Protective Effects Of Curcumin On Liver Fibrosis Through Epigenetic Mechanisms

BackgroundCurcumin is the main active ingredient of turmeric and has been shown to have anti-hepatic fibrosis (HF) effect. However, the exact mechanism is not fully elucidated. The anit-hepatic fibrosis effect of curcumin has been demonstrated to be associated with antioxidant, anti-inflammatory and scavenging free radicals. Existing studies show that the methylation status and expression of the fibrosis genes are obviously different in HF tissues as compare with the normal liver tissues. Therefore, we hypothesized that curcumin might play an anti-fibritic role through epigenetic mechanisms. This study is expected to find new mechanisms of curcumin on HF.Methods1. Experimental animals:Eighteen SPF C57BL/6 mice were randomly divided into three groups:control group (FC), model group (FM) and curcumin treatment group (FJ).2. Induction of liver fibrosis and curcumin treatment:Carbon tetrachloride (CC14) was intraperitoneally injected into the mice of FM and FJ group by the dose of 0.6 ml/kg twice a week for six weeks. Mice in the FC group were injected with an equal volume of olive oil. Meanwhile, each mouse of the FJ group was treated with curcumin by gavage at the dose of 200 mg/kg once daily for six weeks. Mice in the FC and FM group were given an equal volume of PBS.3. Specimen collection:All mice were sacrificed 48 h after the last injection of CC14. Blood samples and liver tissues were collected. The liver enzymes were detected in the serum of the mice. HE staining was performed in the liver tissues. DNA was extracted from the liver tissues for the detection of methylation. Expression of a-SMA, collagen and DNMT was determined by real time-PCR and western blot.4. Screening and identification of methylation profiles:Roche-NimbleGen mouse MeDIP-chip technology was used to screen the different methylated genes in liver tissues of mice. Then, GO signal classification and KEGG pathway analysis was performed. Eight representative genes (Camk4, Fgfr3, FZD10, Gpx4, Hoxd3, Nfkb2, Prkcb and Tcfeb) were validated by MeDIP-qPCR.5. HSC-T6 cells were treated with curcumin and were divided into three groups: control group, TGF-β1-induced group, TGF-β1-treated group. Cell proliferation, cell cycle, the expression of a-SMA, collagen and DNMT, the methylation of Camk4, FZD10, Gpx4 and Hoxd3 genes was detected.6. SPSS 16.0 software was used for statistical analysis. Data were analyzed using Student t test or nonparametric tests when comparing numerical variables between two groups and one-way analysis of variance for comparisons between more than two groups. P<0.05 was considered to be statistically significant.Results1. Serum liver enzymes levels:The serum alanine aminotransferase (ALT) levels of FC, FM and FJ were:(23.0±1.55) U/ml, (2485.8±362.85) U/ml and (386.93± 33.02) U/ml, respectively. The serum aspartate aminotransferase (AST) levels were: (100.8±9.98) U/ml, (1593.8±303.2) U/ml and (322.2±47.56) U/ml, respectively. Serum ALT and AST levels were significantly higher in the FM group than FC group. Curcumin treatment could significantly reduce serum ALT and AST levels in mice.2. Hepatic histological findings:The liver tissues in the mice of FC group showed a normal structure without any abnormal morphological alternations in lobular architecture and hepatocytes. The liver tissues of mice in the FM group showed significantly pericellular and periportal fibrosis, distortion of liver architecture and massive inflammatory cell infiltration. However, oral administration of curcumin significantly attenuated alterations in liver histology.3. α-SMA, Collal and DNMT1 expression in liver tissues of mice:As determined by real time PCR and western blot, the expression of a-SMA, Collal and DNMT1 was significantly increased in the liver tissues of mice in the FM group as compared with FC group. However, their expression was siginifcantly decreased in the FJ group compared to that of FM group.4. Methylation chip analysis:According to hypomethylation of FC group and hypermethylation of FM group,934 differentially methylated genes were screened. According hypomethylation of FJ group and hypermethylation of FM group,550 differentially methylated genes were screened. A total of 75 overlapping portions of both genes were analyzed by GO (gene ontology) and KEGG signaling pathway. The main processes involved in cell regulation, biological processes, cellular metabolic processes and the development process, the most common signaling pathways involved were:tumor-related pathway, NK-kappa B signal, MAPK signal, Wnt signaling pathway, NK cell-mediated cytotoxicity. In accordance to Peak Score> 2, Peak M Value> 0.7, promoter type HCP or ICP, genotype relates to the repair, cell cycle, apoptosis, proliferation and other conditions, eight representative genes (Camk4, Fgfr3, FZD10, Gpx4, Hoxd3, Nfkb2, Prkcb, Tcfeb) were further validatd by MeDIP-qPCR among the 75 differentially expressed genes. The results are consistent with the chip results (P<0.05). These results confirmed the reliability of the chip results.5. In vitro study:Curcumin could siginifcantly inhibit the proliferation of HSC-T6 cells induced by TGF-β1, reduce the percentatage of cell with the G2/M phase, reduce the α-SMA, collagen and DNMT expression, and reverse the hypermethylation status of Camk4, FZD10, Gpx4 and Hoxd3 genes.Conclusions1. Curcumin has antifibrotic effects on hepatic fibrosis.2. Curcumin may alleviate liver fibrosis through regulation of DNA methylation.