Preliminary Study On The Anti-hepatic Fibrosis Pharmacodynamics And Mechanisms Of Trionyx Sinensis

Objective:Based on the preliminary study on the pharmacodynamics of turtle-shell peptide HGRFG,to study the effects of different administration modes on the therapeutic effect of CC4-induced hepatic fibrosis in rats.To investigate whether turtle-shell peptide HGRFG can inhibit the activation of primary HSC by inhibiting the signal transduction of TGF-beta/Smad signaling pathway in primary hepatic stellate cell（HSC）of rats,thereby inhibiting the secretion of extracellular matrix.Quality.Methods:1.Liver fibrosis model of SD rats was established by intraperitoneal injection of carbon tetrachloride olive oil solution（CCl₄:Oil=4:6）.The model rats were randomly divided into normal group,model group,IFN-gamma group,HGRFG（IM）group,HGRFG（IV）group and Union group.Ten rats in each group were given corresponding administration for 8 weeks.Serum biochemical indicators were measured to evaluate liver function;rat body weight and liver weight were weighed to calculate liver index;liver tissue superoxide dismutase（SOD）,malondialdehyde（MDA）and hydroxyproline（Hpy）levels were measured to evaluate the degree of liver injury;liver tissue HE staining was performed to observe the pathological changes of rat liver tissue.2.The activation of primary HSC in rats was induced by TGF-β1.Whether turtle chitin HGRFG could inhibit the activation of primary HSC and inhibit its secretion of extracellular matrix by inhibiting the signal transduction in TGF-beta/Smad signaling pathway was studied.The effects of turtle-shell peptide HGRFG on the expression of alpha-SMA,TGFBR1,CD105,COLⅠ,COLIII,alpha-SMA,the influences of TβR1,COLⅠ,COLIII,Smad2/3 and p-Smad2/3proteins in primary HSC cells of rats were also investigated.Results:1.In the CCl₄-induced rat liver fibrosis model,rats lost weight,had dark hair color,increased liver index,abnormal structure of hepatic lobules in pathological sections,appeared fibroid lesions in different degrees,and formed pseudolobules.Serum levels of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,hyaluronic acid（LN）,procollagen III（PC III）,fibronectin（HA）were significantly increased in hepatic fibrosis model rats,while SOD activity in liver tissue was significantly decreased,contents of MDA and Hyp significant increased.After tail vein injection of turtle-shell peptide HGRFG,the serum standard substances of model rats decreased significantly,and the contents of MDA and Hyp decreased significantly.2.The contents of alpha-SMA,COLⅠ,COLIII m RNA were increased and TGFBR1,CD105 m RNA were significantly decreased after the intervention of turtle-shell peptide HGRFG in primary HSC of rats activated by TGF beta 1.There was no significant change in extracellular collagen type I,collagen type III,TGFBR1,CD105,COLIII,SMAD2,SMAD3 and COLI in low dose group,but no significant change in intracellular TGFBR1,CD105 and COLIII.In the high dose group,extracellular type I collagen content decreased significantly,type III collagen content did not change significantly,intracellular alpha-SMA,TGFBR1 m RNA content decreased significantly,while CD105,SMAD2,SMAD3,COL I,COLIII m RNA content did not change significantly.The expression of Smad2/3 protein and p-Smad2/3 protein decreased significantly in low-dose group and high-dose group,while the expression of TBe RI protein decreased significantly in low-dose group,and the expression of alpha-SMA and TBe RI protein decreased significantly in high-dose group.Conclusion:Combined with the results of HGRFG study,the effects of different administration methods on the efficacy of HGRFG were studied.The tail vein administration of HGRFG could significantly alleviate liver fibrosis in rats,improve liver function and have better anti-hepatic fibrosis effect.The results of pathway study confirmed that inhibitor SB431542 inhibited primary HSC activation in rats by inhibiting TGF-beta type I receptor and blocking signal transduction in TGF-beta/Smad signaling pathway.HGRFG has a weak inhibitory effect on primary HSC activation in rats,probably by regulating the TGF-beta and non-classical Smad pathway,thus exhibiting a weak inhibitory activity.2m M HGRFG can inhibit primary HSC activation in rats,significantly reduce the content of collagen type I in extracellular matrix,and down regulation the expression of Smad2/3 and p-Smad2/3proteins.