敲除let-7a减轻脑缺血再灌注后炎症反应和神经损伤

目的研究微小RNA let-7a在脑缺血再灌注后神经损伤中的作用及机制。方法将大鼠分为假手术组、对照组、anti-let-7a组和GFP组,每组各12只大鼠,假手术组大鼠为假手术组。对照组、anti-let-7a组和GFP组分别经尾静脉给予生理盐水、表达anti-let-7a的AAV-9质粒或者对照空质粒实验,然后通过线栓法建立脑缺血再灌注大鼠模型。模型建立24 h后,TTC法检测梗死体积,tunel法检测细胞凋亡,Western blot检测caspase-3表达,ELISA和qRT-PCR检测脑组织中TNF-α和IL-6的表达。并通过Western blot、qRT-PCR和荧光报告基因验证let-7a对MKP1表达的调控。结果 anti-let-7a组大鼠脑梗死体积明显小于GFP组和对照组,脑组织内凋亡细胞数、caspase-3、TNF-α和IL-6的表达明显低于GFP组和对照组。let-7a可与MKP1 mRNA 3’UTR端结合,下调MKP1在PC12细胞中的蛋白表达。结论敲除let-7a可通过调控MKP1表达,抑制MAPK信号通路的激活从而减轻脑缺血再灌注后的炎症反应和细胞凋亡发挥神经保护作用。

Knockdown of let-7a attenuates neuroinflammation and nerve injury after cerebral ischemia-reperfusion injury

Objective To study the role and mechanism of let-7a in cerebral ischemia-reperfusion injury.Methods Rats were divided into Sham group,Control group,anti-let-7a group and GFP group and 6 rats were in each group.Rats in Control group,anti-let-7a group and GFP group were given saline by tail vein,anti-let-7a expression of AAV-9 plasmid or control empty plasmid experiments respectively,and then establish cerebral ischemia and reperfusion model.Infarct volume was examined using TTC staining and apoptosis was evaluated using Tunel staining and caspase-3 detection.The expression of TNF-α and IL-6 in brain tissue was detected by ELISA and qRT-PCR.The regulation of let-7a on the expression of MKP1 was confirmed by Western blot,qRT-PCR and luciferase assay.Results The infarct volume of anti-let-7a group was significantly smaller than that of GFP group and control group,the number of apoptotic cells,the expression of caspase-3,TNF-α and IL-6 in brain tissue were significantly lower than that of GFP group and Control group.Let-7a binds to the MKP1 mRNA 3'UTR end and down-regulates the expression of MKP1 in PC12 cells.Conclusion Knockout of let-7a can inhibit the activation of MAPK signaling pathway by regulating the expression of MKP1,thus excerting the neuroprotective effect against inflammatory reaction and apoptosis after cerebral ischemia-reperfusion.