Rosiglitazone prevents nutritional fibrosis and steatohepatitis in mice

Scandinavian Journal of Gastroenterology, 2009; 44: 358
365 Rosiglitazone prevents nutritional fibrosis and steatohepatitis in mice YUE-MIN NAN1, NA FU1, WEN-JUAN WU1, BAO-LI LIANG1, RONG-QI WANG1,SU-XIAN ZHAO1, JING-MIN ZHAO2 & JUN YU3,4 1Department of Traditional and Western Medical Hepatology, the Third Hospital of Hebei Medical University, Shijiazhuang,China, 2Department of Pathology, Beijing 302 Hospital, Beijing, China, 3Institute of Digestive Disease and Li Ka ShingInstitute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong,and 4Department of Gastroenterology, the First Hospital of Hebei Medical University, Shijiazhuang, China AbstractObjective. Currently, no agent has been confirmed as preventing the fibrosing progression of non-alcoholic steatohepatitis(NASH). In this study, rosiglitazone was used in the clinical treatment of insulin resistance in patients with type 2 diabetesmellitus. However, its protective effect on non-alchoholic fibrosing steatohepatitis is not clear. The study aimed to elucidatethe effect and the mechanism of rosiglitazone in inhibiting nutrition-related fibrosis in mice. Methods. C57BL6/J mice werefed a high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce hepatic fibrosis, and rosiglitazone was given inthe treated group. The effect of rosiglitazone was assessed by comparing the severity of hepatic fibrosis in liver sections, theactivation of hepatic stellate cells (HSCs) and the expression of TGF-b1 and connective tissue growth factor (CTGF).
Results. At week 8, MCD-diet-induced fibrosing NASH models showed increased serum ALT and AST levels, severehepatic steatosis, and infiltration of inflammation and fibrosis which, associated with down-regulated PPARg mRNA andprotein expression, up-regulated a-SMA protein expression and enhanced TGF-b1, CTGF mRNA and protein expression.
Rosiglitazone significantly lowered serum ALT and AST and it reduced MCD-induced fibrosis by repressing levels of For personal use only.
a-SMA protein expression and pro-fibrosis factors TGF-b1 and CTGF. It also restored expression of PPARg.
Conclusions. The present study provides clear morphological and molecular biological evidence of the protective roleof rosiglitazone in ameliorating nutritional fibrosing steatohepatitis. Rosiglitazone may ameliorate hepatic fibrosis byactivating PPARg, which can inhibit HSC activation and suppress TGF-b1 and CTGF expression.
Key Words: Fibrosing steatohepatitis, hepatic stellate cells, non-alcoholic steatohepatitis, peroxisome proliferator activatedreceptor gamma, rosiglitazone liferation, loss of vitamin A storing capability,expression of a-smooth muscle actin (a-SMA) and The pathological progression of non-alcoholic fatty Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 overproduction of ECM components. Among profi- liver disease (NAFLD) includes simple hepatic brogenic cytokines, transforming growth factor beta steatosis, steatohepatitis, hepatic fibrosis and cirrho-sis. Up to now, the pathogenesis of non-alcoholic 1 (TGF-b1) and its downstream effective factor, steatohepatitis with hepatic fibrosis remains unclear.
connective tissue growth factor (CTGF), are the Hepatic stellate cells (HSCs) are the primary source master factors for promoting HSC activation, ECM of excessive production of extracellular matrix synthesis and secretion of other profibrogenic factors (ECM), and activation of HSCs is the critical event in hepatic fibrosis [1]. HSCs belong to the non- Peroxisome-proliferator-activated receptor gamma parenchymal cells in Disse space. Many profibro- (PPARg) is a soluble transcription factor. Recent studies have demonstrated that PPARg expres- into activated HSCs which transdifferentiate to a sion decreases markedly with the activation of myofibroblast phenotype characterized by cell pro- HSCs, which indicates that PPARg is prominent in Correspondence: Yue-min Nan, Traditional and Western Medical, Department of Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang,China. Tel: 86 311 88602151. Fax: 86 311 87023626. E-mail: [email protected] (Received 9 August 2008; accepted 1 October 2008) ISSN 0036-5521 print/ISSN 1502-7708 online # 2009 Informa UK Ltd.
DOI: 10.1080/00365520802530861 Rosiglitazone and fibrosing steatohepatitis maintaining the quiescent phenotype of HSCs [5].
hepatic steatosis, necroinflammation and fibrosis in This implies that PPARg may be a potential target in accordance with the 2006 Guidelines for Diagnosis preventing hepatic fibrogenesis and the selective and Treatment of Non-Alcoholic Fatty Liver Dis- PPARg agonist may be used as a therapeutic agent eases [7] and the 2001 programme of prevention and for liver fibrosis. Rosiglitazone, an agonist of PPARg, cure for viral hepatitis [8] issued by the Chinese may protect the pathogenesis and progression of Society of Hepatology of the Chinese Medical fibrotic disease by activating PPARg. The aim of this study was to investigate the effect of rosiglitazoneon activation of HSCs and expression of PPARg,TGF-b1 and CTGF in mice with hepatic fibrosis induced by the MCD diet [6].
Immunostaining for CTGF and a-SMA was per-formed in paraffin-embedded liver sections using thespecific antibodies (Santa Cruz, Calif., USA) and an Material and methods avidin-biotin complex (ABC) immunoperoxidase Animals and treatments method. Briefly, endogenous peroxidase activitywas blocked by treating sections with 3% hydrogen Eight-week-old male C57BL6/J mice with body peroxide. After blocking with 10% non-immunized weights between 20 and 25 g were obtained from goat serum, the primary specific antibodies for the Experimental Animal Center of the Chinese CTGF (dilution 1:200) and for a-SMA (dilution Academy of Medical Sciences and were housed in a 1:200) were applied. Primary antibodies were 228C-controlled room under a 12-h light
dark omitted and non-immunized goat serum was used cycle. They had free access to water and were for negative controls. After extensive rinsing, the allowed to adapt to their food and environment for biotinylated secondary antibody and ABC complex 1 week before the start of the experiment. The /HRP were applied. Peroxidase activity was visua- C57BL6/J mice were divided into 3 groups (10 lized by applying diaminobenzidine to the sections, /group) and fed a control diet (ICN, Aurora, Oh., which were then counter-stained with haematoxylin.
USA), a methionine and choline deficient (MCD) Quantitative analysis of CTGF and a-SMA-stained diet (ICN) or an MCD diet supplemented with liver sections was performed by morphometric rosiglitazone (50 mg kg
1 d
1). During the experi- ments, their body weights and rate of diet con-sumption were recorded. After 8 weeks, all of the For personal use only.
animals were killed and blood collected from the Determination of hepatic PPARg, TGF-b1 and CTGF femoral artery. Livers were weighed and fixed in 10% formalin for histological analysis or snap-frozen in lipid nitrogen followed by storage at Total RNA was isolated with TRIzol reagent
808C in a freezer until required. All the protocols (Saibaisheng Biological Technique Institute, Beijing, and procedures were carried out following the China). For RT-PCR, 5 mg total RNA was reverse- guidelines of the Hebei committee for care and transcribed with M-MLV in accordance with the use of laboratory animals, and were approved by manufacturer's instructions. cDNAs were amplified the Animal Experimentation Ethics Committee of using specific sets of primers for PPARg (sense, the Hebei Medical University.
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 gaggcctg-3?), TGF-b1 (sense, 5?-caacgccatctatga-gaaaacc-3?; Measurement of serum ALT and AST c-3?) and CTGF (sense, 5?-caaagcagctgcaaatacca; Serum alanine aminotransferase (ALT) and aspar- antisense, 5?-ggccaaatgtgtcttccagt). The PCR proce- tate aminotransferase (AST) levels were determined dure for PPARg consisted of 30 cycles of denatura- using spectrophotometric assay kits (Sigma, St.
tion at 948C for 45 s, annealing at 578C for 45 s, Louis, Mo., USA) with an automatic biochemical extension at 728C for 5 min, with initial denatura- analyser (Olympus UA2700, Japan).
tion of sample cDNAs at 948C for 5 min before PCRand an additional extension period of 10 minafter the last cycle. The different procedure for Histological examination TGF-b1 and CTGF is annealing at 608C for 45 s.
The fat on the frozen liver sections (5 mm thick) was In parallel, PCR reactions were performed with detected with Sudan IV stain, a conventional primers coding for the housekeeping gene b-actin method for fatty tissue in histology. Haematoxylin and eosin-stained paraffin-embedded liver tissues 5?-gctgatccacatctgctggaa-3?) to control for equal isolated from mice (5 mm thick) were graded for amounts of template cDNAs. A quantity of 6 ml of Y.-M. Nan et al.
PCR products was analysed in an 8% sodium Effect of rosiglitazone on liver inflammatory injury phoresis (SDS-PAGE) with a 100-bp DNA marker.
Densitometric analysis of PCR products was per- As indicated in Table 1, mice fed the MCD diet formed by computer software and standardized by showed significantly higher serum ALT and AST levels compared with control mice, indicating hepa-tic injury ( pB0.05), and a significant reduction wasnoted after rosiglitazone treatment ( pB0.05). How- Western blot analysis of hepatic proteins of PPARg and ever, serum triglyceride and cholesterol concentra- tions were not significantly altered.
Hepatic tissues (10% w/vol) were homogenized inlysis buffer containing 20 mM Tris-HCl (pH 7.4), Effect of rosiglitazone on liver histology 1% Triton X-100, 140 mM NaCl, 1 mM phenyl-methylsulfonyl fluoride (PMSF) and aprotinin, leu- Compared to the controls (Figure 1A and 2A), the peptin, soybean trypsin inhibitors (1 mg/ml each).
liver section from mice fed the MCD diet exhibited Total protein was extracted and concentration was disordered lobule structure, macrosteatosis in Zone measured by the Bradford method (DC protein 3, spot or focal hepatocyte necrosis and inflamma- assay; Bio-Rad, Hercules, Calif., USA) as previously tory infiltration (Figure 1B) and portal and perisi- described [9]. A quantity of 100 mg protein was nusoidal fibrosis (Figure 2B). Treatment with separated by 10% sodium dodecyl sulphate-poly- rosiglitazone markedly improved hepatic steatosis, acrylamide gel electrophoresis and then transferred inflammatory infiltration (Figure 1C) and fibrosis onto equilibrated polyvinylidene difluoride mem- (Figure 2C).
brane (Amersham Biosciences, Buckinghamshire,UK) by electroblotting. Membranes were blocked Effect of rosiglitazone on body weight and liver weight with 5% skim milk in 1 TBST for 1 h, then incubated with specific antibodies against PPARg,TGF-b1 or b-actin (Santa Cruz Biotechnology, Similar to what has been reported previously [10], Santa Cruz, Calif., USA) overnight at 48C. After administration of the MCD diet caused body weight incubation with secondary antibody, proteins were loss and liver weight gain (Table 2). Administering For personal use only.
detected by enhanced chemiluminescence (ECL; rosiglitazone significantly increased body weight by up to 7% ( pB0.05) under the MCD diet andreduced liver weight ( pB0.05).
Statistical analysis Effect of rosiglitazone treatment on CTGF expression The Statistical Package for Social Science software(v. 13.0; SPSS Inc., Chicago, Ill., USA) was used for As shown in Figure 3A, CTGF, a cysteine-rich, statistical analysis. The data are presented as means9SD. Statistical analysis was carried out by dramatically increased mostly in fibrous tissue (Fig- one-way analysis of variance (ANOVA) and the ure 3A2) compared to CTGF in the control mice Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 Student
Newman
Keuls test (for evaluating differ- (Figure 3A1), but was significantly reduced by ences between groups). A p-value of B0.05 was rosiglitazone treatment (Figure 3A3) as determined considered statistically significant.
by a computerized morphometric analysis (Figure 4).
Table 1. The effect of MCD diet with or without rosiglitazone on serum characteristics Triglyceride (mmol/L) Cholesterol (mmol/L) NOTE. Data are mean9SD ( n10/group).
*PB0.05 relative to mice fed the control diet.
#PB0.05 relative to mice fed the MCD diet.

Rosiglitazone and fibrosing steatohepatitis Figure 1. The effect of rosiglitazone on the liver histology in MCD-diet-induced fibrosing steatohepatitis. Haematoxylin and eosin-stainedliver sections from mice fed: (A) the control diet, (B) the MCD diet, (C) the MCD diet supplemented with rosiglitazone (50 mg kg1 d1)(black arrows indicate hepatic steatosis). Experimental duration is 8 weeks. Slides are representative of 10 animals per group.
Figure 2. The effect of rosiglitazone on hepatic fibrosis in mice fed the MCD diet. Masson-trichrome staining for collagen fibres in liversections from mice fed: (A) the control diet, (B) the MCD diet, (C) the MCD diet supplemented with rosiglitazone (50 mg kg1 d1)(black arrows indicate hepatic fibrosis). Experimental duration is 8 weeks. Slides are representative of 10 animals per group.
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Effect of rosiglitazone treatment on HSC activation with that in mice fed the control diet. Rosiglitazonerestored the expression of PPARg (Figure 5). The To provide further evidence of the anti-fibrotic mRNA and protein expression of TGFb1 was property of rosiglitazone in MCD feeding mice, we increased in MCD-feeding mice and was signifi- determine the effects of rosiglitazone on hepatic cantly blunted by rosiglitazone treatment (Figure 5).
a-SMA protein expression, a well-established marker Consistent with the protein expression determined of HSC activation during liver fibrogenesis. As by immunohistochemistry (Figure 3A), treatment shown in Figure 3B and 4, and as compared to with rosiglitazone significantly down-regulated the MCD mice, rosiglitazone significantly suppressed mRNA expression of CTGF induced by MCD a-SMA expression.
feeding (Figure 5).
Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 Analysis of transcripts and proteins of genes associated with HSC activation and liver fibrosis A high fat, methionine-choline-deficient diet in Both mRNA and protein expression of PPARg were mice results in hepatic steatosis, inflammation dramatically decreased in mice fed MCD compared and fibrosis. This is distinct from the high fat Table 2. Weight changes in mice fed the control or MCD diet with or without Rosiglitazone Number of animals Body weight (% change) Relative liver weight (% of body weight) NOTE. Data are mean9SD.
*PB0.05 relative to mice fed the control diet.
#PB0.05 relative to mice fed the MCD diet.

Y.-M. Nan et al.
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Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 Figure 3. Effect of rosiglitazone on protein expression of CTGF and a-SMA. (A) Immunostaining for CTGF protein in (A1) mice fed thecontrol diet, (A2) mice fed the MCD diet and (A3) mice fed the MCD diet supplemented with rosiglitazone 50 mg kg1 d1. (B)Immunostaining for a-SMA protein in (B1) mice fed the control diet, (B2) mice fed the MCD diet and (B3) mice fed the MCD dietsupplemented with rosiglitazone 50 mg kg1 d1. There were inductions in CTGF and a-SMA staining in mice fed MCD for 8 weeks, andadministering rosiglitazone blunted the induction of both CTGF and a-SMA.
diet-induced liver injury, but resembles the hepatic steatohepatitis with liver fibrosis, resulting in char- pathohistological alteration in human non-alcoholic acteristic pathology of steatosis, mixed inflammatory steatohepatitis. We used this representative experi- cell infiltration hepatocyte necrosis, and fibrosis mental model of fibrosing steatohepatitis to investi- in the pericellular, perisinusoidal and portal area.
gate the mechanism behind rosiglitazone preventing Concomitant with increased levels of serum ALT and the progress of hepatic fibrosis.
AST, administering rosiglitazone in MCD-fed mice Following the MCD diet for 8 weeks, mice rapidly resulted in attenuation of the fibrosing steatohepati- and consistently developed a severe pattern of tis, as evidenced by decreased ALT and AST levels

Rosiglitazone and fibrosing steatohepatitis CCl4 and bile duct ligation (BDL) in murine. Ourresults were also supported by the evidence thatrosiglitazone protected induced steatohepatitis [12] and patients withNASH with reduced inflammation and liver injury[13,14]. Restoration of the body weight of mice fedthe MCD diet treated with rosiglitazone, with areduction of liver weight and hepatic fat contentand serum ALT level, suggests a shipment of fat fromthe liver to adipose tissue. The roles of adipose tissueand its PPARg on hepatic fat accumulation have beendemonstrated previously in loss-of-function studies,where it has been shown that genetically engineered Figure 4. Effect of rosiglitazone on quantitative protein expres-sion of CTGF and a-SMA. The expressions of CTGF and mice with no adipose tissue or adipose-tissue-specific a-SMA were estimated by average area density (areas of positive PPARg knockout automatically acquired fatty liver cells/total areas 100%) in each scope (200-fold) to observe [15
17]. On the other hand, activation of PPARg by 10 scopes at every section (2G). Data are shown as mean9SD rosiglitazone promotes lipogenesis in adipose tissue.
( n10/group), *PB0.05 compared with control mice, #PB0.05 Besides enhancing fat redistribution, the ameliorat- rosiglitazone-treated MCD-fed compared with the mice fed theMCD diet.
ing effect of rosiglitazone was also mediated throughmodulating adipokines. Other than inflammatory and diminished histologic evidence of fibrosis.
cytokines in liver induced by lipotoxicity of the In keeping with our finding, Galli et al. [11] accumulated fat, adiponectin derived from adipose tissue certainly plays an anti-inflammatory role in the liver fibrosis induced by dimethyl nitroxide (DMN), progression of steatohepatitis [18].
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Scand J Gastroenterol Downloaded from informahealthcare.com by Nyu Medical Center on 06/25/10 Figure 5. Effects of rosiglitazone on hepatic PPARg, TGF-b1 and CTGF expression in MCD feeding mice. (A) PPARg, TGF-b1 andCTGF mRNA expression was analysed by RT-PCR. The level of PPARg was low and TGF-b1 and CTGF were high in MCD mice.
However, rosiglitazone could restore PPARg and suppress TGF-b1 and CTGF expression. The expression of mRNA was estimated by ratioagainst b-actin. (B) Protein expression of PPARg and TGF-b1 was determined by Western blot. The changes in protein were consistent withthose of the mRNA. Western blots were scanned by densitometry and the data presented as relative intensity units against b-actin. Each barrepresents the mean9SD of four to seven mice. *PB0.05 compared with control mice, #PB0.05, ##PB0.01, ###PB0.001 rosiglitazone-treated MCD-fed compared with the mice fed the MCD diet Y.-M. Nan et al.
HSC activation is key in the early phase of liver mechanisms of its action could be involved in fibrosis and activated HSC is accompanied by a high activating PPARg to reverse activated HSCs into a expression of a-SMA protein. Many kinds of fibro- quiescent phenotype and reduce the expression of genic cytokines are involved in HSC activation in TGF-b1 and CTGF.
keeping HSCs quiescent [5]. We found that reducedexpression of PPARg and enhanced expression of a-SMA in fibrosing steatohepatitis in mice fed MCDand rosiglitazone could dramatically up-regulate This work was supported financially by the Wang PPARg and down-regulate a-SMA. Thus, rosiglita- zone might delay or inhibit the progression of liver 20070021 and by a Research Grants Council fibrosis through agitating PPARg, which might Competitive Earmarked Research Grant (CUHK be the major mechanism of anti-fibrogenesis in non-alcoholic fatty liver disease.
The important role of TGF-b1 in liver fibrosis has Declaration of interest: The authors report no been well documented [19
22]. It has been pro- conflicts of interest. The authors alone are respon- posed that TGF-b1
secreted by fibroblasts, Kupf- sible for the content and writing of the paper.
fer cells and T-lymphocytes [23]
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