Study On The Protective Effect And Mechanism Of Tat-NTS On Cerebral Ischemia-Reperfusion Injury

Background: Previous studies have found that reducing the abnormal nuclear entry of Annexin A1(ANXA1)can effectively inhibit neuronal apoptosis.Thus,we synthesized a small molecule peptide that could prevent nuclear translocation of ANXA1 and named it Tat-NTS.Tat-NTS has been shown to effectively inhibit ANXA1 nuclear translocation in neurons cultured in vitro.On this basis,this study used Middle cerebral artery occlusion/reperfusion(MCAO/R)model mice as the research object to evaluate the protective effect of Tat-NTS on neurons.In vitro experiments,microglia were treated with Oxygen glucose deprivation/ reperfusion(OGD/R)and Tat-NTS,and the microglia conditioned medium(MCM)were collected.Neurons were treated with OGD/R and MCM to observe the effect of MCM from microglia that treated with Tat-NTS.The protective mechanism of Tat-NTS on ischemia-hypoxic neurons from the perspective of microglia was preliminarily discussed.Method:(1)The mouse MCAO/R model was constructed,protein expression level and its distribution in the nucleus and cytoplasm were detected by WB.(2)The cerebral infarction size were detected by TTC staining.(3)The cell apoptosis were detected by TUNEL staining.(4)The neurological function scores,rotarod and open field tests were used to detect the neurological and motor function deficits.(5)The Morris water maze and novel object recognition experiments were used to detect the learning and memory ability.(6)The primary cultured microglia were treated with Tat-NTS and/or OGD/R to obtain microglial conditioned medium under different treatment conditions,and the primary cultured OGD/R-treated neurons were stimulated with MCM.Result: After the MCAO/R model established,Tat-NTS was injected into the lateral ventricle.The results showed that:(1)Tat-NTS reduced the nuclear translocation of ANXA1 in the cortex treated with ischemia and reperfusion by inhibiting the binding of ANXA1 to Importin β.(2)Tat-NTS significantly reduced the cerebral infarction volume in mice with cerebral ischemia-reperfusion injury and reduced the risk of neuronal apoptosis.(3)Tat-NTS significantly improved the neurological and exercise capacity in mice with cerebral ischemia-reperfusion injury.(4)The Morris water maze and the novel object recognition experiment showed that Tat-NTS could significantly improve the learning and memory ability of MCAO/R mice.(5)Tat-NTS significantly reduced the levels of inflammatory factors TNF-α and IL-1β in the cortex of cerebral ischemia-reperfusion injured mice.The results of primary cultured microglia and neurons showed that:(1)Tat-NTS significantly reduced the expression and secretion of microglia-derived inflammatory factors TNF-α and IL-1β after OGD/R treatment.(2)MCM from microglia with Tat-NTS treatment can significantly reduce the levels of apoptosis-related proteins Cleaved caspase-3 and Bid in OGD/R-treated neurons,while knockdown of ANXA1 in microglia can partially reverse the role of Tat-NTS.Conclusion: Tat-NTS has a clear protective effect on cerebral ischemia-reperfusion injured mice,and one of the protective pathways of Tat-NTS on neurons is achieved by inhibiting the nuclear entry of ANXA1 and reducing the expression and secretion of inflammatory factors in microglia.