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Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model
Journal of Ethnopharmacology 140 (2012) 179–185
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Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model Jing-Hua Wang a , Min-Kyung Choi a , Jang-Woo Shin a , Seock-Yeon Hwang b , Chang-Gue Son a,∗ a b
Liver and Immunology Research Center, Daejeon Oriental Hospital of Daejeon University, 22-5 Daeheung-dong, Jung-gu, Daejeon 301-704, Republic of Korea Department of Biomedical Laboratory Science, College of Applied Science and Industry, Daejeon University, 96-3 Yongun-dong, Dong-gu, Daejeon 300-716, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 2 January 2012 Accepted 7 January 2012 Available online 14 January 2012 Keywords: Carbon tetrachloride Artemisia capillaris Artemisia iwayomogi Liver fibrosis Oxidative stress
a b s t r a c t Ethnopharmacological relevance: Artemisia capillaris and Artemisia iwayomogi, both members of the Compositae family, have been indiscriminately used for various liver disorders as traditional hepatotherapeutic medicines in Korea for many years. Aim of the study: In this study, the anti-hepatofibrotic effects of Artemisia capillaris and Artemisia iwayomogi were comparatively analyzed using a carbon tetrachloride (CCl4 )-induced liver fibrosis rat model. Materials and methods: Hepatic fibrosis was induced via a 10-week course of intraperitoneal CCl4 injections (50% dissolved in olive oil, 2 mL/kg, twice per week). Water extract of Artemisia capillaris (AC) or Artemisia iwayomogi (AI) was orally administered six times per week from the 5th to the 10th week. Results: AI (50 mg/kg) significantly attenuated the CCl4 -induced excessive release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum (p < 0.05), and hydroxyproline and malondialdehyde (MDA) contents in liver tissue (p < 0.05). Further, AI markedly ameliorated the depletion of total antioxidant capacity (TAC), glutathione (GSH), and superoxide dismutase (SOD) in liver tissue (p < 0.01). Unexpectedly, AC did not exert any effects on the above parameters. Histopathological and immunohistochemical analyses revealed that AI drastically reduced inflammation, necrosis, fatty infiltration, collagen accumulation, and activation of hepatic satellite cells in liver tissue. These changes were not observed with AC treatment. Several critical genes of fibrosis-related cytokines including transforming growth factor beta (TGF-), platelet-derived growth factor beta (PDGF), and alpha smooth muscle actin (␣-SMA) were more prominently downregulated by AI compared to AC treatment. Conclusion: Our results show that AI exerts greater hepatoprotective and anti-fibrotic effects as compared with AC via enhancing antioxidant capacity and downregulating fibrogentic cytokines. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatic fibrosis is a common chronic disease that occurs prior to the formation of liver cirrhosis and generally accounts for the
Abbreviations: AC, water extract of Artemisia capillaris; AI, water extract of Artemisia iwayomogi; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CCl4 , carbon tetrachloride; CTGF, connective tissue growth factor; DAB, diaminobenzidine; DEPPD, N,N-diethyl-para-phenylendiamine sulfate; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); ECM, extracellular matrix; GSH, glutathione; H&E, hematoxylin and eosin; H2 O2 , hydrogen peroxide; HPLC, high performance liquid chromatography; HSCs, hepatic stellate cells; KCl, potassium chloride; MDA, malondialdehyde; PDGF-, platelet-derived growth factor-beta; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; SOD, superoxide dismutase; SPF, specific pathogen free; TAC, total antioxidant capacity; TBA, thiobarbituric acid; TEP, 1,1,3,3-tetraethoxypropane; TGF-, transforming growth factor-beta; UPLC, ultra performance liquid chromatography; ␣-SMA, alpha-smooth muscle actin; -NADPH, -nicotinamide adenine dinucleotide phosphate. ∗ 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 © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.007
incessant insult to hepatocytes and kupffer cells, followed by the activation of hepatic stellate cells (HSCs) (Fowell and Iredale, 2006). Activated HSCs transform into myofibroblast-like cells and abnormally secrete excessive extracellular matrix (ECM) (Gabele et al., 2003). Continuous accumulation of ECM leads to liver fibrosis and ultimately contributes to liver cirrhosis accompanying liver dysfunction, which has a high mortality rate (Friedman, 2003; Iredale, 2003). Wide range of pathogenic factors, such as: hepatitis B virus (HBV), hepatitis C virus (HCV), hepatotoxins, metabolic disorders, alcoholisms and schistosomiasis are closely related with hepatic fibrogenesis or progression of hepatic cirrhosis (Bataller and Brenner, 2005). Although many therapies are used to treat liver cirrhosis in Western medicine, radical treatments do not exist, excluding living donor liver transplantation (Graziadei, 2007). Consequently, timely inhibition of fibrogenesis at an early stage is considered as a feasible strategy for curing chronic liver diseases (Gines and Cardenas, 2008). Herbal medicines have been frequently investigated for their hepatoprotective and antifibrotic effects in both humans (Yip et al.,
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2007) and animal models (Lin et al., 2011). Artemisia capillaries Thunb. and Artemisia iwayomogi KITAMURA are well-known herbal hepatotherapeutic drugs and have been traditionally used in Korea and/or China for a long time (Jang, 1975; Zhang, 1978). Both of Artemisia capillaris and Artemisia iwayomogi belong to the Compositae family and commonly named as “Injinho” and “Haninjin” respectively in Korea. Previous studies have reported that Artemisia capillaris and Artemisia iwayomogi, or their derived compounds, exhibit various pharmacological activities, such as antioxidant effects (Seo and Yun, 2008), cytoprotection (Hong et al., 2007), choleretic effects (Okuno et al., 1981), hepatoprotection (Choi et al., 2011), antimicrobial effects (Seo et al., 2010), and antiinflammatory effects (Jang et al., 2005; Shin et al., 2006). Several studies separately presented evidence of the hepatoprotective and antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in carbon tetrachloride (CCl4 )-induced acute hepatitis and a model for early stage fibrosis (Lee et al., 2000; Park et al., 2000; Choi et al., 2005). However, investigations on Artemisia capillaris or Artemisia iwayomogi for the treatment of long-term CCl4 -induced liver damage have not been performed. The two herbs belong to the genus Artemisia and have been indiscriminately used in the treatment of various hepatic disorders and are known by the same Korean name, “InJin” in traditional Korea clinics. However, there is a lack of scientific evidence to indicate whether Artemisia capillaris and Artemisia iwayomogi exert a coordinative antifibrotic effect. No scientific information exists as to whether Artemisia capillaris and Artemisia iwayomogi exert similar therapeutic properties. The aim of the present study was to compare the hepatotherapeutic properties of Artemisia capillaris and Artemisia iwayomogi using a fibrotic rat model that was induced by long-term CCl4 administration.
Fig. 1. HPLC fingerprinting analysis comparing Artemisia capillaris and Artemisia iwayomogi. The water extract of Artemisia capillaris and Artemisia iwayomogi and their standard compounds were subjected to HPLC. All chromatograms were produced at a wavelength of 345 nm.
Korea). Artemisia capillaris and Artemisia iwayomogi were washed twice with distilled water. After fully drying in oven at 60 ◦ C, Artemisia capillaris and Artemisia iwayomogi were weighed and boiled separately in distilled water for 30 min and unceasingly concentrated for 120 min. After filtration and lyophilization, the extracts were stored at −70 ◦ C for future use. The yield of water extract of Artemisia capillaris (AC) and Artemisia iwayomogi (AI) was 3.20% and 4.33% (w/w), respectively, and they (Voucher specimen No. AC-2010-364 and AI-2010-361) have been deposited at Liver and Immunology Research Center of Daejeon Oriental Hospital in South Korea.
2. Materials and methods 2.3. HPLC-based fingerprinting analysis 2.1. Reagents and chemicals Carbon tetrachloride was obtained from Showa Chemical Co. (Tokyo, Japan). The chemicals 6,7-dimethoxycoumarin (scoparone), 7-hydroxy-6-methoxycoumarin (scopoletin), p-dimethylaminobenzaldehyde, 1,1,3,3hydroxyproline, tetraethoxypropane (TEP), chloramines-T, potassium chloride (KCl), hydrochloric acid (HCl), n-butanol, N,N-diethyl-paraphenylendiamine sulfate (DEPPD), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), glutathione reductase and -nicotinamide adenine dinucleotide phosphate-reduced form (-NADPH), formic acid, and acetonitrile were all obtained from Sigma (MO, USA); perchloric acid was obtained from GFS Chemical Co. (OH, USA); and thiobarbituric acid (TBA) was obtained from Lancaster Co. (Lancashire, England). Hydrogen peroxide (H2 O2 ) was obtained from Junsei Chemical Co. Ltd. (Tokyo, Japan). Anti␣-smooth muscle actin mouse monoclonal antibody was obtained from Abcam (Cambridge, UK); histofine was purchased from Nichirei Biosciences (Tokyo, Japan); diaminobenzidine (DAB) was purchased from BioGenex (CA, USA); and Mayer’s hematoxylin was obtained from Wako Pure Chemical Industries (Osaka, Japan). 2.2. Extraction process of Artemisia capillaris and Artemisia iwayomogi According to the standards of the Korean Herbal Pharmacopoeia (2002), the dried Artemisia capillaris and Artemisia iwayomogi were obtained from the Jeong-Seoung Oriental Medicine Company (Daejeon, Korea). Artemisia capillaris and Artemisia iwayomogi samples were identified by Professor Seok-Rhin Lim (Daejeon University,
Fingerprints were produced using two-dimensional highperformance liquid chromatography (HPLC) profiles of two herbal extracts with their major components (scopoletin and scoparone) as standards. Briefly, after dissolution (5 mg of AC and AI in 1 mL 50% methanol, 0.1 mg of two standards in 1 mL 50% methanol) and filtration, the drugs were analyzed by HPLC. The HPLC system consisted of the Thermo Accela Ultra Performance LC (UPLC) system, Acquity UPLC Binary Solvent Manager, Acquity sample manager/column heater module, and Acquity Photo-Diode Array detector system (Waters, MA, USA). An Acquity UPLC BEH C18 (1.7 m, 2.1 mm × 100 mm) column was used and the compounds were eluted with solvents A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL/min. Solutions of 95% A and 5% B changed over a 20 min period to 0% A and 100% B, followed by a 25 min changing to 95% A and 5% B. All chromatograms were obtained using a wavelength of 345 nm (Fig. 1). 2.4. Animals and experimental design The design and performance of the experiments were approved by the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-185-1002-02), and conducted in accordance with the Policy on the Humane Care and Use of Laboratory Animals, as adopted and promulgated by the USA National Institute of Health (NIH). In total, 54 pathogen-free Sprague-Dawley male rats (6-weekold, 190–210 g) were purchased from Koatech (Gyeonggi Do, Korea), and acclimated under environmentally controlled conditions at 22 ± 2 ◦ C using a 12/12-h light/dark cycle. Rats were
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randomly divided into six groups (n = 9): normal (control), CCl4 , AC 25 (25 mg/kg), AC 50 (50 mg/kg), AI 25 (25 mg/kg), and AI 50 (50 mg/kg). CCl4 (2 mL/kg, 50% dissolved in olive oil) was intraperitoneally injected twice a week for 10 weeks into all groups, except for the normal group. AC (25 mg/kg or 50 mg/kg) or AI (25 mg/kg or 50 mg/kg) was given six times per week from the 5th to the 10th week via oral gavage. In this experiment, the dose of AC and AI was made reference to the recommended dosage in clinic of Korea. On the final experimental day, after 18 h of fasting all of the animals were weighed for sacrifice preparation. The animals were anesthetized with ethyl ether. Then, blood was collected from abdominal aorta using syringes for biochemical analyses, and the livers removed, photographed, and immediately weighed. Liver tissue was either fixed or stored at −70 ◦ C in Bouin’s solution or RNAlater solution (Ambion, TX, USA) for histopathological examination, related gene expression analysis, and biochemical determination, respectively. 2.5. Serum biochemical analysis Blood was collected from the abdominal aorta. After centrifuging at 3000 × g for 15 min, the serum was separated and stored in −70 ◦ C. The serum levels of total protein, albumin, total bilirubin, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were determined using an Auto Chemistry Analyzer (AU400; Olympus, Tokyo, Japan). 2.6. Histopathological findings and immunohistochemical staining Liver tissues fixed in Bouin’s solution were embedded in paraffin and cut into 4-m-thick sections for histomorphological examination. After drying, liver tissue section slides were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. For immunohistochemical staining of alpha-smooth muscle actin (␣SMA), liver tissue sections were deparaffinized, hydrated, and heated in citrate buffer at 100 ◦ C for 15 min, then treated with normal serum for 30 min. The slides were then treated with an anti-␣-SMA mouse monoclonal antibody (1:100; Abcam, Cambridge, UK) overnight. After washing with PBS, Histofine (Nichirei Biosciences, Tokyo, Japan) was added using DAB as a substrate. The slides were counterstained with Mayer’s hematoxylin (Leica Microsystems, Wetzlar, Germany) and examined under an optical microscope. The percentage areas of collagen staining and positive ␣-SMA staining were analyzed by the image analysis program, ImageJ (NIH, USA). 2.7. Determination of hydroxyproline in liver tissues Hydroxyproline determination was conducted using a slight modification of a method described previously (Fujita et al., 2003). Briefly, liver tissues (200 mg) were homogenized in HCl and hydrolyzed at 110 ◦ C. After filtering the acid hydrolyzates using filter paper (Toyo Roshi Kaisha, Tokyo, Japan), the samples were completely dried in an oven. After dissolving in methanol, 50% isopropanol and chloramine-T solution were added, followed by reaction with Ehrlich’s solution at 50 ◦ C for 90 min. The absorbance was read at 558 nm. A standard curve was established using serial twofold dilutions of 1.0 mg solutions of hydroxyproline. 2.8. Determination of lipid peroxidation in liver tissues Lipid peroxidation levels were evaluated by the thiobarbituric acid reactive substances (TBARS) method, as described previously (Mihara and Uchiyama, 1978). Briefly, liver tissue (200 mg) was homogenized in ice-cold KCl (1.15%), and the homogenate was
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mixed with 1% H3 PO4 and 0.67% TBA solution. The mixture was heated for 45 min at 100 ◦ C, n-butanol was added, and the solution was then mixed and centrifuged at 3000 × g for 15 min. The absorbance of the supernatant was measured at 535 and 520 nm, and compared to a standard value (freshly prepared TEP solution). 2.9. Determination of total antioxidant capacity (TAC), total glutathione (GSH) content, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities in liver tissue Radioimmunoprecipitation assay (RIPA) buffer-based liver tissue homogenate was centrifuged at 10,000 × g for 15 min. The supernatant was transferred and stored at −70 ◦ C until required. The total antioxidant activity was determined using a previously described method (Kambayashi et al., 2009). Total GSH was determined according to the method of Ellman (Evans and Ellman, 1959). SOD activities were determined using a SOD assay kit (Dojindo Laboratories, Japan). Catalase activity in the liver tissue was determined using the method of Beers and Sizer (1952). 2.10. Real-time PCR for analyzing gene expression in liver tissue Total RNA was isolated from RNAlater-treated liver tissues using an RNeasy midi kit (Qiagen, USA). Real-time PCR was performed using IQ 5 (Bio-Rad, USA) after cDNA synthesis. The primer sequences used were as follows (forward and reverse, respectively): -actin, 5 -GTG GGG CGC CCC AGG CAC CA and 5 -CTC CTT AAT GTC ACG CAC GAT TTC; ␣-SMA, 5 -CAT CAG GAA CCT CGA GAA GC and 5 -TCG GAT ACT TCA GGG TCA GG; PDGF-, 5 -CTG CCT CTC TGC TGC TAC CT and 5 -GAT GAG CTT TCC GAC TCG AC; TGF-, 5 TGA GTG GCT GTC TTT TGA CG and 5 -TTC TCT GTG GAG CTG AAG CA; and CTGF, 5 -ATG GAG ACA TGG CGT AAA GC and 5 -TTG CAT GAC AAT GAC ACA CG. The final results are expressed as normalized fold values relative to the normal group. 2.11. Statistical analysis The results were expressed as the mean ± standard deviation (n = 9). Differences between groups were analyzed using a oneway analysis of variance (ANOVA) followed by the least significant difference post hoc test. In all analyses, p-values <0.05 were considered statistically significant. 3. Results 3.1. Changes in body and organ weights CCl4 treatment significantly reduced body and liver weights, while the spleen weight increased as compared with the normal group (p < 0.01). Treatment with AC and AI did not obviously influence changes in body, liver, and spleen as compared with the CCl4 group (Table 1). 3.2. Alterations in serum biochemical parameters CCl4 treatment markedly elevated serum AST, ALT, ALP, and bilirubin, and significantly reduced serum total protein and albumin as compared with the normal group (p < 0.05). AI treatment (50 mg/kg) significantly attenuated the CCl4 -induced increases in serum levels of AST, ALT, and ALP (p < 0.05). Nevertheless, no remarkable differences in serum AST, ALT, and ALP levels were found between the AC and CCl4 treatment. Treatment with AC and AI did induce statistically significant differences in serum bilirubin, total protein, and albumin as compared with the CCl4 group (Table 1).
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Table 1 Comparison of organ weights and serum biochemistry parameters. Groups
Normal
CCl4 only
Artemisia capillaris (mg/kg) CCl4 + 25
Body mass (g) Liver mass (g) Spleen mass (g) AST (IU/L) ALT (IU/L) ALP (IU/L) Bilirubin (g/dL) Total protein (g/dL) Albumin (g/dL) # ## *
401 9.9 0.73 144 52 132 0.2 7.0 3.6
± 19 ± 13.3 ± 0.10 ± 14 ±9 ± 31 ± 0.0 ± 0.5 ± 0.2
340 13.3 1.00 229 141 301 0.3 6.3 3.2
± ± ± ± ± ± ± ± ±
26## 1.5## 0.09## 77# 36## 99# 0.1# 0.5# 0.4#
331 12.6 1.08 227 128 274 0.3 6.6 3.4
± ± ± ± ± ± ± ± ±
21 1.0 0.16 90 69 113 0.1 0.3 0.1
Artemisia iwayomogi (mg/kg)
CCl4 + 50 323 12.6 0.96 261 159 298 0.3 6.4 3.3
± ± ± ± ± ± ± ± ±
35 1.7 0.23 157 64 73 0.1 0.6 0.3
CCl4 + 25 335 13.3 0.87 224 167 305 0.3 6.6 3.4
± ± ± ± ± ± ± ± ±
26 2.6 0.25 101 69 82 0.1 0.5 0.2
CCl4 + 50 324 12.1 0.88 153 103 191 0.3 6.7 3.3
± ± ± ± ± ± ± ± ±
20 1.1 0.17 36* 22* 18* 0.1 0.5 0.2
p < 0.05, compared with the normal. p < 0.01, compared with the normal. p < 0.05, compared with the CCl4 only (n = 9).
3.3. Histopathology and immunohistochemistry The CCl4 -treated livers were shrunken, nodular, and discolored. However, these changes were moderately attenuated by AC and AI treatment (data not shown). Histopathological observations indicated massive fatty infiltration, hepatocellular necrosis, infiltration of lymphocytes, and bridging collagen accumulation
in the CCl4 group, whereas AC and AI treatment evidently alleviated these features (Fig. 2A and B). Based on immunostaining, CCl4 treatment produced a strong positive signal for ␣-SMA around the collagen septa region in liver tissue; however, this effect was obviously weakened by AC and AI treatment (Fig. 2C). AI treatment, rather than AC treatment, produced a statistical significant difference as compared with the CCl4 group in the percentage
Fig. 2. Histopathological analysis and immunohistochemical staining. Following the final time point, all animals were sacrificed and the livers were removed and fixed in Bouin’s solution. After staining with hematoxylin and eosin (A) or Masson’s trichrome (B) and immunohistochemistry for ␣-SMA (C), pathophysiologic examinations were performed under light microscopy at 200× magnification. The percent area of the fibrotic region (E) and the ␣-SMA staining region (F) were analyzed by ImageJ software (NIH, USA). ## p < 0.01, compared with the normal; *p < 0.05, **p < 0.01, compared with CCl4 only.
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Table 2 Comparison of hydroxyproline contents and antioxidant related parameters in liver tissue. Groups
Normal
CCl4 only
Artemisia capillaris (mg/kg) CCl4 + 25
Hydroxyproline (g/g tissue) MDA (M/g tissue) TAC (U/mg tissue) Total GSH (mM/g tissue) SOD (U/mg tissue) Catalase (U/mg tissue) # ## * **
57.7 184.3 271 2.04 0.37 861
± ± ± ± ± ±
6.7 21.3 37 0.27 0.10 37
159.1 362.7 198 0.51 0.22 643
± ± ± ± ± ±
44.8## 117.4## 49## 0.13## 0.05# 97##
144.1 341.7 193 0.44 0.21 582
± ± ± ± ± ±
Artemisia iwayomogi (mg/kg)
CCl4 + 25 18.1 95.2 33 0.19 0.07 97
153.2 346.6 188 0.52 0.22 546
± ± ± ± ± ±
CCl4 + 25 33.8 63.3 42 0.30 0.06 132
119.6 334.3 239 0.90 0.29 688
± ± ± ± ± ±
CCl4 + 50 21.4* 104.4 20* 0.31* 0.06* 76
118.5 247.5 243 1.04 0.35 672
± ± ± ± ± ±
9.1* 60.9* 38* 0.38** 0.09** 97
p < 0.05, compared with the normal. p < 0.01, compared with the normal. p < 0.05, compared with the CCl4 only (n = 9). p < 0.01, compared with the CCl4 only (n = 9).
area of liver fibrosis and positive ␣-SMA staining (Fig. 2D and E). 3.4. Hydroxyproline content in liver tissue Hydroxyproline was examined to evaluate the collagen content in liver tissue. CCl4 treatment dramatically increased the concentration of hydroxyproline as compared with the normal group (p < 0.01). AI treatment (25 and 50 mg/kg) significantly reduced the elevation of hydroxyproline concentration in a dose-dependent manner as compared with the CCl4 group (p < 0.05), whereas AC treatment merely showed a slight positive trend without statistical significance (Table 2). 3.5. MDA content and TAC levels in liver tissue Lipid peroxidation was determined by assessing MDA content in liver tissue. CCl4 treatment elevated the content of MDA as compared with the normal group (p < 0.01), whereas AI treatment (50 mg/kg) significantly reduced the MDA content as compared with the CCl4 group (p < 0.05) (Table 2). There was no obvious difference in the MDA content between AC and CCl4 treatments. TAC levels were significantly lowered by CCl4 treatment as compared with the normal group (p < 0.01). AI treatment (25 and 50 mg/kg) significantly restored the TAC level in a dose-dependent manner as compared with the CCl4 group (p < 0.05) (Table 2). Nonetheless, AI treatment did not alter the severe reduction of TAC. 3.6. GSH content and antioxidant enzyme activity in liver tissue CCl4 treatment significantly decreased the GSH content and SOD and catalase activity in liver tissue as compared with the normal group (p < 0.05 and p < 0.01). Only AI treatment (25 mg/kg and 50 mg/kg) restored the depletion in GSH content and SOD activity as compared with the CCl4 group in a dose-dependent manner (p < 0.05 and p < 0.01; Table 2). Statistical significance was not observed among the AC, AI, and CCl4 treatments with respect to catalase activity. 3.7. Hepatic fibrosis-related gene expression in liver tissue The expression levels of major liver fibrosis-associated genes were analyzed using real-time PCR. CCl4 treatment noticeably upregulated gene expression of ␣-SMA, TGF-, PDGF-, and CTGF as compared with the normal group. In contrast, AC and AI treatment normalized gene expression of ␣-SMA, TGF-, PDGF-, and CTGF, with AI producing a greater effect than AC (Fig. 3A–C). CTGF gene expression was similarly regulated by both AI and AC treatment (Fig. 3D).
4. Discussion In the present study, we induced hepatic fibrosis to allow for the comparative evaluation of two medical herbs that are used indiscriminately, Artemisia capillaris and Artemisia iwayomogi (Lee et al., 2008). Fibrosis was induced in rats by 10 weeks of CCl4 injection (2 mL/kg, 50% dissolved in olive oil, i.p.). CCl4 is a typical hepatoxin and is widely used as an inducer of hepatic injury in scientific studies (Seifert et al., 1994). Toxicity is caused by CCl4 being metabolized by cytochrome P450, leading to the production of trichloromethyl radicals (CCl3 • ) and reactive oxygen species (ROS; Recknagel et al., 1989). Generally, administration of CCl4 over a 4-week period induces early fibrosis, over an 8-week period causes cirrhosis, and over a 12-week period leads to micronodular cirrhosis (Natarajan et al., 2006). The 10-week CCl4 treatment induced classical advanced fibrosis or early cirrhosis characteristics such as shrunken liver with nodular surface. Severe impairment of liver function was demonstrated by the elevated serum levels of ALT, AST, ALP, and total bilirubin. CCl4 treatment also increased the weight of the spleen, together with an obvious reduction in body and liver weight. Serum levels of transaminase (AST and ALT) and ALP are reliable indicators for assessing liver cellular lesions, as they are excessively released into the bloodstream after hepatocellular membrane disruption (Recknagel et al., 1989). Though AC and AI treatment did not substantially influence changes in parameters including body/liver weight, serum albumin, and bilirubin, AI treatment (50 mg/kg) significantly increased the serum levels of the AST, ALT, and ALP (p < 0.05; Table 1). However, AC treatment did not show the same trend in any of these parameters. Moreover, AI treatment decreased the severe hepatocyte necrosis, lymphocyte infiltration, formation of portal to portal septa, and fatty infiltration to a greater extent than AC treatment (Fig. 2A). Continuous accumulation of extracellular matrix (ECM) results in hepatofibrosis; collagen is the main component of the ECM in fibrotic tissue (Friedman, 2008). Hydroxyproline, which is a major component of collagen, was used as an indicator for evaluating the extent of liver fibrosis (Toyoki et al., 1998). AI treatment (25 and 50 mg/kg) significantly lowered collagen accumulation as evidenced by the inhibition of CCl4 -elevated hydroxyproline concentrations and the percentage area of fibrotic tissue (Table 2 and Fig. 2B and D). Although AC treatment partially inhibited the fibrotic effect, there was no statistically significant difference between the AC and CCl4 group based on quantitative analysis results. Therefore, AI treatment may be more beneficial than AC treatment with respect to antihepatofibrotic properties. It is generally recognized that HSC activation plays a critical role in the process of hepatic fibrogenesis, and ␣-SMA is a marker of activated HSCs (Kisseleva and Brenner, 2008). In the present study, immunohistochemical observations of ␣-SMA indicated that AI (25 and 50 mg/kg) and AC treatment (50 mg/kg) noticeably suppressed
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Fig. 3. Hepatic fibrosis-related gene expression in liver tissue. Total RNA was isolated from a portion of liver tissue, followed by cDNA synthesis and real-time PCR amplification. The results are expressed as normalized fold values relative to the normal group.
the activation of HSCs (Fig. 2C). However, only AI (25 and 50 mg/kg) treatment significantly inhibited the activation of HSC in a dosedependent manner based on quantification of the ␣-SMA staining area (p < 0.01, Fig. 2E). Gene suppression of ␣-SMA in liver tissue was also more prominent in the AI treatment group as compared with the AC treatment group (Fig. 3A). Evidence has been published that indicates oxidative stress influences a series of liver diseases including hepatic fibrogenesis (Ha et al., 2010). In addition, antioxidants including various herbal medicines have been reported to have a protective role in various liver diseases (Lieber et al., 2003; Wang et al., 2011). In order to examine differences between AC and AI treatments regarding perturbations of the antioxidant system, oxidative stress-related factors in liver tissue were analyzed. AI treatment significantly reduced lipid peroxidation in liver tissue, whereas AC treatment showed a slight reduction without statistical significance (Table 2). Additionally, AI treatment, but not AC treatment, restored the total antioxidant capacity in a dose-dependent manner. Interestingly, depletion of total GSH content and SOD activity were notably influenced only by AI treatment. Taken together, it was proposed that Artemisia iwayomogi shows preferable antioxidative capacity as compared with Artemisia capillaris. Catalase activity was moderately depleted by CCl4 treatment, and the AI and AC treatments produced the same effects. Gene expression profiles of three major profibrogenic cytokines (TGF-, PDGF-, and CTGF) were analyzed to explore the different mechanisms underlying hepatoprotection and antihepatofibrosis between AC and AI treatments. TGF- and PDGF- are able to potently activate HSCs following ECM synthesis and collagen formation (Borkham-Kamphorst et al., 2007; Brenner, 2009). In this study, both AI and AC treatments suppressed gene expression of TGF-, PDGF-, and CTGF, showing identical trends. AI treatment more prominently normalized the gene expression of TGF- and
PDGF- as compared with the AC treatment. However, both herbs dramatically regulated the gene expression of CTGF. CTGF is considered to be downstream of TGF-, and functions in a profibrotic role (Blom et al., 2002; Gabele et al., 2003). Further studies are needed to explore the pharmaceutical activity of both herbs, as CTGF has been shown to have potential as an antifibrotic target (Blom et al., 2002). However, the results presented here show a more prominent antihepatofibrotic effect for Artemisia iwayomogi as compared with Artemisia capillaris, and the corresponding mechanisms likely involve the regulation of these profibrogenic cytokine genes. To date, few studies have investigated the hepatoprotective effects of Artemisia capillaris and Artemisia iwayomogi in CCl4 induced acute and sub-acute animal models (Lee et al., 2000; Park et al., 2000). It has been established that Artemisia capillaris and Artemisia iwayomogi have different component profiles. As shown by the HPLC analysis results (Fig. 1), scoparone is a main component in Artemisia capillaris and Artemisia iwayomogi, while scopoletin is strongly present in Artemisia iwayomogi (Seo et al., 2010). Both scoparone and scopoletin are known to have antioxidant activity (Thuong et al., 2010; Atmaca et al., 2011), and hepatoprotective effects (Kang et al., 1998; Murat Bilgin et al., 2011). Based on these publications, the two traditional hepatotherapeutic herbs seem to have hepatotherapeutic properties. However, the properties are not the same and are likely to have different effects in different hepatic conditions. These types of queries, relating to which of the two extracts exert preferable hepatotherapeutic properties, are often discussed in traditional clinics. Taken together, our study demonstrated that Artemisia iwayomogi rather than Artemisia capillarisis effective against hepatic fibrogenesis in the CCl4 -induced chronic hepatic injury model. The mechanism of action may involve melioration of the antioxidative defense system and regulation of several critical fibrogenetic genes, triggering HSCs activation. To further elucidate the
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mechanisms involved, additional comparative studies will be required to investigate other liver disease models. Conflicts of interest The authors declare that there are no conflicts of interest. The authors are responsible for the content and writing of this paper. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by theMinistry of Education, Science and Technology, Republic of Korea (No. 2010-0028119). References Anon, 2002. The Korean Herbal Pharmacopoeia, English ed. Korea Food and Drug Administration (KFDA), pp. 20–21. Atmaca, M., Bilgin, H.M., Obay, B.D., Diken, H., Kelle, M., Kale, E., 2011. The hepatoprotective effect of coumarin and coumarin derivates on carbon tetrachloride-induced hepatic injury by antioxidative activities in rats. Journal of Physiology and Biochemistry 67, 569–576. Bataller, R., Brenner, D.A., 2005. Liver fibrosis. The Journal of Clinical Investigation 115, 209–218. Beers Jr., R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. The Journal of Biological Chemistry 195, 133–140. Blom, I.E., Goldschmeding, R., Leask, A., 2002. Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biology 21, 473–482. 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. Brenner, D.A., 2009. Molecular pathogenesis of liver fibrosis. Transactions of the American Clinical and Climatological Association 120, 361–368. Choi, J.H., Kim, D.W., Yun, N., Choi, J.S., Islam, M.N., Kim, Y.S., Lee, S.M., 2011. Protective effects of hyperoside against carbon tetrachloride-induced liver damage in mice. Journal of Natural Products 74, 1055–1060. Choi, W.S., Kim, C.J., Park, B.S., Lee, S.E., Takeoka, G.R., Kim, D.G., Lanpiao, X., Kim, J.H., 2005. Inhibitory effect on proliferation of vascular smooth muscle cells and protective effect on CCl4 -induced hepatic damage of HEAI extract. Journal of Ethnopharmacology 100, 176–179. Evans, J.C., Ellman, G.L., 1959. The ionization of cysteine. Biochimica et Biophysica Acta 33, 574–576. Fowell, A.J., Iredale, J.P., 2006. Emerging therapies for liver fibrosis. Digestive Disease 24, 174–183. Friedman, S.L., 2003. Liver fibrosis – from bench to bedside. Journal of Hepatology 38 (Suppl. 1), S38–S53. 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. Gabele, E., Brenner, D.A., Rippe, R.A., 2003. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Frontiers in Bioscience 8, d69–d77. Gines, P., Cardenas, A., 2008. The management of ascites and hyponatremia in cirrhosis. Seminars in Liver Disease 28, 43–58. Graziadei, I.W., 2007. Living donor liver transplantation. Tropical Gastroenterology 28, 45–50. Ha, H.L., Shin, H.J., Feitelson, M.A., Yu, D.Y., 2010. Oxidative stress and antioxidants in hepatic pathogenesis. World Journal of Gastroenterology 16, 6035–6043. Hong, J.H., Lee, J.W., Park, J.H., Lee, I.S., 2007. Antioxidative and cytoprotective effects of Artemisia capillaris fractions. Biofactors 31, 43–53. Iredale, J.P., 2003. Cirrhosis: new research provides a basis for rational and targeted treatments. British Medical Journal 327, 143–147.
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Antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in a carbon tetrachloride-induced chronic hepatic fibrosis animal model Jing-Hua Wang a , Min-Kyung Choi a , Jang-Woo Shin a , Seock-Yeon Hwang b , Chang-Gue Son a,∗ a b
Liver and Immunology Research Center, Daejeon Oriental Hospital of Daejeon University, 22-5 Daeheung-dong, Jung-gu, Daejeon 301-704, Republic of Korea Department of Biomedical Laboratory Science, College of Applied Science and Industry, Daejeon University, 96-3 Yongun-dong, Dong-gu, Daejeon 300-716, Republic of Korea
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 2 January 2012 Accepted 7 January 2012 Available online 14 January 2012 Keywords: Carbon tetrachloride Artemisia capillaris Artemisia iwayomogi Liver fibrosis Oxidative stress
a b s t r a c t Ethnopharmacological relevance: Artemisia capillaris and Artemisia iwayomogi, both members of the Compositae family, have been indiscriminately used for various liver disorders as traditional hepatotherapeutic medicines in Korea for many years. Aim of the study: In this study, the anti-hepatofibrotic effects of Artemisia capillaris and Artemisia iwayomogi were comparatively analyzed using a carbon tetrachloride (CCl4 )-induced liver fibrosis rat model. Materials and methods: Hepatic fibrosis was induced via a 10-week course of intraperitoneal CCl4 injections (50% dissolved in olive oil, 2 mL/kg, twice per week). Water extract of Artemisia capillaris (AC) or Artemisia iwayomogi (AI) was orally administered six times per week from the 5th to the 10th week. Results: AI (50 mg/kg) significantly attenuated the CCl4 -induced excessive release of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum (p < 0.05), and hydroxyproline and malondialdehyde (MDA) contents in liver tissue (p < 0.05). Further, AI markedly ameliorated the depletion of total antioxidant capacity (TAC), glutathione (GSH), and superoxide dismutase (SOD) in liver tissue (p < 0.01). Unexpectedly, AC did not exert any effects on the above parameters. Histopathological and immunohistochemical analyses revealed that AI drastically reduced inflammation, necrosis, fatty infiltration, collagen accumulation, and activation of hepatic satellite cells in liver tissue. These changes were not observed with AC treatment. Several critical genes of fibrosis-related cytokines including transforming growth factor beta (TGF-), platelet-derived growth factor beta (PDGF), and alpha smooth muscle actin (␣-SMA) were more prominently downregulated by AI compared to AC treatment. Conclusion: Our results show that AI exerts greater hepatoprotective and anti-fibrotic effects as compared with AC via enhancing antioxidant capacity and downregulating fibrogentic cytokines. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatic fibrosis is a common chronic disease that occurs prior to the formation of liver cirrhosis and generally accounts for the
Abbreviations: AC, water extract of Artemisia capillaris; AI, water extract of Artemisia iwayomogi; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CCl4 , carbon tetrachloride; CTGF, connective tissue growth factor; DAB, diaminobenzidine; DEPPD, N,N-diethyl-para-phenylendiamine sulfate; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); ECM, extracellular matrix; GSH, glutathione; H&E, hematoxylin and eosin; H2 O2 , hydrogen peroxide; HPLC, high performance liquid chromatography; HSCs, hepatic stellate cells; KCl, potassium chloride; MDA, malondialdehyde; PDGF-, platelet-derived growth factor-beta; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; SOD, superoxide dismutase; SPF, specific pathogen free; TAC, total antioxidant capacity; TBA, thiobarbituric acid; TEP, 1,1,3,3-tetraethoxypropane; TGF-, transforming growth factor-beta; UPLC, ultra performance liquid chromatography; ␣-SMA, alpha-smooth muscle actin; -NADPH, -nicotinamide adenine dinucleotide phosphate. ∗ 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 © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.007
incessant insult to hepatocytes and kupffer cells, followed by the activation of hepatic stellate cells (HSCs) (Fowell and Iredale, 2006). Activated HSCs transform into myofibroblast-like cells and abnormally secrete excessive extracellular matrix (ECM) (Gabele et al., 2003). Continuous accumulation of ECM leads to liver fibrosis and ultimately contributes to liver cirrhosis accompanying liver dysfunction, which has a high mortality rate (Friedman, 2003; Iredale, 2003). Wide range of pathogenic factors, such as: hepatitis B virus (HBV), hepatitis C virus (HCV), hepatotoxins, metabolic disorders, alcoholisms and schistosomiasis are closely related with hepatic fibrogenesis or progression of hepatic cirrhosis (Bataller and Brenner, 2005). Although many therapies are used to treat liver cirrhosis in Western medicine, radical treatments do not exist, excluding living donor liver transplantation (Graziadei, 2007). Consequently, timely inhibition of fibrogenesis at an early stage is considered as a feasible strategy for curing chronic liver diseases (Gines and Cardenas, 2008). Herbal medicines have been frequently investigated for their hepatoprotective and antifibrotic effects in both humans (Yip et al.,
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2007) and animal models (Lin et al., 2011). Artemisia capillaries Thunb. and Artemisia iwayomogi KITAMURA are well-known herbal hepatotherapeutic drugs and have been traditionally used in Korea and/or China for a long time (Jang, 1975; Zhang, 1978). Both of Artemisia capillaris and Artemisia iwayomogi belong to the Compositae family and commonly named as “Injinho” and “Haninjin” respectively in Korea. Previous studies have reported that Artemisia capillaris and Artemisia iwayomogi, or their derived compounds, exhibit various pharmacological activities, such as antioxidant effects (Seo and Yun, 2008), cytoprotection (Hong et al., 2007), choleretic effects (Okuno et al., 1981), hepatoprotection (Choi et al., 2011), antimicrobial effects (Seo et al., 2010), and antiinflammatory effects (Jang et al., 2005; Shin et al., 2006). Several studies separately presented evidence of the hepatoprotective and antifibrotic effects of Artemisia capillaris and Artemisia iwayomogi in carbon tetrachloride (CCl4 )-induced acute hepatitis and a model for early stage fibrosis (Lee et al., 2000; Park et al., 2000; Choi et al., 2005). However, investigations on Artemisia capillaris or Artemisia iwayomogi for the treatment of long-term CCl4 -induced liver damage have not been performed. The two herbs belong to the genus Artemisia and have been indiscriminately used in the treatment of various hepatic disorders and are known by the same Korean name, “InJin” in traditional Korea clinics. However, there is a lack of scientific evidence to indicate whether Artemisia capillaris and Artemisia iwayomogi exert a coordinative antifibrotic effect. No scientific information exists as to whether Artemisia capillaris and Artemisia iwayomogi exert similar therapeutic properties. The aim of the present study was to compare the hepatotherapeutic properties of Artemisia capillaris and Artemisia iwayomogi using a fibrotic rat model that was induced by long-term CCl4 administration.
Fig. 1. HPLC fingerprinting analysis comparing Artemisia capillaris and Artemisia iwayomogi. The water extract of Artemisia capillaris and Artemisia iwayomogi and their standard compounds were subjected to HPLC. All chromatograms were produced at a wavelength of 345 nm.
Korea). Artemisia capillaris and Artemisia iwayomogi were washed twice with distilled water. After fully drying in oven at 60 ◦ C, Artemisia capillaris and Artemisia iwayomogi were weighed and boiled separately in distilled water for 30 min and unceasingly concentrated for 120 min. After filtration and lyophilization, the extracts were stored at −70 ◦ C for future use. The yield of water extract of Artemisia capillaris (AC) and Artemisia iwayomogi (AI) was 3.20% and 4.33% (w/w), respectively, and they (Voucher specimen No. AC-2010-364 and AI-2010-361) have been deposited at Liver and Immunology Research Center of Daejeon Oriental Hospital in South Korea.
2. Materials and methods 2.3. HPLC-based fingerprinting analysis 2.1. Reagents and chemicals Carbon tetrachloride was obtained from Showa Chemical Co. (Tokyo, Japan). The chemicals 6,7-dimethoxycoumarin (scoparone), 7-hydroxy-6-methoxycoumarin (scopoletin), p-dimethylaminobenzaldehyde, 1,1,3,3hydroxyproline, tetraethoxypropane (TEP), chloramines-T, potassium chloride (KCl), hydrochloric acid (HCl), n-butanol, N,N-diethyl-paraphenylendiamine sulfate (DEPPD), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), reduced glutathione (GSH), glutathione reductase and -nicotinamide adenine dinucleotide phosphate-reduced form (-NADPH), formic acid, and acetonitrile were all obtained from Sigma (MO, USA); perchloric acid was obtained from GFS Chemical Co. (OH, USA); and thiobarbituric acid (TBA) was obtained from Lancaster Co. (Lancashire, England). Hydrogen peroxide (H2 O2 ) was obtained from Junsei Chemical Co. Ltd. (Tokyo, Japan). Anti␣-smooth muscle actin mouse monoclonal antibody was obtained from Abcam (Cambridge, UK); histofine was purchased from Nichirei Biosciences (Tokyo, Japan); diaminobenzidine (DAB) was purchased from BioGenex (CA, USA); and Mayer’s hematoxylin was obtained from Wako Pure Chemical Industries (Osaka, Japan). 2.2. Extraction process of Artemisia capillaris and Artemisia iwayomogi According to the standards of the Korean Herbal Pharmacopoeia (2002), the dried Artemisia capillaris and Artemisia iwayomogi were obtained from the Jeong-Seoung Oriental Medicine Company (Daejeon, Korea). Artemisia capillaris and Artemisia iwayomogi samples were identified by Professor Seok-Rhin Lim (Daejeon University,
Fingerprints were produced using two-dimensional highperformance liquid chromatography (HPLC) profiles of two herbal extracts with their major components (scopoletin and scoparone) as standards. Briefly, after dissolution (5 mg of AC and AI in 1 mL 50% methanol, 0.1 mg of two standards in 1 mL 50% methanol) and filtration, the drugs were analyzed by HPLC. The HPLC system consisted of the Thermo Accela Ultra Performance LC (UPLC) system, Acquity UPLC Binary Solvent Manager, Acquity sample manager/column heater module, and Acquity Photo-Diode Array detector system (Waters, MA, USA). An Acquity UPLC BEH C18 (1.7 m, 2.1 mm × 100 mm) column was used and the compounds were eluted with solvents A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL/min. Solutions of 95% A and 5% B changed over a 20 min period to 0% A and 100% B, followed by a 25 min changing to 95% A and 5% B. All chromatograms were obtained using a wavelength of 345 nm (Fig. 1). 2.4. Animals and experimental design The design and performance of the experiments were approved by the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-185-1002-02), and conducted in accordance with the Policy on the Humane Care and Use of Laboratory Animals, as adopted and promulgated by the USA National Institute of Health (NIH). In total, 54 pathogen-free Sprague-Dawley male rats (6-weekold, 190–210 g) were purchased from Koatech (Gyeonggi Do, Korea), and acclimated under environmentally controlled conditions at 22 ± 2 ◦ C using a 12/12-h light/dark cycle. Rats were
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randomly divided into six groups (n = 9): normal (control), CCl4 , AC 25 (25 mg/kg), AC 50 (50 mg/kg), AI 25 (25 mg/kg), and AI 50 (50 mg/kg). CCl4 (2 mL/kg, 50% dissolved in olive oil) was intraperitoneally injected twice a week for 10 weeks into all groups, except for the normal group. AC (25 mg/kg or 50 mg/kg) or AI (25 mg/kg or 50 mg/kg) was given six times per week from the 5th to the 10th week via oral gavage. In this experiment, the dose of AC and AI was made reference to the recommended dosage in clinic of Korea. On the final experimental day, after 18 h of fasting all of the animals were weighed for sacrifice preparation. The animals were anesthetized with ethyl ether. Then, blood was collected from abdominal aorta using syringes for biochemical analyses, and the livers removed, photographed, and immediately weighed. Liver tissue was either fixed or stored at −70 ◦ C in Bouin’s solution or RNAlater solution (Ambion, TX, USA) for histopathological examination, related gene expression analysis, and biochemical determination, respectively. 2.5. Serum biochemical analysis Blood was collected from the abdominal aorta. After centrifuging at 3000 × g for 15 min, the serum was separated and stored in −70 ◦ C. The serum levels of total protein, albumin, total bilirubin, alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were determined using an Auto Chemistry Analyzer (AU400; Olympus, Tokyo, Japan). 2.6. Histopathological findings and immunohistochemical staining Liver tissues fixed in Bouin’s solution were embedded in paraffin and cut into 4-m-thick sections for histomorphological examination. After drying, liver tissue section slides were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. For immunohistochemical staining of alpha-smooth muscle actin (␣SMA), liver tissue sections were deparaffinized, hydrated, and heated in citrate buffer at 100 ◦ C for 15 min, then treated with normal serum for 30 min. The slides were then treated with an anti-␣-SMA mouse monoclonal antibody (1:100; Abcam, Cambridge, UK) overnight. After washing with PBS, Histofine (Nichirei Biosciences, Tokyo, Japan) was added using DAB as a substrate. The slides were counterstained with Mayer’s hematoxylin (Leica Microsystems, Wetzlar, Germany) and examined under an optical microscope. The percentage areas of collagen staining and positive ␣-SMA staining were analyzed by the image analysis program, ImageJ (NIH, USA). 2.7. Determination of hydroxyproline in liver tissues Hydroxyproline determination was conducted using a slight modification of a method described previously (Fujita et al., 2003). Briefly, liver tissues (200 mg) were homogenized in HCl and hydrolyzed at 110 ◦ C. After filtering the acid hydrolyzates using filter paper (Toyo Roshi Kaisha, Tokyo, Japan), the samples were completely dried in an oven. After dissolving in methanol, 50% isopropanol and chloramine-T solution were added, followed by reaction with Ehrlich’s solution at 50 ◦ C for 90 min. The absorbance was read at 558 nm. A standard curve was established using serial twofold dilutions of 1.0 mg solutions of hydroxyproline. 2.8. Determination of lipid peroxidation in liver tissues Lipid peroxidation levels were evaluated by the thiobarbituric acid reactive substances (TBARS) method, as described previously (Mihara and Uchiyama, 1978). Briefly, liver tissue (200 mg) was homogenized in ice-cold KCl (1.15%), and the homogenate was
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mixed with 1% H3 PO4 and 0.67% TBA solution. The mixture was heated for 45 min at 100 ◦ C, n-butanol was added, and the solution was then mixed and centrifuged at 3000 × g for 15 min. The absorbance of the supernatant was measured at 535 and 520 nm, and compared to a standard value (freshly prepared TEP solution). 2.9. Determination of total antioxidant capacity (TAC), total glutathione (GSH) content, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities in liver tissue Radioimmunoprecipitation assay (RIPA) buffer-based liver tissue homogenate was centrifuged at 10,000 × g for 15 min. The supernatant was transferred and stored at −70 ◦ C until required. The total antioxidant activity was determined using a previously described method (Kambayashi et al., 2009). Total GSH was determined according to the method of Ellman (Evans and Ellman, 1959). SOD activities were determined using a SOD assay kit (Dojindo Laboratories, Japan). Catalase activity in the liver tissue was determined using the method of Beers and Sizer (1952). 2.10. Real-time PCR for analyzing gene expression in liver tissue Total RNA was isolated from RNAlater-treated liver tissues using an RNeasy midi kit (Qiagen, USA). Real-time PCR was performed using IQ 5 (Bio-Rad, USA) after cDNA synthesis. The primer sequences used were as follows (forward and reverse, respectively): -actin, 5 -GTG GGG CGC CCC AGG CAC CA and 5 -CTC CTT AAT GTC ACG CAC GAT TTC; ␣-SMA, 5 -CAT CAG GAA CCT CGA GAA GC and 5 -TCG GAT ACT TCA GGG TCA GG; PDGF-, 5 -CTG CCT CTC TGC TGC TAC CT and 5 -GAT GAG CTT TCC GAC TCG AC; TGF-, 5 TGA GTG GCT GTC TTT TGA CG and 5 -TTC TCT GTG GAG CTG AAG CA; and CTGF, 5 -ATG GAG ACA TGG CGT AAA GC and 5 -TTG CAT GAC AAT GAC ACA CG. The final results are expressed as normalized fold values relative to the normal group. 2.11. Statistical analysis The results were expressed as the mean ± standard deviation (n = 9). Differences between groups were analyzed using a oneway analysis of variance (ANOVA) followed by the least significant difference post hoc test. In all analyses, p-values <0.05 were considered statistically significant. 3. Results 3.1. Changes in body and organ weights CCl4 treatment significantly reduced body and liver weights, while the spleen weight increased as compared with the normal group (p < 0.01). Treatment with AC and AI did not obviously influence changes in body, liver, and spleen as compared with the CCl4 group (Table 1). 3.2. Alterations in serum biochemical parameters CCl4 treatment markedly elevated serum AST, ALT, ALP, and bilirubin, and significantly reduced serum total protein and albumin as compared with the normal group (p < 0.05). AI treatment (50 mg/kg) significantly attenuated the CCl4 -induced increases in serum levels of AST, ALT, and ALP (p < 0.05). Nevertheless, no remarkable differences in serum AST, ALT, and ALP levels were found between the AC and CCl4 treatment. Treatment with AC and AI did induce statistically significant differences in serum bilirubin, total protein, and albumin as compared with the CCl4 group (Table 1).
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Table 1 Comparison of organ weights and serum biochemistry parameters. Groups
Normal
CCl4 only
Artemisia capillaris (mg/kg) CCl4 + 25
Body mass (g) Liver mass (g) Spleen mass (g) AST (IU/L) ALT (IU/L) ALP (IU/L) Bilirubin (g/dL) Total protein (g/dL) Albumin (g/dL) # ## *
401 9.9 0.73 144 52 132 0.2 7.0 3.6
± 19 ± 13.3 ± 0.10 ± 14 ±9 ± 31 ± 0.0 ± 0.5 ± 0.2
340 13.3 1.00 229 141 301 0.3 6.3 3.2
± ± ± ± ± ± ± ± ±
26## 1.5## 0.09## 77# 36## 99# 0.1# 0.5# 0.4#
331 12.6 1.08 227 128 274 0.3 6.6 3.4
± ± ± ± ± ± ± ± ±
21 1.0 0.16 90 69 113 0.1 0.3 0.1
Artemisia iwayomogi (mg/kg)
CCl4 + 50 323 12.6 0.96 261 159 298 0.3 6.4 3.3
± ± ± ± ± ± ± ± ±
35 1.7 0.23 157 64 73 0.1 0.6 0.3
CCl4 + 25 335 13.3 0.87 224 167 305 0.3 6.6 3.4
± ± ± ± ± ± ± ± ±
26 2.6 0.25 101 69 82 0.1 0.5 0.2
CCl4 + 50 324 12.1 0.88 153 103 191 0.3 6.7 3.3
± ± ± ± ± ± ± ± ±
20 1.1 0.17 36* 22* 18* 0.1 0.5 0.2
p < 0.05, compared with the normal. p < 0.01, compared with the normal. p < 0.05, compared with the CCl4 only (n = 9).
3.3. Histopathology and immunohistochemistry The CCl4 -treated livers were shrunken, nodular, and discolored. However, these changes were moderately attenuated by AC and AI treatment (data not shown). Histopathological observations indicated massive fatty infiltration, hepatocellular necrosis, infiltration of lymphocytes, and bridging collagen accumulation
in the CCl4 group, whereas AC and AI treatment evidently alleviated these features (Fig. 2A and B). Based on immunostaining, CCl4 treatment produced a strong positive signal for ␣-SMA around the collagen septa region in liver tissue; however, this effect was obviously weakened by AC and AI treatment (Fig. 2C). AI treatment, rather than AC treatment, produced a statistical significant difference as compared with the CCl4 group in the percentage
Fig. 2. Histopathological analysis and immunohistochemical staining. Following the final time point, all animals were sacrificed and the livers were removed and fixed in Bouin’s solution. After staining with hematoxylin and eosin (A) or Masson’s trichrome (B) and immunohistochemistry for ␣-SMA (C), pathophysiologic examinations were performed under light microscopy at 200× magnification. The percent area of the fibrotic region (E) and the ␣-SMA staining region (F) were analyzed by ImageJ software (NIH, USA). ## p < 0.01, compared with the normal; *p < 0.05, **p < 0.01, compared with CCl4 only.
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Table 2 Comparison of hydroxyproline contents and antioxidant related parameters in liver tissue. Groups
Normal
CCl4 only
Artemisia capillaris (mg/kg) CCl4 + 25
Hydroxyproline (g/g tissue) MDA (M/g tissue) TAC (U/mg tissue) Total GSH (mM/g tissue) SOD (U/mg tissue) Catalase (U/mg tissue) # ## * **
57.7 184.3 271 2.04 0.37 861
± ± ± ± ± ±
6.7 21.3 37 0.27 0.10 37
159.1 362.7 198 0.51 0.22 643
± ± ± ± ± ±
44.8## 117.4## 49## 0.13## 0.05# 97##
144.1 341.7 193 0.44 0.21 582
± ± ± ± ± ±
Artemisia iwayomogi (mg/kg)
CCl4 + 25 18.1 95.2 33 0.19 0.07 97
153.2 346.6 188 0.52 0.22 546
± ± ± ± ± ±
CCl4 + 25 33.8 63.3 42 0.30 0.06 132
119.6 334.3 239 0.90 0.29 688
± ± ± ± ± ±
CCl4 + 50 21.4* 104.4 20* 0.31* 0.06* 76
118.5 247.5 243 1.04 0.35 672
± ± ± ± ± ±
9.1* 60.9* 38* 0.38** 0.09** 97
p < 0.05, compared with the normal. p < 0.01, compared with the normal. p < 0.05, compared with the CCl4 only (n = 9). p < 0.01, compared with the CCl4 only (n = 9).
area of liver fibrosis and positive ␣-SMA staining (Fig. 2D and E). 3.4. Hydroxyproline content in liver tissue Hydroxyproline was examined to evaluate the collagen content in liver tissue. CCl4 treatment dramatically increased the concentration of hydroxyproline as compared with the normal group (p < 0.01). AI treatment (25 and 50 mg/kg) significantly reduced the elevation of hydroxyproline concentration in a dose-dependent manner as compared with the CCl4 group (p < 0.05), whereas AC treatment merely showed a slight positive trend without statistical significance (Table 2). 3.5. MDA content and TAC levels in liver tissue Lipid peroxidation was determined by assessing MDA content in liver tissue. CCl4 treatment elevated the content of MDA as compared with the normal group (p < 0.01), whereas AI treatment (50 mg/kg) significantly reduced the MDA content as compared with the CCl4 group (p < 0.05) (Table 2). There was no obvious difference in the MDA content between AC and CCl4 treatments. TAC levels were significantly lowered by CCl4 treatment as compared with the normal group (p < 0.01). AI treatment (25 and 50 mg/kg) significantly restored the TAC level in a dose-dependent manner as compared with the CCl4 group (p < 0.05) (Table 2). Nonetheless, AI treatment did not alter the severe reduction of TAC. 3.6. GSH content and antioxidant enzyme activity in liver tissue CCl4 treatment significantly decreased the GSH content and SOD and catalase activity in liver tissue as compared with the normal group (p < 0.05 and p < 0.01). Only AI treatment (25 mg/kg and 50 mg/kg) restored the depletion in GSH content and SOD activity as compared with the CCl4 group in a dose-dependent manner (p < 0.05 and p < 0.01; Table 2). Statistical significance was not observed among the AC, AI, and CCl4 treatments with respect to catalase activity. 3.7. Hepatic fibrosis-related gene expression in liver tissue The expression levels of major liver fibrosis-associated genes were analyzed using real-time PCR. CCl4 treatment noticeably upregulated gene expression of ␣-SMA, TGF-, PDGF-, and CTGF as compared with the normal group. In contrast, AC and AI treatment normalized gene expression of ␣-SMA, TGF-, PDGF-, and CTGF, with AI producing a greater effect than AC (Fig. 3A–C). CTGF gene expression was similarly regulated by both AI and AC treatment (Fig. 3D).
4. Discussion In the present study, we induced hepatic fibrosis to allow for the comparative evaluation of two medical herbs that are used indiscriminately, Artemisia capillaris and Artemisia iwayomogi (Lee et al., 2008). Fibrosis was induced in rats by 10 weeks of CCl4 injection (2 mL/kg, 50% dissolved in olive oil, i.p.). CCl4 is a typical hepatoxin and is widely used as an inducer of hepatic injury in scientific studies (Seifert et al., 1994). Toxicity is caused by CCl4 being metabolized by cytochrome P450, leading to the production of trichloromethyl radicals (CCl3 • ) and reactive oxygen species (ROS; Recknagel et al., 1989). Generally, administration of CCl4 over a 4-week period induces early fibrosis, over an 8-week period causes cirrhosis, and over a 12-week period leads to micronodular cirrhosis (Natarajan et al., 2006). The 10-week CCl4 treatment induced classical advanced fibrosis or early cirrhosis characteristics such as shrunken liver with nodular surface. Severe impairment of liver function was demonstrated by the elevated serum levels of ALT, AST, ALP, and total bilirubin. CCl4 treatment also increased the weight of the spleen, together with an obvious reduction in body and liver weight. Serum levels of transaminase (AST and ALT) and ALP are reliable indicators for assessing liver cellular lesions, as they are excessively released into the bloodstream after hepatocellular membrane disruption (Recknagel et al., 1989). Though AC and AI treatment did not substantially influence changes in parameters including body/liver weight, serum albumin, and bilirubin, AI treatment (50 mg/kg) significantly increased the serum levels of the AST, ALT, and ALP (p < 0.05; Table 1). However, AC treatment did not show the same trend in any of these parameters. Moreover, AI treatment decreased the severe hepatocyte necrosis, lymphocyte infiltration, formation of portal to portal septa, and fatty infiltration to a greater extent than AC treatment (Fig. 2A). Continuous accumulation of extracellular matrix (ECM) results in hepatofibrosis; collagen is the main component of the ECM in fibrotic tissue (Friedman, 2008). Hydroxyproline, which is a major component of collagen, was used as an indicator for evaluating the extent of liver fibrosis (Toyoki et al., 1998). AI treatment (25 and 50 mg/kg) significantly lowered collagen accumulation as evidenced by the inhibition of CCl4 -elevated hydroxyproline concentrations and the percentage area of fibrotic tissue (Table 2 and Fig. 2B and D). Although AC treatment partially inhibited the fibrotic effect, there was no statistically significant difference between the AC and CCl4 group based on quantitative analysis results. Therefore, AI treatment may be more beneficial than AC treatment with respect to antihepatofibrotic properties. It is generally recognized that HSC activation plays a critical role in the process of hepatic fibrogenesis, and ␣-SMA is a marker of activated HSCs (Kisseleva and Brenner, 2008). In the present study, immunohistochemical observations of ␣-SMA indicated that AI (25 and 50 mg/kg) and AC treatment (50 mg/kg) noticeably suppressed
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Fig. 3. Hepatic fibrosis-related gene expression in liver tissue. Total RNA was isolated from a portion of liver tissue, followed by cDNA synthesis and real-time PCR amplification. The results are expressed as normalized fold values relative to the normal group.
the activation of HSCs (Fig. 2C). However, only AI (25 and 50 mg/kg) treatment significantly inhibited the activation of HSC in a dosedependent manner based on quantification of the ␣-SMA staining area (p < 0.01, Fig. 2E). Gene suppression of ␣-SMA in liver tissue was also more prominent in the AI treatment group as compared with the AC treatment group (Fig. 3A). Evidence has been published that indicates oxidative stress influences a series of liver diseases including hepatic fibrogenesis (Ha et al., 2010). In addition, antioxidants including various herbal medicines have been reported to have a protective role in various liver diseases (Lieber et al., 2003; Wang et al., 2011). In order to examine differences between AC and AI treatments regarding perturbations of the antioxidant system, oxidative stress-related factors in liver tissue were analyzed. AI treatment significantly reduced lipid peroxidation in liver tissue, whereas AC treatment showed a slight reduction without statistical significance (Table 2). Additionally, AI treatment, but not AC treatment, restored the total antioxidant capacity in a dose-dependent manner. Interestingly, depletion of total GSH content and SOD activity were notably influenced only by AI treatment. Taken together, it was proposed that Artemisia iwayomogi shows preferable antioxidative capacity as compared with Artemisia capillaris. Catalase activity was moderately depleted by CCl4 treatment, and the AI and AC treatments produced the same effects. Gene expression profiles of three major profibrogenic cytokines (TGF-, PDGF-, and CTGF) were analyzed to explore the different mechanisms underlying hepatoprotection and antihepatofibrosis between AC and AI treatments. TGF- and PDGF- are able to potently activate HSCs following ECM synthesis and collagen formation (Borkham-Kamphorst et al., 2007; Brenner, 2009). In this study, both AI and AC treatments suppressed gene expression of TGF-, PDGF-, and CTGF, showing identical trends. AI treatment more prominently normalized the gene expression of TGF- and
PDGF- as compared with the AC treatment. However, both herbs dramatically regulated the gene expression of CTGF. CTGF is considered to be downstream of TGF-, and functions in a profibrotic role (Blom et al., 2002; Gabele et al., 2003). Further studies are needed to explore the pharmaceutical activity of both herbs, as CTGF has been shown to have potential as an antifibrotic target (Blom et al., 2002). However, the results presented here show a more prominent antihepatofibrotic effect for Artemisia iwayomogi as compared with Artemisia capillaris, and the corresponding mechanisms likely involve the regulation of these profibrogenic cytokine genes. To date, few studies have investigated the hepatoprotective effects of Artemisia capillaris and Artemisia iwayomogi in CCl4 induced acute and sub-acute animal models (Lee et al., 2000; Park et al., 2000). It has been established that Artemisia capillaris and Artemisia iwayomogi have different component profiles. As shown by the HPLC analysis results (Fig. 1), scoparone is a main component in Artemisia capillaris and Artemisia iwayomogi, while scopoletin is strongly present in Artemisia iwayomogi (Seo et al., 2010). Both scoparone and scopoletin are known to have antioxidant activity (Thuong et al., 2010; Atmaca et al., 2011), and hepatoprotective effects (Kang et al., 1998; Murat Bilgin et al., 2011). Based on these publications, the two traditional hepatotherapeutic herbs seem to have hepatotherapeutic properties. However, the properties are not the same and are likely to have different effects in different hepatic conditions. These types of queries, relating to which of the two extracts exert preferable hepatotherapeutic properties, are often discussed in traditional clinics. Taken together, our study demonstrated that Artemisia iwayomogi rather than Artemisia capillarisis effective against hepatic fibrogenesis in the CCl4 -induced chronic hepatic injury model. The mechanism of action may involve melioration of the antioxidative defense system and regulation of several critical fibrogenetic genes, triggering HSCs activation. To further elucidate the
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