research article the effects of high-dose qinggan huoxue...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 905715 8 pageshttpdxdoiorg1011552013905715

Research ArticleThe Effects of High-Dose Qinggan Huoxue Recipe onAcute Liver Failure Induced by D-Galactosamine in Rats

Hong Zhu1 Yang Zhang2 Xiaoyu Hu2 Cheng Yi1 Sen Zhong2

Yanyan Wang2 and Fang Yang3

1 Department of Abdominal Cancer West China Hospital Sichuan University Chengdu 610041 Sichuan China2Department of Infectious Diseases Affiliated Hospital of Chengdu University of Traditional Chinese MedicineChengdu 610072 Sichuan China

3Department of Clinical Medicine Chengdu University of Traditional Chinese Medicine Chengdu 610072 Sichuan China

Correspondence should be addressed to Xiaoyu Hu meddmailyahoocomcn

Received 27 November 2012 Revised 3 February 2013 Accepted 10 February 2013

Academic Editor H Balaji Raghavendran

Copyright copy 2013 Hong Zhu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Qinggan Huoxue Recipe is a traditional Chinese medicine which has been usually used to improve liver function in hepatitis Inorder to investigate the effects of high-dose Qinggan Huoxue Recipe on acute liver failure and explore the potential mechanism wehad built acute liver failure models in rats by intraperitoneal injection of D-galactosamine (D-GalN) High-dose Qinggan HuoxueRecipe was delivered by gavage After treatment the blood alanine aminotransferase (ALT) aspartate aminotransferase (AST) totalbilirubin (TBIL) albumin (ALB) cholinesterase (CHE) and prothrombin time (PT) were determined The pathological score ofliver tissue was recorded Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and fluorescence quantitativereverse transcription polymerase chain reaction (qRT-PCR) of high mobility group box 1 (HMGB1) toll-like receptor 4 (TLR4)nuclear factor-kappa B (NF-120581B) and Caspase-3 were performed The survival curve was also depicted Our results demonstratedthat high-dose QingganHuoxue Recipe could significantly improve liver function and increase survival rates in rats with acute liverfailure These effects were supposed to be mediated by suppressing inflammatory reaction and apoptosis

1 Introduction

Acute liver failure (ALF) is a life-threatening medical emer-gency and occurs when the liver rapidly loses its functionwithin a short period ALF can develop secondary to a varietyof causes and occurs when the extent of hepatocyte deathexceeds the liverrsquos regenerative capacity [1] Currently livertransplantation is the ldquoGold Standardrdquo therapy for the diseaseHowever due to the limited availability of donor organsand rapid progression of the disease the mortality of ALFremains high [2] Therefore it is imperative to develop noveltherapeutic reagents for ALF

Qinggan Huoxue Recipe is a traditional Chinesemedicine prescription which has been used in China for along time [3] Previous literature had indicated that it couldimprove the liver function in alcohol liver disease models[4ndash6] but the effects of Qinggan Huoxue Recipe on acuteliver failure were rarely explored

In one of our previous prospective clinical cohort studies[7] we found that high-dose Qinggan Huoxue Recipe couldsignificantly improve liver function and coagulation func-tion reduce complications and reduce mortality in patientswith hepatitis B-related acute-on-chronic liver failure Inorder to confirm the effects of high-dose Qinggan HuoxueRecipe on acute liver failure and explore the potential mech-anism we had conducted this experiment

2 Methods

21 Materials and Methods All specific pathogen-free (SPF)male Wistar rats weighing 150 plusmn 20 g were purchased fromShanghai Experimental Animal Co Ltd (Shanghai China)D-galactosamine (D-GalN) a commonly used liver injuryinducing drug [8 9] was purchased from Hongbang Med-ical Technology CO Ltd (Shanghai China) Stronger Neo-Minophagen C (SNMC) a classic liver protection drug [10]

2 Evidence-Based Complementary and Alternative Medicine

Table 1 The primer sequences used in fluorescence quantitative RT-PCR

Parameters 51015840 primer sequence 31015840 primer sequenceHMGB1 51015840TGTTCTGAGTACCGCCCAAA31015840 51015840TTTCGCTGCATCAGGTTTTC31015840

TLR4 51015840CCAGGAAGGCTTCCACAAGA31015840 51015840AATTCGACCTGCTGCCTCAG31015840

NF-120581B 51015840GCACGAGGCTCCTTTTCTCAA31015840 51015840CGTTTTTCTTCAATCCGGTGG31015840

Caspase-3 51015840ACCGATGTCGATGCAGCTAA31015840 51015840AGGTCCGTTCGTTCCAAAAA31015840

120573-Actin 51015840AAGGAGGCAAAGGACACCAA31015840 51015840AATGGCCCCCTTCACAGTTA 31015840

was purchased from Minophagen Pharmaceutical Co Ltd(Tokyo Japan) and fixed into a concentration of 156mgmLwith distilled water Qinggan Huoxue Recipe which wasboiled using Artemisia capillaris Patrinia Scutellaria baical-ensis Polygonum cuspidatum rhubarb and red Peony (2 4 4 4 1 4) was purchased fromAffiliatedHospital of ChengduUniversity of Traditional Chinese Medicine (ChengduChina) with a concentration of 297 gmL

22 Design of Animal Experiment 70 rats were randomizedinto four groups negative control group (Control 10 rats)fed with distilled water by gavage model group (Model 20rats) fed with distilled water by gavage and injected with D-GalN 14 gkg intraperitoneally three days after the gavageStronger Neo-Minophagen C group (SNMC 20 rats) fedwith SNMC 156mgkgd by gavage and injected with D-GalN 14 gkg intraperitoneally three days after the gavageQinggan Huoxue Recipe group (Experiment 20 rats) fedwith Qinggan Huoxue Recipe 297 gkgd (which equals625times clinical dose) by gavage and injected with D-GalN14 gkg intraperitoneally three days after the gavage Thegavage last for 5 days The dosage of Qinggan HuoxueRecipe used on rats was calculated by the formula Doserat= Dosehuman times (habeas indexrathabeas indexhuman) times (bodyweighthumanbody weightrat) times 23 [11] Based on this for-mula the translational coefficient 625 was produced In ourprevious clinical trial the dose of Qinggan Huoxue Recipeused on human was 285 g60 kgd (475 gkgd) At last thedose of 297 gkgd was achieved through the multiplicationof human dose (475 gkgd) and translational coefficient(625) The dose of 297 gkgd is a relatively very high-doseused in rats compared with the other studies of QingganHuoxue Recipe reported [4 12] Besides a dose responsestudy was carried out in preexperiment to confirm theusage of this high-dose (data not shown) 36 hours afterthe D-GalN injection 6mL blood was collected throughfemoral artery of alive rats for detection of serum alanineaminotransferase (ALT) aspartate aminotransferase (AST)total bilirubin (TBIL) albumin (ALB) cholinesterase (CHE)and prothrombin time (PT)Then the rats were sacrificed andthe left lobe of the liver was collected for further studies suchas hematoxylin and eosin (HE) staining proliferating cellnuclear antigen (PCNA) immunohistochemistry assay andfluorescence quantitative reverse transcription polymerasechain reaction (qRT-PCR)

23 Serum ALT AST TBIL ALB CHE and PT Determina-tion The serum biochemical parameters ALT AST TBIL

ALB CHE and PT which closely reflect the liver function [4]were analyzed by the Department of Laboratory MedicineAffiliated Hospital of Chengdu University of TraditionalChinese Medicine (Chengdu China)

24 Pathological Scores The liver specimens were fixedparaffin embedded and cut into 3- to 5-120583m sections Thesections were used for HE and PCNA staining The methodof HE staining has been introduced in the previous literature[13 14] All slides were read by three investigators who wereblinded to the allocation arm of the animal They were askedto grade the microscopic injuries seen in the liver using asemiquantitative scoring system [15] with zero indicating nodiscernable injury and 4 indicating the presence of severeinjuryThese scores were allocated based on their assessmentof the following histological features cellular oedema inter-stitial oedema neutrophil infiltration capillary congestionand structural distortion For each slide the average score ofat least three observations was considered as the pathologicalscore

25 PCNA Immunohistochemistry The PCNA immunohis-tochemistry kit was purchased from Boster BioengineeringCO Ltd (Wuhan China) Immunohistochemical stainingof PCNA was performed according to the manufacturerrsquosinstructions PCNA-positive cells were counted in 5 randomvisual fields under 40x magnifications for each section andthe number was expressed as the percentage of PCNA posi-tive cells to the total number of cells counted [16] Sectionswere examined microscopically for specific staining andphotographs were taken with a digital image-capture system(Olympus CX40 Tokyo Japan)

26 Fluorescence Quantitative RT-PCR The expression ofhigh mobility group box 1 (HMGB1) toll-like receptor 4(TLR4) nuclear factor kappa B (NF-120581B) and Caspase-3 wasdetected by qRT-PCR Total RNAs were extracted with Trizolreagent (Invitrogen) and reverse transcribed into cDNA bythe ABI Step-One Plus Real-Time PCR System (AppliedBiosystemCo CA USA)The primer sequences of the aboveparameters including 120573-actin were listed in Table 1

27 Survival Curves Another 55 rats were divided into theabove four groups with animal numbers of 10 15 15 and 15respectively for observation of survival The treatments werethe same as mentioned before The observation was begunwith the time of D-GalN injection while the endpoint wasset at 96 hours after the injection

Evidence-Based Complementary and Alternative Medicine 3

0

200

400

600

Con

trol

Mod

el

SNM

C

Expe

rimen

t

ALT

(UL

)

dagger

daggerlowastDagger

daggerlowast

Groups

(a)

0

500

1000

1500

Con

trol

Mod

el

SNM

C

Expe

rimen

t

AST

(UL

) dagger

daggerlowastDaggerdaggerlowast

Groups

(b)

0

10

20

30

40

50

Con

trol

Mod

el

SNM

C

Expe

rimen

t

dagger

daggerlowastDagger

daggerlowast

Groups

TBIL

(120583m

olL

)

(c)

0

10

20

30

40

50

Con

trol

Mod

el

SNM

C

Expe

rimen

t

ALB

(gL

)

Groups

ampamp amp

(d)

0

200

400

600

800

Con

trol

Mod

el

SNM

C

Expe

rimen

t

Groups

CHE

(UL

)

ampamp

amp

(e)

0

10

20

30

40

Con

trol

Mod

el

SNM

C

Expe

rimen

t

Groups

PT (S

)

ampamp

amp998771

(f)

Figure 1 The serum ALT AST TBIL ALB and CHE levels and PT after treatment (a) ALT (b) AST (c) TBIL (d) ALB (e) CHE (f)PT Control negative control group Model the group injected with D-GalN SNMC the group injected with D-GalN and treated withStronger Neo-Minophagen C Experiment the group injected with D-GalN and treated with Qinggan Huoxue Recipe For Control ModelSNMC and Experiment groups the ALT levels were 3515 plusmn 601UL 44110 plusmn 6036UL 26718 plusmn 4145UL and 14322 plusmn 2296ULrespectively the AST levels were 15161 plusmn 2087UL 88780 plusmn 12847UL 38049 plusmn 5538UL and 28736 plusmn 6897UL respectively theTBIL levels were 155 plusmn 043 120583molL 3804 plusmn 684 120583molL 2437 plusmn 403 120583molL and 1865 plusmn 296 120583molL respectively the ALB levelswere 3525 plusmn 419 gL 2367 plusmn 321 gL 2665 plusmn 450 gL and 2946 plusmn 419 gL respectively the CHE levels were 55740 plusmn 4323UL34392 plusmn 6893UL 43050 plusmn 8353UL and 51582 plusmn 7331UL respectively and the PT was 1360 plusmn 173 s 3180 plusmn 502 s 2993 plusmn 383 sand 2446 plusmn 425 s respectively For (a) (b) and (c) dagger119875 lt 005 comparing Control group lowast119875 lt 005 comparing Model group and Dagger119875 lt 005comparing SNMC group For (d) (e) amp119875 lt 005 comparing Control group and

119875 lt 005 comparing Model group For (f) amp119875 lt 005comparing Control group and 998771119875 lt 005 comparing Model group and SNMC group

28 Statistical Analysis The biochemical parameters patho-logical scores PCNA immunohistochemistry and mRNAexpressions were analyzed by one-way ANOVA followed bythe Studentrsquos 119905-test Survival curves were plotted using theKaplan-Meier method and analyzed applying the Log-ranktest All statistical analyses were performed using the SPSS170 software package All 119875 values were two sided and 119875 lt005 was considered as the significant level of difference

3 Results

31 The Serum ALT AST TBIL ALB and CHE Levels andPT The serumALT AST TBIL ALB andCHE levels and PTwere shown in Figure 1 From the results we could find thatthe ALT AST and TBIL were significantly increased whileALB and CHEwere significantly decreased after the injectionof D-GalN PT was remarkably elongated All these parame-ters have indicated severe liver damage However Stronger

Neo-Minophagen C and high-dose Qinggan Huoxue Recipecould improve the liver function by decreasing ALT ASTTBIL andPT and increasingALBCHE levels And the effectswere more significant in Qinggan Huoxue Recipe group

32 Pathological Scores As shown in Figure 2 there weremassive necroses in the liver tissues of the model group Thenecroses were reduced in the SNMC and experiment groupsespecially in the experiment group

33 PCNA Immunohistochemistry The PCNA positive rateswere 748 plusmn 090 1755 plusmn 24 2557 plusmn 294 and3568 plusmn 475 respectively in control model SNMC andexperiment groups (Figure 3) The PCNA positive rate ofexperiment groupwas significantly larger than the other threegroups indicating high regeneration rates after the treatmentof high-dose Qinggan Huoxue Recipe


Control Model

SNMC Experiment

(a)

0

1

2

3

4

5

Control Model SNMC ExperimentGroups

Path

olog

y sc

ores

amp

amp

amp998771

(b)

Figure 2 Pathological scores (a) HE staining (b) pathological scores Control negative control group Model the group injected with D-GalN SNMC the group injected with D-GalN and treated with Stronger Neo-Minophagen C Experiment the group injected with D-GalNand treated with Qinggan Huoxue Recipe The pathological scores were 0 369 plusmn 038 278 plusmn 031 and 172 plusmn 021 respectively in ControlModel SNMC and Experiment groups amp119875 lt 005 comparing Control group 119875 lt 005 comparing Model group and 998771119875 lt 005 comparingSNMC group

Control Model

SNMC Experiment

(a)

0

10

20

30

40

50


PCN

A (

)

amp

amp

amp998771

(b)

Figure 3 PCNA immunohistochemistry (a) PCAN immunohistochemistry (b) histogram of immunohistochemistry Control negativecontrol group Model the group injected with D-GalN SNMC the group injected with D-GalN and treated with Stronger Neo-MinophagenC Experiment the group injected with D-GalN and treated with Qinggan Huoxue Recipe The PCNA positive rates were 748 plusmn 0901755 plusmn 24 2557 plusmn 294 and 3568 plusmn 475 respectively in Control Model SNMC and Experiment groups amp119875 lt 005 comparingControl group 119875 lt 005 comparing Model group and 998771119875 lt 005 comparing SNMC group

34 The mRNA Expressions As shown in Figure 4 high-dose Qinggan Huoxue Recipe could remarkably decrease themRNA expressions of HMGB1 TLR4 NF-120581B and Caspase-3 indicating that it could decrease inflammatory reaction andapoptosis of liver tissues

35 Survival Curves We excluded the control group fromsurvival curve because there was no rat died at the endpointFrom Figure 5 we could found that high-dose QingganHuoxue Recipe could improve the survival of acute liverfailure model significantly


0

01

02

03


HM

GB1

amp

ampamp998771

(a)

0

02

04

06

08


TLR4

amp

amp

amp998771

(b)

0

1

2

3

4

5


NF-120581

B

amp

amp

amp998771

(c)

0

1

2

3

4

5


Casp

ase-

3

amp

amp

amp998771

(d)

Figure 4ThemRNA expression (a)HMGB1 (b) TLR4 (c) NF-120581B (d) Caspase-3 Control negative control groupModel the group injectedwith D-GalN SNMC the group injected with D-GalN and treated with Stronger Neo-Minophagen C Experiment the group injected withD-GalN and treated with Qinggan Huoxue Recipe For Control Model SNMC and Experiment groups the HMGB1 mRNA expressionswere 001 plusmn 001 025 plusmn 004 011 plusmn 004 and 007 plusmn 006 respectively the TLR4 mRNA expressions were 004 plusmn 002 041 plusmn 022 022 plusmn 008and 008 plusmn 004 respectively the NF-120581B mRNA expressions were 049 plusmn 025 268 plusmn 135 178 plusmn 064 and 098 plusmn 063 respectively and theCaspase-3 mRNA expressions were 136 plusmn 026 341 plusmn 085 257 plusmn 104 and 164 plusmn 081 respectively amp119875 lt 005 comparing Control group119875 lt 005 comparing Model group and 998771119875 lt 005 comparing SNMC group

4 Discussion

In this study the remarkable increased serum ALT ASTand TBIL levels elongated PT decreased serum ALB CHElevels increased pathological scores and rapid death hadconfirmed that acute liver failure models were successfullybuilt by D-GalN injection SNMC and high-dose QingganHuoxue Recipe could both ameliorate liver function andincrease survival times however high-doseQingganHuoxueRecipe had significant stronger effects

The Qinggan Huoxue Recipe used in this study hasbeen practiced for many years in the Affiliated Hospital ofChengduUniversity of Traditional ChineseMedicine andwasfound to be very effective in relieving hepatic complicationsHowever the protective effects of QingganHuoxue Recipe onacute liver failure patients were unsatisfactory when used ingeneral dose So we enhanced the dosage of Qinggan Huoxue

Recipe in acute liver failure patients gradually and we foundthat liver functions were remarkably improved without dis-covering any drug-related side effects In order to confirm theprotective effects of high-dose Qinggan Huoxue Recipe onacute liver failure we had carried out our previous prospec-tive clinical trial [7] For the Chinese medicine included inthis recipe Artemisia capillaris could ameliorate the hydro-philic bile acids-induced hepatic injury which is probablyrelated to a reduced oxidant stress and degree of hepaticfibrosis [17] Patrinia could inhibit the biomarkers related toinflammation through the blocking of NF-120581B activationand potentiate anti-inflammatory effects [18] Scutellariabaicalensis could inhibit cyclooxygenase-2 overexpressionand therefore alleviates cantharidin-induced rat hemorrhagiccystitis [19] Polygonum cuspidatum was found to be an effec-tive hepatoprotective agent and a promising candidate forthe treatment of oxidative stress- and inflammation-related


Time after D-GalN injection (hours)10080604020

Surv

ival

rate

1

08

06

04

02

0

SNMCModel

Survival functions

Experimental

Figure 5 The survival curve Model the group injected with D-GalN SNMC the group injected with D-GalN and treated withStronger Neo-Minophagen C Experiment the group injected withD-GalN and treated with Qinggan Huoxue Recipe

119875 lt 005

comparing Model group

diseases [20 21] Rhubarb was found to have antioxidantantiplatelet and anticoagulant activities and could be usedto treat experimental jaundice in rats [22] Red Peony couldinhibit inflammation and scavengs free radicals and wasfound to be effective in treating severe acute pancreatitis [2324] Qinggan Huoxue Recipe has combined these Chineseherb medicines following the principle of clearing heat andresolving stasis Finally our previous clinical trial provedthat high-dose Qinggan Huoxue Recipe could significantlyimprove hepatic function in acute liver failure [7] but thepotential mechanism is still not for sure

HMGB1 a highly conserved ubiquitous protein thatpresents in the nuclei and cytoplasm of nearly all cell types isa necessary and sufficient mediator of inflammation duringsterile- and infection-associated responses [25] Most cellsconstitutively express HMGB1 and release it on injury ordeath [26] It has been suggested that HMGB-1 itself cansignal through receptor for advanced glycation end products(RAGEs) and through the toll-like receptors TLR2 TLR4and TLR9 Activation of these receptors results ultimately inthe activation of nuclear factor-kappa B (NF-120581B) inducingthe upregulation of leukocyte adhesion molecules produc-tion of proinflammatory cytokines and angiogenic factors inboth hematopoietic and endothelial cells thereby promotinginflammation [27] Although HMGB1 exerts its cellularand biologic inflammatory responses by binding to threemembers of TLRs family namely TLR2 TLR4 and TLR9 aswell as RAGE TLR4 is the primary receptor of endogenousHMGB1 in mediating cytokine release and tissue damagein various conditions such as ischemiareperfusion injury

hemorrhage and trauma and this mechanism of injury isattenuated or prevented by deficiency in TLR4 [28] So wehad detected the mRNA expressions of HMGB1 TLR4 andNF-120581B in this study We found that the mRNA expressionsof HMGB1 TLR4 and NF-120581B were remarkably increasedin acute liver failure model group However they weresignificantly decreased after the treatment of SNMC or high-dose Qinggan Huoxue Recipe especially in Qinggan HuoxueRecipe treatment group Therefore we think that high-doseQinggan Huoxue Recipe could improve liver function inacute liver failure by suppressing inflammation and thiseffect was most probably mediated by inhibiting HMGB1TLR4NF-120581B pathway though further studies were neededto exclude the interactions between HMGB1 with otherreceptors Caspases are crucial mediators of programmedcell death (apoptosis) Among them Caspase-3 frequentlyactivated death protease catalyzing the specific cleavage ofmany key cellular proteins [29] After the activation ofNF-120581Bthe activities of antigen presenting cells could be enhancedand then cytotoxic T lymphocytes (CTL) were largely acti-vated which mediated the apoptosis of hepatocytes [30] Sohigh-dose Qinggan Huoxue Recipe could also improve liverfunction in acute liver failure by decreasing the activation ofNF-120581B and therefore decrease the apoptosis of hepatocytes

In this study we had also found that the PCNA pos-itive rates were significantly higher in high-dose QingganHuoxue Recipe group indicating high regeneration ratesHowever whether the promotion of regeneration was pro-duced by direct effects of high-dose Qinggan Huoxue Recipeor the subsequent effects of inflammation suppression wasunknown Besides further studies were needed to explore therelationship of inflammation apoptosis and regeneration inthe treatment of acute liver failure

Although high-dose Qinggan Huoxue Recipe was usedin this study no drug-related side effects were discovered Inour previous prospective clinical study the incidence rates ofadverse events in the treatment group and the control groupwere 000 and 1250 respectively and the difference wasstatistically significant No drug-related adverse events werefound in blood urine and stool routine tests renal functiontest and electrocardiography [7] During this experiment wealso observed the ratsrsquo appetite behavior change reactionto stimulation and so on We found that the appetites andbehaviors of rats in acute liver failure model group weredecreased evidently Besides they had developed a seriesof symptoms such as urinary incontinence yellow urinelistlessness lethargy irritability convulsion and hemor-rhage However these symptoms were remarkably decreasedafter the treatment of high-dose Qinggan Huoxue Recipeand more important no treatment-related side effects werediscovered

5 Conclusion

High-dose Qinggan Huoxue Recipe could significantlyimprove liver function and increase survival rates in rats withacute liver failureThese effects were supposed to bemediatedby suppressing inflammatory reaction and apoptosis


Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

H Zhu and Y Zhang equally contributed to the paper

Acknowledgments

All animal procedures were approved by the Animal Careand Scientific Committee of Chengdu University of Tradi-tional Chinese Medicine This work was supported by grantsfrom the The National Key Technology RampD Program (no2008ZX10005 no 2009ZX10005)

References

[1] N J Taylor A Nishtala G K Vijay et al ldquoCirculating neu-trophil dysfunction in acute liver failurerdquo Hepatology 2012

[2] W F Tan R H Steadman D G Farmer et al ldquoPretrans-plant neurological presentation and severe posttransplant braininjury in patients with acute liver failurerdquo Transplantation vol94 no 7 pp 768ndash774 2012

[3] G Ji Y Q Wang and C L Cao ldquoClinical study on treatmentof alcoholic liver disease by qinggan huoxue reciperdquo ZhongguoZhong Xi Yi Jie He Za Zhi vol 24 no 1 pp 13ndash16 2004

[4] G Ji L Wang S H Zhang J W Liu P Y Zheng and T LiuldquoEffect of Chinese medicine Qinggan Huoxuefang on inducingHSC apoptosis in alcoholic liver fibrosis ratsrdquo World Journal ofGastroenterology vol 12 no 13 pp 2047ndash2052 2006

[5] J M Chen LWang and L J Xing ldquoRegulatory effects of Qing-gan Huoxue Recipe on matrix metalloproteinases of alcoholicliver fibrosis ratsrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 31no 11 pp 1538ndash1544 2011

[6] G Ji ldquoClinical study on treatment of alcoholic liver disease byQinggan Huoxue Reciperdquo Zhong Xi Yi Jie He Xue Bao vol 1 no2 pp 103ndash124 2003

[7] X Y Hu Y Zhang G Chen S Zhong and X J Fan ldquoAprospective cohort study on the influence of high doses of herbsfor clearing heat and resolving stasis on survival rates in patientswith hepatitis B-related acute-on-chronic liver failurerdquo ZhongXi Yi Jie He Xue Bao vol 10 no 2 pp 176ndash185 2012

[8] S Banu B Bhaskar and P Balasekar ldquoHepatoprotective andantioxidant activity of Leucas aspera against d-galactosamineinduced liver damage in ratsrdquo Pharmaceutical Biology vol 2012 pp 1592ndash1595 2012

[9] J W Kang S J Kim H Y Kim et al ldquoProtective effects of HV-P411 complex against D-galactosamine-induced hepatotoxicityin ratsrdquo The American Journal of Chinese Medicine vol 40 no3 pp 467ndash480 2012

[10] B S Yang Y J Ma YWang et al ldquoProtective effect andmecha-nism of stronger neo-minophagen C against fulminant hepaticfailurerdquo World Journal of Gastroenterology vol 13 no 3 pp462ndash466 2007

[11] J S Li Y Li S Y Li et al ldquoLong-term effects of Tiaobu Feishentherapies on systemic and local inflammation responses in ratswith stable chronic obstructive pulmonary diseaserdquo Zhong Xi YiJie He Xue Bao vol 10 no 9 pp 1039ndash1048 2012

[12] T Wu T Liu P Y Zheng L J Xing and G Ji ldquoEffects ofQinggan Huoxue Recipe and its separated recipes on the

expression of tumor necrosis factor-120572 in rats with alcoholic liverinjuryrdquo Journal of Chinese Integrative Medicine vol 6 no 11 pp1145ndash1151 2008

[13] Z Z Chen Z L Wang C Y Deng et al ldquo(Z)-5-(4-me-thoxybenzylidene)thiazolidine-2 4-dione protects rats fromcarbon tetrachloride-induced liver injury and fibrogenesisrdquoWorld Journal of Gastroenterology vol 18 no 7 pp 654ndash6612012

[14] X Luan W Liao X Lai Y He Y Liu J Gong et al ldquoDynamicchanges of indoleamine 2 3-dioxygenase of Kupffer cells inrat liver transplant rejection and tolerancerdquo TransplantationProceedings vol 44 no 4 pp 1045ndash1047 2012

[15] C K Ho C W Lee J Lu et al ldquoNew hope for an old cure apilot animal study on selective venesection in attenuating thesystemic effects of ischaemic-reperfusion injuryrdquo Annals of theAcademy ofMedicine Singapore vol 38 no 7 pp 569ndash577 2009

[16] H Zeng Z Yuan H Zhu et al ldquoExpression of hPNAS-4 radio-sensitizes lewis lung cancerrdquo International Journal of RadiationOncologylowastBiologylowastPhysics vol 84 no 4 pp e533ndashe540 2012

[17] T Y Lee H H Chang J H Chen M L Hsueh and J JKuo ldquoHerb medicine Yin-Chen-Hao-Tang ameliorates hepaticfibrosis in bile duct ligation ratsrdquo Journal of Ethnopharmacologyvol 109 no 2 pp 318ndash324 2007

[18] E J Lee C Kim J Y Kim et al ldquoInhibition of LPS-inducedinflammatory biomarkers by ethyl acetate fraction of Patriniascabiosaefolia through suppression of NF-kappaB activation inRAW 2647 cellsrdquo Immunopharmacology and Immunotoxicol-ogy vol 34 no 2 pp 282ndash291 2012

[19] S K Huan K T Wang S D Yeh et al ldquoScutellaria baicalensisalleviates cantharidin-induced rat hemorrhagic cystitis throughinhibition of cyclooxygenase-2 overexpressionrdquo Molecules vol17 no 6 pp 6277ndash6289 2012

[20] H Zhang C H Yu Y P Jiang et al ldquoProtective effects ofpolydatin from polygonum cuspidatum against carbon tetra-chloride-induced liver injury in micerdquo PLoS One vol 7 no 9Article ID e46574 2012

[21] H Ghanim C L Sia S Abuaysheh et al ldquoAn antiinflammatoryand reactive oxygen species suppressive effects of an extractof Polygonum cuspidatum containing resveratrolrdquo Journal ofClinical Endocrinology andMetabolism vol 95 no 9 pp E1ndashE82010

[22] J Lv S Fu J Guo Y Liu H Yuan and X Xiao ldquoPrimaryresearch on daily administration times of rhubarb used to treatexperimental jaundice in ratsrdquoZhongguo Zhong Yao Za Zhi vol36 no 24 pp 3506ndash3510 2011

[23] L Qin X Peng S H Zhang L Wang and F Liu ldquoInfluenceof monkshood root-peony root combination on inflamation-induced agents and free radicalsrdquo Zhongguo Zhong Yao Za Zhivol 25 no 6 pp 370ndash373 2000

[24] M Zhang D Z Zhu Z S Li and X B Zhan ldquoRed peony rootdecoction in treatment of severe acute pancreatitis a random-ized controlled trialrdquo Zhong Xi Yi Jie He Xue Bao vol 6 no 6pp 569ndash575 2008

[25] U Andersson and K J Tracey ldquoHMGB1 is a therapeutictarget for sterile inflammation and infectionrdquo Annual Review ofImmunology vol 29 pp 139ndash162 2011

[26] P Kubes and W Z Mehal ldquoSterile inflammation in the liverrdquoGastroenterology vol 143 no 5 pp 1158ndash1172 2012

[27] J A Nogueira-Machado and C M de Oliveira Volpe ldquoHMGB-1 as a target for inflammation controllingrdquo Recent Patents onEndocrine Metabolic amp Immune Drug Discovery vol 6 no 3pp 201ndash209 2012


[28] Q Zhang C Wang Z Liu et al ldquoNotch signal suppresses Toll-like receptor-triggered inflammatory responses inmacrophagesby inhibiting extracellular signal-regulated kinase 12-mediatednuclear factor kappaB activationrdquo The Journal of BiologicalChemistry vol 287 no 9 pp 6208ndash6217 2012

[29] A G Porter and R U Janicke ldquoEmerging roles of caspase-3 inapoptosisrdquo Cell Death and Differentiation vol 6 no 2 pp 99ndash104 1999

[30] B Poligone D J Weaver Jr P Sen A S Baldwin Jr and RTisch ldquoElevated NF-120581B activation in nonobese diabetic mousedendritic cells results in enhanced APC functionrdquo Journal ofImmunology vol 168 no 1 pp 188ndash196 2002

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 The primer sequences used in fluorescence quantitative RT-PCR

Parameters 51015840 primer sequence 31015840 primer sequenceHMGB1 51015840TGTTCTGAGTACCGCCCAAA31015840 51015840TTTCGCTGCATCAGGTTTTC31015840

TLR4 51015840CCAGGAAGGCTTCCACAAGA31015840 51015840AATTCGACCTGCTGCCTCAG31015840

NF-120581B 51015840GCACGAGGCTCCTTTTCTCAA31015840 51015840CGTTTTTCTTCAATCCGGTGG31015840

Caspase-3 51015840ACCGATGTCGATGCAGCTAA31015840 51015840AGGTCCGTTCGTTCCAAAAA31015840

120573-Actin 51015840AAGGAGGCAAAGGACACCAA31015840 51015840AATGGCCCCCTTCACAGTTA 31015840

was purchased from Minophagen Pharmaceutical Co Ltd(Tokyo Japan) and fixed into a concentration of 156mgmLwith distilled water Qinggan Huoxue Recipe which wasboiled using Artemisia capillaris Patrinia Scutellaria baical-ensis Polygonum cuspidatum rhubarb and red Peony (2 4 4 4 1 4) was purchased fromAffiliatedHospital of ChengduUniversity of Traditional Chinese Medicine (ChengduChina) with a concentration of 297 gmL

22 Design of Animal Experiment 70 rats were randomizedinto four groups negative control group (Control 10 rats)fed with distilled water by gavage model group (Model 20rats) fed with distilled water by gavage and injected with D-GalN 14 gkg intraperitoneally three days after the gavageStronger Neo-Minophagen C group (SNMC 20 rats) fedwith SNMC 156mgkgd by gavage and injected with D-GalN 14 gkg intraperitoneally three days after the gavageQinggan Huoxue Recipe group (Experiment 20 rats) fedwith Qinggan Huoxue Recipe 297 gkgd (which equals625times clinical dose) by gavage and injected with D-GalN14 gkg intraperitoneally three days after the gavage Thegavage last for 5 days The dosage of Qinggan HuoxueRecipe used on rats was calculated by the formula Doserat= Dosehuman times (habeas indexrathabeas indexhuman) times (bodyweighthumanbody weightrat) times 23 [11] Based on this for-mula the translational coefficient 625 was produced In ourprevious clinical trial the dose of Qinggan Huoxue Recipeused on human was 285 g60 kgd (475 gkgd) At last thedose of 297 gkgd was achieved through the multiplicationof human dose (475 gkgd) and translational coefficient(625) The dose of 297 gkgd is a relatively very high-doseused in rats compared with the other studies of QingganHuoxue Recipe reported [4 12] Besides a dose responsestudy was carried out in preexperiment to confirm theusage of this high-dose (data not shown) 36 hours afterthe D-GalN injection 6mL blood was collected throughfemoral artery of alive rats for detection of serum alanineaminotransferase (ALT) aspartate aminotransferase (AST)total bilirubin (TBIL) albumin (ALB) cholinesterase (CHE)and prothrombin time (PT)Then the rats were sacrificed andthe left lobe of the liver was collected for further studies suchas hematoxylin and eosin (HE) staining proliferating cellnuclear antigen (PCNA) immunohistochemistry assay andfluorescence quantitative reverse transcription polymerasechain reaction (qRT-PCR)

23 Serum ALT AST TBIL ALB CHE and PT Determina-tion The serum biochemical parameters ALT AST TBIL

ALB CHE and PT which closely reflect the liver function [4]were analyzed by the Department of Laboratory MedicineAffiliated Hospital of Chengdu University of TraditionalChinese Medicine (Chengdu China)

24 Pathological Scores The liver specimens were fixedparaffin embedded and cut into 3- to 5-120583m sections Thesections were used for HE and PCNA staining The methodof HE staining has been introduced in the previous literature[13 14] All slides were read by three investigators who wereblinded to the allocation arm of the animal They were askedto grade the microscopic injuries seen in the liver using asemiquantitative scoring system [15] with zero indicating nodiscernable injury and 4 indicating the presence of severeinjuryThese scores were allocated based on their assessmentof the following histological features cellular oedema inter-stitial oedema neutrophil infiltration capillary congestionand structural distortion For each slide the average score ofat least three observations was considered as the pathologicalscore

25 PCNA Immunohistochemistry The PCNA immunohis-tochemistry kit was purchased from Boster BioengineeringCO Ltd (Wuhan China) Immunohistochemical stainingof PCNA was performed according to the manufacturerrsquosinstructions PCNA-positive cells were counted in 5 randomvisual fields under 40x magnifications for each section andthe number was expressed as the percentage of PCNA posi-tive cells to the total number of cells counted [16] Sectionswere examined microscopically for specific staining andphotographs were taken with a digital image-capture system(Olympus CX40 Tokyo Japan)

26 Fluorescence Quantitative RT-PCR The expression ofhigh mobility group box 1 (HMGB1) toll-like receptor 4(TLR4) nuclear factor kappa B (NF-120581B) and Caspase-3 wasdetected by qRT-PCR Total RNAs were extracted with Trizolreagent (Invitrogen) and reverse transcribed into cDNA bythe ABI Step-One Plus Real-Time PCR System (AppliedBiosystemCo CA USA)The primer sequences of the aboveparameters including 120573-actin were listed in Table 1

27 Survival Curves Another 55 rats were divided into theabove four groups with animal numbers of 10 15 15 and 15respectively for observation of survival The treatments werethe same as mentioned before The observation was begunwith the time of D-GalN injection while the endpoint wasset at 96 hours after the injection


0

200

400

600

Con

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Mod

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Expe

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ALT

(UL

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(a)

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TBIL

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rimen

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Groups

ampamp amp

(d)

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400

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SNM

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CHE

(UL

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ampamp

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(e)

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PT (S

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3 Results






Control Model

SNMC Experiment

(a)

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Control Model

SNMC Experiment

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PCN

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HM

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4 Discussion






Surv

ival

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1

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02

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SNMCModel

Survival functions

Experimental


119875 lt 005







5 Conclusion







Acknowledgments


References






































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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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3 Results






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SNMC Experiment

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Control Model

SNMC Experiment

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4 Discussion






Surv

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Survival functions

Experimental


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5 Conclusion







Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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SNMC Experiment

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Control Model

SNMC Experiment

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Survival functions

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119875 lt 005







5 Conclusion







Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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Survival functions

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119875 lt 005







5 Conclusion







Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Surv

ival

rate

1

08

06

04

02

0

SNMCModel

Survival functions

Experimental


119875 lt 005







5 Conclusion







Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


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 the effects of high-dose qinggan huoxue...

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