Study On The Effect Of MiR-7^def CD4⁺T Cells On Acute Liver Injury In Mice

Objective:To observe the effects of MiR-7 knock down CD4⁺T cells(MiR-7^defCD4⁺T cells)on Concanavalin A?Con A?-induced acute liver injury in mice and explore its potential molecular mechanism.Methods:ConA-induced acute liver injury model was established in wild-type?WT?mice by intraperitoneal injection?i.p?of 30 mg/kg ConA;The relative expression levels of MiR-7 in liver tissues and liver cells and liver infiltrating lymphocytes,as well as spleen CD4⁺T cells,were analyzed by Real-time PCR assay.CD4⁺T cells were deleted by i.p injection of anti-CD4 antibody.7 days later,then,ALI mode was established by intraperitoneal injection?i.p?of 30 mg/kg ConA.The change of body weight was observed.Histopathological changes of liver were observed by HE staining;The level of ALT in serum was detected by continuous monitoring assay.CD62L^hiT cells from CD45.2⁺MiR-7^defef and CD45.2⁺WT mice were obtained by immunomagnetic bead assay?MACS?,respectively.Then,1×10⁶ cells were adoptively transferred into CD45.1 WT mice through the tail vein.After 12 hrs,ALI model was established by intraperitoneal injection with 30 mg/kg ConA.Next,the change of body weight was observed.Histopathological changes of liver were observed by HE staining;The level of ALT in serum was detected by continuous monitoring assay.Flow cytometry was used to detect the proportion of CD4⁺T cells?CD45.1⁺CD4⁺T cells and CD45.2⁺CD4⁺T cells?and CD45.2⁺CD4⁺T cells in liver tissues.Moreover,the levels of CD62L and CD69 associated with lymphocyte activation membrane molecules,cell proliferation marker Ki67,and expression of the cytokine IFN-?in CD4⁺T cells also were analyzed by FACS.Finally,CD4⁺CD62L^hiT cells in the spleen of MiR-7^defmice and MAPK4^-/-MiR-7^defmice were further obtained by MACS,respectively.Then,cells were adoptively transferred into Rag1^-/-mice and ALI model was established as above description,respectively.The level of ALT in serum was detected by continuous monitoring assay.Flow cytometry was used to detect the expression level of CD69 and CD62L,as well as IFN-?,in CD4⁺T cells.Results:Compared with the control group,the expression level of MiR-7 was significantly increased in liver tissues of WT mice with acute liver injury?p<0.05?;The expression level of MiR-7 was not significantly changed in liver cells of WT mice with acute liver injury,while the relative expression level of MiR-7 was significantly increased in liver tissue infiltrating lymphocytes and spleen cells?p<0.05?;Although MiR-7 changes were not significant in spleen cells with CD4⁺T cells removed?p>0.05?,the relative expression level of MiR-7 in CD4⁺T cells was significantly increased?p<0.01?;The weight loss rate of MiR-7^defef mice with CD4⁺T cells cleared was significantly reduced?p<0.05?,Serum alanine aminotransferase level in MiR-7^defef mice cleared of CD4⁺T cells was significantly reduced?p<0.05?,and the inflammatory cell infiltration in the liver tissues was significantly reduced.Compared with control group,adoptive transfer of CD45.2⁺MiR-7^defef CD4⁺CD62L^hii T cells to CD45.1 WT mice group significantly reduced body weight?p<0.05?.Pathological lesions were more severeand serum ALT level was significantly increased?p<0.05?.CD45.2⁺MiR-7^defCD4⁺CD62L^hiT cells transferred to CD45.1⁺WT mice under the acute liver injury model,the proportion of CD45.2⁺CD4⁺T cells and total CD4⁺T cells?CD45.1⁺CD4⁺T cells and CD45.2⁺CD4⁺T cells?in peripheral immune organ spleen was significantly increased?p<0.01,p<0.01?;The expression ratio of Ki67,a marker of CD45.2⁺CD4⁺T cells and total CD4⁺T cells?CD45.1⁺CD4⁺T cells and CD45.2⁺CD4⁺T cells?proliferation in peripheral immune organ spleen,was significantly increased?p<0.05?;The expression level of IFN-?in CD45.2⁺CD4⁺T cells and total CD4⁺T cells?CD45.1⁺CD4⁺T cells and CD45.2⁺CD4⁺T cells?in peripheral immune organ spleen was significantly increased?p<0.05,p<0.05?;The proportion of CD45.2⁺CD4⁺T cells and total CD4⁺T cells?CD45.1⁺CD4⁺T cells and CD45.2⁺CD4⁺T cells?in peripheral immune organ spleen expressing CD62L was significantly decreased?p<0.05?,while that of CD69 was significantly increased?p<0.01?.Finally,in adoptive transfer of MAPK4^-/-MiR-7^defCD4⁺CD62L^hiT cell to Rag1^-/-mice group,serum ALT level significantly decreased?p<0.01?;The expression level of IFN-?in CD4⁺T cells was significantly decreased?p<0.01?;The level of CD62L,the marker of CD4⁺T cell expression and activation,was significantly increased in mouse liver tissues,while the level of CD69 was significantly decreased?p<0.05?.Conclusion:MiR-7 deficicency can significantly elevate the activation,proliferation and cytokine secretion of CD4⁺T cells and promote the pathology of ALI,which is related to its target MAPK4.