The Molecular Mechanism Of Recovery Liver Fibrosis Induced By CCl₄ And The Regulatory Effect Of TRAIL

Liver fibrosis is a liver repair response to chronic injury, also is the common pathological changes of persistent liver injury. liver cells sustained, repeated necrosis or inflammation, fibrosis accompanied by a large number of fiber degradation in relative or absolute lack of ECM deposition in the liver and eventually evolved into a large number of liver cirrhosis and even liver failure. Recent studies suggest that liver fibrosis is a reversible disease, remove damage factors or application anti fibrosis drug, with the recovery time is extended, liver fibrosis can partially or completely reverse recovery, showing deposition of collagen degradation, the liver structure and liver function recovery. The hepatic stellate cells (hepatic stellate cell, HSC) apoptosis play a key role In hepatic fibrosis , therefore, induce apoptosis of hepatic stellate cells promote the liver fibrosis as an important way to recovery. Research shows that after liver fibrosis formation and the recovery phase, Kupffer cells (Kupffer, KC) play the role of a double-edged sword. In liver fibrosis progression period can promote HSC activation, promote the formation of liver fibrosis; In recovery, KC and the activation of HSC expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and other apoptosis stimulating factor so as to promote HSC apoptosis, promoting ECM degradation, thereby inhibiting the formation of hepatic fibrosis.However, various cytokines and cell signaling pathways mechanism is unclear. In order to further reveal the TRAIL during the recovery process of liver fibrosis and the role of MAPK signaling pathways. This study was to choose CCl₄ induction rat liver fibrosis spontaneous reversals build recovery model, to study the mechanisms of hepatic fibrosis reverse with TRAIL and MAPK signaling pathway, discusses the relationship between signaling pathways involved in this process a possible signaling pathways and TRAIL within the influence of this pathway. The study includes the following three parts:1.Preparation of animal models about liver fibrosis and reverse phasesCCl₄ subcutaneous injection animal models is the classical method of liver fibrosis,stop after injection can be spontaneous reversals. This lab reflect liver damage and index of liver fibrosis ALT, AST, Hyp LN, Pâ…¢NP, CCl₄ subcutaneously in increased significantly, after 12 weeks 2,4,6,8 weekend in spontaneous recovery, and at the same time, gradually reduce the pathological and histological Masson collagen dyeing also showed remove made after rats mould factors, hepatic pathology performance and liver fibrosis stage gradually improved, suggesting that rat liver fibrosis and that recovery model had built successfully.2.Study the dynamic expression of TRAIL and MAPK signaling pathway proteins and the correlations of Hyp in the whole animal models of RecoveryExperimental results show that, p-ERK1 / 2 in the model group was the lowest, with the normal group there were significant differences (** p <0.01), restored the expression of 4 weeks, the highest difference compared with the model group was significantly (## p <0.01) , Then gradually decreased to normal. p-JNK1 / 2 was the lowest in the model group (** p <0.01), recovery groups increased (# p <0.05). p-P38 expression of the highest in the model group (** p <0.01), recovery gradually decreased (# p <0.05). TRAIL expression in the model group the lowest (** p <0.01), the highest expression in Recovery2 weeks (## p <0.01), recovery for 4 weeks, 6 weeks, 6 weeks gradually reduce (## p <0.01). Correlation studies show that TRAIL negatively correlated with Hyp and difference was statistically significant (r = - 34, p = 0.019); P - ERK/ERK negatively correlated with Hyp difference was statistically significant (r = - 0.46, p = 0.003); P - JNK/JNK negatively correlated with Hyp car garment be statistically significant (r = - 0.53, p = 0.0004); P - P38 / P38 positively correlated with Hyp and difference was statistically significant (r = 0.81, p = 0.0001).3.The regulation of TRAIL on the recovery of HSC proliferation in vitroThis experiment used Western blot to detect proteins expression of MAPK signaling pathways in HSC from different groups and flow cytometric analysis method to detect apoptosis of experimental HSC cell cycle found that, (1) primary isolated HSC resume four weeks after TRAIL (400μg/L), PD98059 (30μM) pretreatment after 24h; The results showed that compared with model group TRAIL (400μg/L) pretreatment significantly reduced the recovery HSC in p-ERK1/2 expression, suggesting that recovery of liver fibrosis TRAIL reduce the p-ERK1/2 expression, inhibition of HSC proliferation and promote apoptosis of Recovery HSC . Around the separation of primary HSC resume by TRAIL (400μg/L), SP600125 (100ng/ml) 24h after Pretreatment; results compared with model group TRAIL (400μg/L) pretreatment significantly reduced the recovery of p-JNK expression, suggesting that TRAIL in recovery of liver fibrosis reducie the expression of p-JNK, inhibition of HSC proliferation and promote apoptosis of Recovery HSC . Around the separation of primary HSC resume by TRAIL (400μg/L), SB203580 (50μM) pretreatment after 24h; results compared with model group TRAIL (400μg/L) pretreatment significantly reduced the recovery of p-P38 expression in HSC, suggesting that TRAIL in recovery of liver fibrosis reduce the expression of p-P38, suppressed HSC proliferation, and promote recovery HSC apoptosis. (2) AV/PI double staining showed that, compared with the normal group, there is reversal of spontaneous apoptosis of HSC, 10.1% or so, TRAIL (100, 200, 400μg/L) pretreatment group could promote the apoptosis of HSC (p<0.05), percentage of apoptosis was 19.7%, 25.1% and 33.1%. Prompt recovery of liver fibrosis TRAIL can promote the apoptosis of HSC. (3) PI single-stained cell cycle results, TRAIL (100, 200, 400μg/L) pretreated cells, after 24h, compared with the recovery HSC, TRAIL pretreatment at different concentrations, G0/G1 cells was significantly increased (p<0.01), S phase cells decreased significantly (P <0.01), cells were arrested in G1/S phase.In summary, this study demonstrated the relevance of the proteins expression of MAPK signaling pathway and TRAIL and the reverse hepatic fibrosis in rat; demonstrated that TRAIL can promote cell apoptosis in Recovery HSC, and affect the cell cycle; Preliminary illuminates that TRAIL may regulates the reversal of liver fibrosis by MAPK signaling pathways; applied for the TRAIL in vitro liver fibrosis treatment provides experimental basis; as evaluation, selection of liver fibrosis drug provides a new concept.