The Research Of The Change And Significance Of Histone Modification In Hepatic Fibrosis Pathogenetic Mechanism

Objective: Histone modification is an important content in the epigenetic regulation,while,the abnormal histone modifications associated with the occurrence and development of many diseases.It is unclear that histone modification palyed a role in changes of liver fibrosis. Here,we investigated that the changes of histone modifications in CCl4 induced rat liver fibrosis and observed the changes of histone modifications on activated primary hepatic stellate cells(HSCs)of rat. To explore the role of histone modification in the mechanism of liver fibrosis. Methods:(1) Twenty male Wistar rats were randomly divided into liver fibrosis group and normal control group(180g~200g).The liver fibrosis model was established by subcutaneous injection of 40% CCl4(mixed with vegetable oil)(0.3ml/100g)and interval three days, and the normal control group was injected vegetable oils. All rats were killed at the end of 8 weeks.The liver index was analyzed, the liver fibrosis degree and the morphological change of live were detected by HE and Masson staining. The levels of histone 3 lysine 4 dimethyation(H3K4me2), histone 3 lysine 9 dimethyation(H3K9me2), histone 3 lysine 9 acetylation(ac H3K9) and histone 4 lysine 12 acetylation(ac H4K12) were detected by western-blot.(2) The HSCs of rat was isolated by in situ perfusion of collagenase combined with density gradient centrifugation, cultured in vitro and identified by immunofluorescence staining. The morphological features of the cells were observed under inverted microscope. The changes of desmin and α-smooth muscle actin(α-SMA) during the activation of HSCs were detected by western blot. The levels of H3K4me2, H3K9me2, ac H3K9 and ac H4K12 in quieted HSCs and activated HSCs were determined by western blot. Results:(1)The liver index of rats in liver fibrosis group was higher than that in control group(5.98±0.69/3.81±0.17,P<0.01). Hepatic pathological examination showed that the liver of liver fibrosis group had much more mass inflammatory cell infiltration and collagen deposition. Part of the region appeared pseudolobuli formation,according to the liver fibrosis grading catalogue of Baoen Wang. These results proved that the liver fibrosis model was successful. Expression of extracellular matrix was significantly higher than the normal group by Masson staining.(2)The levels of ac H4K12,ac H3K9,H3K4me2 and H3K9me2 of rats liver between liver fibrosis and control group were detected by western blot.Compared to the control group,the level of ac H4K12 was decreased(1.11±0.19/0.55±0.27,P<0.01),ac H3K9 and H3K9me2 was increased(0.379±0.01/2±0.20,P<0.01;0.277±0.03/1.6±0.23,P<0.01),and H3K4me2 had no significant changes(P>0.05).(3)We obtained rat primary HSCs by collagenase circulation perfusion combing with density gradient centrifugation.Desmin positive cells were more than 90% by immunofluorescence.Freshly isolated rat HSCs presenting strong refractivity, HSCs partly adhered to the dish of culturing after 6h and the parapodium stretched out after culturing 24 h.After culturing 10 days to 15 days,HSCs lack both lipid droplets and long processes,and display proliferative and fibrogenic myofibroblast-like phenotype.(4)Western blot showed that the levels of α-SMA expression were gradually increased and reached maximum at 15 d. While the expression of desmin was opposite. According to the results of cell morphology and western blot, the cells cultured for 24 h and 15 d were quieted and activated HSCs respectively.(5) Compared with quieted HSCs, there were higher H3K4me2 and lower H3K9me2, ac H3K9, ac H4K12 modification levels in activated HSCs(P<0.01). Conclusion:In this study we found that the levels of ac H4K12 in the liver of rats of liver fibrosis decreased,but the levels of ac H3K9 and H3K9me2 increased.These results implied that ac H4K12,ac H3K9 and H3K9me2 may play essential roles in pathogenesis of liver fibrosis and these histone modifications may regulate gene expression associated with ECM metabolism.Furthermore,during HSCs activation process,we found that the levels of ac H4K12 decreased and H3K4me2 increased,which may contribute to the transdifferentiation of HSCs and the ECM accumulation.