantitative analysis of the anthraquinones emodin andphyscion formed in in vitro propagated shoots and plantsrdquoBiological and Pharmaceutical Bulletin vol 26 no 10 pp 1467ndash1471 2003

[10] T Yi K S Y Leung G-H Lu H Zhang and K ChanldquoIdentification and determination of the major constituentsin traditional Chinese medicinal plant Polygonum multiflorumThunbbyHPLC coupledwith PADandESIMSrdquoPhytochemicalAnalysis vol 18 no 3 pp 181ndash187 2007

[11] G J-H Park S P Mann and M C Ngu ldquoAcute hepatitisinduced by Shou-Wu-Pian a herbal product derived fromPolygonummultiflorumrdquo Journal of Gastroenterology and Hepa-tology vol 16 no 1 pp 115ndash117 2001

[12] K A Jung H J Min S S Yoo et al ldquoDrug-induced liverinjury twenty five cases of acute hepatitis following ingestionof Polygonum multiflorum thunbrdquo Gut and Liver vol 5 no 4pp 493ndash499 2011

[13] L Dini ldquoClearance of apoptotic lymphocytes by humanKupffercells Phagocytosis of apoptotic cells in the liver role of lectinreceptors and therapeutic advantagesrdquo inApoptosis and ItsMod-ulation by Drugs R G Cameron and G Feuer Eds Handbookof Experimental Pharmacology pp 319ndash341 Springer BerlinGermany 2000

[14] M G Neuman ldquoApoptosis in diseases of the liverrdquo CriticalReviews in Clinical Laboratory Sciences vol 38 no 2 pp 109ndash166 2001

[15] L Dini L Falasca A Lentini et al ldquoGalactose-specific receptormodulation related to the onset of apoptosis in rat liverrdquoEuropean Journal of Cell Biology vol 61 no 2 pp 329ndash337 1993

[16] Y Ossawa E Seki and D A Brenner ldquoApoptosis in liver injuryand liver diseasesrdquo in Essentials of Apoptosis Z Dong and X MYin Eds pp 547ndash564 Humana Press 2009

[17] D Schrenk H-J Schmitz S Bohnenberger B Wagner andW Worner ldquoTumor promoters as inhibitors of apoptosis inrat hepatocytesrdquo Toxicology Letters vol 149 no 1ndash3 pp 43ndash502004

[18] T-Y Wu C-P Chen and T-R Jinn ldquoTraditional Chinesemedicines and Alzheimerrsquos diseaserdquo Taiwanese Journal ofObstetrics and Gynecology vol 50 no 2 pp 131ndash135 2011

[19] P LiuMW Kong S H Yuan J Liu and PWang ldquoHistory andexperience a survey of traditional Chinese medicine treatmentfor Alzheimerrsquos diseaserdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 642128 5 pages 2014

[20] K A OrsquoBrien ldquoAlternative perspectives how Chinese medicineunderstands hypercholesterolemiardquo Cholesterol vol 2010 Arti-cle ID 723289 9 pages 2010

[21] Y F Yang ldquoHerbs that tonifyrdquo in Chinese Herbal MedicineComparisons and Characteristics Y F Yang Ed pp 157ndash185Elsevier 2nd edition 2010

[22] V Scheid D Bensky R Barolet et al ldquoFormulas that tonifyrdquoin Chinese Herbal Medicine Formulas and Strategies V ScheidD Bensky A Ellis and R Barolet Eds pp 307ndash418 EastlandPress Seattle Wash USA 2nd edition 2009

[23] B Singh J R Nadkarni R A Vishwakarma S B BharateM Nivsarkar and S Anandjiwala ldquoThe hydroalcoholic extract


of Cassia alata (Linn) leaves and its major compound rheinexhibits antiallergic activity via mast cell stabilization andlipoxygenase inhibitionrdquo Journal of Ethnopharmacology vol141 no 1 pp 469ndash473 2012

[24] S K Agarwal S S Singh S Verma and S Kumar ldquoAntifungalactivity of anthraquinone derivatives from Rheum emodirdquoJournal of Ethnopharmacology vol 72 no 1-2 pp 43ndash46 2000

[25] C Jong-Chol M Tsukasa A Kazuo K Hiroaki Y Haruki andO Yasuo ldquoAnti-Bacteroides fragilis substance from rhubarbrdquoJournal of Ethnopharmacology vol 19 no 3 pp 279ndash283 1987

[26] D L Barnard J H Huffman J L B Morris S G Wood B GHughes and R W Sidwell ldquoEvaluation of the antiviral activityof anthraquinones anthrones and anthraquinone derivativesagainst human cytomegalovirusrdquo Antiviral Research vol 17 no1 pp 63ndash77 1992

[27] A Delpino M G Paggi P F Gentile et al ldquoProtein syntheticactivity and adenylate energy charge in Rhein-treated culturedhuman glioma cellsrdquoCancer Biochemistry Biophysics vol 12 no4 pp 241ndash252 1992

[28] S Castiglione M Fanciulli T Bruno et al ldquoRhein inhibitsglucose uptake in Ehrlich ascites tumor cells by alteration ofmembrane-associated functionsrdquoAnti-Cancer Drugs vol 4 no3 pp 407ndash414 1993

[29] S LinM Fujii andD-XHou ldquoRhein induces apoptosis inHL-60 cells via reactive oxygen species-independent mitochondrialdeath pathwayrdquoArchives of Biochemistry andBiophysics vol 418no 2 pp 99ndash107 2003

[30] Q Du X-L Bian X-L Xu B Zhu B Yu and Q Zhai ldquoRole ofmitochondrial permeability transition in human hepatocellularcarcinomaHep-G2 cell death induced by rheinrdquoFitoterapia vol91 pp 68ndash73 2013

[31] M A Davis ldquoIntroductionrdquo in Apoptosis Methods in Phar-macology and Toxicology Approaches to Measurements andQuantitation M A Davis Ed pp 1ndash9 Humana Press TotowaNJ USA 2002

[32] A H Wyllie J F R Kerr and A R Currie ldquoCell death thesignificance of apoptosisrdquo International Review of Cytology vol68 pp 251ndash306 1980

[33] M D Jacobson M Weil and M C Raff ldquoProgrammed celldeath in animal developmentrdquo Cell vol 88 no 3 pp 347ndash3541997

[34] G Kasof K Degenhardt D Perez T Anju and E WhiteldquoOverview a matter of life and deathrdquo in Signaling Pathways inApoptosis D Watters and M Lavin Eds pp 1ndash30 HarwoodAcademic Publishers Herston Australia 1999

[35] S Fulda and K-M Debatin ldquoExtrinsic versus intrinsic apop-tosis pathways in anticancer chemotherapyrdquo Oncogene vol 25no 34 pp 4798ndash4811 2006

[36] K Kagedal D Bironalte and K Ollinger ldquoAnthraquinone cyto-toxicity and apoptosis in primary cultures of rat hepatocytesrdquoFree Radical Research vol 31 no 5 pp 419ndash428 1999

[37] R E Schwartz H E Fleming S R Khetani and S NBhatia ldquoPluripotent stem cell-derived hepatocyte-like cellsrdquoBiotechnology Advances vol 32 no 2 pp 504ndash513 2014

[38] E L LeCluyse ldquoHuman hepatocyte culture systems for the invitro evaluation of cytochrome P450 expression and regula-tionrdquo European Journal of Pharmaceutical Sciences vol 13 no4 pp 343ndash368 2001

[39] J Luo H Zhou F Wang et al ldquoThe hepatitis B virus X proteindownregulates NF-120581B signaling pathways through decreasingthe Notch signaling pathway in HBx-transformed L02 cellsrdquo

International Journal of Oncology vol 42 no 5 pp 1636ndash16432013

[40] N Yang L Chen M-K Yang et al ldquoIn vitro study ofthe interactions of galactosylated thermo-responsive hydrogelswith cellsrdquo Carbohydrate Polymers vol 88 no 2 pp 509ndash5162012

[41] X Hu T Yang C Li et al ldquoHuman fetal hepatocyte line L-02exhibits good liver function in vitro and in an acute liver failuremodelrdquo Transplantation Proceedings vol 45 no 2 pp 695ndash7002013

[42] M Ma Z Z Jiang J Ruan et al ldquoThe furano norclerodanediterpenoid disobulbin-D induces apoptosis in normal humanliver L-02 cellsrdquoExperimental and Toxicologic Pathology vol 64no 6 pp 611ndash618 2012

[43] X-M Chen J Liu T Wang and J Shang ldquoColchicine-inducedapoptosis in human normal liver L-02 cells by mitochondrialmediated pathwaysrdquo Toxicology in Vitro vol 26 no 5 pp 649ndash655 2012

[44] D-Y Shi F-Z Xie C Zhai J S Stern Y Liu and S-L Liu ldquoTherole of cellular oxidative stress in regulating glycolysis energymetabolism in hepatoma cellsrdquoMolecular Cancer vol 8 article32 2009

[45] J Hu H Ma W Zhang Z Yu G Sheng and J Fu ldquoEffectsof benzene and its metabolites on global DNA methylation inhuman normal hepatic L02 cellsrdquo Environmental Toxicologyvol 29 no 1 pp 108ndash116 2014

[46] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[47] A Cossarizza M Baccarani-Contri G Kalashnikova andC Franceschi ldquoA new method for the cytofluorometricanalysis of mitochondrial membrane potential using theJ-aggregate forming lipophilic cation 551015840661015840-tetrachloro-111015840331015840-tetraethylbenzimidazolcarbocyanine iodide (JC-1)rdquoBiochemical and Biophysical Research Communications vol 197no 1 pp 40ndash45 1993

[48] C Fleury B Mignotte and J-L Vayssiere ldquoMitochondrialreactive oxygen species in cell death signalingrdquo Biochimie vol84 no 2-3 pp 131ndash141 2002

[49] S Sreelatha P R Padma andMUmadevi ldquoProtective effects ofCoriandrum sativum extracts on carbon tetrachloride-inducedhepatotoxicity in ratsrdquo Food and Chemical Toxicology vol 47no 4 pp 702ndash708 2009

[50] S Ghosh S Mitra and A Paul ldquoPhysiochemical studies ofSodium Chloride onMungbean (Vigna radiate L Wilczek) andits possible recovery with Spermine and Gibberellic Acidrdquo TheScientific World Journal vol 2015 Article ID 858016 8 pages2015

[51] S K Powers J C Quindry and A N Kavazis ldquoExercise-induced cardioprotection against myocardial ischemia-reperfusion injuryrdquo Free Radical Biology and Medicine vol 44no 2 pp 193ndash201 2008

[52] Z-Q Jiang X-J Yan L Bi J-P Chen Q Zhou andW-P ChenldquoMechanism for hepato-protective action of Liangxue HuayuRecipe (LHR) blockade of mitochondrial cytochrome c releaseand caspase activationrdquo Journal of Ethnopharmacology vol 148no 3 pp 851ndash860 2013

[53] M Furukawa S Kasajima Y Nakamura et al ldquoToxic hepatitisinduced by Show-Wu-Pian a Chinese herbal preparationrdquoInternal Medicine vol 49 no 15 pp 1537ndash1540 2010


[54] D Bironaite and K Ollinger ldquoThe hepatotoxicity of rheininvolves impairment of mitochondrial functionsrdquo Chemico-Biological Interactions vol 103 no 1 pp 35ndash50 1997

[55] A A Mahbub C L Le Maitre S L Haywood-Small G JMcDougall N A Cross and N Jordan-Mahy ldquoDifferentialeffects of polyphenols on proliferation and apoptosis in humanmyeloid and lymphoid leukemia cell linesrdquo Anti-Cancer Agentsin Medicinal Chemistry vol 13 no 10 pp 1601ndash1613 2013

[56] R Rej ldquoAspartate aminotransferase activity and isoenzymeproportions in human liver tissuesrdquo Clinical Chemistry vol 24no 11 pp 1971ndash1979 1978

[57] J D Y Chow R T Lawrence M E Healy et al ldquoGenetic inhi-bition of hepatic acetyl-CoA carboxylase activity increases liverfat and alters global protein acetylationrdquoMolecular Metabolismvol 3 no 4 pp 419ndash431 2014

[58] K Kakisaka S C Cazanave C D Fingas et al ldquoMechanismsof lysophosphatidylcholine-induced hepatocyte lipoapoptosisrdquoThe American Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 302 no 1 pp G77ndashG84 2012

[59] F R Maxfield and I Tabas ldquoRole of cholesterol and lipidorganization in diseaserdquoNature vol 438 no 7068 pp 612ndash6212005

[60] E P Beltroy J A Richardson J D Horton S D Turley and JM Dietschy ldquoCholesterol accumulation and liver cell death inmice with Niemann-Pick type C diseaserdquo Hepatology vol 42no 4 pp 886ndash893 2005

[61] H Tsukamoto H She S Hazra J Cheng and J Wang ldquoFatparadox of steatohepatitisrdquo Journal of Gastroenterology andHepatology vol 23 supplement 1 pp S104ndashS107 2008

[62] D F Calvisi C Wang C Ho et al ldquoIncreased lipogenesisinduced by AKT-mTORC1-RPS6 signaling promotes develop-ment of human hepatocellular carcinomardquo Gastroenterologyvol 140 no 3 pp 1071ndash1083 2011

[63] N Yahagi H Shimano K Hasegawa et al ldquoCo-ordinateactivation of lipogenic enzymes in hepatocellular carcinomardquoEuropean Journal of Cancer vol 41 no 9 pp 1316ndash1322 2005

[64] C A Nagle J An M Shiota et al ldquoHepatic overexpression ofglycerol-sn-3-phosphate acyltransferase 1 in rats causes insulinresistancerdquo Journal of Biological Chemistry vol 282 no 20 pp14807ndash14815 2007

[65] K R Feingold M H Wiley A H Moser and M D SipersteinldquoAltered activation state of hydroxymethylglutaryl-coenzymeA reductase in liver tumorsrdquo Archives of Biochemistry andBiophysics vol 226 no 1 pp 231ndash241 1983

[66] N Li Z Chen X Mao J Yu and R Zhao ldquoEffects of lipidregulation using raw and processed radix polygoni multifloriin rats fed a high-fat dietrdquo Evidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 329171 10 pages 2012

[67] W Wang Y He P Lin et al ldquoIn vitro effects of active compo-nents of Polygonum multiflorum radix on enzymes involved inthe lipid metabolismrdquo Journal of Ethnopharmacology vol 153no 3 pp 763ndash770 2014

[68] S Desagher and J-C Martinou ldquoMitochondria as the centralcontrol point of apoptosisrdquo Trends in Cell Biology vol 10 no 9pp 369ndash377 2000

[69] Y Hatefi ldquoThe mitochondrial electron transport and oxidativephosphorylation systemrdquo Annual Review of Biochemistry vol54 pp 1015ndash1069 1985

[70] M Ott J D Robertson V Gogvadze B Zhivotovsky and SOrrenius ldquoCytochrome c release from mitochondria proceedsby a two-step processrdquo Proceedings of the National Academy of

Sciences of the United States of America vol 99 no 3 pp 1259ndash1263 2002

[71] M Ott V Gogvadze S Orrenius and B Zhivotovsky ldquoMito-chondria oxidative stress and cell deathrdquo Apoptosis vol 12 no5 pp 913ndash922 2007

[72] H L Xu X F Yu S C Qu X R Qu Y F Jiang and D Y SuildquoJuglone from Juglans mandshruica Maxim inhibits growthand induces apoptosis in human leukemia cell HL-60 througha reactive oxygen species-dependent mechanismrdquo Food andChemical Toxicology vol 50 no 3-4 pp 590ndash596 2012

[73] H Raza A John and S Benedict ldquoAcetylsalicylic acid-inducedoxidative stress cell cycle arrest apoptosis and mitochondrialdysfunction in human hepatoma HepG2 cellsrdquo European Jour-nal of Pharmacology vol 668 no 1-2 pp 15ndash24 2011

[74] H Wiseman and B Halliwell ldquoDamage to DNA by reactiveoxygen and nitrogen species role in inflammatory disease andprogression to cancerrdquo Biochemical Journal vol 313 no 1 pp17ndash29 1996

[75] H Fang Y Wu J Guo et al ldquoT-2 toxin induces apoptosis indifferentiated murine embryonic stem cells through reactiveoxygen species-mediated mitochondrial pathwayrdquo Apoptosisvol 17 no 8 pp 895ndash907 2012

[76] A Goel V Dani and D K Dhawan ldquoProtective effects ofzinc on lipid peroxidation antioxidant enzymes and hepatichistoarchitecture in chlorpyrifos-induced toxicityrdquo Chemico-Biological Interactions vol 156 no 2-3 pp 131ndash140 2005

[77] A-GWang T Xia Q-L Chu et al ldquoEffects of fluoride on lipidperoxidation DNA damage and apoptosis in human embryohepatocytesrdquoBiomedical and Environmental Sciences vol 17 no2 pp 217ndash222 2004

[78] Y Y Ye J W Liu J H Xu L J Sun M C Chen and M BLan ldquoNano-SiO

2induces apoptosis via activation of p53 and

Bax mediated by oxidative stress in human hepatic cell linerdquoToxicology in Vitro vol 24 no 3 pp 751ndash758 2010

[79] B Sha W Gao S Wang F Xu and T Lu ldquoCytotoxicity oftitanium dioxide nanoparticles differs in four liver cells fromhuman and ratrdquo Composites Part B Engineering vol 42 no 8pp 2136ndash2144 2011

[80] N P Gomes and JM Espinosa ldquoGene-specific repression of thep53 target gene PUMA via intragenic CTCF-Cohesin bindingrdquoGenes and Development vol 24 no 10 pp 1022ndash1034 2010

[81] J R Jeffers E Parganas Y Lee et al ldquoPuma is an essentialmedi-ator of p53-dependent and -independent apoptotic pathwaysrdquoCancer Cell vol 4 no 4 pp 321ndash328 2003

[82] W W Tsai and M C Barton ldquop53rdquo in Signaling Pathways inLiver Diseases J F Durfour and P A Clavien Eds pp 345ndash357Springer Berlin Germany 2nd edition 2010

[83] E A Slee M T Harte R M Kluck et al ldquoOrdering thecytochrome c-initiated caspase cascade hierarchical activationof caspases-2-3-6-7-8 and -10 in a caspase-9-dependentmannerrdquo Journal of Cell Biology vol 144 no 2 pp 281ndash2921999

[84] G-C Lee J H Lee B Y Kim and C H Lee ldquoMitochondria-targeted apoptosis in human cytomegalovirus-infected cellsrdquoJournal of Microbiology and Biotechnology vol 23 no 11 pp1627ndash1635 2013

[85] Y Peng J-G Sun and G-J Wang ldquoPharmacokinetic study ofrhein and its carboxyl-esterification derivatives in ratsrdquoChineseJournal of Natural Medicines vol 7 no 3 pp 228ndash233 2009

[86] M-L Hou L-W Chang C-H Lin L-C Lin and T-H TsaildquoDetermination of bioactive components in Chinese herbal


formulae and pharmacokinetics of Rhein in rats by UPLC-MSMSrdquoMolecules vol 19 no 4 pp 4058ndash4075 2014

[87] K Hao Q Qi P Wan et al ldquoPrediction of human pharma-cokinetics frompreclinical information of rhein an antidiabeticnephropathy drug using a physiologically based pharmacoki-netic modelrdquo Basic amp Clinical Pharmacology amp Toxicology vol114 no 2 pp 160ndash167 2014

[88] S Reagan-Shaw M Nihal and N Ahmad ldquoDose translationfrom animal to human studies revisitedrdquo The FASEB Journalvol 22 no 3 pp 659ndash661 2008

[89] Center for Drug Evaluation and Research (CDER) and Centerfor Biologics Evaluation and Research (CBER) Estimatingthe Safe Starting Dose in Clinical Trials for Therapeutics inAdult Healthy Volunteers US Food and Drug AdministrationRockville Md USA 2002

[90] L Tan Y S Yuan J W Yang and Q Qiu ldquoDeterminationof rhein in human plasma by HPLC and the study of itspharmacokineticsrdquo Bulletin of Jinling Hospital vol 11 pp 112ndash115 1998

[91] W Zhu X-MWang L Zhang X-Y Li and B-XWang ldquoPhar-macokinetic of Rhein in healthy male volunteers following oraland retention enema administration of rhubarb extract a singledose studyrdquo The American Journal of Chinese Medicine vol 33no 6 pp 839ndash850 2005

[92] J-Y Jiang M-W Yang W Qian et al ldquoQuantitative determi-nation of rhein in human plasma by liquid chromatography-negative electrospray ionization tandem massmass spectrom-etry and the application in a pharmacokinetic studyrdquo Journal ofPharmaceutical and Biomedical Analysis vol 57 no 1 pp 19ndash252012

[93] Pharmacopoeia of the Peoplersquos Republic of China First DivisionChina Chemical Industry Press Beijing China 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


OH

OHOH O

O

O

Figure 1 Chemical structure of rhein (4 5-dihydroxyanthraquin-one-2-carboxylic acid) Molecular formula C

15H8O6 Molecular

weight 28422 gmol

liver injury chronic viral hepatitis cholestatic liver diseasesand hepatic fibrosis [8 16 17]

With thousands of years of medical practice TraditionalChinese Medicine (TCM) has accumulated rich theories(including yin yang chrsquoi (qi) meridian five-phase (or five-element) and zang-fu theories) and a great deal of valuableexperience in the prevention and treatment of several diseasesor medical condition [18 19] According to the theory ofTCM the liver is regarded as a special organ in the bodyit is related to the eyes via the meridian connections andits condition is reflected in the nails It is involved by virtueof its role in regulating or ensuring the free flow of qiaround the body and its role in regulating blood volume(ldquostoring bloodrdquo) [20] Based on long period of traditionaland clinical practice TCM considered that the rhizome ofPolygonum multiflorum possesses sweet and slightly warmproperties making it especially suitable for treating patientswho have not only blood deficiency but also a mild Yangand Qi deficiency of the body It opens the meridians andcollaterals able to tonify the blood as well the kidney essence(Jing) strengthens the tendons and bones and improves sleep[21] TCM theory said that both graying of hair and hairloss may be due to lack of essential essences explaining whyPolygonummultiflorum has been used to promote the growthof hair and to treat premature greying of hair which is doneby nourishing the kidneyrsquos ldquoyinrdquo energy replenishing andcooling down the blood [22] However the long-term use ofPolygonum multiflorummay lead to liver and kidney toxicityas several clinical cases of hepatotoxicity have been linked toits consumption

Rhein (4 5-dihydroxyanthraquinone-2-carboxylic acidFigure 1) is one of the most important bioactive componentsof PMT Mounting published reports demonstrated thatits pharmacological effects including anti-inflammatory [8]antiallergic [23] antifungal [24] antibacterial [25] antiviral[26] and anticancer ones [27ndash29] Recently the suppressionof Hep-G2 cells proliferation induced by rhein was expressedvia mitochondrial permeability transition but the oxidativestress injury mechanism was not investigated in this study[30] Mostly the mechanism of rhein antitumor activity incancer cells listed previously is commonly due to its abilityto induce apoptosis in corresponding cancer cells [28 29]

Apoptosis or programmed cell death (PCD) a geneticallycontrolled process whereby cells die in response to environ-mental or developmental cues contributes to the pathogene-sis of disease or removal of cells in adult organism [31] It is

characterized by the activation of biochemical pathways thatlead to changes in cellular morphology DNA fragmentationperturbation of mitochondrial membrane function decreaseof mitochondrial membrane potential translocation of phos-phatidylserine (PS) to the external cell surface and changesin the plasma membrane [32] Understanding and regula-tion of apoptosis are critical for normal development andtissue homeostasis and disruption of this process can havesevere consequences [33] Too much cell death may produceneurodegenerative diseases and impaired development whileinsufficient cell death can lead to increased susceptibility tocancer and sustained viral infection [34] Numerous scientificdata have proven that apoptotic signaling within the cellmay occur by two fundamental pathways (1) death receptoror extrinsic pathway and (2) the mitochondria or intrinsicpathway [35] Rhein has been investigated and shown toinduce cytotoxicity and apoptosis in primary cultures ofrat hepatocytes [36] Measurement of apoptosis has becomean essential component of the evaluation of cytotoxicity ofchemicals [31]

Liver cells especially hepatocytes are notable for theirwide variety of metabolic and other functional capacitiesspanning over 500 classes of functions such as energymetabolism bile production and synthetic or detoxificationfunctions [37] Primary human hepatocytes remain differ-entiated and sustain the major drug-metabolizing enzymesactivities they represent a unique in vitro system and serveas a ldquogold standardrdquo for studies of drug metabolism andtoxicity [38] HL-7702 cell expressed a distinct ultrastructurecompared to hepatic carcinoma and is considered an ideal invitromodel of a primary Chinese nonmalignant liver [39] Invitro and in vivo studies have been conducted to assess thebiosynthetic activities of HL-7702 cells in order to explore thepossibility to use this cell line for a liver support system Ina 7-day in vitro study done by Yang et al [40] the authorsfound that HL-7702 cells could keep their function of proteinsynthesis by culturing on thermoresponsive hydrogen Albu-min secretion continuously grew and the function of ureasynthesis was significantly increased with in time delayedfrom (016 plusmn 002) 120583molmL to (041 plusmn 004) 120583molmL [40]Furthermore the proliferation state of cells by cell cycleanalysis was proven to not be damaged In another studyconducted by Hu and colleagues [41] the biosynthetic func-tion of HL-7702 cells in terms of albumin uridine diphos-phate glucuronosyltransferase (UGT) and cytochrome P4503A4 gene and protein was significantly expressed In thesame study in an in vivo acute liver failure (ALF) modelestablished by 90 partial hepatectomy rats transplantedwith HL-7702 cells showed significantly improved survivalof 70 versus 0 in controls (119875 lt 001) Moreover theenzymatic analysis of various enzymes or liver markers suchas albumin alanine transaminase aspartate transaminaseserum ammonia alkaline phosphates and total and directbilirubin revealed a significant improvement compared to thecontrol groups [41] These evidences support that HL-7702cells could proliferate and keep their biosynthetic functionsat the same time suggesting them to be a feasible sourcefor liver support system and ideal for pharmacological andtoxicological studies















































020406080

100120

012 037 12 37 12 37 120 370

Cel

l via

bilit

y (

)


24h48h


3 Results








05

101520253035404550

Control

LDH

rele

ase (

o

f con

trol)

Rhein24h48h

50120583M 100120583M 200120583M 400120583M10120583M


lowastlowast

lowastlowast


(a)

Control

Rhein

50120583M25120583M 100120583M

(b)

020406080

100120140

Karm

en u

nit

ASTALT

Control

Rhein

50120583M 100120583M 200120583M 400120583M10120583M

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(c)













119875 lt 001









Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)



0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































020406080

100120

012 037 12 37 12 37 120 370

Cel

l via

bilit

y (

)


24h48h


3 Results








05

101520253035404550

Control

LDH

rele

ase (

o

f con

trol)

Rhein24h48h

50120583M 100120583M 200120583M 400120583M10120583M


lowastlowast

lowastlowast


(a)

Control

Rhein

50120583M25120583M 100120583M

(b)

020406080

100120140

Karm

en u

nit

ASTALT

Control

Rhein

50120583M 100120583M 200120583M 400120583M10120583M

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(c)













119875 lt 001









Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)



0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05

101520253035404550

Control

LDH

rele

ase (

o

f con

trol)

Rhein24h48h

50120583M 100120583M 200120583M 400120583M10120583M


lowastlowast

lowastlowast


(a)

Control

Rhein

50120583M25120583M 100120583M

(b)

020406080

100120140

Karm

en u

nit

ASTALT

Control

Rhein

50120583M 100120583M 200120583M 400120583M10120583M

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(c)













119875 lt 001









Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)



0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























119875 lt 001









Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)



0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

PI

8623

7 624

9062

655

022

284

03

075

674

6597

2655

5492

4069

061

379

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

Annexin V-FITC100 101 102 103 104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

PI50120583

M

PI25120583

MPI

100120583

M

(a)

05

1015202530354045

Apop

tosis

rate

()

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583MControl

(b)



0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250


lowastlowast

lowast

Rhein

50120583M25120583M 100120583MControl

(120583m

olL

)

(a)

005

115

225

335

4

HMG-CoARGPATACoAC

lowastlowast

lowastlowastlowast

Rhein

50120583M25120583M 100120583MControl

2minusΔΔ

Ct

(b)


02468

101214161820


Red

gree

n flu

ores

cenc

ein

tens

ity ra

tio

Rhein

50120583M25120583M 100120583M

lowastlowastlowast







020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















020406080

100120140160180


ROS

( o

f con

trol)

Rhein (120583M)

100 + NAC

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30lowastlowast

lowast

Control

Rhein

50120583M25120583M 100120583M

MD

A (120583

M120583

gpr

otei

n)

(b)



0

05

1

15

2

25

3

Control

lowastlowast

lowastlowast

Rhein

50120583M25120583M 100120583M

SOD

activ

ity (u

nits120583

gpr

otei

n)



4 Discussion







50of rhein was


50values




0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

ControlCasp

ase a

ctiv

ity (U

mg

prot

ein)


Rhein

50120583M25120583M 100120583M

lowastlowast

lowastlowast


lowastlowast

(a)

05

1015202530354045

Apop

totic

cells

()


minusminusminus

minus

minusminus

minus

+

minus

minus

++

minus

minus+

+minusminus

+


Rhein (100120583M)

(b)








005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















005

115

225

335

4

Control

Fasp53PUMA

Rhein

50120583M25120583M 100120583M



lowast

2minusΔΔ

Ct

(a)

005

115

225

335

4

Control


Caspase-9Caspase-3

Rhein


lowastlowast

50120583M25120583M 100120583M

2minusΔΔ

Ct

(b)






Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Rhein

Cytosolic Cyt-c

Mitochondrial Cyt-c

Fas

Control 50120583M25120583M 100120583M

120573-actin

120573-actin

120573-actin

(a)

Rhein

050

100150200250300350

Control

Rela

tive i

nten

sity

( o

f con

trol)


50120583M25120583M 100120583M

lowastlowast

lowast

lowast

(b)











119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















119898value





119898

Human 119870119898

(1)



5 Conclusion


Abbreviations







dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















dehydrogenase





References












































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article rhein elicits in vitro cytotoxicity in...

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