Changes Of Nrf2,NOX4mRNA Levels And Serum ROS Levels In Liver Tissues During The Formation Of Liver Fibrosis

Objective:The changes of Nrf2,NOX4 mRNA and serum ROS in liver tissue during the formation of hepatic fibrosis induced by carbon tetrachloride(CCL4)were studied.To explore the significance of Nrf2 in the process of liver fibrosis formation,and to provide a new target for clinical delay and prevention of liver fibrosis development.Methods:1.Thirty-two healthy male rats were randomly divided into 2 groups: six in the normal control group and twenty-four in the model group.Rats in the model group were given subcutaneous injections of 40% CCL4 oil twice a week for 8 weeks.The first time is 5ml/kg and the next 3ml/kg.The normal group was given the same dose of oil by subcutaneous injection.Both groups of rats were given a normal diet.Six rats were killed in normal and model 2,4,6,and 8 weeks,and the liver tissue and serum of the same site were taken for use.The liver tissues of each group were stained with HE and Masson.The pathological changes of liver in each group were observed under microscope.The expression of NOX4 mRNA and Nrf2 mRNA in liver tissues of each group were determined by Real-time PCR.Serum ROS was determined by colorimetry.concentration.2.The plasma samples of normal healthy and chronic cirrhosis patients were collected in twenty cases.The concentration of ROS in each group was determined by colorimetric method.Results:(1)pathological changes: HE and Masson after three-color staining.Under light microscopy,the normal control group showed intact hepatic lobule structure,neat lineof hepatocytes,and no pathological changes in hepatocytes.In the early stage of the model group,hepatocyte edema,balloon-like changes and partial fat changes;with the prolonged modeling time,the cell fat became more obvious,the inflammation of the portal area was aggravated,accompanied by varying degrees of hepatocyte necrosis,the center of the hepatic lobules Fibrous deposition around the vein and in the portal area increased significantly;by the 8th week,the hepatic lobule structure was disordered,forming pseudolobules.There was no significant difference in the percentage of liver collagen fiber between the model group(0.024±0.016)and the control group(0.018±0.014)at 2 weeks(P>0.05),and compared with the control group,the model time was prolonged.The degree of liver fibrosis gradually increased,and the percentage of liver collagen fiber area gradually increased(0.210±0.031,0.310±0.037,0.390±0.036,respectively),which was higher than that of the control group(0.018±0.014).The difference was statistically significant(P<0.05)6 or 8 weeks is more obvious.(2)Real-time PCR results showed that: The expression of Nrf2 mRNA in the model group increased gradually with the prolongation of modeling time(0.53±0.49,1.51±1.63,3.35±2.68,4.01±3.40)were significantly higher than the control group(0.12±0.11),the difference was statistically significant(P<0.05);the expression levels of NOX4 mRNA were(6.53±0.89,5.49±1.24,14.49±2.39,31.78±3.96,respectively).The difference was statistically significant(P<0.05)compared with the control group(2.03±0.31)(3)With the prolongation of modeling time,the ROS levels in the model group were(1.84±0.69)ug/ml,(1.72±0.65)ug/ml,(2.48±1.14)ug/ml,(1.82±0.46)ug/ml was increased compared with the control group(1.56±0.84)ug/ml,(P<0.05),the difference was statistically significant.(4)Pearson correlation analysis showed a positive correlation between Nrf2 and ROS,Nrf2 and NOX4,NOX4 and ROS(r = 0.862,0.091,0.315,P < 0.05).(5)The serum ROS concentration(90.32±56.74)ug/ml in patients with chronic cirrhosis was significantly higher than that in the normal group(55.01±40.09)ug/ml.(P<0.05).Conclusion:(1)During the formation of rat liver fibrosis the expression of Nrf2 mRNA and NOX4 mRNA in liver tissue increased gradually,and the expression of serum ROS also showed a trend of increasing gradually.At the same time,serum ROS levels in patients with chronic cirrhosis are gradually higher than those in healthy controls,which further indicates that the occurrence and development of liver fibrosis may be related to NOX4 and ROS.(2)Nrf2,NOX4 and ROS are positively correlated,indicating that the possible presence of NOX4 to affect the expression of Nrf2 by the production of ROS-mediated profibrotic factors.