Intervention Effect And Mechanism Of Metallothionein 2 On Liver Fibrosis In Mice

Objective:To observe the role of MT2 deficiency in liver function damage and liver fibrosis induced by carbon tetrachloride in mice,explore the anti-liver fibrosis effect and possible mechanism of MT2,and provide experimental evidence for clinical prevention and treatment of liver function and liver fibrosis caused by trauma and chronic inflammation.Methods:1.Preparation and grouping of animal models12 healthy male SPF?18-22g?male C57BL wild type mice and 12 mice with low expression of MT2 were constructed by CRISPR/Cas9 knockout MT2,and were randomly divided into 4 groups:normal control group?WT??6?,MT2 low expression control group?MT-KO??6?,wild type carbon tetrachloride group?WT-CCl₄??6?,MT2low expression carbon tetrachloride group?MT-K0-CCl₄??6?.The experimental group was induced by repeated intraperitoneal injection of olive oil diluted carbon tetrachloride solution for 4 weeks?carbon tetrachloride and olive oil mixed at a ratio of1:4,5 ml/kg twice a week?to establish liver fibrosis.Liver fibrosis model.The control group was injected with an equal volume of olive oil.After weighing at the end of the4th week,4%chloral hydrate?0.15mL/20g?was intraperitoneally injected and the blood was collected from the heart.The serum and liver samples were collected and stored at-80°C for use.2.Serological indicators checkThe serum levels of liver function and related inflammatory factors in each group were detected:alanine aminotransferase?ALT?,aspartate aminotransferase?AST?and total bilirubin?TB?were detected by serology.The changes of serum IL-6 and TNF-?were detected by ELISA.3.Pathological examination of tissue specimensHE,Masson and Sirius red staining were used to observe the pathological and fibrotic changes of liver tissue,and Western Blot was used to detect?-SAM in liver tissue.4.Changes in MT2 and related inflammatory factors in liverThe changes of MT2,IL-6,TNF-?and IL-10 in liver tissue were detected by RT-PCR.The expression of F4/80 in liver tissue was detected by immunohistochemistry.The expression levels of P65 and p-p65 were detected by Western Blot.5.Expression of Nrf2/HO-1 signaling pathway-related proteins in liverThe changes of MDA and SOD in liver tissues were detected by ELISA,and the expressions of Nrf2,catalase,NQO-1 and HO-1 in liver tissues were detected by Western Blot.Result:1.MT2 protects against liver fibrosis induced by CCl4?1?The expression of MT2 mRNA in the liver of wild type mice was significantly higher than that of knockout mice,and the difference was statistically significant?P<0.05?.After injection of CCl₄,the MT2 content in wild-type mice increased significantly,and had no significant effect on knockout mice.?2?Compared with the control group,both genotype mice showed impotence after injection of CCl₄,and their feeding and activities decreased,and the body weight did not increase significantly.?3?There were no significant differences in serum AST,ALT and TB between the two genotype mice in the control group.The levels of CCl₄ increased after different injections.The knockout genotype mice were significantly higher than the wild type mice,and the difference was statistically significant?P<0.05?.?4?In the control group,the liver lobules of the two genotype mice were arranged neatly,without obvious inflammatory cell infiltration,and only a small amount of blue fiber was found in the portal area.After injection of CCl₄,the mice in the two groups showed different degrees of hepatic lobular structural disorder,inflammatory cell infiltration and fibrosis,which were more severe in the knockout genome.?5?The expression of?-SAM in the liver tissues of the two genotype mice in the control group was low,and there was no significant difference between the two groups.After injection of CCl₄,the levels were increased.The knockout genotype mice were significantly higher than the wild type mice,and the difference was statistically significant?P<0.05?.2.MT2 reduces liver damage by regulating inflammation?1?ELISA detection The serum levels of pro-inflammatory factors IL-6 and TNF-?in the two genotype mice of the control group were low,and there was no significant difference between the two groups.After injection of CCl₄,the levels were increased.The knockout genotype mice were significantly higher than the wild type mice,and the difference was statistically significant?P<0.05?.This is consistent with the results of RT-PCR in liver tissue.?2?Immunohistochemical detection In the control group,the F4/80+macrophages in the liver tissues of the two genotype mice were scattered in a small amount.After injection of CCl4,they increased to different extents and aggregated in the portal area.More significant.?3?Western Blot detected that the expression of P-P65 in the liver tissues of the two genotype mice was low,and increased to different degrees after injection of CCl4.The knockout genotype mice were more obvious,the difference was statistically significant?P<0.05?.3.MT2 attenuates liver oxidative stress damage by activating Nrf2/HO-1signaling pathway?1?In the control group,the MDA and SOD contents in the liver of the two genotype mice were similar.After injection of CCl₄,the MDA increased significantly and the SOD expression decreased significantly,which was more significant in knockout genotype mice.?2?Western Blot showed no significant difference in the expression of Nrf2 in the liver tissues of the two genotype mice.After injection of CCl₄,the expression of Nrf2 increased,and the expression of downstream antioxidant proteins catalase,NQO-1 and HO-1 increased.It is more pronounced in wild-type mice.Conclusion:1.Metallothionein 2?MT2?deficiency aggravates inflammatory cell infiltration,fibrosis and liver tissue damage induced by CCl₄ in liver tissue;2.The regulation of hepatic fibrosis by metallothionein 2?MT2?may be achieved by down-regulating NF-?B P65 signaling pathway and anti-inflammatory and up-regulating Nrf2/HO-1 signaling pathway.