Self-developed MyD88 Inhibitor Protects Against Cerebral Ischemia-reperfusion Injury

[Objective]To investigate the role and mechanism of TLR/My D88 signaling pathway in cerebral ischemia-reperfusion injury in mice by using self-developed My D88 inhibitor TJ-5.[Methods]In vivo,adult male C57BL/6 mice were used as experimental objects,and the middle cerebral artery embolism(MCAO)model was established.After ischemia for 1 hour,different concentrations of TJ-5 were given intravenously during reperfusion and detected after 24 hours.They were divided into five groups: Sham group,I/R group,I/R+TJ-5 5mg/kg group,I/R+TJ-5 10mg/kg group and I/R+TJ-5 15mg/kg group.Six rats in each group were evaluated for neurological function after 24 hours reperfusion.After anesthesia and perfusion,the brain was taken,the area of cerebral infarction was measured,and the best drug concentration group was selected.The follow-up experiment was divided into four groups: Sham group,I/R group,Vehicle group and I/R+TJ-5 15mg/kg group.Ten mices in each group were taken after 24 hours reperfusion,and TUNEL fluorescence staining and western blot test were performed to detect the apoptosis level of nerve cells.Immunohistochemical staining was used to measure the neuronal damage and activation level of glial cells.The expression levels of TNF-α,IL-1β,IL-6 and i NOS were detected by ELISA and RT-PCR.The expression level of My D88 signaling pathway protein in brain tissue was detected by western blot.The immune cells in the hemisphere of cerebral infarction area of mice were extracted to detect the proportion changes of CD11b+CD45hi Ly6G+ central granulocyte,CD11b+CD45hi Ly6G-mononuclear macrophage and CD11b+CD45int Ly6G-resting microglia.In vitro,BV-2 mouse microglia cell line was used to study TLR4/My D88 pathway of microglia,and LPS stimulation model was established,which was divided into Control group,LPS group,LPS+10μM TJ-5 group and LPS+20μM TJ-5 group.drugs were added 2h before stimulation,and cell morphology was observed 24 h after stimulation with 1μg/ml LPS.The morphological changes of cell activation were observed by microscope.The expression levels of My D88 signaling pathway protein and i NOS in glial cells were detected by western blot.The secretion levels of TNF-α,IL-1β and IL-6 were detected by ELISA.[Results]In vivo,compared with the I/R group,the cerebral infarction range of mice in TJ-5group was significantly reduced,and the neurological function was significantly improved and changed in a concentration-dependent manner.The I/R+TJ-5 15mg/kg group had the best effect,and the cerebral infarction volume was reduced by about 80%.TJ-5 can decrease the apoptosis level of brain tissue in infarcted area after 24 h reperfusion.Neu N protein was down-regulated,indicating that neuronal damage was alleviated.The expression level of Iba-1 decreased,indicating that the activation of microglia was inhibited.Glial cells and lymphocytes in cerebral infarction hemisphere of mice in each group were extracted and labeled with CD11 b,CD45 and Ly6 G.The results showed that TJ-5 reduced the infiltration ratio of peripheral myeloid cells in cerebral infarction area after 24 h reperfusion,and increased the proportion of resting microglia.ELISA and RT-PCR results showed that TJ-5 significantly decreased the expression levels of inflammatory factors TNF-α,IL-1β and IL-6 in brain infarction area after 24 h reperfusion.At the level of m RNA and protein,TJ-5 decreased the level of oxidative stress,such as i NOS expression,and inhibited TLR4/My D88 signaling pathway in the infarcted area of brain tissue after 24 h reperfusion.In vitro,compared with LPS group,the activation of microglia in TJ-5 group was inhibited,the secretion level of inflammatory factors decreased,the expression level of i NOS decreased,and the expression level of TLR4/My D88 signaling pathway protein decreased.[Conclusion]TJ-5 can alleviate cerebral ischemia-reperfusion injury and inhibit excessive inflammation injury in mice.Protective effect is due to the inhibition of excessive activation of TLR4/My D88 signaling pathway,which makes the activation of endogenous microglia down-regulated and the chemotaxis of peripheral myeloid cells into the brain reduced,thus down-regulating the local excessive inflammatory reaction level and oxidative stress level,reducing neuronal apoptosis and narrowing the scope of inflammatory injury.