| Nonalcoholic fatty liver disease(NAFLD) is a chronic liver disease with a wide spectrum of liver damage ranging from simple steatosis , steatohepatitis(NASH) to advanced fibrosis and cirrhosis. NASH has been implicated as a major risk factor for cryptogenic cirrhosis. NAFLD is a clinical syndrome with lesion of steatosis and fat storage in hepatic lobules but without alcohol abuse. Obesity, type 2(non-insulin-dependent) diabetes mellitus, and hyperlipidemia which comprise the metabolic syndrome, are coexisting conditions frequently associated with NAFLD. It is becoming a medical and social problem with common concern. Many of the risk factors for NAFLD are well defined, but the underlying pathogenesis is not well understood. At present, therapy is aimed at correcting the risk factors, but there are no proven therapies at this point.NASH is characterized by sinusoidal fibrosis, which is mediated by activated hepatic stellate cells(Ac-HSC). It has been reported that angiotensinⅡ(ANGⅡ) plays an important role in the pathogenesis of organ fibrosis, and some studies have demonstrated that AT1-R blockers(ARB) significantly attenuated experimental liver fibrosis along with suppression of the Ac-HSC and hepatic TGF-βexpression. Recently, telmisartan, an angiotensinⅡreceptor blocker(ARB) is discovered that, beyond just blockade of the type 1 angiotensinⅡreceptor, it is also a partial agonist of PPARγand has the capacity to improve glucose and lipid metabolism and retard weight gain while promoting formation of small efficient adipocytes. In this study, we explore the antifibrotic effect of telmisartan on rats with NASH in order to offer a new way for the treatment of NASH.Objectives: To establish the model of nonalcoholic steatohepatitis ( NASH ) and investigate the effects of telmisartan on body weight,levels of serum transaminases and lipids,insulin resistance and histological presentation including fibrosis in rats with NASH, and to explore the possible mechanism of telmisartan in the treatment of NASH. Methods: Thirty-five male Sprague Dawley rats were randomized into three groups after seven days of normal feeding: Normal control group(NC group,n=10)were fed with common diet; High fat-fed control group(FC group,n=15)were fed with high fat diet(88%common food plus 2%cholesterol and 10% lard); High fat-fed with telmisartan group(FT group,n=10) were fed with high fat diet. After 12 weeks, five rats were selected from FC group for hematoxylin-eosin staining to determine if model was successed. Then FT group were given telmisartan(5mg.kg-1.d-1) by intragastric administration and continued high-fat diet. NC group and FC group was given the same normal sodium by intragastric administration. The diets and water were given freely. By the end of 16th week, all rats were killed. Body weight and liver wet weight were weighted from electronic blance, calculating liver index: liver wet weight/body weight%. Liver samples (size: 1cm×1cm×0.5cm) were from right lobe of liver and fixed in 4%paraform. Slices were made and stained by Masson and HE. The extents of fatty degeneration and inflammatory cell infiltration of slices of liver tissue were detected by light microscope. Levels of serum TG,TC,HDL-C,ALT and AST were detected. Fasting blood glucose(FBG), fasting plasma insulin(FINS) and insulin index(HOMA-IR) were also assayed. The expression ofα-SMA was observed by immunohistochemical method. Levels of TIMP-1, MMP-2, TGF-β1mRNA expressions in liver tissues were detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR).Results:1 Body weight,liver weight and liver index: The NASH rat model was successfully established with high fat diet for sixteen weeks. The mean body weight, liver weight and liver index of rats in FC group(609.56±37.29, 25.4±4.71 and 4.29±0.49%, respectively) were increased significantly compared with NC group(516.29±37.77, 13.03±2.74 and 2.72±0.57%, respectively)(p﹤0.01). Compared with FC group, those in FT group(558.15±46.12, 18.19±2.73 and 3.79±0.57%,respectively) were decreased markedly(p﹤0.01, 0.01 and 0.05, respectively). 2 Histological analysis: Steatosis and inflammation were not seen in NC group, while severe lipid degeneration were found in FC group(p﹤0.01), especially around central vein. Lobular inflammation, periportal inflammation and degeneration, focal necrosis were found in FC group. There has significant difference between NC group and FC group(p﹤0.01). It was shown that steatosis and inflammation significant improved in FT group compared with FC group(p﹤0.01). No fibrosis was found at 12th week in FC group, while fibrosis appeared at 16th weeks around central vein and perisinusoid. Compared with FC group,there was no fibrosis in FT group(p﹤0.01).3 Serum biochemical index: (1) Serum ALT and AST (U/L) of FC group(114.00±19.75 and 265.33±52.16, respectively) were increased significantly compared with NC group(48.20±10.99 and 153.00±45.06) (p﹤0.01). Compared with FC group, serum ALT level of FT group(78.80±15.64) were decreased markedly (p﹤0.01). Serum AST level of FT group(211.83±65.51) have no significant difference compared with FC group(p>0.05). (2) Serum TC and TG level(mmol/L) of FC group(2.65±0.77 and 0.53±0.27,respectively) were increased significantly compared with NC group (1.44±0.24 and 0.29±0.14, respectively) (p﹤0.01, 0.05, respectively). The two values of FT group (2.22±0.55 and 0.39±0.18, respectively) have no statistical difference(p>0.05) compared with FC group;Serum HDL-C level (mmol/L) of FC group (0.30±0.09) was significantly decreased compared with that of NC group(0.46±0.05) (p﹤ 0.01).Serum HDL-C level (mmol/L) of FT group(0.31±0.11) has no significant difference compared with that of FC group(p>0.05).4 Insulin resistance and fasting blood glucose: Fasting blood glucose(mmol/L),fasting plasma insulin(μU/ml) and HOMA-IR of FC group(6.58±0.86,20.73±0.91 and 6.23±0.19, respectively) were increased markedly compared with NC group (4.58±1.00, 10.48±1.46 and 2.13±0.29, respectively) (p﹤0.01). Compared with FC group, the values of FT group (5.38±0.88,15.02±1.22 and 3.59±0.29, respectively) were significantly decreased, have statistical difference(p﹤0.01).5 Immuneohistochemistry: Liver samples from NC group exhibited only fewα-SMA-positive cells in smooth muscle cells of blood vessel wall. Liver samples from FC group not only exhibitedα-SMA-positive cells in smooth muscle cells of blood vessel wall, but also exhibited in hepatic tissue, with statistical difference compared with NC group (p﹤0.01). Compared with FC group, the expression ofα-SMA-positive cells in FT group were decreased remarkably (p﹤0.01).6 Expression of TIMP-1,MMP-2 and TGF-β1mRNA: The expression of TIMP-1, MMP-2 and TGF-β1mRNA in FC group (0.93±0.08,0.88±0.09 and 0.36±0.04, respectively) were increased significantly compared with NC group (0.32±0.03, 0.47±0.05 and 0.12±0.01, respectively) (p﹤0.01). Compared with FC group, these values in FT group (0.77±0.07, 0.69±0.07 and 0.26±0.03, respectively)were decreased remarkably(p﹤ 0.01), and also had significant difference with NC group(p﹤0.01).Conclusion: Model of NASH rats has been successfully established by fat-rich diet after 16 weeks. Telmisartan can improve glucose and lipid metabolism and insulin resistance while attenuating weight gain, alleviating steatosis and inflammation, and has antifibrotic effect. Its therapeutic molecular mechanisms may be related to activing PPAR-γreceptor and inhibiting angiotensinⅡtype-1 receptor, presenting the protective and therapeutic effect for NASH.
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