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Antifibrotic effects of CGX, a traditional herbal formula, and its mechanisms in rats
Journal of Ethnopharmacology 127 (2010) 534–542
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Antifibrotic effects of CGX, a traditional herbal formula, and its mechanisms in rats Jing-Hua Wang, Jang-Woo Shin, Jin-Young Son, Jung-Hyo Cho, Chang-Gue Son ∗ Liver-Immune Research Center, Daejeon Oriental Hospital of Daejeon University, 22-5 Daeheung-dong, Jung-gu, Daejeon 301-704, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 February 2009 Received in revised form 25 September 2009 Accepted 5 October 2009 Available online 13 October 2009 Keywords: Antifibrosis Antioxidant Superoxide dismutase Catalase Glutathione Glutathione peroxidase
a b s t r a c t Aim: CGX is a modification of a traditional herbal medicine for “liver cleaning,” which is used to treat various chronic liver disorders in oriental clinics. This study investigated the antifibrotic effects and associated mechanisms of CGX. Materials and methods: Liver fibrosis was induced in rats by dimethylnitrosamine (DMN; 10 mg kg−1 , ip) injection on 3 consecutive days per week for 4 weeks. CGX (100 or 200 mg kg−1 , po) was administrated once a day for 4 weeks. Three cell lines (HepG2, RAW 264.7, and HSC-T6) were used to examine its mechanisms. Results: CGX treatment dramatically ameliorated the change in liver and spleen weight and serum albumin (p < 0.01), aspartate transaminase (p < 0.01), alanine transaminase (p < 0.01), alkaline phosphatase (p < 0.01), and total bilirubin (p < 0.01) levels. Histopathologically, CGX administration decreased necrosis, inflammatory cell infiltration, and collagen accumulation. The antifibrotic effects of CGX were confirmed from hydroxyproline determination and the reduction in the numbers of activated hepatic stellate cells. In addition, antioxidant proteins, glutathione content, and glutathione peroxidase, catalase, and superoxide dismutase activities were maintained in the CGX-treated groups compared with the DMN group. CGX downregulated fibrosis-related genes (inducible nitric oxide synthase, tumor necrosis factor-alpha, transforming growth factor-beta, connective tissue growth factor, and platelet-derived growth factorbeta) and decreased the protein levels of profibrotic cytokines (transforming growth factor-beta and platelet-derived growth factor-beta) in liver tissues. In the cell line-based studies, CGX showed supportive effects, such as the protection of hepatocytes from CCl4 -toxicity, inhibition of NO production in RAW 264.7 cells, and inactivation of hepatic stellate cells. Conclusion: These results demonstrated the antifibrotic effects of CGX and the corresponding mechanisms associated with sustaining the antioxidative system and inhibiting hepatic stellate cell activation via the downregulation of fibrogenic cytokines. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The chronic destruction of liver tissue can cause gradual, progressive fibrotic change, and the development of liver cirrhosis is
Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CCl4 , carbon tetrachloride; CTGF, connective tissue growth factor; DMN, dimethylnitrosamine; ECM, extracellular matrix; GSH, glutathione; GSH-px, glutathione peroxidase; HE, hematoxylin and eosin; HGF, hepatocyte growth factor; HPLC, high-performance liquid chromatography; HSCs, hepatic stellate cells; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MMP-2, matrix metalloproteinase 2; PDGF-, plateletderived growth factor-beta; SOD, superoxide dismutase; TBA, thiobarbituric acid; TGF-, transforming growth factor-beta; TIMP-1, tissue inhibitor of matrix metalloprotease-1; TIMP-2, tissue inhibitor of matrix metalloprotease-2; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +82 42 229 6723; fax: +82 42 257 6398. E-mail address: [email protected] (C.-G. Son). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.10.001
a critical event in determining the consequences of chronic hepatic disorders and their clinical outcomes (Friedman, 2003; Gines et al., 2004). The roles of various key cytokines, cellular effectors including hepatic stellate cells (HSCs), and the determinants of extracellular matrix (ECM) turnover in the fibrotic process have been well elucidated (Iredale, 2007; Kisseleva and Brenner, 2008). Currently, it is believed that liver fibrosis is reversible, which increases the hope of treatments for liver fibrosis and cirrhosis (Fallowfield et al., 2006; Fowell and Iredale, 2006). However, because no universally effective therapeutic drug for chronic liver injury and fibrotic progression is available, the search for antifibrotic agents, including herbal-derived ingredients, is a major medical objective (Levy et al., 2004; Yang et al., 2008). CGX is an herbal drug that originated from a traditional Korean formula composed of 13 herbs and other items. It is prescribed for patients who suffer from various chronic liver diseases, such as alcoholic liver disorders, chronic viral hepatitis, and liver cirrhosis (Cho et al., 2001; Son et al., 2001). Previously, we reported
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the safety of CGX in a beagle dog-based toxicological study and its hepatoprotective effects in acute and chronic liver injury models (Choi et al., 2006; Shin et al., 2006; Hu et al., 2008). However, the antifibrotic properties of CGX and mechanisms responsible for those effects require further experimental evaluation. Here, we investigated the antifibrotic effects of CGX using a modified schedule with a different animal strain from a previous model and cell lines derived from the three main cellular components of liver tissue. We explored the potential mechanisms of these effects, focusing on antioxidative actions and HSC-related profibrogenic cytokines. 2. Materials and methods 2.1. Reagents and chemicals Dimethylnitrosamine (N-nitrosodimethylamine or DMN), hydroxyproline, p-dimethylaminobenzaldehyde, 1,1,3,3tetraethoxypropane (TEP), chloramine-T, potassium chloride (KCl), n-butanol, 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), reduced glutathione, glutathione reductase, glutathione peroxidase (GSH-px), reduced -nicotinamide adenine dinucleotide phosphate (-NADPH), and lipopolysaccharide (LPS, Salmonella typhosa) were purchased from Sigma (St. Louis, MO, USA); perchloric acid was obtained from GFS Chemical (Columbus, OH, USA); thiobarbituric acid (TBA) was purchased from Lancaster (Lancashire, England); and carbon tetrachloride (CCl4 ) was purchased from Junsei Chemical (Tokyo, Japan). Anti-␣-SMA mouse monoclonal antibody was from Abcam (Cambridge, UK); histofine was from Nichirei Biosciences (Tokyo, Japan); diaminobenzidine (DAB) was from BioGenex (CA, USA); and Mayer’s hematoxylin was from Wako Pure Chemical Industries (Japan). 2.2. CGX and high-performance liquid chromatography (HPLC)-based fingerprinting All the herbs used met the Korean Pharmacopoeia standards. CGX was manufactured by Samik Pharmacy (Seoul, Korea) using the approved Good Manufacturing Practice of the Korea Food and Drug Administration. Briefly, 120 kg of the 13 herbs (Table 1) were boiled in 1200 L of distilled water for 4 h at 100 ◦ C and then filtered using a 300-mesh filter (50 m). Part of this batch was filtered through filter paper (Advantec, Toyo Roshi Kaisha, Tokyo, Japan) and lyophilized in our laboratory for this study. The final extraction gave a yield of 10.71% (w/w), and the batch used in this experiment was stored for future use (voucher specimen No. CGX-2007-09WE-SI). The final CGX extract satisfied the criteria of the Korean Table 1 The herbal prescription of CGX. Scientific name
Part used
Voucher specimen number
Relative amount (g)
Artemisia capillaries Thunberg Trionyx sinensis Wiegmann Raphanus sativus Linne Atractylodes macrocephala Koidzumi Poria cocos Wolf Alisma orientalis Juzepczuk Atractylodes chinensis Koidzumi Salvia miltiorrhiza Bunge Polyporus umbellatus Fries Poncirus trifoliate Rafinesqui Amomum villosum Loureiro Glycyrrhiza uralensis Fischer Aucklandia lappa Decne
Herba Carapax Semen Rhizoma
AC-2006-01-He TS-2006-01-Ca RS-2006-01-Se AM-2006-01-Rh-Al
5 5 5 3
Poria Rhizoma Rhizoma
PC-2006-01-Po AO-2006-01-Rh AC-2006-02-Rh
3 3 3
Radix Polyporus Fructus Fructus Radix Radix
SM-2006-01-Ra PU-2006-01-Po PT-2006-01-Fr AV-2006-01-Fr GU-2006-01-Ra AL-2006-01-Ra
3 2 2 2 1 1
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Pharmaceutical Codex, including the quantity of each herb, contamination by heavy metals, general bacteria, fungi, and specific pathogens, and the quantity of ingredients. A CGX fingerprint was produced using the two-dimensional high-performance liquid chromatography (HPLC) profile of five major compositional herbs and their main compounds: Glycyrrhiza uralensis root, Artemisia capillaries, Raphanus sativus seeds, Salvia miltiorrhiza root, and Poncirus trifoliate fruit vs. liquiritin and glycyrrhizin, 6,7-dimethoxycoumarin, naringin, rosmarinic acid, and poncirin, respectively (Fig. 1). Briefly, after dissolution (20 mg of CGX and 2 mg of the five herbal extracts in 1 mL of water; 0.01 mg of the six standards in 1 mL of water or 50% methanol) and filtration, these drugs were subjected to HPLC analysis. The HPLC system consisted of an SCL-10A system controller, LC-10AD pump, SPD-10MVP diode array detector, and CTO-10AS column temperature controller (Shimadzu, Kyoto, Japan). A Phenomenex Prodigy C18 (2.0 mm × 150 mm) column was eluted with solvents A (10% acetonitrile in water containing 0.05% formic acid) and B (90% acetonitrile in water) at a flow rate of 0.4 mL/min. Solutions 100% A and 0% B were changed to 75% A/25% B at 30 min, and to 25% A/75% B at 60 min. All chromatograms were obtained at a wavelength of 230 nm. 2.3. Cell lines and cell culture In order to study the effects of CGX on the main cellular components of liver tissue, three cell lines were used for in vitro experiments. Human hepatocellular carcinoma cell line HepG2 and mouse leukemic monocyte macrophage cell line RAW 264.7 were purchased from the Korea Cell Line Bank (KCLB); rat hepatic stellate cell line HSC-T6 was obtained from Dr. S.L. Friedman. HepG2 and RAW 264.7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (WelGENE, Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS) (WelGENE, Daegu, South Korea) at 37 ◦ C in a 5% CO2 humidified incubated environment, and HSC-T6 cells were cultured in DMEM containing 5% FBS. Logarithmic growth phase cells were subcultured three times a week for in vitro experiments. 2.4. Animals and experimental design Forty male Sprague–Dawley rats (6 weeks old, 160–180 g) were purchased from Korea Animal Tech (Gyeonggi-do, Korea). After 7 days of acclimation at 22 ± 2 ◦ C under a 12-h light/12-h dark cycle with free access to water and food, the rats were divided into five groups (normal, DMN, CGX100, CGX200, and silymarin) of eight animals each. The rats were injected intraperitoneally with DMN (10 mg kg−1 ) on 3 consecutive days per week for 4 weeks. The normal group was injected with normal saline instead of DMN. CGX (100 or 200 mg kg−1 ) or silymarin (50 mg kg−1 ) as a positive control was given orally to the respective groups seven times per week for 4 weeks, and distilled water was administered to the normal and DMN groups. On the final day, all of the rats were sacrificed under ether anesthesia, and the blood, liver, and spleen were collected for biochemical analyses and other measurements. The experiments were conducted in accordance with the Policy on the Humane Care and Use of Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health (NIH). 2.5. Histopathologic examination with immunohistochemistry and biochemical analyses The liver tissue was evaluated histomorphologically with hematoxylin and eosin (HE) and Masson’s trichrome stains. The histopathologic features, such as necrosis or inflammatory cell infiltration and collagen fiber changes, were examined using light microscopy in a blind manner. Immunohistochemistry for alpha
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Fig. 1. HPLC-based fingerprinting for CGX. The five major component herbs of CGX and their main compounds were subjected to HPLC. All chromatograms were obtained at a wavelength of 230 nm (details in the text). GRE, Glycyrrhiza uralensis root; AHE, Artemisia capillaries; RSE, Raphanus sativus seeds; SRE, Salvia miltiorrhiza root; PFE Poncirus trifoliate fruit; A, liquiritin; B, 6,7-dimethoxycoumarin; C, naringin; D, rosmarinic acid; E, poncirin; F, glycyrrhizin.
smooth muscle actin (␣-SMA) was performed. Briefly, the tissue sections were deparaffinized, hydrated, and treated with normal serum for 30 min. Next, the tissue slides were treated with anti-␣SMA mouse monoclonal antibody (1:200) overnight. After washing three times with PBS, two drops of Histofine (Nichirei Biosciences, Tokyo, Japan) were added on the slide. The slides were washed three times with PBS and DAB for 30 min. After counterstaining with Mayer’s hematoxylin, the slides were mounted. In addition, the serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), serum albumin, total protein, and bilirubin levels were determined using an Auto Chemistry Analyzer (Chiron, Emeryville, CA, USA). 2.6. Assay of lipid peroxidation products in liver tissues Lipid peroxidation in liver tissues was examined using the thiobarbituric acid reactive substances (TBARS) method, as described previously (Uchiyama and Mihara, 1978). Briefly, liver tissue (0.2 g) was homogenized in 2 mL of ice-cold KCl (11.5 g L−1 ), and then 0.13 mL of homogenate were mixed with 0.08 mL of 10 g L−1 phosphoric acid and 0.26 mL of 0.67% TBA. After heating the mixture for 45 min at 100 ◦ C, 1.03 mL of n-butanol were added to the mixture, which was then mixed vigorously and centrifuged at 3000 rpm for 15 min. The absorbance of the upper organic layer was measured at 535 and 520 nm and compared against freshly prepared TEP as a standard. 2.7. Hydroxyproline determination in liver tissues Hydroxyproline was determined with a slight modification of a reported method (Fujita et al., 2003). Briefly, liver tissues (0.2 g) were homogenized in 2 mL of 6 M hydrochloric acid and incubated overnight at 110 ◦ C. The acid hydrolysates were filtered and dried. After dissolving the product with 50 L methanol, 1.2 mL
of 50% isopropanol and 200 L of chloramines-T solution were added sequentially. Next, 1.3 mL of Ehrlich’s solution was added to the mixture, which was incubated at 50 ◦ C for 90 min, and then the absorbance was read at 558 nm. A standard curve was constructed using serial 1:2 dilutions of 1.0 mg solutions of hydroxyproline. 2.8. Determination of the SOD, catalase, and GSH-Px activities and GSH content of liver tissues After RIPA buffer-based tissue homogenization, the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Sigma) and used to determine the SOD, catalase, GSH-Px, and GSH activities. The SOD activity in the liver tissues was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol, using bovine erythrocyte SOD (Sigma) as a standard. Catalase activity in liver tissue was determined using the method of Beers and Siezer (1952). Briefly, the supernatant fraction was diluted 200-fold with 50 mM potassium phosphate buffer (pH 7.0 at 25 ◦ C). Then, 100-L tissue sample solutions and a standard solution were mixed with 2.9 mL of substrate solution (0.0036% [w/w] H2 O2 solution), and the absorbance was measured at 240 nm after 5 min. The GSH-Px activity in liver tissue was determined with a Glutathione Peroxidase Cellular Activity assay kit (Sigma, St Louis, USA) according to the manufacturer’s protocol. Total GSH in liver tissues was determined using the Ellman method (Evans and Ellman, 1959), which measures the reduction of DTNB (Ellman’s reagent) by sulfhydryl groups to 2-nitro-5mercaptobenzoic acid, which has an intense yellow color. Briefly, duplicate 50-L aliquots of the supernatant (GSH standard) were combined with 80 L of a previously prepared DTNB/NADPH mixture (10 L of 4 mM DTNB and 70 L of 0.3 mM NADPH) in a 96-well microtiter plate. After adding 20 L (0.06 U) of GSH reduc-
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Table 2 Organ weights and serum biochemistry. Groups Body mass (g) Liver mass (g) Spleen mass (g) AST (IU L−1 ) ALT (IU L−1 ) ALP (IU L−1 ) Bilirubin (g dL−1 ) Albumin (g dL−1 ) # **
Normal 369.2 13.52 0.83 85.83 46.67 291.8 0.20 3.57
± ± ± ± ± ± ± ±
DMN 29.4 1.33 0.11 7.99 6.06 59.7 0.00 0.15
278.7 6.85 1.75 286.1 215.5 1015 2.64 2.91
CGX100 ± ± ± ± ± ± ± ±
#
27.8 2.99# 0.43# 82.9# 64.4# 338# 1.89# 0.25#
351.5 12.23 1.14 102.1 67.25 474.3 0.26 3.56
± ± ± ± ± ± ± ±
CGX200 **
19.7 0.70** 0.16** 6.01** 6.90** 71.1** 0.05** 0.11**
335.9 11.66 0.99 97.00 54.88 373.4 0.20 3.50
± ± ± ± ± ± ± ±
Silymarin **
17.6 0.72** 0.09** 8.80** 5.51** 64.9** 0.20** 0.08**
337.6 12.03 1.18 94.75 69.50 425.4 0.23 3.59
± ± ± ± ± ± ± ±
19.0** 0.84** 0.19** 11.9** 11.9** 76.7** 0.05** 0.08**
p < 0.01, compared with the normal group. p < 0.01, compared with the DMN group (n = 8).
tase solution to each well and incubating the solution for 5 min, the absorbance at 450 nm was measured using a microtiter plate reader.
Raw 264.7 cells (2 × 105 per well) were pretreated with CGX (0, 10, 50, or 100 g mL−1 ) for 4 h in 24-well plates and then with added LPS (0.1 g mL−1 ) for 24 h. The culture supernatant was used to determine NO production using to Green et al.’s (1982) protocol.
2.9. Determination of profibrotic cytokines in liver tissue by ELISA After RIPA buffer-based tissue homogenization, the levels of TGF- and PDGF-BB were measured using commercial ELISA kits according to the manufacturers’ instructions (Biosource, Camarillo, CA, USA; R&D Systems, Minneapolis, MN, USA). 2.10. Lactate dehydrogenase (LDH) leakage assay and the determination of nitro oxide (NO) HepG2 cells were adjusted to 2 × 104 cells/well in 24-well plates. After pretreatment with various concentrations of CGX (0, 10, 50, and 100 g mL−1 ) for 2 h, the cells were treated with 2 mM of CCl4 dissolved in dimethyl sulfoxide (DMSO) (final concentration 0.5%) for 2 h. The LDH leakage assay was performed by measuring the LDH activity in cell culture supernatants using a CytoTox 96 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Two groups were treated with DMSO (0.5%) or lysis buffer only as spontaneous control and maximum groups, respectively. The death rate (%) was calculated using the following formula: death rate (%) =
ODsample − ODcontrol ODmaximum − ODcontrol
× 100
2.11. RT-PCR and real-time PCR for analyzing gene expression in tissue or HSC-T6 cells Total RNA was extracted from frozen rat liver tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Midi Kits (QIAGEN, Valencia, CA, USA). RT-PCR was performed on 10 genes, including the -actin gene, using standard protocols for DNA synthesis and PCR amplification. The primer sequences used were as follows (shown 5 → 3 ): for -actin, GTG GGG CGC CCC AGG CAC CA and CTC CTT AAT GTC ACG CAC GAT TTC; for inducible nitric oxide synthase (iNOS), TGA GTG GCT GTC TTT TGA CG and TTC TCT GTG GAG CTG AAG CA; for tumor necrosis factor-alpha (TNF-␣), TGG TGG TGA CAA GCA CAT TT and CTG AGT TCG TCC CCT TCT CC; for transforming growth factor-beta (TGF-), ATG GAG ACA TGG CGT AAA GC and TTG CAT GAC AAT GAC ACA CG; for connective tissue growth factor (CTGF), CTC CCA GGT TCT CTT CAA GG and TGG AAG ACT CCT CCC AGG TA; for platelet-derived growth factor-beta (PDGF-), GAG TCG AGT CGG AAA GCT CA and CTG CTG CAG CCA GAG ACC; for matrix metalloproteinase 2 (MMP-2), GAA ACC GTG GAT GAT GCT TT and CAT CAC TGC GAC CAG TFT CT; for hepatocyte growth factor (HGF), ACA CAT CTG TGG GGG ATC AT and TGG TGC TGA CTG CAT TTC TC; for tissue inhibitor of matrix metalloprotease-1 (TIMP-1),
Fig. 2. Gross findings and histopathological examination of the liver. At necropsy, livers from each group (n = 8) were examined with the naked eye (A) and then fixed in Bouin’s solution. After staining with hematoxylin and eosin (B) or Masson’s trichrome (C) and after immunohistochemistry for ␣-SMA (D), histological examinations were performed at 200× magnification using light microscopy.
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Fig. 3. Hydroxyproline, MDA, and antioxidant enzyme activities in liver tissues. At the end of the experiment, hydroxyproline, MDA, SOD, catalase, GSH-Px, and GSH were determined in the liver homogenates (n = 8). *p < 0.05 and **p < 0.01 compared with the DMN group.
CGG ACC TGG TTA TAA GGG CT and ACT CTC CAG TTT GCA AGG GA; and for tissue inhibitor of matrix metalloprotease-2 (TIMP-2), GCA TCA CCC AGA AGA AGA GC and GTT TCC AGG AAG GGA TGT CA. In order to examine the effect of CGX on the inactivation of hepatic stellate cells, the gene expression of procollagen types I and III and ␣-SMA in HSC-T6 cells was examined. HSC-T6 cells (5 × 105 cells/well) were treated with CGX (0, 10, 50, or 100 g mL−1 ) for 4 h. Total RNA was isolated using an RNeasy mini kit (QIAGEN, Valencia, CA, USA). Real-time PCR was performed after cDNA synthesis using AccuPower RT premix (Bioneer, Daejeon, Korea). The primers sequences used were as follows (shown
5 → 3 ): for procollagen type I, CGA GAC CCT TCT CAC TCC TG and CAC CCC TTC TGC GTT GTA TT; for procollagen type III, GTA CAG CTG GCC TTC CTC AG and TTT TGT TTT GCT GGG GTT TC; and for ␣-SMA, CAT CAG GAA CCT CGA GAA GC and TCG GAT ACT TCA GGG TCA GG. These sequences were also used for -actin. 2.12. Statistical analysis All data are expressed as the mean ± SD (n = 8). Statistically significant differences between the groups were analyzed using one-way analysis of variance (ANOVA) followed by a paired
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Student’s t-test. p < 0.05 was regarded as statistically significant. 3. Results 3.1. CGX attenuated the changes in organ weights and biochemical parameters In the DMN group, the weights of the body, liver, and spleen were 45.9, 50.7, and 209.4% of the respective normal values at the 4-week endpoint. CGX (100 or 200 mg kg−1 ) and silymarin administration attenuated these severe changes in bodyweight by 87.4, 77.3, and 78.2%, in liver weight by 86.3, 88.1, and 89.0%, and in spleen weight by 136.7, 119.7, and 141.8% of the normal values, respectively. CGX treatment increased body weight significantly compared to the DMN group and alleviated the decrease in liver weight with DMN treatment (p < 0.01) (Table 2). Conversely, CGX treatment significantly suppressed the DMNinduced increases in the serum AST, ALT, ALP, and total bilirubin levels (p < 0.05 or p < 0.01). The moderately low serum albumin level of the DMN group was significantly restored to normal in the CGX100 and CGX200 groups (p < 0.01) (Table 2). 3.2. CGX prevented inflammation, ECM accumulation, and HSC activation in liver tissues At the end of the experiment, the livers of the animals given DMN were shrunken and congested compared to those of the control group, and they showed features of inflammation, whereas the CGX treatment significantly ameliorated these changes (Fig. 2A and B). The collagen accumulation was severe in the DMN group, whereas the CGX treatment reduced it significantly (Fig. 2C). In particular, markedly fewer ␣-SMA-positive cells were observed in the CGX groups (Fig. 2D).
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3.3. CGX decreased collagen accumulation in liver tissues To examine the collagen content of liver tissue, hydroxyproline was determined from frozen liver tissues. DMN treatment dramatically increased the amount of hydroxyproline by 6-fold, whereas CGX (100 and 200 mg kg−1 ) administration significantly decreased the hydroxyproline content in liver tissue compared to the DMN group (p < 0.01) (Fig. 3A). 3.4. CGX prevented lipid peroxidation and the depletion of antioxidant enzymes in liver tissues The lipid peroxidation was examined by determining malondialdehyde (MDA) content in frozen liver tissue homogenate. The MDA content in the DMN group was significantly higher than that in the normal group (approximately 375% of normal). In the CGX100, CGX200, and silymarin groups, MDA decreased by 36.7, 46.4, and 53.4% respectively, compared to the DMN group (Fig. 3B). We determined the major antioxidant enzymes GSH, GSH-Px, SOD, and catalase in liver tissues. We found notably sustained levels of GSH components in the CGX-treated groups compared to the DMN group (p < 0.01) (Fig. 3E). In addition, CGX treatment significantly inhibited the depletion of GSH-Px, catalase, and SOD caused by DMN toxicity compared to the DMN group (p < 0.05 or p < 0.01) (Fig. 3C, D and F). 3.5. CGX inhibited profibrotic cytokines, TGF-ˇ, and PDGF-ˇ in liver tissue The main profibrotic cytokines were determined using an ELISA method. CGX significantly inhibited the levels of TGF- and PDGF (PDGF-BB) in liver tissue compared with the DMN group (p < 0.05 or p < 0.01) (Fig. 4B and C).
Fig. 4. Expression of fibrosis-related genes and profibrotic cytokines in liver tissue. Total RNA was extracted from liver tissues, and cDNA was synthesized. PCR was performed for 10 genes, and the products were visualized with UV illumination (A). TGF- (B) and PDGF-BB (C) were determined in the liver lysates using ELISA kits. *p < 0.05 and **p < 0.01 compared with the DMN group.
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Fig. 5. Mechanism of action of CGX in vitro. The LDH leakage assay was performed using HepG2 cells after CGX pretreatment followed by CCl4 exposure for 2 h (A). NO production by RAW 264.7 cells was measured after pretreatment with CGX and then LPS treatment for 24 h (B). *p < 0.05 and **p < 0.01 compared with the control. Gene expression changes in HSC-T6 cells triggered by CGX were examined using real-time PCR. The results are expressed as normalized fold values relative to the control (C).
3.6. CGX restored the altered gene expression associated with liver fibrosis In order to investigate the hepatoprotective mechanism of CGX, the expression of the iNOS, TGF-, TNF-␣, CTGF, PDGF-, MMP2, HGF, TIMP-1, and TIMP-2 genes was analyzed using RT-PCR. DMN treatment induced high expression of the iNOS, TGF-, TNF-␣, CTGF, PDGF-, HGF, and TIMP-1 genes compared with the normal group, whereas their activation was dramatically inhibited by CGX treatment. CGX did not affect the expression of the TIMP-2 gene (Fig. 4A). 3.7. CGX affected hepatocyte protection, the inhibition of NO production, and HSC inactivation in vitro In vitro, 100 g mL−1 of CGX protected hepatocytes against CCl4 treatment by about 40% compared to the CCl4 control (p < 0.01) (Fig. 5A). CGX treatment also significantly inhibited NO production in RAW 264.7 cells in a concentration-dependent manner (p < 0.01) (Fig. 5B). In addition, CGX drastically downregulated expression of the procollagen types I and III genes by 42 and 55%, respectively, at CGX 50 g mL−1 , and moderately downregulated ␣-SMA in HSC-T6 cells (Fig. 5C). 4. Discussion Chronic liver injury commonly leads to liver fibrosis resulting from imbalance between synthesis and degradation of the ECM
(Benyon and Iredale, 2000). Some drugs have antifibrotic effects by reducing the accumulation of ECM in chronic hepatic injury models (Hammel et al., 2001; Bataller and Brenner, 2005). Based on our previous results for CGX, we further examined the protective effects of CGX against liver fibrosis and the compartment enzymes playing roles in the antioxidative process to identify the mechanisms responsible. We also investigated the effects of CGX on individual cellular components of liver tissue using hepatocyte-, Kupffer cell-, and stellate cell-derived cell lines. As we expected, CGX treatment significantly ameliorated the DMN-induced histopathologic deterioration in liver tissues, as well as serum parameters (Fig. 2A and B, and Table 2). In addition, the amount of ECM was very low in the CGX-treated groups in contrast to the severe ECM accumulation seen in the DMN group (Fig. 2C). Preventing the underlying destructive condition is optimal antifibrotic strategy, and antioxidants protect against liver injury (Vitaglione et al., 2004; Medina and MorenoOtero, 2005). Our in vitro data showed the protective effect of CGX against hepatocyte destruction caused by CCl4 treatment (Fig. 5A). During fibrosis progression, the main ECM-producing cells are the activated HSCs, which secrete ECM, including collagen types I and III. The inhibition of this step is a major strategy in the development of antifibrotic drugs (Bataller and Brenner, 2001; Reeves and Friedman, 2002; Safadi and Friedman, 2002). We found that CGX inhibited stellate cell activation by DMN treatment using immunostaining for ␣-SMA, a marker of stellate cell activation (Fig. 2D). This concurred with the in vitro results from the rat hepatic stellate cell
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line HSC-T6 (Fig. 5C). CGX drastically downregulated the key gene expression corresponding to activation of HSC, collagen types I and III, and ␣-SMA. The hydroxyproline measurement also showed that CGX treatment significantly inhibited the accumulation of collagen compared to the DMN group (Fig. 3A). To investigate the molecular mechanisms underlying these effects, we examined the levels of lipid peroxides and the activity of antioxidant enzymes in liver tissues. The combination of excessive oxygen stressors and decreased antioxidant levels contributes to the initiation or progression of hepatic damage and liver fibrosis (Parola and Robino, 2001; Rockey, 2005). Our results suggest that CGX has the pharmacological capacity to reduce oxidative stress, as evidenced by the decreased MDA (an important indicator of oxidative damage) concentrations in the CGX-treated groups compared to in the DMN group (Fig. 3B). DMN treatment drastically lowered the glutathione content and GSH-Px, SOD, and catalase activities in hepatic tissues, whereas the CGX-treated groups sustained levels near the normal range (Fig. 3C–F). We examined the changes in the expression of several genes known to be key factors in the hepatic fibrosis process (Fig. 4A). Of the nine genes examined, the iNOS, TNF-␣, TGF- (only in CGX200), CTGF, and PDGF- genes were dramatically normalized by CGX treatment compared to the DMN group. These are prominent inflammatory or profibrogenic cytokines (MacDonald et al., 2001; Breitkopf et al., 2006; Friedman, 2008). In particular, PDGF is the most potent mitogen and activator of HSCs (Pinzani et al., 1989; Borkham-Kamphorst et al., 2007), and CTGF is a potent profibrotic factor that mediates the stimulatory actions of TGF-induced ECM synthesis (Ihn, 2002). The levels of TGF- and PDGF- proteins in liver tissues were drastically lower in the CGX treatment groups (Fig. 4B and C). The upregulation of the HGF and MMP-2 genes in the DMN group only might be a response to compensate for the severe hepatic injury and excess accumulation of ECM in liver tissues. DMN treatment upregulated the expression of two genes, TIMP-1 and TIMP-2, which is another characteristic of hepatic fibrosis (Benyon et al., 1996; Rippe and Brenner, 2004). No difference in TIMP-2 and only slight inhibition of TIMP-1 was observed in the CGX-treated groups. These positive results for lipid peroxidation, the SOD, catalase, GSH-Px, and GSH contents of liver tissues, the gene expression patterns of iNOS and TNF-␣, and inhibition of NO production by RAW 264.7 cells (Fig. 5B) are very consistent with the antioxidant action of CGX. The over-produced NO and profibrogenic cytokines by activated Kupffer cells and apoptotic hepatocytes direct the transformation of HSCs into myofibroblasts secreting ECM (Watanabe et al., 2007; Friedman, 2008). Taken together, our results lead us to conclude that CGX protects against liver fibrosis during chronic liver damage, and the responsible mechanisms are associated with both hepatocyte protection and the suppression of HSC activation via antioxidative stress and antifibrotic cytokines. Acknowledgments We gratefully thank Dr. S.L. Friedman for providing the HSC-T6 cell line. This work was supported by the Oriental Medicine R&D Project of the Ministry of Health and Welfare, Republic of Korea (No. B070031). References Bataller, R., Brenner, D.A., 2001. Hepatic stellate cells as a target for the treatment of liver fibrosis. Seminars in Liver Disease 21, 437–451. Bataller, R., Brenner, D.A., 2005. Liver fibrosis. The Journal of Clinical Investigation 115, 209–218.
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Beers, R.F., Siezer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry 195, 133–140. Benyon, R.C., Iredale, J.P., Goddard, S., Winwood, P.J., Arthur, M.J., 1996. Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver. Gastroenterology 110, 821–831. Benyon, R.C., Iredale, J.P., 2000. Is liver fibrosis reversible? Gut 46, 443–446. Borkham-Kamphorst, E., van Roeyen, C.R., Ostendorf, T., Floege, J., Gressner, A.M., Weiskirchen, R., 2007. Pro-fibrogenic potential of PDGF-D in liver fibrosis. Journal of Hepatology 46, 1064–1074. Breitkopf, K., Godoy, P., Ciuclan, L., Singer, M.V., Dooley, S., 2006. TGFbeta/Smad signaling in the injured liver. Zeitschrift für Gastroenterologie 44, 57–66. Cho, J.H., Lee, Y.Y., Seo, S.H., Yoo, H.S., Choi, W.J., Lee, Y.W., Son, C.G., Cho, C.K., 2001. A clinical report about 57 patients with chronic liver disease. Korean Journal of Oriental Medicine 21, 112–121. Choi, W.J., Shin, J.W., Son, J.Y., Seo, D.S., Park, H.S., Han, S.H., Sung, H.J., Cho, J.H., Cho, C.K., Yoo, H.S., Lee, Y.W., Son, C.G., 2006. Toxicological study of the hepatotherapeutic herbal formula, CGX, in beagle dogs. World Journal of Gastroenterology 12, 7497–7502. Evans, J.C., Ellman, G.L., 1959. The ionization of cysteine. Biochimica et Biophysica Acta 33, 574–576. Fallowfield, J.A., Kendall, T.J., Iredale, J.P., 2006. Reversal of fibrosis: no longer a pipe dream? Clinics in Liver Disease 10, 481–497. Fowell, A.J., Iredale, J.P., 2006. Emerging therapies for liver fibrosis. Digestive Diseases 24, 174–183. Friedman, S.L., 2003. Liver fibrosis-from bench to bedside. Journal of Hepatology 38, S38–53. Friedman, S.L., 2008. Mechanisms of hepatic fibrogenesis. Gastroenterology 134, 1655–1669. Fujita, M., Shannon, J.M., Morikawa, O., Gauldie, J., Hara, N., Mason, R.J., 2003. Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta. American Journal of Respiratory Cell and Molecular Biology 29, 669–676. Gines, P., Cardenas, A., Arroyo, V., Rodes, J., 2004. Management of cirrhosis and ascites. The New England Journal of Medicine 350, 1646–5164. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Hammel, P., Couvelard, A., O’Toole, D., Ratouis, A., Sauvanet, A., Fléjou, J.F., Degott, C., Belghiti, J., Bernades, P., Valla, D., Ruszniewski, P., Lévy, P., 2001. Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of the common bile duct. The New England Journal of Medicine 344, 418–423. Hu, X.P., Shin, J.W., Wang, J.H., Cho, J.H., Son, J.Y., Cho, C.K., Son, C.G., 2008. Antioxidative and hepatoprotective effect of CGX, an herbal medicine, against toxic acute injury in mice. Journal of Ethnopharmacology 120, 51–55. Ihn, H, 2002. Pathogenesis of fibrosis: role of TGF- and CTGF. Current Opinion in Rheumatology 14, 681–685. Iredale, J.P., 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. The Journal of Clinical Investigation 117, 539–548. Kisseleva, T., Brenner, D.A., 2008. Mechanisms of fibrogenesis. Experimental Biology and Medicine 233, 109–122. Levy, C., Seeff, L.D., Lindor, K.D., 2004. Use of herbal supplements for chronic liver disease. Clinical Gastroenterology and Hepatology 2, 947–956. MacDonald, G.A., Bridle, K.R., Ward, P.J., Walker, N.I., Houglum, K., George, D.K., Smith, J.L., Powell, L.W., Crawford, D.H., Ramm, G.A., 2001. Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3. Journal of Gastroenterology and Hepatology 16, 599–606. Medina, J., Moreno-Otero, R., 2005. Pathophysiological basis for antioxidant therapy in chronic liver disease. Drugs 65, 2445–2461. Parola, M., Robino, G., 2001. Oxidative stress-related molecules and liver fibrosis. Journal of Hepatology 35, 297–306. Pinzani, M., Gesualdo, L., Sabbah, G.M., Abboud, H.E., 1989. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. The Journal of Clinical Investigation 84, 1786–1793. Reeves, H.L., Friedman, S.L., 2002. Activation of hepatic stellate cells—a key issue in liver fibrosis. Frontiers in Bioscience 7, 808–826. Rippe, R.A., Brenner, D.A., 2004. From quiescence to activation: gene regulation in hepatic stellate cells. Gastroenterology 127, 1260–1262. Rockey, D.C., 2005. Antifibrotic therapy in chronic liver disease. Clinical Gastroenterology and Hepatology 3, 95–107. Safadi, R., Friedman, S.L., 2002. Hepatic fibrosis-role of hepatic stellate cell activation. Medscape General Medicine 4, 27. Shin, J.W., Son, J.Y., Oh, S.M., Han, S.H., Wang, J.H., Cho, J.H., Cho, C.K., Yoo, H.S., Lee, Y.W., Lee, M.M., Hu, X.P., Son, C.G., 2006. An herbal formula, CGX, exerts hepatotherapeutic effects on dimethylnitrosamine-induced chronic liver injury model in rats. World Journal of Gastroenterology 12, 6142–6148. Son, C.G., Choi, B.L., Shin, J.W., Choi, W.J., Cho, J.H., Cho, C.K., 2001. Effect of gamichunggantang on alcoholic metabolism and alcoholic liver disease. Korean Journal of Oriental Medicine 2, 89–98. Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278.
542
J.-H. Wang et al. / Journal of Ethnopharmacology 127 (2010) 534–542
Vitaglione, P., Morisco, F., Caporaso, N., Fogliano, V., 2004. Dietary antioxidant compounds and liver health. Critical Reviews in Food Science and Nutrition 44, 575–586. Watanabe, A., Hashmi, A., Gomes, D.A., Town, T., Badou, A., Flavell, R.A., Meha, W.Z., 2007. Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9. Hepatology 46, 1509–1518.
Yang, Y., Yang, S., Chen, M., Zhang, X., Zou, Y., Zhang, X., 2008. Compound Astragalus and Salvia miltiorrhiza Extract exerts anti-fibrosis by mediating TGFbeta/Smad signaling in myofibroblasts. Journal of Ethnopharmacology 118, 264–270.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antifibrotic effects of CGX, a traditional herbal formula, and its mechanisms in rats Jing-Hua Wang, Jang-Woo Shin, Jin-Young Son, Jung-Hyo Cho, Chang-Gue Son ∗ Liver-Immune Research Center, Daejeon Oriental Hospital of Daejeon University, 22-5 Daeheung-dong, Jung-gu, Daejeon 301-704, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 February 2009 Received in revised form 25 September 2009 Accepted 5 October 2009 Available online 13 October 2009 Keywords: Antifibrosis Antioxidant Superoxide dismutase Catalase Glutathione Glutathione peroxidase
a b s t r a c t Aim: CGX is a modification of a traditional herbal medicine for “liver cleaning,” which is used to treat various chronic liver disorders in oriental clinics. This study investigated the antifibrotic effects and associated mechanisms of CGX. Materials and methods: Liver fibrosis was induced in rats by dimethylnitrosamine (DMN; 10 mg kg−1 , ip) injection on 3 consecutive days per week for 4 weeks. CGX (100 or 200 mg kg−1 , po) was administrated once a day for 4 weeks. Three cell lines (HepG2, RAW 264.7, and HSC-T6) were used to examine its mechanisms. Results: CGX treatment dramatically ameliorated the change in liver and spleen weight and serum albumin (p < 0.01), aspartate transaminase (p < 0.01), alanine transaminase (p < 0.01), alkaline phosphatase (p < 0.01), and total bilirubin (p < 0.01) levels. Histopathologically, CGX administration decreased necrosis, inflammatory cell infiltration, and collagen accumulation. The antifibrotic effects of CGX were confirmed from hydroxyproline determination and the reduction in the numbers of activated hepatic stellate cells. In addition, antioxidant proteins, glutathione content, and glutathione peroxidase, catalase, and superoxide dismutase activities were maintained in the CGX-treated groups compared with the DMN group. CGX downregulated fibrosis-related genes (inducible nitric oxide synthase, tumor necrosis factor-alpha, transforming growth factor-beta, connective tissue growth factor, and platelet-derived growth factorbeta) and decreased the protein levels of profibrotic cytokines (transforming growth factor-beta and platelet-derived growth factor-beta) in liver tissues. In the cell line-based studies, CGX showed supportive effects, such as the protection of hepatocytes from CCl4 -toxicity, inhibition of NO production in RAW 264.7 cells, and inactivation of hepatic stellate cells. Conclusion: These results demonstrated the antifibrotic effects of CGX and the corresponding mechanisms associated with sustaining the antioxidative system and inhibiting hepatic stellate cell activation via the downregulation of fibrogenic cytokines. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The chronic destruction of liver tissue can cause gradual, progressive fibrotic change, and the development of liver cirrhosis is
Abbreviations: ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CCl4 , carbon tetrachloride; CTGF, connective tissue growth factor; DMN, dimethylnitrosamine; ECM, extracellular matrix; GSH, glutathione; GSH-px, glutathione peroxidase; HE, hematoxylin and eosin; HGF, hepatocyte growth factor; HPLC, high-performance liquid chromatography; HSCs, hepatic stellate cells; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MMP-2, matrix metalloproteinase 2; PDGF-, plateletderived growth factor-beta; SOD, superoxide dismutase; TBA, thiobarbituric acid; TGF-, transforming growth factor-beta; TIMP-1, tissue inhibitor of matrix metalloprotease-1; TIMP-2, tissue inhibitor of matrix metalloprotease-2; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +82 42 229 6723; fax: +82 42 257 6398. E-mail address: [email protected] (C.-G. Son). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.10.001
a critical event in determining the consequences of chronic hepatic disorders and their clinical outcomes (Friedman, 2003; Gines et al., 2004). The roles of various key cytokines, cellular effectors including hepatic stellate cells (HSCs), and the determinants of extracellular matrix (ECM) turnover in the fibrotic process have been well elucidated (Iredale, 2007; Kisseleva and Brenner, 2008). Currently, it is believed that liver fibrosis is reversible, which increases the hope of treatments for liver fibrosis and cirrhosis (Fallowfield et al., 2006; Fowell and Iredale, 2006). However, because no universally effective therapeutic drug for chronic liver injury and fibrotic progression is available, the search for antifibrotic agents, including herbal-derived ingredients, is a major medical objective (Levy et al., 2004; Yang et al., 2008). CGX is an herbal drug that originated from a traditional Korean formula composed of 13 herbs and other items. It is prescribed for patients who suffer from various chronic liver diseases, such as alcoholic liver disorders, chronic viral hepatitis, and liver cirrhosis (Cho et al., 2001; Son et al., 2001). Previously, we reported
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the safety of CGX in a beagle dog-based toxicological study and its hepatoprotective effects in acute and chronic liver injury models (Choi et al., 2006; Shin et al., 2006; Hu et al., 2008). However, the antifibrotic properties of CGX and mechanisms responsible for those effects require further experimental evaluation. Here, we investigated the antifibrotic effects of CGX using a modified schedule with a different animal strain from a previous model and cell lines derived from the three main cellular components of liver tissue. We explored the potential mechanisms of these effects, focusing on antioxidative actions and HSC-related profibrogenic cytokines. 2. Materials and methods 2.1. Reagents and chemicals Dimethylnitrosamine (N-nitrosodimethylamine or DMN), hydroxyproline, p-dimethylaminobenzaldehyde, 1,1,3,3tetraethoxypropane (TEP), chloramine-T, potassium chloride (KCl), n-butanol, 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), reduced glutathione, glutathione reductase, glutathione peroxidase (GSH-px), reduced -nicotinamide adenine dinucleotide phosphate (-NADPH), and lipopolysaccharide (LPS, Salmonella typhosa) were purchased from Sigma (St. Louis, MO, USA); perchloric acid was obtained from GFS Chemical (Columbus, OH, USA); thiobarbituric acid (TBA) was purchased from Lancaster (Lancashire, England); and carbon tetrachloride (CCl4 ) was purchased from Junsei Chemical (Tokyo, Japan). Anti-␣-SMA mouse monoclonal antibody was from Abcam (Cambridge, UK); histofine was from Nichirei Biosciences (Tokyo, Japan); diaminobenzidine (DAB) was from BioGenex (CA, USA); and Mayer’s hematoxylin was from Wako Pure Chemical Industries (Japan). 2.2. CGX and high-performance liquid chromatography (HPLC)-based fingerprinting All the herbs used met the Korean Pharmacopoeia standards. CGX was manufactured by Samik Pharmacy (Seoul, Korea) using the approved Good Manufacturing Practice of the Korea Food and Drug Administration. Briefly, 120 kg of the 13 herbs (Table 1) were boiled in 1200 L of distilled water for 4 h at 100 ◦ C and then filtered using a 300-mesh filter (50 m). Part of this batch was filtered through filter paper (Advantec, Toyo Roshi Kaisha, Tokyo, Japan) and lyophilized in our laboratory for this study. The final extraction gave a yield of 10.71% (w/w), and the batch used in this experiment was stored for future use (voucher specimen No. CGX-2007-09WE-SI). The final CGX extract satisfied the criteria of the Korean Table 1 The herbal prescription of CGX. Scientific name
Part used
Voucher specimen number
Relative amount (g)
Artemisia capillaries Thunberg Trionyx sinensis Wiegmann Raphanus sativus Linne Atractylodes macrocephala Koidzumi Poria cocos Wolf Alisma orientalis Juzepczuk Atractylodes chinensis Koidzumi Salvia miltiorrhiza Bunge Polyporus umbellatus Fries Poncirus trifoliate Rafinesqui Amomum villosum Loureiro Glycyrrhiza uralensis Fischer Aucklandia lappa Decne
Herba Carapax Semen Rhizoma
AC-2006-01-He TS-2006-01-Ca RS-2006-01-Se AM-2006-01-Rh-Al
5 5 5 3
Poria Rhizoma Rhizoma
PC-2006-01-Po AO-2006-01-Rh AC-2006-02-Rh
3 3 3
Radix Polyporus Fructus Fructus Radix Radix
SM-2006-01-Ra PU-2006-01-Po PT-2006-01-Fr AV-2006-01-Fr GU-2006-01-Ra AL-2006-01-Ra
3 2 2 2 1 1
535
Pharmaceutical Codex, including the quantity of each herb, contamination by heavy metals, general bacteria, fungi, and specific pathogens, and the quantity of ingredients. A CGX fingerprint was produced using the two-dimensional high-performance liquid chromatography (HPLC) profile of five major compositional herbs and their main compounds: Glycyrrhiza uralensis root, Artemisia capillaries, Raphanus sativus seeds, Salvia miltiorrhiza root, and Poncirus trifoliate fruit vs. liquiritin and glycyrrhizin, 6,7-dimethoxycoumarin, naringin, rosmarinic acid, and poncirin, respectively (Fig. 1). Briefly, after dissolution (20 mg of CGX and 2 mg of the five herbal extracts in 1 mL of water; 0.01 mg of the six standards in 1 mL of water or 50% methanol) and filtration, these drugs were subjected to HPLC analysis. The HPLC system consisted of an SCL-10A system controller, LC-10AD pump, SPD-10MVP diode array detector, and CTO-10AS column temperature controller (Shimadzu, Kyoto, Japan). A Phenomenex Prodigy C18 (2.0 mm × 150 mm) column was eluted with solvents A (10% acetonitrile in water containing 0.05% formic acid) and B (90% acetonitrile in water) at a flow rate of 0.4 mL/min. Solutions 100% A and 0% B were changed to 75% A/25% B at 30 min, and to 25% A/75% B at 60 min. All chromatograms were obtained at a wavelength of 230 nm. 2.3. Cell lines and cell culture In order to study the effects of CGX on the main cellular components of liver tissue, three cell lines were used for in vitro experiments. Human hepatocellular carcinoma cell line HepG2 and mouse leukemic monocyte macrophage cell line RAW 264.7 were purchased from the Korea Cell Line Bank (KCLB); rat hepatic stellate cell line HSC-T6 was obtained from Dr. S.L. Friedman. HepG2 and RAW 264.7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (WelGENE, Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS) (WelGENE, Daegu, South Korea) at 37 ◦ C in a 5% CO2 humidified incubated environment, and HSC-T6 cells were cultured in DMEM containing 5% FBS. Logarithmic growth phase cells were subcultured three times a week for in vitro experiments. 2.4. Animals and experimental design Forty male Sprague–Dawley rats (6 weeks old, 160–180 g) were purchased from Korea Animal Tech (Gyeonggi-do, Korea). After 7 days of acclimation at 22 ± 2 ◦ C under a 12-h light/12-h dark cycle with free access to water and food, the rats were divided into five groups (normal, DMN, CGX100, CGX200, and silymarin) of eight animals each. The rats were injected intraperitoneally with DMN (10 mg kg−1 ) on 3 consecutive days per week for 4 weeks. The normal group was injected with normal saline instead of DMN. CGX (100 or 200 mg kg−1 ) or silymarin (50 mg kg−1 ) as a positive control was given orally to the respective groups seven times per week for 4 weeks, and distilled water was administered to the normal and DMN groups. On the final day, all of the rats were sacrificed under ether anesthesia, and the blood, liver, and spleen were collected for biochemical analyses and other measurements. The experiments were conducted in accordance with the Policy on the Humane Care and Use of Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health (NIH). 2.5. Histopathologic examination with immunohistochemistry and biochemical analyses The liver tissue was evaluated histomorphologically with hematoxylin and eosin (HE) and Masson’s trichrome stains. The histopathologic features, such as necrosis or inflammatory cell infiltration and collagen fiber changes, were examined using light microscopy in a blind manner. Immunohistochemistry for alpha
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J.-H. Wang et al. / Journal of Ethnopharmacology 127 (2010) 534–542
Fig. 1. HPLC-based fingerprinting for CGX. The five major component herbs of CGX and their main compounds were subjected to HPLC. All chromatograms were obtained at a wavelength of 230 nm (details in the text). GRE, Glycyrrhiza uralensis root; AHE, Artemisia capillaries; RSE, Raphanus sativus seeds; SRE, Salvia miltiorrhiza root; PFE Poncirus trifoliate fruit; A, liquiritin; B, 6,7-dimethoxycoumarin; C, naringin; D, rosmarinic acid; E, poncirin; F, glycyrrhizin.
smooth muscle actin (␣-SMA) was performed. Briefly, the tissue sections were deparaffinized, hydrated, and treated with normal serum for 30 min. Next, the tissue slides were treated with anti-␣SMA mouse monoclonal antibody (1:200) overnight. After washing three times with PBS, two drops of Histofine (Nichirei Biosciences, Tokyo, Japan) were added on the slide. The slides were washed three times with PBS and DAB for 30 min. After counterstaining with Mayer’s hematoxylin, the slides were mounted. In addition, the serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), serum albumin, total protein, and bilirubin levels were determined using an Auto Chemistry Analyzer (Chiron, Emeryville, CA, USA). 2.6. Assay of lipid peroxidation products in liver tissues Lipid peroxidation in liver tissues was examined using the thiobarbituric acid reactive substances (TBARS) method, as described previously (Uchiyama and Mihara, 1978). Briefly, liver tissue (0.2 g) was homogenized in 2 mL of ice-cold KCl (11.5 g L−1 ), and then 0.13 mL of homogenate were mixed with 0.08 mL of 10 g L−1 phosphoric acid and 0.26 mL of 0.67% TBA. After heating the mixture for 45 min at 100 ◦ C, 1.03 mL of n-butanol were added to the mixture, which was then mixed vigorously and centrifuged at 3000 rpm for 15 min. The absorbance of the upper organic layer was measured at 535 and 520 nm and compared against freshly prepared TEP as a standard. 2.7. Hydroxyproline determination in liver tissues Hydroxyproline was determined with a slight modification of a reported method (Fujita et al., 2003). Briefly, liver tissues (0.2 g) were homogenized in 2 mL of 6 M hydrochloric acid and incubated overnight at 110 ◦ C. The acid hydrolysates were filtered and dried. After dissolving the product with 50 L methanol, 1.2 mL
of 50% isopropanol and 200 L of chloramines-T solution were added sequentially. Next, 1.3 mL of Ehrlich’s solution was added to the mixture, which was incubated at 50 ◦ C for 90 min, and then the absorbance was read at 558 nm. A standard curve was constructed using serial 1:2 dilutions of 1.0 mg solutions of hydroxyproline. 2.8. Determination of the SOD, catalase, and GSH-Px activities and GSH content of liver tissues After RIPA buffer-based tissue homogenization, the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Sigma) and used to determine the SOD, catalase, GSH-Px, and GSH activities. The SOD activity in the liver tissues was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol, using bovine erythrocyte SOD (Sigma) as a standard. Catalase activity in liver tissue was determined using the method of Beers and Siezer (1952). Briefly, the supernatant fraction was diluted 200-fold with 50 mM potassium phosphate buffer (pH 7.0 at 25 ◦ C). Then, 100-L tissue sample solutions and a standard solution were mixed with 2.9 mL of substrate solution (0.0036% [w/w] H2 O2 solution), and the absorbance was measured at 240 nm after 5 min. The GSH-Px activity in liver tissue was determined with a Glutathione Peroxidase Cellular Activity assay kit (Sigma, St Louis, USA) according to the manufacturer’s protocol. Total GSH in liver tissues was determined using the Ellman method (Evans and Ellman, 1959), which measures the reduction of DTNB (Ellman’s reagent) by sulfhydryl groups to 2-nitro-5mercaptobenzoic acid, which has an intense yellow color. Briefly, duplicate 50-L aliquots of the supernatant (GSH standard) were combined with 80 L of a previously prepared DTNB/NADPH mixture (10 L of 4 mM DTNB and 70 L of 0.3 mM NADPH) in a 96-well microtiter plate. After adding 20 L (0.06 U) of GSH reduc-
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537
Table 2 Organ weights and serum biochemistry. Groups Body mass (g) Liver mass (g) Spleen mass (g) AST (IU L−1 ) ALT (IU L−1 ) ALP (IU L−1 ) Bilirubin (g dL−1 ) Albumin (g dL−1 ) # **
Normal 369.2 13.52 0.83 85.83 46.67 291.8 0.20 3.57
± ± ± ± ± ± ± ±
DMN 29.4 1.33 0.11 7.99 6.06 59.7 0.00 0.15
278.7 6.85 1.75 286.1 215.5 1015 2.64 2.91
CGX100 ± ± ± ± ± ± ± ±
#
27.8 2.99# 0.43# 82.9# 64.4# 338# 1.89# 0.25#
351.5 12.23 1.14 102.1 67.25 474.3 0.26 3.56
± ± ± ± ± ± ± ±
CGX200 **
19.7 0.70** 0.16** 6.01** 6.90** 71.1** 0.05** 0.11**
335.9 11.66 0.99 97.00 54.88 373.4 0.20 3.50
± ± ± ± ± ± ± ±
Silymarin **
17.6 0.72** 0.09** 8.80** 5.51** 64.9** 0.20** 0.08**
337.6 12.03 1.18 94.75 69.50 425.4 0.23 3.59
± ± ± ± ± ± ± ±
19.0** 0.84** 0.19** 11.9** 11.9** 76.7** 0.05** 0.08**
p < 0.01, compared with the normal group. p < 0.01, compared with the DMN group (n = 8).
tase solution to each well and incubating the solution for 5 min, the absorbance at 450 nm was measured using a microtiter plate reader.
Raw 264.7 cells (2 × 105 per well) were pretreated with CGX (0, 10, 50, or 100 g mL−1 ) for 4 h in 24-well plates and then with added LPS (0.1 g mL−1 ) for 24 h. The culture supernatant was used to determine NO production using to Green et al.’s (1982) protocol.
2.9. Determination of profibrotic cytokines in liver tissue by ELISA After RIPA buffer-based tissue homogenization, the levels of TGF- and PDGF-BB were measured using commercial ELISA kits according to the manufacturers’ instructions (Biosource, Camarillo, CA, USA; R&D Systems, Minneapolis, MN, USA). 2.10. Lactate dehydrogenase (LDH) leakage assay and the determination of nitro oxide (NO) HepG2 cells were adjusted to 2 × 104 cells/well in 24-well plates. After pretreatment with various concentrations of CGX (0, 10, 50, and 100 g mL−1 ) for 2 h, the cells were treated with 2 mM of CCl4 dissolved in dimethyl sulfoxide (DMSO) (final concentration 0.5%) for 2 h. The LDH leakage assay was performed by measuring the LDH activity in cell culture supernatants using a CytoTox 96 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Two groups were treated with DMSO (0.5%) or lysis buffer only as spontaneous control and maximum groups, respectively. The death rate (%) was calculated using the following formula: death rate (%) =
ODsample − ODcontrol ODmaximum − ODcontrol
× 100
2.11. RT-PCR and real-time PCR for analyzing gene expression in tissue or HSC-T6 cells Total RNA was extracted from frozen rat liver tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Midi Kits (QIAGEN, Valencia, CA, USA). RT-PCR was performed on 10 genes, including the -actin gene, using standard protocols for DNA synthesis and PCR amplification. The primer sequences used were as follows (shown 5 → 3 ): for -actin, GTG GGG CGC CCC AGG CAC CA and CTC CTT AAT GTC ACG CAC GAT TTC; for inducible nitric oxide synthase (iNOS), TGA GTG GCT GTC TTT TGA CG and TTC TCT GTG GAG CTG AAG CA; for tumor necrosis factor-alpha (TNF-␣), TGG TGG TGA CAA GCA CAT TT and CTG AGT TCG TCC CCT TCT CC; for transforming growth factor-beta (TGF-), ATG GAG ACA TGG CGT AAA GC and TTG CAT GAC AAT GAC ACA CG; for connective tissue growth factor (CTGF), CTC CCA GGT TCT CTT CAA GG and TGG AAG ACT CCT CCC AGG TA; for platelet-derived growth factor-beta (PDGF-), GAG TCG AGT CGG AAA GCT CA and CTG CTG CAG CCA GAG ACC; for matrix metalloproteinase 2 (MMP-2), GAA ACC GTG GAT GAT GCT TT and CAT CAC TGC GAC CAG TFT CT; for hepatocyte growth factor (HGF), ACA CAT CTG TGG GGG ATC AT and TGG TGC TGA CTG CAT TTC TC; for tissue inhibitor of matrix metalloprotease-1 (TIMP-1),
Fig. 2. Gross findings and histopathological examination of the liver. At necropsy, livers from each group (n = 8) were examined with the naked eye (A) and then fixed in Bouin’s solution. After staining with hematoxylin and eosin (B) or Masson’s trichrome (C) and after immunohistochemistry for ␣-SMA (D), histological examinations were performed at 200× magnification using light microscopy.
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Fig. 3. Hydroxyproline, MDA, and antioxidant enzyme activities in liver tissues. At the end of the experiment, hydroxyproline, MDA, SOD, catalase, GSH-Px, and GSH were determined in the liver homogenates (n = 8). *p < 0.05 and **p < 0.01 compared with the DMN group.
CGG ACC TGG TTA TAA GGG CT and ACT CTC CAG TTT GCA AGG GA; and for tissue inhibitor of matrix metalloprotease-2 (TIMP-2), GCA TCA CCC AGA AGA AGA GC and GTT TCC AGG AAG GGA TGT CA. In order to examine the effect of CGX on the inactivation of hepatic stellate cells, the gene expression of procollagen types I and III and ␣-SMA in HSC-T6 cells was examined. HSC-T6 cells (5 × 105 cells/well) were treated with CGX (0, 10, 50, or 100 g mL−1 ) for 4 h. Total RNA was isolated using an RNeasy mini kit (QIAGEN, Valencia, CA, USA). Real-time PCR was performed after cDNA synthesis using AccuPower RT premix (Bioneer, Daejeon, Korea). The primers sequences used were as follows (shown
5 → 3 ): for procollagen type I, CGA GAC CCT TCT CAC TCC TG and CAC CCC TTC TGC GTT GTA TT; for procollagen type III, GTA CAG CTG GCC TTC CTC AG and TTT TGT TTT GCT GGG GTT TC; and for ␣-SMA, CAT CAG GAA CCT CGA GAA GC and TCG GAT ACT TCA GGG TCA GG. These sequences were also used for -actin. 2.12. Statistical analysis All data are expressed as the mean ± SD (n = 8). Statistically significant differences between the groups were analyzed using one-way analysis of variance (ANOVA) followed by a paired
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Student’s t-test. p < 0.05 was regarded as statistically significant. 3. Results 3.1. CGX attenuated the changes in organ weights and biochemical parameters In the DMN group, the weights of the body, liver, and spleen were 45.9, 50.7, and 209.4% of the respective normal values at the 4-week endpoint. CGX (100 or 200 mg kg−1 ) and silymarin administration attenuated these severe changes in bodyweight by 87.4, 77.3, and 78.2%, in liver weight by 86.3, 88.1, and 89.0%, and in spleen weight by 136.7, 119.7, and 141.8% of the normal values, respectively. CGX treatment increased body weight significantly compared to the DMN group and alleviated the decrease in liver weight with DMN treatment (p < 0.01) (Table 2). Conversely, CGX treatment significantly suppressed the DMNinduced increases in the serum AST, ALT, ALP, and total bilirubin levels (p < 0.05 or p < 0.01). The moderately low serum albumin level of the DMN group was significantly restored to normal in the CGX100 and CGX200 groups (p < 0.01) (Table 2). 3.2. CGX prevented inflammation, ECM accumulation, and HSC activation in liver tissues At the end of the experiment, the livers of the animals given DMN were shrunken and congested compared to those of the control group, and they showed features of inflammation, whereas the CGX treatment significantly ameliorated these changes (Fig. 2A and B). The collagen accumulation was severe in the DMN group, whereas the CGX treatment reduced it significantly (Fig. 2C). In particular, markedly fewer ␣-SMA-positive cells were observed in the CGX groups (Fig. 2D).
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3.3. CGX decreased collagen accumulation in liver tissues To examine the collagen content of liver tissue, hydroxyproline was determined from frozen liver tissues. DMN treatment dramatically increased the amount of hydroxyproline by 6-fold, whereas CGX (100 and 200 mg kg−1 ) administration significantly decreased the hydroxyproline content in liver tissue compared to the DMN group (p < 0.01) (Fig. 3A). 3.4. CGX prevented lipid peroxidation and the depletion of antioxidant enzymes in liver tissues The lipid peroxidation was examined by determining malondialdehyde (MDA) content in frozen liver tissue homogenate. The MDA content in the DMN group was significantly higher than that in the normal group (approximately 375% of normal). In the CGX100, CGX200, and silymarin groups, MDA decreased by 36.7, 46.4, and 53.4% respectively, compared to the DMN group (Fig. 3B). We determined the major antioxidant enzymes GSH, GSH-Px, SOD, and catalase in liver tissues. We found notably sustained levels of GSH components in the CGX-treated groups compared to the DMN group (p < 0.01) (Fig. 3E). In addition, CGX treatment significantly inhibited the depletion of GSH-Px, catalase, and SOD caused by DMN toxicity compared to the DMN group (p < 0.05 or p < 0.01) (Fig. 3C, D and F). 3.5. CGX inhibited profibrotic cytokines, TGF-ˇ, and PDGF-ˇ in liver tissue The main profibrotic cytokines were determined using an ELISA method. CGX significantly inhibited the levels of TGF- and PDGF (PDGF-BB) in liver tissue compared with the DMN group (p < 0.05 or p < 0.01) (Fig. 4B and C).
Fig. 4. Expression of fibrosis-related genes and profibrotic cytokines in liver tissue. Total RNA was extracted from liver tissues, and cDNA was synthesized. PCR was performed for 10 genes, and the products were visualized with UV illumination (A). TGF- (B) and PDGF-BB (C) were determined in the liver lysates using ELISA kits. *p < 0.05 and **p < 0.01 compared with the DMN group.
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Fig. 5. Mechanism of action of CGX in vitro. The LDH leakage assay was performed using HepG2 cells after CGX pretreatment followed by CCl4 exposure for 2 h (A). NO production by RAW 264.7 cells was measured after pretreatment with CGX and then LPS treatment for 24 h (B). *p < 0.05 and **p < 0.01 compared with the control. Gene expression changes in HSC-T6 cells triggered by CGX were examined using real-time PCR. The results are expressed as normalized fold values relative to the control (C).
3.6. CGX restored the altered gene expression associated with liver fibrosis In order to investigate the hepatoprotective mechanism of CGX, the expression of the iNOS, TGF-, TNF-␣, CTGF, PDGF-, MMP2, HGF, TIMP-1, and TIMP-2 genes was analyzed using RT-PCR. DMN treatment induced high expression of the iNOS, TGF-, TNF-␣, CTGF, PDGF-, HGF, and TIMP-1 genes compared with the normal group, whereas their activation was dramatically inhibited by CGX treatment. CGX did not affect the expression of the TIMP-2 gene (Fig. 4A). 3.7. CGX affected hepatocyte protection, the inhibition of NO production, and HSC inactivation in vitro In vitro, 100 g mL−1 of CGX protected hepatocytes against CCl4 treatment by about 40% compared to the CCl4 control (p < 0.01) (Fig. 5A). CGX treatment also significantly inhibited NO production in RAW 264.7 cells in a concentration-dependent manner (p < 0.01) (Fig. 5B). In addition, CGX drastically downregulated expression of the procollagen types I and III genes by 42 and 55%, respectively, at CGX 50 g mL−1 , and moderately downregulated ␣-SMA in HSC-T6 cells (Fig. 5C). 4. Discussion Chronic liver injury commonly leads to liver fibrosis resulting from imbalance between synthesis and degradation of the ECM
(Benyon and Iredale, 2000). Some drugs have antifibrotic effects by reducing the accumulation of ECM in chronic hepatic injury models (Hammel et al., 2001; Bataller and Brenner, 2005). Based on our previous results for CGX, we further examined the protective effects of CGX against liver fibrosis and the compartment enzymes playing roles in the antioxidative process to identify the mechanisms responsible. We also investigated the effects of CGX on individual cellular components of liver tissue using hepatocyte-, Kupffer cell-, and stellate cell-derived cell lines. As we expected, CGX treatment significantly ameliorated the DMN-induced histopathologic deterioration in liver tissues, as well as serum parameters (Fig. 2A and B, and Table 2). In addition, the amount of ECM was very low in the CGX-treated groups in contrast to the severe ECM accumulation seen in the DMN group (Fig. 2C). Preventing the underlying destructive condition is optimal antifibrotic strategy, and antioxidants protect against liver injury (Vitaglione et al., 2004; Medina and MorenoOtero, 2005). Our in vitro data showed the protective effect of CGX against hepatocyte destruction caused by CCl4 treatment (Fig. 5A). During fibrosis progression, the main ECM-producing cells are the activated HSCs, which secrete ECM, including collagen types I and III. The inhibition of this step is a major strategy in the development of antifibrotic drugs (Bataller and Brenner, 2001; Reeves and Friedman, 2002; Safadi and Friedman, 2002). We found that CGX inhibited stellate cell activation by DMN treatment using immunostaining for ␣-SMA, a marker of stellate cell activation (Fig. 2D). This concurred with the in vitro results from the rat hepatic stellate cell
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line HSC-T6 (Fig. 5C). CGX drastically downregulated the key gene expression corresponding to activation of HSC, collagen types I and III, and ␣-SMA. The hydroxyproline measurement also showed that CGX treatment significantly inhibited the accumulation of collagen compared to the DMN group (Fig. 3A). To investigate the molecular mechanisms underlying these effects, we examined the levels of lipid peroxides and the activity of antioxidant enzymes in liver tissues. The combination of excessive oxygen stressors and decreased antioxidant levels contributes to the initiation or progression of hepatic damage and liver fibrosis (Parola and Robino, 2001; Rockey, 2005). Our results suggest that CGX has the pharmacological capacity to reduce oxidative stress, as evidenced by the decreased MDA (an important indicator of oxidative damage) concentrations in the CGX-treated groups compared to in the DMN group (Fig. 3B). DMN treatment drastically lowered the glutathione content and GSH-Px, SOD, and catalase activities in hepatic tissues, whereas the CGX-treated groups sustained levels near the normal range (Fig. 3C–F). We examined the changes in the expression of several genes known to be key factors in the hepatic fibrosis process (Fig. 4A). Of the nine genes examined, the iNOS, TNF-␣, TGF- (only in CGX200), CTGF, and PDGF- genes were dramatically normalized by CGX treatment compared to the DMN group. These are prominent inflammatory or profibrogenic cytokines (MacDonald et al., 2001; Breitkopf et al., 2006; Friedman, 2008). In particular, PDGF is the most potent mitogen and activator of HSCs (Pinzani et al., 1989; Borkham-Kamphorst et al., 2007), and CTGF is a potent profibrotic factor that mediates the stimulatory actions of TGF-induced ECM synthesis (Ihn, 2002). The levels of TGF- and PDGF- proteins in liver tissues were drastically lower in the CGX treatment groups (Fig. 4B and C). The upregulation of the HGF and MMP-2 genes in the DMN group only might be a response to compensate for the severe hepatic injury and excess accumulation of ECM in liver tissues. DMN treatment upregulated the expression of two genes, TIMP-1 and TIMP-2, which is another characteristic of hepatic fibrosis (Benyon et al., 1996; Rippe and Brenner, 2004). No difference in TIMP-2 and only slight inhibition of TIMP-1 was observed in the CGX-treated groups. These positive results for lipid peroxidation, the SOD, catalase, GSH-Px, and GSH contents of liver tissues, the gene expression patterns of iNOS and TNF-␣, and inhibition of NO production by RAW 264.7 cells (Fig. 5B) are very consistent with the antioxidant action of CGX. The over-produced NO and profibrogenic cytokines by activated Kupffer cells and apoptotic hepatocytes direct the transformation of HSCs into myofibroblasts secreting ECM (Watanabe et al., 2007; Friedman, 2008). Taken together, our results lead us to conclude that CGX protects against liver fibrosis during chronic liver damage, and the responsible mechanisms are associated with both hepatocyte protection and the suppression of HSC activation via antioxidative stress and antifibrotic cytokines. Acknowledgments We gratefully thank Dr. S.L. Friedman for providing the HSC-T6 cell line. This work was supported by the Oriental Medicine R&D Project of the Ministry of Health and Welfare, Republic of Korea (No. B070031). References Bataller, R., Brenner, D.A., 2001. Hepatic stellate cells as a target for the treatment of liver fibrosis. Seminars in Liver Disease 21, 437–451. Bataller, R., Brenner, D.A., 2005. Liver fibrosis. The Journal of Clinical Investigation 115, 209–218.
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Beers, R.F., Siezer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry 195, 133–140. Benyon, R.C., Iredale, J.P., Goddard, S., Winwood, P.J., Arthur, M.J., 1996. Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver. Gastroenterology 110, 821–831. Benyon, R.C., Iredale, J.P., 2000. Is liver fibrosis reversible? Gut 46, 443–446. Borkham-Kamphorst, E., van Roeyen, C.R., Ostendorf, T., Floege, J., Gressner, A.M., Weiskirchen, R., 2007. Pro-fibrogenic potential of PDGF-D in liver fibrosis. Journal of Hepatology 46, 1064–1074. Breitkopf, K., Godoy, P., Ciuclan, L., Singer, M.V., Dooley, S., 2006. TGFbeta/Smad signaling in the injured liver. Zeitschrift für Gastroenterologie 44, 57–66. Cho, J.H., Lee, Y.Y., Seo, S.H., Yoo, H.S., Choi, W.J., Lee, Y.W., Son, C.G., Cho, C.K., 2001. A clinical report about 57 patients with chronic liver disease. Korean Journal of Oriental Medicine 21, 112–121. Choi, W.J., Shin, J.W., Son, J.Y., Seo, D.S., Park, H.S., Han, S.H., Sung, H.J., Cho, J.H., Cho, C.K., Yoo, H.S., Lee, Y.W., Son, C.G., 2006. Toxicological study of the hepatotherapeutic herbal formula, CGX, in beagle dogs. World Journal of Gastroenterology 12, 7497–7502. Evans, J.C., Ellman, G.L., 1959. The ionization of cysteine. Biochimica et Biophysica Acta 33, 574–576. Fallowfield, J.A., Kendall, T.J., Iredale, J.P., 2006. Reversal of fibrosis: no longer a pipe dream? Clinics in Liver Disease 10, 481–497. Fowell, A.J., Iredale, J.P., 2006. Emerging therapies for liver fibrosis. Digestive Diseases 24, 174–183. Friedman, S.L., 2003. Liver fibrosis-from bench to bedside. Journal of Hepatology 38, S38–53. Friedman, S.L., 2008. Mechanisms of hepatic fibrogenesis. Gastroenterology 134, 1655–1669. Fujita, M., Shannon, J.M., Morikawa, O., Gauldie, J., Hara, N., Mason, R.J., 2003. Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta. American Journal of Respiratory Cell and Molecular Biology 29, 669–676. Gines, P., Cardenas, A., Arroyo, V., Rodes, J., 2004. Management of cirrhosis and ascites. The New England Journal of Medicine 350, 1646–5164. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry 126, 131–138. Hammel, P., Couvelard, A., O’Toole, D., Ratouis, A., Sauvanet, A., Fléjou, J.F., Degott, C., Belghiti, J., Bernades, P., Valla, D., Ruszniewski, P., Lévy, P., 2001. Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of the common bile duct. The New England Journal of Medicine 344, 418–423. Hu, X.P., Shin, J.W., Wang, J.H., Cho, J.H., Son, J.Y., Cho, C.K., Son, C.G., 2008. Antioxidative and hepatoprotective effect of CGX, an herbal medicine, against toxic acute injury in mice. Journal of Ethnopharmacology 120, 51–55. Ihn, H, 2002. Pathogenesis of fibrosis: role of TGF- and CTGF. Current Opinion in Rheumatology 14, 681–685. Iredale, J.P., 2007. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. The Journal of Clinical Investigation 117, 539–548. Kisseleva, T., Brenner, D.A., 2008. Mechanisms of fibrogenesis. Experimental Biology and Medicine 233, 109–122. Levy, C., Seeff, L.D., Lindor, K.D., 2004. Use of herbal supplements for chronic liver disease. Clinical Gastroenterology and Hepatology 2, 947–956. MacDonald, G.A., Bridle, K.R., Ward, P.J., Walker, N.I., Houglum, K., George, D.K., Smith, J.L., Powell, L.W., Crawford, D.H., Ramm, G.A., 2001. Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3. Journal of Gastroenterology and Hepatology 16, 599–606. Medina, J., Moreno-Otero, R., 2005. Pathophysiological basis for antioxidant therapy in chronic liver disease. Drugs 65, 2445–2461. Parola, M., Robino, G., 2001. Oxidative stress-related molecules and liver fibrosis. Journal of Hepatology 35, 297–306. Pinzani, M., Gesualdo, L., Sabbah, G.M., Abboud, H.E., 1989. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. The Journal of Clinical Investigation 84, 1786–1793. Reeves, H.L., Friedman, S.L., 2002. Activation of hepatic stellate cells—a key issue in liver fibrosis. Frontiers in Bioscience 7, 808–826. Rippe, R.A., Brenner, D.A., 2004. From quiescence to activation: gene regulation in hepatic stellate cells. Gastroenterology 127, 1260–1262. Rockey, D.C., 2005. Antifibrotic therapy in chronic liver disease. Clinical Gastroenterology and Hepatology 3, 95–107. Safadi, R., Friedman, S.L., 2002. Hepatic fibrosis-role of hepatic stellate cell activation. Medscape General Medicine 4, 27. Shin, J.W., Son, J.Y., Oh, S.M., Han, S.H., Wang, J.H., Cho, J.H., Cho, C.K., Yoo, H.S., Lee, Y.W., Lee, M.M., Hu, X.P., Son, C.G., 2006. An herbal formula, CGX, exerts hepatotherapeutic effects on dimethylnitrosamine-induced chronic liver injury model in rats. World Journal of Gastroenterology 12, 6142–6148. Son, C.G., Choi, B.L., Shin, J.W., Choi, W.J., Cho, J.H., Cho, C.K., 2001. Effect of gamichunggantang on alcoholic metabolism and alcoholic liver disease. Korean Journal of Oriental Medicine 2, 89–98. Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278.
542
J.-H. Wang et al. / Journal of Ethnopharmacology 127 (2010) 534–542
Vitaglione, P., Morisco, F., Caporaso, N., Fogliano, V., 2004. Dietary antioxidant compounds and liver health. Critical Reviews in Food Science and Nutrition 44, 575–586. Watanabe, A., Hashmi, A., Gomes, D.A., Town, T., Badou, A., Flavell, R.A., Meha, W.Z., 2007. Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9. Hepatology 46, 1509–1518.
Yang, Y., Yang, S., Chen, M., Zhang, X., Zou, Y., Zhang, X., 2008. Compound Astragalus and Salvia miltiorrhiza Extract exerts anti-fibrosis by mediating TGFbeta/Smad signaling in myofibroblasts. Journal of Ethnopharmacology 118, 264–270.