Role Of SIRT1 In Mouse With Liver Fibrosis And Gene Expression Profiling And Proteomics Study

Objective: 1. To establish a mouse model with liver-specific knockout of SIRT1(SIRT1-LKO). 2. To observe liver fibrosis induced by carbon tetrachloride(CCL4) in SIRT1-LKO mice and wild type(WT) littermates. 3. To screen differential molecules associated with SIRT1 and explore the possible roles of them in the pathologic process of liver fibrosis systematically. 4. To observe the protective effects of resveratrol(RES) in mice with liver fibrosis. Methods: 1. SIRT1-LKO mouse model was established by using a Cre-lox P approach. Mice genotypes were identified by agarose gel electrophoresis. Real-time quantitative polymerase chain reaction(Q-PCR) was used to validate SIRT1 gene expression in mice livers. SIRT1 protein expression was tested by Western Blot and immunohistochemistry. 2. Liver fibrosis was induced by intraperitoneal injection of CCL4 solution repetitively. Liver function was measured by serum biochemistry. Sirius red staining was used to observe collagen fibers of liver. The expression of α-smooth muscle actin(α-SMA) was tested by immunohistochemistry and Western Blot, which showed the activation of hepatic stellate cells(HSCs). 3. The c DNA microarray technology and liquid chromatography tandem mass spectrometry(LC-MS/MS) were performed to screen SIRT1 related genes and proteins respectively in SIRT1-LKO and WT mice with CCL4 or olive oil treatment. Screening principles were formulated and differential molecules were selected in the fibrosis models. Differential molecules were analysed by DAVID Bioinformatics Resources 6.7 system. The candidate molecules were screened and selected with proteomics databases. Q-PCR was used to validate the expression of candidate genes. 4. CCL4 treatment was performed by intraperitoneal injection repetitively to induce mice liver fibrosis, and resveratrol solution was injected in the same way. Liver function was measured by serum biochemistry. Hematoxylin-eosin(HE) staining was used to observe the morphology of liver tissues. Masson’s trichromic staining was performed to observe collagen fibers of liver. Results: 1. Through propagation, hybridization and identification of mice, SIRT1-LKO mouse model was established and WT littermate was obtained successfully. 2. After 8 weeks of CCL4 administration, the levels of serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the CCL4 groups rose significantly compared with the olive oil groups(P<0.01), with increased collagen fibers of liver. Expression of α-SMA protein elevated. More importantly, the injury and fibrosis of liver in SIRT1-LKO mice were more severe compared with their WT littermates with CCL4 treatment(P<0.05), and these results were discovered firstly. 3. Through screening and bioinformatics analysis of differential genes and proteins(Foldchange > 2), a group of key molecules were discovered, which may be related with SIRT1 in mouse liver fibrosis. Eight candidate genes(AFP, OIP5, CCNB2, FHL2, GPC3, POC1 A, CCNB1 and PHGDH) were validated by Q-PCR. The results of Q-PCR were consistent with the c DNA microarray basically. 4. After 8 weeks, the levels of serum ALT and AST in CCL4 groups rose significantly compared with the olive oil groups(P<0.01). Hepatocytes was injured and hepatic collagen fibers increased. However, the liver injury and fibrosis were significantly reduced in mice injected with CCL4 and resveratrol solution together(P<0.05). Conclusions: 1. The SIRT1-LKO enhanced liver injury and promoted liver fibrosis induced by CCL4 in mice. 2. SIRT1 may cooperate with the above-mentioned molecules and be involved in the occurrence and development of liver fibrosis. 3. Resveratrol had protective effect on liver injury and fibrosis in mice induced by CCL4 significantly.