DNA Methylation And MeCP2Determine Fibroblast Activation And Fibrogenesis In The Rat Liver And Its Molecular Mechanism Of Action

Hepatic fibrosis is a wound-healing process that occurs when the liver is injuredchronically. Hepatic stellate cells （HSC） are responsible for the excess production ofextracellular matrix （ECM） components. Therefore, inhibition of HSC activation and itsrelated subsequent events, such as increased production of ECM components andenhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesiscascade. To explore the cellular and molecular mechanisms of liver fibrosis, we chooseHSC as a target for the pharmacological, molecular, and other novel therapeutics forhepatic fibrosis. One focus of this review is the inhibition of DNA methylation.Platelet-derived growth factor, which are important in hepatic fibrogenesis. The maincontents are divided into three sections, as follows:1. The expression of RASAL1in rat liver fibrosisLiver fibrosis was generated by12-weeks treatment of adult male Sprague-Dawley（200-220g） rats with CCl₄（CCl₄/olive oil,1:1（vol/vol） per kg body weight byintraperitoneal injection twice weekly） as previously described. Vehicle control animalswere treated intraperitoneally with1ml of olive oil/kg body weight at same timeintervals. At24h after the final CCl₄administration, animals were culled by CO2asphyxiation and liver samples prepared. After12-Weeks, the pathological and histological Masson collagen dyeing were also detected. At five time points, theexpression of RASAL1in rat liver tissues was measured by immunohistochemicalstaining, Western blotting, RT-PCR respectively. These data show that liver fibrosismodel had bulit successfully. RASAL1expression was decreased during hepaticfibrosis.2. Experimental study of proliferation and activation of HSC-T6cells through DNAmethylation inhibitor and MeCP2To investigate the effect of cell proliferation in HSC-T6cells by RNA interferenceinhibiting expression of MeCP2gene and DNA methylation inhibitor. Hepatic stellatecells（HSC-T6cells） were divided into3groups and incubated with10ng/ml PDGF.5-AzadC at different concentrations was added into the cell culture media, and after48hof incubation, HSC cell proliferation was determinated by MTT, the mRNA levels ofα-SMA，Collagenâ…  in the supernatant of the cell culture were measured by RT-PCR.The expression of MeCP2and α-SMA in rat liver tissues was measured byimmunohistochemical staining, Western blotting. siRNA targeting MeCP2weredesigned and synthesized according to its mRNA, and their corresponding expressionvectors were constructed and transfected into HSC-T6cells with LipofectamineTM2000.Then observe the corpuscular transfection efficiency by inverted microscope. HSC cellproliferation was determinated by MTT. The mRNA levels of α-SMA, MeCP2in thesupernatant of the cell culture were measured by Quantitative Real-Time PCR andRT-PCR. HSC cell cycle was detected by flow cytometry of DNA content in each phase.The protein level of α-SMA and MeCP2were measured by western blot.MeCP2-siRNA effectively inhibited the cell proliferation, MeCP2-siRNA significantlydecreased the levels of α-SMA and MeCP2, suggesting that DNA methylation andMeCP2may be a potential target gene in the hepatic fibrosis.3. MeCP2controls the expression of RASAL1Hepatic stellate cells （HSCs） activation is an essential event during liverfibrogenesis. A major pathway is conversion of HSCs into hepatic myofibroblasts. Treatment of HSCs with DNA methylation inhibitor5-aza-2′-deoxycytidine （5-AzadC）blocked proliferation.5-AzadC also prevented loss of Ras GTPase activating-likeprotein1（RASAL1） expression that occurs during HSCs proliferation. In a search forunderlying molecular mechanisms, we hypothesized that this perpetuation offibrogenesis is caused by DNA methylation.It was demonstrated that hypermethylationof RASAL1is associated with the perpetuation of fibroblast activation and fibrogenesisin the liver. siRNA knockdown of MeCP2increased the expressions of RASAL1mRNAand protein in myofibroblasts. It was demonstrated that RASAL1occurs methylationassociated with MeCP2. These studies demonstrated that MeCP2and DNA methylationmay provide molecular mechansims for perpetuated fibroblast activation andfibrogenesis in the liver.