The Effects Of Ethyl Pyruvate On The Liver Fibrosis Induced By Bile Duct Ligation In Rats

Liver fibrosis is a common result of liver to repeated injury, which characterized by the deposition of extracellular matrix(ECM). Oxidative stress(OS) has been confirmed a key step in onset and progression of liver fibrosis. Ethyl pyruvate(EP) has been widely accepted as a protective agent with its anti-oxidant properties and anti-inflammatory. However, the effects of EP on liver fibrosis induced by common bile duct ligation(BDL) is not clear. Objective1. To make a liver fibrosis model by BDL, and proved by the serum enzyme and pathological histology detection;2. To observe the effect of EP on the changes of liver function and fibrosis indexes.3. To investigate the influence of Nrf2、SOD2 and α-SMA with EP compound administration, and the influence on Nrf2 signaling pathway-related genes(SOD2, NQO1 and GSH-Px).4. To observe the changes of HMGB1, IL-1β, TNF-α and HSP27 with EP compound administration. Explore the most possible protective mechanisms of EP contribute to anti-fibrosis in liver. Materials and Methods70 Rats were randomly divided into three groups: Sham group(n=6), BDL group(n=37) and EP group(n=37), which with and without EP administration. Based on the progression of liver fibrosis induced by bile duct ligation, BDL and EP groups were respectively subgrouping for 2, 4, and 6 weeks groups. The rats that underwent a laparotomy but without common bile duct ligation(BDL) in Sham group. For BDL group, rats were underwent BDL to develop liver fibrosis, and rats were underwent BDL and were treated by intraperitoneal injection of EP(40 mg/kg per day) in EP group. Over the course of 2 weeks, 4 weeks and 6 weeks of BDL, the specimens were respectively collected and detected as follows:1.Liver function was evaluated by measuring serum ALT and AST.2.The liver pathology morphology and fibrosis degrees ware judged by hematoxylin and eosin staining and Masson’s trichromic staining.3.Immunohistochemistry staining was used to measure the expression of α-SMA, Nrf2 and HMGB1 in rats.α-SMA4.ELISA was used to detect Nrf2 and Western-blotting was used to detect SOD2 in rats.5.Nrf2 signaling pathway-related antioxidant enzymes, inflammatory cytokines and HSP27 were determined by the real-time fluorescence quantitative PCR.6.With the bioinformatics methods to explore whether there is a link between Nrf2 and HSP27 genes.7.Data analyses were performed with SPSS 17.0 software. Quantitative data were determined by one-way analysis of variance(ANOVA) and expressed as mean ± standard error of the mean(S.E.M), P<0.05 was considered as statistical significance. Results1.Liver fibrosis model was established successfully by the BDL method.With BDL adminstration, the serum ALT and AST levels substantially increased at the 2 weeks, 4 weeks and 6 weeks.Whlie EP treatment group, the serum ALT and AST levels decreased, especially at 2 weeks and 6 weeks(p<0.05).2.The histopathological examination showed that more and more collagen fibers was observed in a time-dependent manner from 2 to 6 weeks.3.ELISA and Western blot detection showed that the Nrf2 and SOD2 levels were significantly increased in liver tissue of BDL group compared to the Sham group, and higher levels presented in EP group than the BDL group.4.RT-PCR detection showed that compared to the BDL group, the expression of Nrf2, SOD2, NQO1 and GSH-Px was significantly increased in liver tissue of EP group. While the expression of HMGB1, IL-1β, TNF-α and HSP27 were significantly decreased in EP group.5.Bioinformatics methods suggested that Nrf2 was indirectly related to HSP27 via the transcription factor Jun and zinc finger protein 36(b ZFP36L1) in the rat. Conclusion1.EP administration diminished fibrosis index at 2w and 4w, which indicated that EP could inhibit hepatic fibrosis in the cholestasis liver fibrosis.2.EP administration could upregulate the Nrf2 signaling pathway related genes SOD2, NQO1 and GSH-Px expressions at 2w and 4w, suggested that EP improved the activity of endogenous antioxidant system in the development of cholestasis hepatic fibrosis;3.EP administration downregulated the gene expression of HMGB1, IL-1β and TNF-α, suggesting that EP could inhibit the inflammatory response in the cholestasis liver fibrosis.4.EP downregulated the gene expression of HSP27 in the cholestasis liver fibrosis, which may contribute to the protective effects of EP on liver fibrosis.