Experimental Study Of Xiong-shao Decoction In Preventing And Treating Liver Fibrosis And The Related TRAIL-FADD/caspase8 Cell Apoptosis Pathway

This experiment was composed of three parts:“establishment of rat model of hepatic fibrosis induced by comprehensive factors of CCL₄","Xiong-Shao decoction in prevention and treatment of hepatic fibrosis and the related TRAIL-FADD/caspase8 apoptosis pathway"and"effect of xiong-shao decoction on hepatocyte apoptosis in Rats"ObjectiveThis experiment analyzes the prevention and treatment of hepatic fibrosis from the perspective of warming the spleen and kidney,which establishes the progressive and recovery phase of hepatic fibrosis in rats by establishing CCL₄comprehensive factors,observes the effect of Xiong-Shao decoction on hepatic fibrosis,and reveals the relationship between xiong-shao decoction,liverfibrosis,apoptosispathwayofhepatocytesand TRAIL-FADD/caspase8.Then to explore the Xiong-Shao decoction’s prevention and treatment of hepatic fibrosis in apoptotic pathways,to provide new ideas and methods for the study of prevention and treatment of liver fibrosis by traditional Chinese medicine.MethodsExperiment 1 30 healthy male adult SD rats were subcutaneously injected with a mixture of CCL₄ and peanut oil（The ratio of the two is 4:6）,which two times a week,injections on Tuesday and Friday,respectively,with an injection volume of 3ml/kg/times.（Note:The ratio of the first subcutaneous injection of CCl₄ and peanut oil is 3:1,the injection dose is5ml/kg）,and the high-fat and low-protein food with cornmeal+0.5%cholesterol+20%lard is given for 2 weeks.High fat and low protein food which contains corn meal+0.5%cholesterol feed for 4 weeks,drinks only30%alcohol.On the 0th,4th,6th,and 8th weekends of the experiment,6serum samples and pathological specimens were randomly selected and tested for pathological changes in the liver,hydroxyproline（HyP）content,serum aspartate transferase（ALT,alanine aminotransferase（AST）,hyaluronic acid（HA）,laminin（LN）,type IV-C collagen（CIV）,and type III procollagen（PCIII）and other indicators.Which ensure of the advanced and convalescent stage of rat liver fibrosis model induced by CCL₄（The recovery period is 4weeks after modeling）.Experiment 2 64 male SD rats,randomly divided into six groups.Normal group 6 weeks:8 subcutaneous injections of sterile peanut oil,injection time with CCl₄ injection time,free consumption of standard feed and water,and the prevention group Xiong-Shao decoction,reciprocally fed purified water;Model group 6 weeks:twelve rats were fed with purified water with xiong-shao decoction in the prevention group.The model was made according to the comprehensive factors of CCL₄ in experiment 1 for 6 weeks,and the control group was fed with xiong-shao decoction in the same way;Prevention group 6 weeks:12 on the basis of model 6 daily Xiong-Shao decoction gavage（administered dose 7 times the adult clinical daily dose Xiong-Shao decoction 8.050g/kg）;normal group 10 weeks:normal feeding on the six weeks-basic and then normal feeding for 4 weeks,and the treatment group Xiong-Shao decoction equivalently fed with purified water;restore group 10 weeks:twelve,model six based on,Normal feeding naturally resumed for 4 weeks,and the treatment group Xiong-Shao decoction was reciprocally fed with purified water;Treatmentgroup 10 weeks:12,on the basis of model group 6 weeks,normal feeding and Xiong-Shao decoction for 4weeks at the same time（injection with prevention 6）.At the end of the 6th week,the specimens of normal serum,liver and liver of Normal group 6weeks,model group 6 weeks,prevention group 6 weeks of rats were collected.At the end of the tenth,specimens of Normal group 10 weeks,restore group10 weeks,prevent group 10 weeks of serum and liver were prevented.Ultraviolet-lactate dehydrogenase was used.UV-malate dehydrogenase method was used to detect the changes of ALT and AST in serum of rats in each group.The levels of HA,LN,CIV and PCIII in serum of rats in each group were detected by radioimmunoassay.HyP in liver tissues of rats in each group was detected by sample hydrolysis method.HE and Masson staining were used to observe the pathological changes of liver tissue in each group.Immunohistochemistry was used to detect the liver tissueα-smooth muscle actin（α-SMA）and 5-lipoxygenase（5-LO）in each group.The expression of5）-LO,α-SMA,matrix metalloproteinase inhibitor-1（TIMP1）,matrix metalloproteinase-13（MMP13）and tumor necrosis factor,related apoptosis-inducing ligand（TRAIL）,Fas-coupled death domain protein（FADD）,proteolytic enzyme-8（Caspase-8）,B-lymphoma-2（BCL-2）,Cyt-c（cytochrome C）mRNA and/or protein expression were detected by PCR and Western blot.Experiment 3 Rat hepatocytes（BRL）were cultured and passaged and randomly divided into six groups.They were co-cultured with the serum of the second part of the normal group 6 weeks,model group 6 weeks,prevention group 6 weeks,normal group 10 weeks,recovery group 10 weeks,and treatment group 10 weeks,using flow cytometry.Apoptosis of rat hepatocytes in each group was detected by flow cytometry.ResultsExperiment 1 The changes of serum ALT,AST,HA,LN,PCIII,CIV and liver tissue Hyp index at 0,4,6 and 8 weeks of the experiment:Compared with 0 weeks,the indexes at 4th,6th,and 8th weeks were significantly higher.Statistical significance（P<0.01）;With the prolongation of modelling time,the indicators at 4,6,and 8 weeks increased gradually,compared with 6 weeks,there was a statistically significant difference（P<0.01）.The indicators also showed statistically significant increases（P<0.05）.The results of HE staining of rats at 0,4,6,and 8 weeks showed extensive microvesicular steatosis at 4weeks,connective tissue hyperplasia in portal area,lymphocyte infiltration,and fibrinous segmentation in liver tissue;more fibroblasts appeared in 6weeks.Cells,fibrous connective tissue hyperplasia,fibrous septa increased;typical pseudo-lobules appeared 8 weeks later.Masson staining of liver tissue and grading of image proplus liver Fibrosis in Rats at 0,4,6 and 8 weeks:0weeks,6 were all 0 degree;4 weeks:1 degree in 4 rats,2 degree in 2 rats;6weeks:3 rats each for 2 degree and 3 degree;8 weeks,1rat for 3 degree,5 rats for 4 degree.Experiment 2 Serum ALT,AST,HA,LN,PCIII,CIV and Hyp index in liver tissue were changed in each group.Compared with normal group 6weeks:Model group 6 weeks indicators all increased,the difference was statistically significant（P<0.01）;Compared with model group 6 weeks:prevention group 6 weeks indicators were decreased,the difference was statistically significant（P<0.01）;Compared with normal group 10 weeks:The recovery group 10 weeks indicators all increased,the differences were statistically significant（P<0.01）;Compared with recovery group 10 weeks:the treatment group 10 weeks indicators were decreased,the differences are all statistics Significance（P<0.01）.Results of HE and masson staining in liver tissue of rats in each group.Normal group 6 weeks:No liver fibrosis;model group 6 weeks:Hepatocyte necrosis,hepatic steatosis,fibroblasts,fibrous connective tissue,increased collagen fibers,hepatic lobular destruction,etc.Liver fibrosis;prevention group 6 Weeks:Compared with the model group 6 weeks,liver fibrosis performance significantly reduced;recovery group 10 weeks:no significant improvement compared with recovery group 10 weeks;treatment group 10weeks:Compared with recovery group 10 weeks,liver fibrosis was markedly alleviated.Rat liver tissue 5-LO,α-SMA,TIMP-1,MMP13（single Western Blot）,TRAIL,FADD,Caspase-8,BCL-2（single-hand PCR）,Cyt-c（single Western Blot）Western Blot and PCR results:Compared with the normal group,the index expression of model group 6 weeks and recovery group 10 weeks increased significantly（P<0.01）;compared with model group 6 weeks,prevention group The expression of 6 weeks’indicators was weakened and the difference was statistically significant（P<0.01）.Compared with recovery group 10 weeks,the expression of treatment group 10 weeks was decreased and the difference was statistically significant.（P<0.05）.Experiment 3 Compared with normal group 6 weeks,the difference in BRL apoptosis of model group 6 weeks was statistically significant（P<0.01）.Compared with model group 6 weeks,the difference in BRL apoptosis of prevention group 6 weeks was statistically significant（P<0.01）.<0.01),There was a statistically significant difference in the BRL apoptosis reduction of recovery group 10 weeks（P<0.01）.Compared with normal group 10 weeks,the difference in BRL apoptosis of recovery group 10 weeks was statistically significant（P<0.01）;Compared with recovery group 10 weeks,treatment group 10 weeks BRL apoptosis decreased significantly.（P<0.01）.Conclusion1 Xiong-Shao decoction can promote the recovery of hepatic fibrosis induced by CCL₄ and slow down the progression of hepatic fibrosis induced by CCL₄:Xiong-Shao decoction has the effect of preventing and treating hepatic fibrosis.2 The mechanism of prevention and treatment of hepatic fibrosis by xiong-shao decoction is as follows:Xiong-Shao decoction can prevent liver fibrosis by protecting HC;inhibiting HSC activation;increasing MMP13activity;inhibiting TIMP1 activity;inhibiting 5-LO activity;inhibiting TRAIL-FADD/caspase8 apoptosis pathway.3 Xiong-Shao decoction drug-containing serum can inhibit the apoptosis of rat hepatocytes;Xiong-Shao decoction may play a role in the prevention and treatment of hepatic fibrosis by inhibiting the cells apoptosis pathway of TRAIL-FADD/caspase8.