Preliminary Study On The Effect And Mechanism Of Periplaneta Americana Extract PA-B On Experimental Hepatic Fibrosis In Rats

Objective:The aim of this study was to establish a rat model of liver fibrosis induced by carbon tetrachloride（CCl₄）,to study the effect of anti fibrosis extract of Periplaneta americana（PA-B）on liver fibrosis,and to explore its mechanism of action,so as to provide reference for the research on anti-liver fibrosis drugs of Periplaneta americana.Objective:Methods:1.The rat liver fibrosis model induced by CCl₄and intervene with PA-B.HE and Masson staining were used to observe the pathological changes of liver fibrosis in rats.microplate method to determine liver function indexes ALT and AST in rat serum;Four liver fibrosis indexes,HA,LN PC-ⅢCol-Ⅳ,in serum were determined by ELISA.2.Immunohistochemistry was used to detect the expression ofα-SMA and Col-I protein in liver tissue.The mRNA expression levels ofα-SMA,Col-I,Col-III,TGF-β1,TβR I,TβR II,Smad2,Smad3 and Smad7 were determined by RT-qPCR.The protein expressions of TβR I,TβR II,Smad2,Smad3 and Smad7 were detected by Western blot to investigate the effect of PA-B on collagen expression and TGF-β1/Smad pathway in experimental hepatic fibrosis rats.3.Immunohistochemistry was used to detect the protein expression of Bcl-2 and Caspase9 in liver tissues,Western blot was used to detect the protein expression of Bax,and RT-qPCR was used to detect the mRNA expression of Bcl-2,Bax and Caspase9 in liver tissues.To evaluate the effect of PA-B on the Bcl-2/Bax pathway.Results:1.Effect of PA-B on experimental hepatic fibrosis in ratsHe and Masson results showed that the normal liver cell structure in the model group was destroyed,and a large number of fibrous connective tissue hyperplasia could be seen around the manifold,and the fibrous connective tissue extended and connected into obvious pseudoblobules in different portal areas.The hepatic cell cords were arranged disorderly,and there were obvious inflammatory cell infiltration.After PA-B treatment,compared with the model group,the proliferation of fibrous connective tissue in colcoline group and PA-B dose groups was improved to varying degrees,the liver cell structure tended to be normal,and the infiltration of inflammatory cells was significantly reduced.Compared with the model group,the content of AST in the serum of PA-B middle and high dose groups was significantly decreased（P<0.05）;the content of ALT in the serum of rats in each dose group of PA-B was significantly reduced（P<0.01）;The expression levels of HA,LN,PC-Ⅲ,Col-Ⅳwere also significantly decreased（P<0.01）.2.Effects of PA-B on collagen expression and TGF-β1/Smad signaling pathway in experimental hepatic fibrosis ratsThe results of immunohistochemistry showed thatα-SMA and Col-I were expressed in strips in the central vein area and portal area of liver tissue.Compared with the normal group,the positive expression ofα-SMA and Col-I in the liver tissue of the model group increased significantly.Compared with the model group,the positive expression ofα-SMA and Col-I in liver tissue was significantly reduced.RT-qPCR results showed that the mRNA expression levels ofα-SMA,Col-I and Col-III in the liver tissues of PA-B group were decreased compared with the model group（P<0.05 or P<0.01）;PA-B can decrease the mRNA expressions of TGF-β1,TβR I,TβR II,Smad2and Smad3 in liver fibrosis tissues（P<0.01）,and the mRNA expression of Smad7 was increased（P<0.05 or P<0.01）.Western Blot results showed that compared with the model group,the TβR I,TβR I and Smad3 protein expression level in the PA-B medium and high dose groups was decreased（P<0.01）,and the Smad2 protein level in the PA-B high dose group was significantly decreased（P<0.01）,The expression level of Smad7protein in each PA-B dose group significantly decreased（P<0.05）.PA-B groups showed dose-dependent expression of the above indexes.3.Effect of PA-B on Bcl-2/Bax signaling pathway in experimental hepatic fibrosis ratsThe results of immunohistochemistry showed that compared with the normal group,the positive expression of Bcl-2 in the liver tissue of the model group was significantly reduced.The positive expression area of each dose group of PA-B increased significantly compared with the model group.Caspase9 is mainly expressed in hepatic tissue within hepatic fibrous septum.The positive expression of Caspase9 in the liver tissue of the model group increased significantly.Compared with the model group,the positive expression of Caspase9 in liver tissue was significantly reduced.Western Blot results showed that the Bax protein expression level in the liver tissue of the model group was significantly increased（P<0.01）.Compared with the model group,the expression level of Bax protein in the PA-B medium and high dose groups was significantly reduced（P<0.01）.RT-qPCR results showed that compared with the model group,the Caspase-9 mRNA expression level in the administration group was significantly decreased（P<0.01）,and the Bax mRNA expression level in the PA-B medium and high dose groups was decreased（P<0.05 or P<0.01）).The mRNA expression level of Bcl-2 in PA-B medium and high dose groups was significantly increased（P<0.01）,and the mRNA expression level of each gene in PA-B group showed a dose-dependent change.Conclusion:1.PA-B can reduce liver tissue lesions and liver index in rats with experimental hepatic fibrosis,down-regulate the expressions of ALT,AST,HA,LN,PC-Ⅲand Col-Ⅳ,and it has a good protective effect on rats with experimental hepatic fibrosis.2.PA-B can improve the mRNA and protein expression levels ofα-SMA、Col-I and Col-III in liver tissues of rats with experimental hepatic fibrosis,suggesting that PA-B can inhibit the synthesis and secretion of collagen in rats with experimental hepatic fibrosis,and the inhibition of TGF-β1/Smad signaling pathway may be related to this mechanism.3.The anti-fibrosis effect of PA-B may be related to the up-regulation of Bcl-2mRNA and protein expression,down-regulation of Bax,Caspase9 mRNA and protein expression.