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Journal of Ethnopharmacology 124 (2009) 263–269

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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eneficial effects of Chinese prescription Kangen-karyu on diabetes associatedith hyperlipidemia, advanced glycation endproducts, and oxidative stress in

treptozotocin-induced diabetic rats

yun Young Kima, Takuya Okamotob, Takako Yokozawac,∗

Department of Food Science, Jinju National University, Jinju 660-758, Republic of KoreaIskra Industry Co., Ltd., 1-14-2 Nihonbashi, Chuo-ku, Tokyo 103-0027, JapanInstitute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

r t i c l e i n f o

rticle history:eceived 7 November 2008eceived in revised form 3 April 2009ccepted 20 April 2009vailable online 3 May 2009

eywords:angen-karyuxidative stressdvanced glycation endproducttreptozotocin

a b s t r a c t

Aim of the study: In the present study, we investigated the effects of Kangen-karyu, a traditional Chineseprescription comprising six herbs, on diabetes.Materials and methods: Kangen-karyu extract (50, 100, or 200 mg/kg body weight) was administeredto streptozotocin (STZ)-induced diabetic rats and serum and hepatic biochemical factors, and proteinexpressions associated with oxidative stress and advanced glycation endproduct (AGE) formation weremeasured.Results: The oral administration of Kangen-karyu significantly ameliorated hypertriglyceridemia inducedby STZ injection, while serum levels of glucose and total cholesterol were mildly affected. Kangen-karyualso markedly reduced the levels of AGEs and malondialdehyde (MDA), a lipid peroxide product usedas an indicator of oxidative stress in both serum and hepatic tissue. In addition, Kangen-karyu dose-dependently lowered the expression levels of Nε-(carboxymethyl) lysine, one of the major component ofAGEs closely associated with the pathogenesis of diabetes and liver cirrhosis, and receptor for AGEs, as

well as the expression levels of nuclear factor-�B, inducible nitric oxide synthase, and cyclooxygenase-2(COX-2) associated with oxidative stress. Especially, MDA levels in both serum and hepatic tissue andCOX-2 expression increased by STZ were recovered by Kangen-karyu (200 mg/kg body weight) to normallevels.Conclusions: Kangen-karyu showed favorable effects on hypertriglycemia, AGE formation, and oxida-tive stress in STZ-treated rats, suggesting beneficial effects on diabetes, diabetic hepatopathy, and liver

, as w

diseases such as cirrhosis

. Introduction

Oxidative stress is characterized by an imbalance betweenrooxidant generation and the antioxidant defense system owing toxcessive free radical production, a loss of antioxidant defenses, oroth. An increase in oxidative stress is implicated in the pathogene-is of numerous diseases, including diabetes and its complications,iver diseases such as alcoholic liver disease, nonalcoholic steato-epatitis, liver cirrhosis, and so on (Baynes and Thorpe, 1999;

est, 2000; Tanikawa and Torimura, 2006). In addition, advanced

lycation endproducts (AGE) formed by nonenzymatic glycationeactions such as Amadori, Schiff base, and Maillard, induce freeadical formation, accumulate during the normal aging process and

∗ Corresponding author. Tel.: +81 76 434 7670; fax: +81 76 434 5068.E-mail address: yokozawa@inm.u-toyama.ac.jp (T. Yokozawa).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.04.032

ell as cardiovascular and cerebrovascular diseases.© 2009 Elsevier Ireland Ltd. All rights reserved.

at accelerated rates during the course of diabetes, and are associ-ated with the pathogenesis of chronic diseases such as arthritis,atherosclerosis, and liver cirrhosis (Lapolla et al., 2005; Yagmur etal., 2006). Therefore, recently, antioxidant therapy has been thoughtto be effective for the prevention and treatment of various diseasesincluding diabetes and liver diseases, because oxidative stress playsa key role in the pathogenesis of human diseases (Da Ros et al.,2004; Medina and Moreno-Otero, 2005). AGE inhibitors workingvia various mechanisms at different steps in AGE formation andAGE-mediated damage such as AGE breakers, RAGE, and receptorsignaling blockers may also be effective in diseases such as diabetesand diabetic complications, Alzheimer’s disease, and rheumatoidarthritis (Rahbar and Figarola, 2002; Peyroux and Sternberg, 2006).

So, studies on the development of prevention and treatment agentsoriginating from natural products and organic synthesis for variousdiseases, including diabetic and liver diseases, have been activelyconducted regarding targets associated with oxidative stress andAGEs. Especially, traditional Chinese prescriptions are considered

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64 H.Y. Kim et al. / Journal of Ethn

seful for the treatment of various diseases, and some prescrip-ions showed marked protection against hepatotoxicity (Lin et al.,001).

Kangen-karyu (Guan-Yuan-Ke-Li), a crude drug developed fromtraditional Chinese prescription consisting of six herbs (Paeoniaeadix, Cnidii Rhizoma, Carthami Flos, Cyperi Rhizoma, Saussureaeadix, and Salviae Miltiorrhizae Radix), has been clinically useds a treatment for cardiovascular diseases such as angina pectorisnd cerebrovascular diseases. Kangen-karyu showed biologicalctivities such as platelet aggregation inhibition, hypertensionuppression, and the recovery of learning and memory impair-ent induced by senescence (Takahashi et al., 1992; Gao et al.,

001; Makino et al., 2002). In our previous studies, Kanken-aryu showed favorable ameliorative effects on fructose-inducedetabolic syndrome, such as hyperglycemia, hyperlipidemia, and

ypertension, through the reduction of triglyceride and cholesterolevels with the regulation of hepatic sterol regulatory element-inding protein-1 expression, and also exhibited protective effectsgainst diet-induced hypercholesterolemia in rats (Yokozawa et al.,006, 2007a,b).

In this study, we evaluated the effects of Kangen-karyu on serumnd hepatic biochemical factors and protein expression associatedith oxidative stress and AGE formation in STZ-treated rats.

. Materials and methods

.1. Reagents

The following reagents were purchased from Wako Purehemical Industries, Ltd. (Osaka, Japan): 4,6-dihydroxy-2-ercaptopyrimidine (2-thiobarbituric acid (TBA)), oxalic acid,

ovine serum albumin (BSA), 2-amino-2-hydroxymethyl-1,3-ropadiol (Tris (hydroxymethyl) aminomethane), Tween 20,henylmethyl sulfonyl fluoride (PMSF), protease inhibitor mixtureMSO solution, and skim milk powder. Dithiothreitol (DTT) wasurchased from BioVision Inc. (Mountain View, CA, USA). The

io-Rad protein assay kit was purchased from Bio-Rad LaboratoriesTokyo, Japan). Polyclonal anti-receptor for AGE (RAGE) antibody,olyclonal antibody for nuclear factor-�B (NF-�B) p65, monoclonalntibodies for cyclooxygenase-2 (COX-2), and inducible nitricxide synthase (iNOS), goat anti-rabbit IgG horseradish peroxidase

Fig. 1. Three-dimensional HPLC of Kangen-

acology 124 (2009) 263–269

(HRP)-conjugated secondary antibody, and goat anti-mouse IgGHRP-conjugated secondary antibody were purchased from SantaCruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyclonal anti-CMLantibody was kindly provided by Dr. R. Nagai (Kumamoto Univer-sity, Japan). STZ, glycerol, Nonidet P 40 (NP-40), and anti-mouse�-actin antibody were purchased from Sigma–Aldrich (St. Louis,MO, USA). ECL Western blotting detection reagent was purchasedfrom GE Healthcare (Piscataway, NJ, USA).

2.2. Preparation of Kangen-karyu extract

The composition of Kangen-karyu used in this study was: 2.25 gPaeoniae Radix (Paeonia lactiflora Pallas root), 2.25 g Cnidii Rhi-zoma (Cnidium officinale Makino rhizome), 2.25 g Carthami Flos(Carthamus tinctrius L. petal), 1.125 g Cyperi Rhizoma (Cyperusrotundus L. rhizome), 1.125 g Aucklandiae Radix (Aucklandia lappaDcne. root), and 4.5 g Salviae Miltiorrhizae Radix (Salvia miltior-rhiza Bunge root). This prescription was extracted with 25 volumesof water at 100 ◦C for 1 h. After filtration, the solution was evapo-rated under reduced pressure to give an extract at a yield of 44%, byweight, of the starting materials. A typical high-performance liquidchromatogram (HPLC) of Kangen-karyu is illustrated in Fig. 1. Eachsample was dissolved in 50% aqueous ethanol with sonication, andfiltered through a Cosmonice filter (PVDF, 0.45 �m, Nacalai Tesque,Inc., Japan). Reverse-phase HPLC analysis was performed using aCosmosil 5C18-AR II column (250 mm × 4.6 mm i.d., Nacalai Tesque,Inc.) with elution gradients of 4–30% (39 min) and 30–75% (15 min)CH3CN in 50 mM H3PO4 at a flow rate of 0.8 ml/min. The ultraviolet(UV) absorbance from 200 to 400 nm was monitored with a JASCOphotodiode array detector MD-910. Peak areas were quantified at311 nm for lithospermic acid B and 331 nm for rosmarinic acid. Avoucher specimen is deposited in the herbarium of the Universityof Toyama.

2.3. Animals and treatment

The Guidelines for Animal Experimentation approved by theUniversity of Toyama were followed in all experimental studies.Five-week-old male Wistar rats (120–130 g) were obtained fromJapan SLC, Inc. (Hamamatsu, Japan), and kept in wire-bottomedcages under a 12-h light/dark cycle. The room temperature and

karyu showing its major compounds.

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H.Y. Kim et al. / Journal of Ethn

umidity were maintained automatically at about 25 ◦C and 60%,espectively. They were given free access to laboratory pellethow (CLEA Japan Inc., Tokyo, Japan; comprising 24.0% protein,.5% lipids, and 60.5% carbohydrate) and water. After several daysf adaptation, the rats were injected intraperitoneally with STZ50 mg/kg body weight) in 10 mM citrate buffer (pH 4.5), and nor-

al group rats were given a sham injection of citrate buffer withoutTZ. Ten days after STZ injection, blood samples were obtainedetween 10 and 11 a.m., avoiding the influence of food consump-ion, to determine glucose levels, and then rats with glucose levels400 mg/dl were randomly divided into five experimental groupsomposed of five rats each. The diabetic control group was givenater, while the other diabetic groups were orally administered0, 100, or 200 mg/kg body weight daily of extract dissolved inater via gavage for 20 days. After 20 days of the administrationeriod, blood samples were obtained between 10 and 11 a.m. fromhe abdominal aorta under etherization, and then the serum wasmmediately separated from the blood samples by centrifugation.fter perfusion through the abdominal artery with ice-cold phys-

ological saline, the liver was extirpated for assays, immediatelymmersed in liquid nitrogen, and kept at −80 ◦C until analysis.

.4. Serum levels of biochemical factors

Serum levels of glucose, triglycerides, and total cholesterol werexamined using commercial reagents (Glucose CII-Test, Triglyceride-Test, and Cholesterol E-Test) obtained from Wako Pure Chemicalndustries, Ltd. (Osaka, Japan). Serum glycosylated protein was col-rimetrically measured by determining 5-hydroxymethylfurfural5-HMF), released form of nonenzymatically bound glucose inerum (McFarland et al., 1979). Malondialdehyde (MDA) levels wereetermined using the methods of Naito and Yamanaka (1978).

.5. AGE level in hepatic tissue

The hepatic AGE level was determined by the method ofakayama et al. (1993). In brief, minced hepatic tissue was delip-

dated with chloroform and methanol (2:1, v/v) overnight. Afterashing, the tissue was homogenized in 0.1 N NaOH, followed by

entrifugation at 8000 × g for 15 min at 4 ◦C. The amounts of AGEsn these alkali-soluble samples were determined by measuring theuorescence at an emission wavelength of 440 nm and an excita-ion wavelength of 370 nm. A native BSA preparation (1 mg/ml of.1 N NaOH) was used as a standard, and its fluorescence intensityas defined as one unit of fluorescence. The fluorescence valuesf samples were measured at a protein concentration of 1 mg/mlnd expressed in arbitrary units (AU) compared with a native BSAreparation.

.6. MDA level in hepatic mitochondria

The MDA level in hepatic mitochondria was assayed using ourrevious experimental method (Yokozawa et al., 2007a,b). The liveras homogenized in a 9-fold volume of ice-cold 0.9% NaCl solu-

ion. Mitochondria were prepared from hepatic homogenate byifferential centrifugation (800 and 12,000 × g, respectively) at 4 ◦Cor 15 min. Each pellet was resuspended in preparation medium,nd then 250 �l of each pellet suspension was added to 750 �lBA–TCA–HCl solution (0.4% TBA, 15% TCA, and 2.5% HCl) and it

as heated at 95 ◦C for 20 min. After cooling in an ice-bath, sam-les were centrifuged at 1000 × g at room temperature for 10 mino transfer supernatants from the denatured protein. The MDA levelas determined by measuring the absorbance at 532 nm and was

xpressed in nmol/mg protein. The protein level was examinedsing BSA as the standard.

acology 124 (2009) 263–269 265

2.7. Protein source

Each liver was homogenized by a Potter–Elvehjem homogenizerin 4 volumes (w/v) of buffer A containing 25 mM Tris–HCl (pH 7.5),250 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM DTT, and proteaseinhibitor mixture (100 mM 4-(2-aminoethyl) benzenesulfonyl flu-oride, 0.08 mM aprotinin, 2 mM leupeptin, 5 mM bestatin, 1 mMpepstatin A, and 1.5 mM E-64). Homogenates were incubated for15 min on ice, 10% NP-40 was added, and then they were cen-trifuged at 4000 × g at 4 ◦C for 5 min. Supernatants were used foriNOS and COX-2 protein determination. For the purpose of NF-�Bprotein detection, the liver was homogenized by a Potter–Elvehjemhomogenizer in 4 volumes (w/v) of buffer B containing 10 mM 2-[4-(2-hydroxyethyl)-1-piperazyl] ethanesulfonic acid (HEPES) (pH7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and pro-tease inhibitor mixture as above. Homogenates were incubated for15 min on ice, 10% NP-40 was added, and then were centrifuged at4000 × g at 4 ◦C for 5 min. Pellets were resuspended in 2 volumes(w/v) of buffer C containing 20 mM HEPES, 400 mM NaCl, 1 mMEDTA, 1 mM DTT, 1 mM PMSF, and the protease inhibitor mixture.Homogenates were kept for 15 min at 4 ◦C and then centrifuged at14,000 × g for 15 min at 4 ◦C. Supernatants were collected in micro-centrifuge tubes and used for NF-�B protein determination. Further,the liver was homogenized with ice-cold lysis buffer (pH 7.5) con-taining 137 mM NaCl, 20 mM Tris–HCl, 1% (v/v) Tween 20, 10% (v/v)glycerol, 1 mM PMSF, and protease inhibitor mixture DMSO solu-tion, and then centrifuged at 2000 × g for 10 min at 4 ◦C to obtainthe CML and RAGE protein sources. The protein concentration wasdetermined by the Bio-Rad protein assay kit using BSA as a stan-dard. Each sample was denatured by boiling in Laemmli samplebuffer and stored at −80 ◦C until the assay.

2.8. Western blot analyses

Western blot analyses were carried out using 30 �g of eachsample through 8 or 10% sodium dodecylsulfate-polyacrylamidegel electrophoresis to determine the iNOS, COX-2, NF-�B, CML,and RAGE protein expressions. The separated proteins were elec-trophoretically transferred to a nitrocellulose membrane, blockedwith 5% (w/v) skim milk solution for 1 h, and then incubatedovernight at 4 ◦C with primary antibodies to iNOS, COX-2, NF-�B,CML, RAGE, and �-actin, respectively. After the blots were washed,they were incubated with goat anti-rabbit and/or goat anti-mouseIgG HRP-conjugated secondary antibody for 90 min at room tem-perature. Each antigen–antibody complex was visualized using ECLWestern Blotting Detection Reagents and detected by chemilumi-nescence with LAS-1000 Plus (Fujifilm, Japan). Band densities weredetermined by Scion image software (Scion Corporation, Frederick,MD, USA) and quantified as the relative ratio to �-actin. The evalu-ation of these protein levels at mean values against normal rats isrepresented as 1, and the corresponding values for the diabetic ratsare expressed as the ratios of these values.

2.9. Statistical analysis

The results are expressed as means ± S.E. The effect on eachparameter was examined using one-way analysis of variance. Indi-vidual differences among groups were analyzed by Dunnett’s test.p < 0.05 was considered significant.

3. Results and discussion

The changes in the body weight, food intake, water intake, andurine volume are summarized in Table 1. The body weight in STZ-induced diabetic rats was significantly lower than that of normalrats, but the administration of Kangen-karyu led to a tendency

266 H.Y. Kim et al. / Journal of Ethnopharmacology 124 (2009) 263–269

Table 1Characteristics of experimental rats.

Group Body weight (g) Food intake (g/day) Water intake (ml/day) Urine volume (ml/day)

Normal rats 250.1 ± 2.5* 18.2 ± 0.5* 40.7 ± 4.2 16.2 ± 3.0*

Diabetic ratsControl 188.2 ± 10.2 30.6 ± 0.7 150.5 ± 11.0 125.3 ± 8.2Kangen-karyu (50 mg/kg B.W.) 190.0 ± 9.7* 30.1 ± 0.4 150.0 ± 9.4 124.4 ± 7.1Kangen-karyu (100 mg/kg B.W.) 194.6 ± 8.2* 28.1 ± 0.8* 148.0 ± 9.5 120.0 ± 6.7Kangen-karyu (200 mg/kg B.W.) 195.9 ± 8.3* 27.0 ± 0.4* 142.2 ± 7.4 116.9 ± 7.4

* p < 0.05 vs. each diabetic control value.

Table 2Serum biochemical parameters.

Group Glucose (mg/dl) Glycosylated protein(nmol/mg protein)

Triglycerides (mg/dl) Total cholesterol (mg/dl) MDA (nmol/ml)

Normal rats 111.1 ± 9.5* 14.2 ± 0.5* 42.7 ± 4.4 47.4 ± 2.0* 2.23 ± 0.17

Diabetic ratsControl 562.2 ± 16.2 19.3 ± 0.4 240.1 ± 72.0 88.4 ± 8.2 3.46 ± 0.23Kangen-karyu (50 mg/kg B.W.) 516.9 ± 12.0* 18.6 ± 0.4 164.0 ± 65.4 86.3 ± 7.0 3.40 ± 0.32

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resulting in the improvement of diabetic pathological conditionssuch as diabetic hepatopathy.

CML, one of the well-known major component of AGEs detectedin serum and tissue, is a product of both lipid peroxidation andglycosylation reactions (Fu et al., 1996). The serum CML level may

Table 3AGEs and MDA in hepatic tissue.

Group AGEs (AU) MDA (nmol/mg protein)

Normal rats 0.495 ± 0.012* 0.194 ± 0.010

Diabetic rats

Kangen-karyu (100 mg/kg B.W.) 498.4 ± 15.1* 17.9 ± 0.3*

Kangen-karyu (200 mg/kg B.W.) 479.0 ± 10.6* 16.6 ± 0.4*

* p < 0.05 vs. each diabetic control value.

oward an increase. The food intake, water intake, and urine vol-me of control rats with diabetes were much higher than those oformal rats. However, these parameters of rats with STZ-inducediabetes given Kangen-karyu orally for 20 days showed a tendencyo decline without significance.

Diabetes induced by STZ in animal models is associated withype 1, characterized by a loss of �-cells of islets of Langerhansn the pancreas, leading to insulin deficiency. In our experi-

ents, STZ-treated rats showed markedly increased serum glucose,riglyceride, and total cholesterol levels. Hyperglycemia is widelyecognized as the pathogenesis of diabetes and diabetic complica-ions, and enhances the formation and accumulation of AGEs as wells oxidative stress (Rolo and Palmeira, 2006). Hypertriglyceridemiand hypercholesterolemia are associated with abnormalities ofipoprotein levels in the blood, and STZ increased the levels ofhylomicron, VLDL, and LDL in the blood, and decreased the HDLevel (Gylling et al., 2004). As shown in Table 2, the levels of serumlucose and triglycerides in the STZ-treated rats were markedly ele-ated by 5 times compared to those of normal rats, and the totalholesterol level was also increased by 1.87 times. However, thelevated serum triglyceride level was significantly reduced by theral administration of Kangen-karyu (50, 100, or 200 mg/kg) in aose-dependent manner, whereas serum levels of glucose and totalholesterol were mildly affected. These results imply that Kangen-aryu can prevent diabetic pathological conditions induced byyperglycemia and hyperlipidemia in diabetes.

Lipid peroxide is a well-established mechanism of cellular injuryn humans, and is used an indicator of oxidative stress in cellsnd tissues. MDA, lipid peroxide derived from polyunsaturatedatty acids, is widely employed as an indicator of oxidative stresss well as lipid peroxidation because it is more abundant thanther reactive carbonyl compounds. Lipid peroxidation is associ-ted with diabetes and diabetic complications as well as liver injuryMaellaro Casini et al., 1990; Davì et al., 2005). Table 2 shows thaterum levels of MDA markedly increased on STZ injection, andhey were significantly reduced by oral Kangen-karyu adminis-ration in a dose-dependent manner. Especially, this serum MDA

evel reduced by Kangen-karyu is comparable to that of normalats. Also, Table 3 shows levels of both AGEs and MDA in hepaticissue were increased 1.5-fold in STZ-treated rats compared to nor-

al rats. Oral Kangen-karyu administration significantly reducedhese two hepatic biochemical factors increased by STZ in a dose-

138.0 ± 20.9 76.6 ± 4.7 3.30 ± 0.24115.3 ± 39.4 76.0 ± 7.8 2.35 ± 0.24

dependent manner. Especially, the hepatic MDA level was reducedto that of normal rats. MDA levels in both serum and hepatic tissueincreased by STZ were reduced by Kangen-karyu administration tothose of normal rats. Serum levels of glycosylated protein and MDAalso markedly increased on STZ injection, and these biochemicalfactors were significantly reduced by oral Kangen-karyu adminis-tration in a dose-dependent manner. Especially, the serum MDAlevel reduced by Kangen-karyu is comparable to that of normal rats.These results suggest that Kangen-karyu can alleviate oxidativestress under diabetic pathological conditions through the inhibitionof lipid peroxidation.

Serum AGEs such as glycosylated albumin and hemoglobinhave been produced by the glycation reaction, which resulted ina decrease in free protein levels (Schleicher et al., 1984). So, serumAGEs are clinically used as an index of glucose control in diabetes.AGE levels in serum and organs such as the kidney, liver, retina,and brain are also associated with normal aging, inflammation,liver disease, Alzheimer’s disease, and diabetes and its compli-cations such as diabetic retinopathy and nephropathy (Lapolla etal., 2005; Ramasamy et al., 2005; Yagmur et al., 2006). In ourstudies, the levels of AGEs in serum and hepatic tissue showeda significant increase in STZ-treated rats, but Kangen-karyu sig-nificantly decreased AGE levels in both serum and hepatic tissue(Tables 2 and 3). These results indicate that the administration ofKangen-karyu might prevent the pathogenesis of diabetic compli-cations caused by the glycosylation of serum proteins, eventually

Control 0.742 ± 0.015 0.282 ± 0.046Kangen-karyu (50 mg/kg B.W.) 0.684 ± 0.025* 0.269 ± 0.024Kangen-karyu (100 mg/kg B.W.) 0.674 ± 0.014* 0.235 ± 0.021Kangen-karyu (200 mg/kg B.W.) 0.611 ± 0.016* 0.208 ± 0.019

* p < 0.05 vs. each diabetic control value.

H.Y. Kim et al. / Journal of Ethnopharmacology 124 (2009) 263–269 267

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rovide a supplementary diagnostic marker for the advanced stagesf liver cirrhosis (Yagmur et al., 2006). In addition, RAGE, receptoror AGEs, is activated by CML, which results in the activation of NF-B and production of proinflammatory cytokines (Yan et al., 1994;aslbeck et al., 2005). On the other hand, a number of proteins

n the liver have been identified as AGE receptors, such as OST-8, 80K-H, galectin-3, RAGE, SR-B1, CD36, and LOX-1 (Miyazaki etl., 2002; Horiuchi et al., 2003). Galectin-3, a multifunctional pro-ein of the carbohydrate-binding lectin family, plays a crucial rolen the AGE–receptor complex which mediates protection againstGE-induced tissue injury, and is associated with hepatic fibrosis,onalcoholic fatty liver, cirrhotic liver, and hepatocellular carci-oma (Hsu et al., 1999; Henderson et al., 2006; Nomoto et al., 2006).D36, a member of the class B scavenger receptor family, is a recep-or for oxidized LDL and HDL, and regulates fatty acid transportHoriuchi et al., 2003; Thorne et al., 2007). SR-B1, a multiligandDL receptor, plays crucial roles in reverse cholesterol transportnd the atheroprotective functions of HDL (Kriger, 2001). LOX-1as identified as the major receptor for oxidized LDL in endothelial

ells, and is associated with the pathogenesis of vascular disorders,articularly atherosclerosis (Chen et al., 2002). Finally, RAGE is asso-iated with hepatic ischemia and reperfusion injury (Zeng et al.,004). Therefore, AGE inhibitors and agonists and/or antagonistsf one or a number of receptors of the scavenger receptor familyould be considered as useful targets for the treatment of diabetesnd diabetic complications, atherosclerosis, and liver diseases. Inur Western blotting analyses, the expression volumes of CML andAGE in STZ-treated rats were significantly increased compared toormal rats, but Kangen-karyu effectively reduced these elevatedolumes (Fig. 2). These results suggest that the expressions of hep-tic AGE receptors such as OST-48, 80K-H, galectin-3, RAGE, SR-B1,D36, and LOX-1 may also be affected by Kangen-karyu, whichrotects against liver damage induced by STZ.

On the other hand, NF-�B is a heterodimeric protein composedf different combinations of members of the Rel family of transcrip-ion factors, including NF-�B1 (p105/p50), NF-�B2 (p100/p52), RelAp65), RelB, and c-Rel. The Rel/NF-�B family is involved mainly intress-induced, immune, and inflammatory responses, and NF-�Ban be activated by the exposure of cells to lipopolysaccharidesr inflammatory cytokines such as tumor necrosis factor (TNF)r interleukin-1 (IL-1), viral infection, UV irradiation, B- or T-

ell activation, and by other physiological and nonphysiologicaltimuli (Baldwin, 1996). NF-�B activated by TNF modulates celleath in the liver (Luedde et al., 2006). Also, nitric oxide (NO)roduced by iNOS in activated macrophages is one of the most

mportant inflammatory mediators and induces various adverse

separate experiments and the means ± S.E. of the results obtained from the tissues

responses such as tissue injury and apoptosis. The iNOS gene isexpressed by hepatocytes in a number of physiologic and patho-physiologic conditions affecting the liver, including septic andhemorrhagic shock. The regulation of iNOS expression is com-plex and occurs at multiple levels in the gene expression pathway.Cytokines such as TNF-�, IL-1�, and interferon-� synergisticallyactivate iNOS expression in the liver. iNOS expression requires thetranscription of NF-�B and is down-regulated by steroids, trans-forming growth factor-�, heat shock response, p53, and NO itself(Taylor et al., 1998). COX-2 is expressed in response to a varietyof proinflammatory agents and cytokines, and is associated withliver pathogenesis, including fibrosis and cancer. COX-2 inhibitorsexhibited significant anti-proliferative effects on a hepatocellularcarcinoma cell line by inducing apoptosis and cell cycle arrest andblocking growth signaling pathways (Hu, 2003; Cheng and Hada,2005). NF-�B, iNOS, and COX-2 are involved in hepatic cell death,hepatic ischemia–reperfusion injury, fibrosis, cirrhosis, and cancer(Baldwin, 1996; Hu, 2003; Cheng and Hada, 2005; Luedde et al.,2006). In our Western blotting analyses, STZ treatment also resultedin the increased expression of NF-�B, iNOS, and COX-2, proteinsassociated with oxidative stress, while the expressions of thesethree proteins were markedly reduced by Kangen-karyu admin-istration in a dose-dependent manner. Of note, COX-2 was fullyrecovered by Kangen-karyu at a concentration of 200 mg/kg to theexpression level of normal rats (Fig. 3).

Our Western blotting analyses suggested that inhibitions of CMLand RAGE by Kangen-karyu resulted in a decrease in oxidative stressand NF-�B activation, and NF-�B translocation inhibition led to adecrease in the induction of iNOS and COX-2 expressions. Kangen-karyu might show favorable ameliorative effects on liver damageinduced by STZ via the mechanisms shown in the scheme in Fig. 4on the basis of references.

In conclusion, Kangen-karyu significantly reduced the serumtriglyceride and AGE levels, the lipid peroxide levels in both serumand hepatic tissue, and the expression volumes of CML, RAGE, NF-�B, iNOS, and COX-2 increased by STZ injection. These effects ofKangen-karyu may be attributed to its antioxidant activities, result-ing from the harmonization of its six components, because allcomponents are commonly known to be rich in antioxidant com-pounds (Ohta et al., 1990; Lee et al., 2005; Zhou et al., 2005; Kim etal., 2007; Pandey et al., 2007; Yazdanparast and Ardestani, 2007).

Antioxidant agents are well-known to effectively regulate biochem-ical factors as gene expression increases due to oxidative stress(Scott and King, 2004; Aragno et al., 2005; Lapolla et al., 2005;Yagmur et al., 2006). Our results suggested that Kangen-karyushowed beneficial effects on diabetes and diabetic complications

268 H.Y. Kim et al. / Journal of Ethnopharmacology 124 (2009) 263–269

Fig. 3. NF-�B, iNOS, and COX-2 expressions in hepatic tissue. Data shown are representative of five separate experiments and the means ± S.E. of the results obtained fromthe tissues of five rats from each group. Significance: *p < 0.05 vs. each diabetic control value.

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Fig. 4. Proposed effect of Kangen-ka

uch as atherosclerosis, liver disease, as well as cardiovascular anderebrovascular diseases. Further studies regarding the effect ofangen-karyu on hepatic AGE receptors, including scavenger recep-

ors, will help to elucidate the hepatoprotective mechanism of thishinese prescription.

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