Mutation Of Nuclear Autoantigen Sperm Protein (NASP) Gene Aggravates ConA-induced Liver Injury In Mice

Objective:To investigate the effect of mutation of nuclear autoantigen sperm protein（NASP）gene on liver injury in mice and its mechanism.Methods:The experimental animals were divided into two groups,with wild type B6（B6-WT）mice were used as the control group and the NASP mutant B6（B6-NASP^M）mice were used as the experimental group.First,the survival rates of mice in the two groups were determined by tail vein injection of lethal dose ConA 25mg/kg;Secondly,two kinds of liver injury models were established by tail vein injection of ConA 12.5mg/kg,that is,the acute hepatitis induced 8 h after injection and the chronic liver fibrosis induced by one injection per week for 8 weeks.HE staining was used to observe liver histopathology,TUNEL staining was used to observe liver cell apoptosis,Masson staining was used to determine collagen fiber deposition and Microplate method was used to estimate serum liver enzymes,then the liver injury degree of mice in the two groups was compared.QRT-PCR was used to detect the m RNA expression levels of TNF-α,IFN-γ,collagen I and collagen III in liver tissue,ELISA was used to estimate the serum protein expression levels of TNF-αand IFN-γ,Immunohistochemical staining was used to evaluate the positive expression ofα-SMA in liver tissue,Flow cytometry was used to assess the T lymphocyte subsets in liver tissue,and Western blot was used to detect the protein expression of liver injury-related signaling pathways,so as to explore the mechanism of the mutation of nuclear autoantigen sperm protein（NASP）gene leading to differences in liver injury.Results:（1）Survival rate:The survival rate of B6-NASP^Mgroup was significantly lower than that of B6-WT group.（2）In acute liver injury model,there was no significant difference in liver index between the two groups.Compared with the B6-WT group,the mice in the B6-NASP^Mgroup had severe liver pathological damage injury and hepatocyte apoptosis,the levels of serum TNF-αand IFN-γwere higher,while the m RNA expression levels of TNF-αand IFN-γin liver were not significantly changed.The results of flow cytometry showed that the number of leucocyte in liver was not significantly changed in B6-NASP^Mgroup compared with B6-WT group,the proportion and number of T lymphocytes were decreased,and the number of T lymphocyte subsets(CD3⁺T,CD3⁺CD4⁺T,CD3⁺CD8⁺T,CD4⁺CD69⁺T,CD4⁺CD44^hiT,and CD4^-CD8^-T lymphocytes)were decreased,while the proportion of CD4^-CD8^-T and CD4⁺CD44^hiT lymphocytes were increased.Western blot results showed that the non-phosphorylation and phosphorylation levels of STAT1 protein and p65 protein in B6-NASP^Mgroup were significantly higher than those in B6-WT group,while the non-phosphorylation level of JNK protein was increased,but the phosphorylation level was not significantly different.（3）In the chronic fibrosis model,there was no significant difference in liver index between the two groups.Compared with the B6-WT group,the mice in the B6-NASP^Mgroup had severe liver pathological damage and collagen deposition,the levels of serum ALT,and TNF-αwere higher,the positive expression ofα-SMA in liver tissue was significantly higher,while the m RNA expression levels of collagen I and collagen III were not obvious change.The results of flow cytometry showed that the proportion and number of CD4⁺CD44^hiT lymphocyte population in NASP^Mgroup was significantly lower than that in B6-WT group,while the number of other lymphocyte subsets and numbers were not significantly changed.Conclusion:（1）Mutation of nuclear autoantigen sperm protein（NASP）gene aggravates ConA-induced acute liver injury and chronic liver fibrosis in B6-WT mice.（2）Mutation of nuclear autoantigen sperm protein（NASP）gene aggravates acute liver injury by promoting the release of TNF-αand IFN-γinflammatory factors,changing the proportion of T lymphocyte subsets in the liver and up-regulating the protein levels of NF-κB（p65）,JAK-STAT1 and JNK signaling pathways.（3）Mutation of nuclear autoantigen sperm protein（NASP）gene aggravates liver fibrosis by increasing the release of TNF-αinflammatory factors,changing the proportion of T lymphocyte subsets in the liver and promoting the activation of hepatic stellate cells.