二甲双胍对创伤性脑损伤后神经元凋亡的影响及机制

Effect and mechanism of metformin on neuronal apoptosis after traumatic brain injury

目的探讨二甲双胍(MET)对创伤性脑损伤(TBI)后神经元凋亡的影响及可能机制。方法选择小鼠海马神经元(HT22)细胞进行实验,将HT22细胞随机分为对照组、TBI组、MET1、MET2组,除对照组外,其余组通过机械划痕构建TBI体外模型。Westem blotting法测定Bcl-2腺病毒E1B-19 kD相互作用蛋白3(BNIP3)、微管相关蛋白1轻链3(LC3)、p62、线粒体外模转位酶20(TOMM20)、细胞色素C氧化酶4(COXⅣ)表达,免疫荧光双染观察线粒体和溶酶体共定位情况,TUNEL染色法检测细胞凋亡率,LDH试剂盒检测细胞上清液LDH活性。应用小干扰RNA(siRNA)抑制BNIP3表达,并随机分为对照siRNA组、BNIP3-siRNA组、对照siRNA+TBI组、BNIP3-siRNA+TBI组,实验终点观察上述指标变化。结果TBI组神经元凋亡率高于对照组(P<0.05)。TBI组BNIP3表达及LC3-Ⅱ/Ⅰ值均高于对照组(P均<0.05),而p62、TOMM20、COXⅣ表达均低于对照组(P均<0.05)。激光共聚焦显微镜观察发现,TBI组细胞线粒体溶酶体共定位较对照组增加。MET 1组和2组神经元凋亡率均低于TBI组(P均<0.05),且MET 2组细胞凋亡率较MET 1组进一步降低(P<0.05)。MET 1组BNIP3表达及LC3-Ⅱ/Ⅰ值均较TBI组降低(P均<0.05);MET 2组BNIP3表达及LC3-Ⅱ/Ⅰ值较MET 1组进一步降低(P均<0.05)。MET 1组p62、TOMM20、COXⅣ表达均较TBI组升高(P均<0.05);MET 2组p62、TOMM20、COXⅣ表达较MET 1组进一步升高(P均<0.05)。激光共聚焦显微镜观察发现,MET 1组细胞线粒体溶酶体共定位较TBI组减少,MET 2组细胞线粒体溶酶体共定位较MET 1组进一步减少。对照siRNA+TBI组及BNIP3-siRNA+TBI组细胞凋亡率分别为(32.57±2.68)%、(16.59±1.80)%,差异有统计学意义(P<0.05)。对照siRNA+TBI组BNIP3表达水平及LC3-Ⅱ/Ⅰ值分别为0.66±0.01、1.46±0.02,高于BNIP3-siRNA+TBI组(P均<0.05);而对照siRNA+TBI组p62、TOMM20、COXⅣ相对表达量分别为0.06±0.01、0.14±0.01、0.09±0.01,低于BNIP3-siRNA+TBI组(P均<0.05)。激光共聚焦显微镜观察发现,对照siRNA+TBI组线粒体溶酶体共定位较BNIP3-siRNA+TBI组增加。结论MET可抑制TBI后神经元凋亡,其机制可能与抑制BNIP3介导的线粒体自噬有关。

Objective To investigate the effect and mechanism of metformin(MET)on neuronal apoptosis after traumatic brain injury(TBI).Methods HT22(mouse hippocampal neurons)cells were selected for the experiment.HT22 cells were randomly divided into the control group,TBI group,MET1 group,and MET2 group.Except for the control group,the in vitro models of TBI were constructed by mechanical scratching in the other groups.We utilized Western blotting to examine the expression of Bcl-2 and adenovirus E1B19kDa-interacting protein 3(BNIP3),LC3,p62,TOMM20,and COX IV.We utilized immunofluorescence double staining to observe the colocalization of mitochondria and lysosome,TUNEL staining to detect apoptosis rate,and LDH kit to detect LDH activity of cell supernatant.The small interfering RNA(siRNA)was used to inhibit the expression of BNIP3.The cells were randomly divided into the control siRNA group,BNIP3-siRNA group,control siRNA+TBI group,and BNIP3-siRNA+TBI group,and the changes of the above parameters were detected at the end of the experiment.Results Compared with the control group,the neuronal apoptosis rate increased in the TBI group(P<0.05).Compared with the control group,the expression of BNIP3 and LC3-Ⅱ/Ⅰincreased,while the expression of p62,TOMM20,and COX IV decreased in the TBI group(all P<0.05).Compared with the control group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy increased in the TBI group.Compared with the TBI group,the neuronal apoptosis rates were lower in the MET 1 group and MET 2 group(all P<0.05),and the apoptosis rate was lower in the MET 2 group than in MET 1 group(P<0.05).Compared with the TBI group,the expression of BNIP3 and LC3-Ⅱ/Ⅰdecreased in the MET 1 group(all P<0.05),and the expression of BNIP3 and LC3-Ⅱ/Ⅰwas lower in MET 2 group than in MET 1 group(all P<0.05).Compared with the TBI group,the expression of p62,TOMM20,and COX IV increased in the MET 1 group(all P<0.05),and the expression of p62,TOMM20,and COX IV were significantly higher in the MET 2 group than in the MET 1 group(all P<0.05).Compared with the TBI group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy decreased in the MET 1 group,and the colocalization of mitochondria and lysosomes was lower in the MET 2 group than in the MET 1 group.The neuronal apoptosis rates in the control siRNA+TBI group and BNIP3-siRNA+TBI group were 32.57%±2.68%and 16.59%±1.80%,respectively,with significant difference between the two groups(P<0.05).The expression levels of BNIP3 and the LC3-Ⅱ/Ⅰvalue in the control siRNA+TBI group were 0.66±0.01 and 1.46±0.02 respectively,which were significantly higher than those in the BNIP3-siRNA+TBI group(all P<0.05).The expression levels of p62,TOMM20,and COX IV in the control siRNA+TBI group were 0.06±0.01,0.14±0.01,and 0.09±0.01,respectively,which were significantly lower than those in the BNIP3-siRNA+TBI group(all P<0.05).Compared with the BNIP3-siRNA+TBI group,the colocalization of mitochondria and lysosomes observed by laser confocal microscopy increased in the control siRNA+TBI group.Conclusion MET can inhibit neuronal apoptosis after TBI,and its mechanism may be related to the inhibition of BNIP3-mediated mitophagy.