Study On The Effect And Mechanism Of MDL-1 On Immune Injury Caused By Cerebral Ischemia Reperfusion

Objective:As the innate immune cells of the central nervous system,microglia cells are involved in the regulation of inflammation after stroke.DNAX-activating protein of 12 kDa(DAP 12)expressed on the surface of microglia cells has been demonstrated to be involved in the immunopathological mechanism of ischemic stroke by regulating the activity of microglia cells.Since DAP 12 has no ligand binding ability,it needs to bind to DAP 12-related receptors and then bind to ligands.Therefore,the role of DAP 12-related receptors in ischemic stroke needs further study.Myeloid DAP12-associated lectin-1(MDL-1),one of the DAP12-related receptors,has been shown to be significantly associated with inflammation in other diseases.The purpose of this study was to clarify the effect of MDL-1 on immune injury and immune microenvironment after cerebral ischemia reperfusion,and to further explore the specific mechanism of MDL-1 regulating inflammation after cerebral ischemia reperfusion.Methods:Middle cerebral artery occlusion(MCAO)in mice for 60min was used as the experimental stroke model.First,the neurological function score,the relative infarct volume calculated by TTC staining and the detection of apoptosis were performed.Subsequently,the number of activated microglia and astrocytes in the MDL-1 group was observed by immunofluorescence assay.Next,mRNA levels of pro-inflammatory factors(TNF-α,IL-1β and IL-6),anti-inflammatory factors(IL-10 and Arg-1),and chemokines(CCL-2,CCL-3,CCL-5 and CCL-10)were detected by real-time PCR.Flow cytometry was used to detect the number of CD4+T cells,CD8+T cells,macrophages,neutrophils and natural killer cells in the brain and spleen of MDL-1 group.The effect of MDL-1 on the expression and activation of STAT3 was further studied by Western blot.Results:Neurological function scores showed that the neurological defects in the MDL-1 group were significantly less than those in the control group.TTC staining showed that the infarct volume in the MDL-1 group was smaller than that in the control group.Immunofluorescence assay showed that the number of activated microglia and astrocytes in the MDL-1 group was significantly lower than that in the control group.real-time PCR results showed that the mRNA levels of inflammatory cytokines(TNF-α,IL-1β,IL-6)were significantly decreased.The mRNA levels of anti-inflammatory factors(IL-10,Arg-1)were increased.mRNA levels of chemokines(CCL-2,CCL-3,CCL-5 and CCL-10)decreased.Flow cytometry results showed that the proportion of apoptotic cells at the infarction side,compared with the control group,was decreased in the MDL-1 group,and the number of CD4+T cells,CD8+T cells,macrophages,neutrophils and natural killer cells in the spleen of mice in the MDL-1 group were increased to varying degrees.The numbers of CD4+T cells,CD8+T cells,macrophages,neutrophils and natural killer cells were all reduced to varying degrees.Western blot results showed that STAT3 expression and activation were decreased in MDL-1 mice.Conclusion:The results indicate that MDL-1 plays a protective role in immune injury caused by cerebral ischemia reperfusion.MDL-1 inhibited the activation of microglia around the infarct and reduced the number of reactive astrocytes around the infarct.MDL-1 can regulate the transformation of neuroinflammation to anti-inflammatory state after cerebral ischemia reperfusion and improve the intracerebral immune microenvironment after cerebral infarction.MDL-1 can inhibit the infiltration of peripheral immune cells into the center.In addition,MDL-1 may play an immunoregulatory role by inhibiting STAT3 expression and phosphorylation.