| ObjectiveTo study the effect of Yangying Huoxue Decoction on NOX4-mediated PI3K/AKT signal pathway in treating liver fibrosis in rats,further clarify the mechanism of anti-liver fibrosis,and provide experimental basis for clinical application.MethodsAfter 7 days of feeding at normal room temperature,60 SD male rats were randomly divided into control group(n = 10)and model group(n = 50).The control group was fed a normal diet,while the model group was fed a diet of 10% ethanol and high fat and low protein(795g/kg corn meal +200g/kg lard +5g/kg cholesterol).The model group was treated with carbon tetrachloride(CCL4)complex method(40%CCL4 and paraffin oil mixed solution combined with pig serum)to induce liver fibrosis model.After 6 weeks,1 rats in the control group were randomly selected,and 3 rats in the model group were taken pathological sections of liver tissue to observe the success of the liver fibrosis model.After successful modeling,the modeling rats were randomly divided into model group,colchicine group,Bijiajian pill group and Yangying Huoxue Decoction group,with 10 rats in each group.The Yangying Huoxue Tang group was given 4.734g/ml daily and gavaged at a ratio of 2ml/200g·d,the colchicine group was given 0.2mg/kg suspension gavage,and the Bijiajian pill group was given 540mg/kg gavage daily.Both the model group and the control group were given physiological saline by gavage,and the behavior state of the rats was recorded in detail during the whole experiment.After 6 weeks,the samples were taken,and ALT and AST were detected by automatic biochemical analyzer.Enzyme-linked immunoassay(ELISA)was used to detect the levels of serum Ⅳ-C,PⅢP,HA and LN.The pathological changes of liver tissue were observed by HE and Masson staining.TUNEL staining was used to detect apoptosis of liver cells in rats.The expression of α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1 were detected by immunohistochemistry(IHC).Western blot was used to detect the expression of α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1 proteins,and statistical software SPSS20.0 was used for statistical analysis.Results1.Detection of serum levels in liver tissue: Compared with the control group,the contents of AST,ALT,HA,LN and serum PⅢP and Ⅳ-C in model group were significantly increased(P<0.01);Compared with model group,AST,ALT and liver levels in colchicine group,Yangying Huoxue Decoction group and Bijiajian pill group treatment group were significantly decreased(P<0.05).Compared with Yangying Huoxue Decoction group,AST,ALT and Ⅳ-C in colchicine group had statistical significance(P<0.05),but HA,LN and PⅢP had no statistical significance(P>0.05).There were no significant differences in serum factors between Yangying Huoxue Decoction group and Bijiajian pill group(P>0.05).2.Histopathological examination of liver: HE staining showed that the structure of liver lobules of rats in the control group was complete,the cord of liver cells was arranged radially,and the nucleus and cytoplasm were clearly visible without degeneration and necrosis.In model group,the structure of hepatic lobules and hepatic cord was disordered,with different sizes and shapes,hepatic cell steatosis,hyperplasia of connective tissue in the portal area,thickened and obvious hyperplasia of fibrous septa,and a large number of inflammatory cell infiltration near the central vein.Masson staining showed that a large number of collagen fibers were deposited near the portal area and central vein in the model group,which were connected with each other and gradually formed false lobules.Compared with model group,the degree of liver fibrosis in Yangying Huoxue Decoction group,Bijiajian pill group and colchicine group was significantly reduced after drug intervention.3.Hepatocyte apoptosis was detected by TUNEL method: Compared with the control group,the apoptosis rate of liver cells in the model group was significantly increased(P<0.01);Compared with model group,Yangying Huoxue Decoction group,Bijiajian pill group and colchicine group had significant differences,All three groups showed a significant decrease(P<0.01);Compared with Yangying Huoxue Decoction group,colchicine group had a significant difference(P<0.01),but there was no statistical significance compared with Bijiajian pill group(P>0.05).The study found that Yangying Huoxue Decoction group had a lower rate of hepatocyte apoptosis and a significant therapeutic effect.4.α-SMA,collagen I,NOX4,p-PI3K/PI3 K,p-AKT1/AKT1 protein expression: westernblot detection results showed that,compared with control group,α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1 protein expression in liver tissue of model group were significantly increased(P<0.01).Compared with model group,Yangying Huoxue Decoction group,Bijiajian pill group and colchicine group were all able to effectively down-regulate the expression of α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1 in rat liver tissue,showing statistical significance(P<0.05).Compared with Yangying Huoxue Decoction group,There were significant differences in colchicine group(P<0.05);There was no statistical significance between Yangying Huoxue Decoction group and Bijiajian pill group(P>0.05).Immunohistochemical results showed that α-SMA,collagen I,NOX4,p-PI3K/PI3 K,p-AKT1 /AKT1 target proteins were mainly expressed in cytoplasm and cytoplasm.Compared with the control group,the histone expression in the model was significantly increased(P<0.01).Compared with model group,Yangying Huoxue Decoction group,Bijiajian pill group and colchicine group were all able to effectively down-regulate the expression of α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1 in rat liver tissue(P<0.05).Compared with Yangying Huoxue decoction group,colchicine had significant difference(P<0.05);There was no statistical significance between Yangying Huoxue Decoction group and Bijiajian pill group(P>0.05).Conclusion1.Yangying Huoxue Decoction has the effect of protecting liver and alleviating liver fibrosis.2.One of the mechanisms of Yangying Huoxuxue Decoction anti liver fibrosis effect is through downregulation of protein expression of α-SMA,collagen I,NOX4,p-PI3K/PI3 K and p-AKT1/AKT1,retard the PI3K/AKT signaling pathway mediated by NOX4,and inhibit the proliferation and activation of HSC. |
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