The Research Of Prevention-treatment And Related Mechanism In Rat Liver Fibrosis By Resveratrol

Hepatic fibrosis results from the disequilibrium between synthesis and degradation of extracellular matrix(ECM) components.Intense research has established that hepatic stellate cells(HSC) play a central role in the process.During fibrogenesis,quiescent HSC are transformed into myofibroblast-like cells contribute significantly to the process of fibrous scar formation.Recent efforts to understand liver fibrosis focus on oxidative stress.Oxidative stress has been postulated as a important mechanism involved in activation of HSC,which the reactive oxygen species play an important role in the pathogenesis of hepatic fibrosis.Depletion of oxidative-free radicals and inhibition of lipid peroxidation is expected to be a new target for inhibiting hepatic fibrosis.In China,liver fibrosis,liver cirrhosis are usually caused by the developing of chronic viral hepatitis.To aim directly at the pathological features of chronic hepatitis and liver fibrosis.This paper proposed the basis of previous work,using pig serum-induced liver fibrosis model,to elucidate the effectiveness of anti-hepatic fibrosis from pathological observations and serum biochemical indicators.This research intends to adopt the phenotype HSC-T6 cells model in vitro,use the molecular biology methods(Real Time-PCR technology) to research the mechanism of action for anti-hepatic fibrosis treatment by resveratrol from apoptosis,H202-induced lipid peroxidation HSC and so on. This study provide the experimental basis for the other anti-liver fibrosis to study the mechanism for the further exploitation and application of the resveratrol.1.Through the pig serum-induced rat model of liver fibrosis was observed in rat liver tissue morphology Resveratrol Liver tissue of the impact of HA,LN,C-â…£,Pâ…¢NP and the impact of HyP.The results showed that:(1) high doses of resveratrol and a marked improvement in the dose group,the District a significant reduction in connective tissue fibers;and model group,rat liver tissue within the lobules,between lobular inflammatory cell infiltration;Health District due to fiber desmoplastic significantly expanded. Resveratrol can significantly improve the rat liver fibrosis,prevention group and treatment group intervention resveratrol grading the degree of rat liver fibrosis model group compared with the respective retaining reduced significantly(P＜0.01).(2) resveratrol 30mg/kg,15mg/kg intraperitoneal injection can significantly reduce the serum ALT,AST, ALP level,to improve A/G ratio(P＜0.01).(3) resveratrol 30gm/kg,15mg/kg intraperitoneal injection can significantly reduce the liver fibrosis serum HA,LN,Pâ…¢NP, C-â…£levels(P＜0.01).(4) compared with the model group,hepatic fibrosis in rats during and after the formation of liver fibrosis in oral administration of resveratrol 30mg/kg, 15mg/kg,7.5mg/kg can be reduced TGF-β1,TIMP-1 expression(P＜0.01).2.Through the pig serum-induced rat liver fibrosis model,rat liver tissue homogenate, were used to detect the superoxide dismutase(SOD),malondialdehyde(MDA), hydroxyproline(Hyp) content.Pig serum-induced liver fibrosis model in rat liver lipid peroxidation indicators increased MDA content,SOD content was decreased(compared with normal group P＜0.01),and high-dose resveratrol were able to reduce the dose of MDA content,increased SOD content(compared with the model group P＜0.05,P＜0.01); model rats Hyp content in liver tissue significantly increased(compared with normal group P＜0.01),resveratrol in high-dose group and dose group Hyp content can be significantly reduced(compared with the model group P＜0.01,P＜0.05).3.Observation of rat Fas/FasL,TNF-α-apoptotic gene mRNA expression and the role of resveratrol intervention.The results showed that:Resveratrol can significantly increase rat liver tissue and in vitro HSC Fas/FasL gene mRNA expression in vitro in vivo role in the increase are better than the positive control drug.That resveratrol,through regulation of Fas/FasL system-induced apoptosis of activated HSC in order to reverse liver fibrosis.4.Observation of resveratrol on hepatic stellate cells(HSC-T6) inhibit proliferation and matrix metalloproteinase inhibitor mRNA(TIMP-1mRNA) expression to explore the resveratrol high,medium and low dose group of anti-fibrosis effect and role.In vitro HSC-T6,were randomly divided into 4 groups:control group,high-dose resveratrol group, middle dose group and small dose group.Dosing by MTT assay after each change in the proliferation of HSC-T6,and the use of RT-PCR method in each group of HSC-T6 expression of TIMP-1mRNA.The results showed that:resveratrol drug in each group on the proliferation of HSC-T6 significantly inhibited,and a more stable effect.Expression of TIMP-1mRNA control group TIMP-1mRNA/GAPDH(0.936±0.01,P＜0.01) higher than the 30mg group(0.646±0.007,P＜0.01),15mg(0.765±0.02,P＜0.01),7.5mg(0.718±0.04,P＜0.01) group.Normal control group,high-dose group,middle dose group and low-dose group,the drug group was significantly lower than the control group.Resveratrol can be confirmed by inhibiting TIMP-I of this role to play its role in hepatic fibrosis,and the more obvious role.5.The use of typeâ…£collagenase perfusion isolated rat liver cells in primary culture, with H₂O₂-induced liver cell injury in vitro,MTT assay cell survival and proliferation. Experiments show that,H₂O₂ injury led to changes in liver enzymes,such as the reflection of liver cell injury in AST,ALT were significantly increased.Liver injury in H₂O₂ cells after addition of resveratrol,with the dose of resveratrol increased cell culture supematant of the liver to reduce sexual activity,suggesting that resveratrol can reduce liver cell injury, ALT and reduction in AST release(P＜0.01).Liver injury in H₂O₂ cells after addition of resveratrol,resveratrol decreased hepatic MDA content in cells,but also to improve the content of GSH,suggesting that resveratrol can be effective against lipid peroxidation caused by H₂O₂ with a view to reducing the formation of lipid peroxides.Resveratrol tips on H₂O₂-induced liver cell injury has a direct protective effect.6.HSC with H₂O₂-induced lipid peroxidation,resveratrol(0.125～1mg·ml-1) can be significantly reduced in culture supernatants of MDA content and collagen typeâ… levels, compared with control group,model group,HSC significantly higher proliferation;with the model control group,the dose of resveratrol in varying degrees to reduce the proliferation of HSC,Dose-response relationship was significantly(P＜0.01).Model group,MDA levels were significantly increased,and SOD significantly decreased vitality.After given resveratrol,MDA levels were significantly lower,SOD significantly increased vitality,and the dose-response relationship was significantly(P＜0.01).Resveratrol tips for HSC proliferation and collagen-â… -mediated inhibition as with the role may be related to antioxidant effects.In this paper,the research uses the pig serum-induced rat liver fibrosis model and evaluate the treatment effect of resveratrol to experimental liver fibrosis from the serum indicators of liver,liver liver HyP content and morphological changes.The result shows that the resveratrol had the action of prevention and treatment for the pig serum-induced rat experimental liver fibrosis.Its mechanism of action is correlated with collagen degradation; inhibit hepatic stellate cell activation and to promote the activation of hepatic stellate cell, which caused by cytokine TGF-βrivalry,TIMP-1 expession refraining increasedThe extracorporeal experiments use the HSC-T6 cells as a model,exerting MTT to assay the action of resveratrol on the HSC-T6 proliferation.It further confirmed that resveratrol can inhibit HSC proliferation and activation of the role.In addition,this research approach apoptosis induced action and the way apoptosis for HSC cell.The result indicated that resveratrol may be mediated apoptosis in HSC play a role in the treatment of liver fibrosis through the Fas/FasL system.In extracorporeal experiments,use HSC-T6 cells as a research platform to explore the Effects of Resveratrol on H₂O₂-induced lipid peroxidation in HSC,and confirm that resveratrol may inhibit lipid peroxidation in order to inhibit HSC activation,proliferation and extracellular matrix(ECM) generation,plays the role of anti-hepatic fibrosis.