The Expression Of NOX4 In Liver Fibrosis Model Mice And Its Relationship With Liver Fibrosis

Objective:liver fibrosis(HF)is a kind of repair reaction of chronic liver injury,and it is also a necessary stage of liver cirrhosis,liver cancer and other malignant diseases.Hepatic stellate cell(HSC)activation is the central link of HF,resulting in the deposition of extracellular matrix(ECM).ROS produced by NADPH Oxidase 4(NOX4)can regulate a variety of signal pathways and related factors in HSCs,thus affecting the process of liver fibrosis.In this study,the liver fibrosis model mice were taken as the research object to explore the expression level and mechanism of NOX4 in liver fibrosis tissue,aiming at prevention and treatment of liver fibrosis.Methods:C57BL/6J mice(n=30)were selected and allocated in control group(n1=10),model group(n2=10)and knockout group(n3=10).The model group mice(n2)were injected with carbon tetrachloride(CC14)intraperitoneally for twice a week to induce liver fibrosis model;the knockout group(n3)first used CRISPR/cas9 technology to knock out the NOX4 gene(Western blot was used to prove the significant loss of ASPH protein in liver tissue after knockout,indicating the success of knockout),and then intraperitoneal cavity The liver fibrosis model was induced by injecting 2 UL/g carbon tetrachloride(CC14)twice a week;the control group mice(n1)were given corn oil in same dose twice a week.The living conditions of mice in each group were observed and recorded.At the third week after modeling,the operator extracted the blood of mouse femoral artery and killed the mice to take the liver with weighted liver index and changes compared.Automatic biochemical instrument was applied to detect the content of serum ALT and AST.The radioimmunoassay was conducted to detect the content of serum hyaluronidase(HA),laminin(LN),type III procollagen(PCIII),type IV collagen(IV-c).Western blot was used to detect the expression of NOX4,TGF-β1、Smad in the liver tissues of three groups of mice.The expression levels of NOX4,TGF-β1 and Smad mRNA in liver tissues of three groups of mice were detected by RT-PCR,the localization expression of NOX4,TGF-β1 and Smad in liver tissues was observed by immunohistochemistry(S-P),and the pathological changes of liver tissues of three groups of mice w by HE staining and Masson staining.the severity of fibrosis in three groups of mice was evaluated,and the fibrosis related components in liver tissues of three groups of mice were detected by RT-PCR The relative expression of mRNA of son SMA,collagen I,TIMP-1,MMP-2,TGF-β1.Results:The results of serological and radioimmunoassay showed that the liver index,alt,AST,ha,LN,IV-C and PC Ⅲ levels in the model group were higher than those in the control group and knockout group(P<0.05);the staining showed normal liver tissue structure in the control group,but significantly damaged liver lobules in the model group,and cells appeared under the microscope.In the knockout group,the degree of liver injury was less than that in the model group,the arrangement of liver cords was more orderly,the degree of liver lobule damage was also lower,the infiltration was reduced,and there was no significant change in edema.Masson staining showed that the collagen fibers in the model group localized in the portal area,and the area was larger.The number of liver fibrosis in model group was significantly higher than that in knockout group and control group(P<0.05),the difference was statistically significant(P<0.05);immunohistochemistry results showed that the positive expressions of NOX4,TGF-β1 and Smad were brown,and mainly expressed in the cytoplasm of liver cells;Ishak liver fibrosis score showed that the liver fibrosis in model group was significantly higher than that in knockout group and control group,the difference was statistically significant(P<0.05).Western blot revealed the expression levels of NOX4,TGF-β1 and Smad in the model group were significantly higher than those in the knockout group and the control group(P<0.05).RT-PCR showed that the expression levels of NOX4,TGF-β1 and Smad mRNA and fibrosis related factors SMA,collagen I,TIMP-1,MMP-2mRNA in the model group were higher than those in the control group and the knockout group(P<0.05)There was statistical significance(P<0.05).Conclusion:NOX4 is highly expressed in the liver tissue of mice and has the effect of promoting fibrosis.The mechanism may be related to its effect on the production of ROS mediated TGF-β/Smad signaling pathway and the expression of fibrosis related molecules SMA,collagen I,TIMP-1,MMP-2,.However,in the liver fibrosis mice without NOX4 gene,the liver fibrosis is reduced,suggesting that inhibition of NOX4 expression can inhibit the expression of liver fibrosis It is of great significance to study the medicine of Uygur and chemical.