Study On The Dynamic Expression And Biological Role Of OX40 On CD4~+T Cells In Hepatic Fibrosis

Objective:To explore and verify the difference in the expression of costimulatory molecule OX40 between the normal group and the liver fibrosis disease group,and to explore the expression and role of OX40 in the development of liver fibrosis.To enrich the information on immunomodulation in the occurrence and development of liver fibrosis,and to provide a theoretical basis and new ideas for clinical treatment and management of liver fibrosis.Methods:Firstly,the data set GSE55747 in the GEO database was used to analyze the expression of OX40 between the normal group and the liver fibrosis disease group.Then the CCl₄-induced liver fibrosis model was established in mice,BALB/C mice were randomly divided into a blank control group and a model group.The blank group without any treatment.The model group received intraperitoneal injections of 20%CCl₄5ml/kg twice a week.The mice in the model group were killed at 2,4,6 and 8 weeks after injection respectively,and the samples were taken and the related indexes were detected.AST and ALT were detected to evaluate the degree of liver injury.HE staining was used to observe the degree of liver injury and inflammatory cell infiltration.Masson staining was used to observe the changes in collagen deposition.The expression ofα-SMA was detected by liver immunohistochemistry which relate with hepatic stellate cell activation.The dynamic expression of CD4⁺T lymphocytes and OX40 on CD4⁺T lymphocytes in spleen lymphocytes during liver fibrosis was detected by flow cytometry.ELISA was used to detect the expression of cytokines IL-2and IL-4 before and after interfering with the OX40 signal.Results:Analysis of the GEO Database dataset GSE55747 data suggested that OX40molecular expression was up-regulated in liver fibrosis disease samples compared with healthy samples（P<0.01）.Compared with the blank group mice,the model group mice’s liver surface appeared rough particles,the texture hardened,and the lower edge of the liver became thick and rounded blunt.The levels of AST and ALT in CCl₄-induced liver fibrosis model group were significantly higher than those in control group.The results of HE staining and Masson staining showed,with the extension of molding time,the collagen fibers were diffusely deposited,the fibers were thickened and the fiber spacing was significant.The expression ofα-SMA in the liver of mice increased continuously after the onset of the model.After 6 weeks,the expression level ofα-SMA in the model group was significantly higher than that in the control group（P<0.01）It is further suggested that hepatic stellate cells are activated and hepatic fibrosis continues to develop.These results suggest that intraperitoneal injection of CCl₄ can produce a stable liver fibrosis model.The flow cytometry showed that CD4⁺T lymphocytes decreased sharply after 2 weeks of modeling（P<0.01）,and the reduction of CD4⁺T lymphocytes slowed down in subsequent experiments.The expression of OX40was up-regulated on CD4⁺T lymphocytes after 2 weeks of modeling,and there was a significant difference compared with the blank group after 4 weeks of modeling（P<0.01）,and the up-regulation lasted until the end of the experiment.CD4⁺T lymphocyte decreased rapidly when OX40 expression was down-regulated or slowly up-regulated,while CD4⁺T lymphocyte decreased inhibited when OX40 expression was up-regulated rapidly,suggesting that OX40 is crucial to the survival of CD4⁺T lymphocyte.The up-regulation trend of OX40was the same as that of Masson staining for collagen fibers,and the same as that ofα-SMA in immunohistochemistry,the expression level of CD4⁺OX40⁺T cells was positively correlated with that ofα-SMA（R=0.687,P<0.001）.These results suggest that OX40 may be involved in the immunomodulation of hepatic fibrosis.The results of ELISA showed that the expression of IL-2 was down-regulated（P<0.01）and the expression of IL-4 was up-regulated（P<0.01）in vivo,suggesting the imbalance of anti-inflammatory factors and pro-inflammatory factors.After blocking the OX40-OX40L signaling pathway in vitro,IL-2 expression was up-regulated（P<0.01）,and IL-4 expression was down-regulated（P<0.01）.It is suggested that interfering with OX40 can affect the secretion of cytokines,and blocking the OX40-OX40L signaling pathway can regulate the direction of immune deviation to some extent.Conclusion:In a CCl₄-induced liver fibrosis model,OX40 molecules contribute to CD4⁺T lymphocyte survival,and OX40 molecules are involved in immune imbalance events in the development of liver fibrosis.Blocking the OX40-OX40L signaling pathway can affect the expression of cytokines and regulate the direction of immune deviation.Therefore,OX40may serve as an effective immune target to regulate the development of liver fibrosis.