干扰NIX通过抑制自噬缓解脊髓神经元的缺血再灌注损伤

Interference of NIX alleviates spinal cord neurons ischemia-reperfusion injury by inhibiting autophagy

目的观察线粒体外膜蛋白NIX能否通过抑制自噬缓解脊髓神经元缺血再灌注损伤。方法利用H_(2)O_(2)(200μmol/mL)处理分离的原代脊髓神经元,以构建体外缺血再灌注模型,通过转染shNIX干扰NIX的表达,并通过流式凋亡检测与CCK-8增殖检测观察脊髓神经元的凋亡情况,PCR检测NIX基因的转录水平,蛋白免疫印迹法检测NIX、LC3与Beclin蛋白的表达水平。结果shNIX能显著下调NIX基因的转录水平与蛋白的表达水平,抑制效果明显,用于后续的实验操作。流式凋亡检测与CCK-8增殖检测表明,缺血再灌注损伤诱导脊髓神经元出现凋亡,降低其增殖速度。缺血再灌注损伤促进NIX、LC3和Beclin的表达。shNIX使缺血再灌注损伤诱导脊髓神经元的凋亡减少,促进了细胞的增殖,并且使LC3和Beclin的蛋白表达减少。结论干扰NIX通过抑制自噬减弱了缺血再灌注诱导的脊髓神经元损伤。

Objective This study aimed to observe whether interference of mitochondrial outer membrane protein NIX could alleviate spinal cord neurons ischemia reperfusion injury by inhibiting autophagy.Methods Primary spinal cord neurons were isolated and treated with H_(2)O_(2)(200μmol/mL)to construct the ischemia-reperfusion cell model in vitro.The expression of NIX was knocked down by transfection with shNIX.The apoptosis and proliferation of spinal cord neurons were observed by flow cytometry proliferation of CCK-8 proliferation detection.The transcriptional level of NIX gene was detected by PCR,and the protein expression of NIX,LC3,and Beclin were detected by Western blotting.Results shNIX significantly downregulated the transcription level and protein expression levels of NIX,and the inhibition effect is obvious,which is used for subsequent experimental operations.Flow cytometry proliferation of CCK-8 proliferation detection showed that ischemia-reperfusion injury induced apoptosis of spinal cord neurons with reduced proliferation rate.In addition,ischemia-reperfusion injury promoted the expressions of NIX,LC3,and Beclin.More importantly,shNIX reduced the apoptosis of spinal cord neurons,promoted cell proliferation,as well as reduced the protein expressions of LC3 and Beclin.Conclusion Interference of NIX attenuated ischemia-reperfusion induced spinal cord neurons injury by inhibiting autophagy.