LMO3基因在卒中后抑郁大鼠认知功能障碍中的作用

The role of LMO3 gene in cognitive dysfunction of post-stroke depression rats

目的探讨LMO3基因在卒中后抑郁(PSD)大鼠认知功能障碍中的作用.方法选择清洁级SD雄性大鼠40只,随机分为对照组、模型组、阴性对照组(control-shRNA组)、LMO3抑制组(LMO3 shRNA组),每组10只,除对照组外,其余各组采用线栓法构建PSD模型.对各组大鼠神经功能缺损评价和Morris水迷宫实验;采用苏木精-伊红染色法观察大鼠脑海马组织病理形态学变化;采用免疫组化检测各组大鼠脑组织LMO3的表达水平;采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)检测神经元凋亡水平;采用蛋白印迹法(Western blot)检测脑组织Bax、Bcl-2蛋白表达水平;采用实时荧光定量PCR(qRT-PCR)法检测脑组织LMO3、Bax、Bcl-2 mRNA表达水平.结果与对照组相比,模型组和control-shRNA组大鼠逃避潜伏期长,象限停留时间短,穿越平台次数减少(P<0.05),差异有统计学意义;LMO3 shRNA组与control-shRNA组相比,大鼠逃避潜伏期短,象限停留时间长、穿越平台次数增加(P<0.05).模型组和control-shRNA组大鼠神经元凋亡数均高于对照组;抑制LMO3基因的表达后神经元凋亡数减少.与对照组相比,模型组和control-shRNA组大鼠脑组织LMO3、Bax蛋白及mRNA表达水平均显著升高,Bcl-2蛋白及mRNA表达水平下降(均P<0.05);与control-shRNA组相比,LMO3 shRNA组大鼠脑组织LMO3蛋白及mRNA表达水平显著降低,抑制LMO3表达明显下调Bax蛋白及mRNA表达水平,上调Bcl-2蛋白及mRNA表达水平(P<0.05).结论抑制LMO3基因表达可改善PSD大鼠认知功能障碍,其作用机制可能是通过减少海马组织神经元凋亡水平.

Objective To explore the role of LMO3 gene in cognitive dysfunction of post-stroke depression(PSD)rats.Methods 40 SD male rats were randomly divided into control group,model group,negative control group(control shRNA group)and LMO3 inhibition group(LMO3 shRNA group).There were 10 rats in each group.And except for the control group,the other groups used the thread bolt method to build PSD model.Evaluation of neurological deficit and Morris water maze test in rats of each group.The histopathology of hippocampus in rats was observed by Hematoxylin Yihong staining.Immunohistochemistry was used to detect the expression of LMO3 in brain tissue of rats in each group.Detection of neuron apoptosis by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL).Western blot was used to detect the expression of Bax and Bcl-2 protein,and real time fluorescence quantitative PCR(q RT-PCR)was used to detect the expression of LMO3,Bax and Bcl-2 mRNA.Results Compared with the control group,rats in the model group and control shRNA group had longer escape latency,shorter quadrant stay time and fewer crossing platform times(P<0.05).Rats in LMO3 shRNA group had shorter escape latency,longer quadrant stay time and more crossing platform times than the control shRNA group(P<0.05).The number of neuron apoptosis in the model group and control shRNA group was higher than that in the control group.The expression level of LMO3,Bax protein and mRNA in brain tissue of model group and control shRNA group were significantly increased than the control group,and the expression level of Bcl-2 protein and mRNA was decreased(all P<0.05);compared with control shRNA group,the expression level of LMO3 protein and mRNA in brain tissue of LMO3 shRNA group was significantly decreased,the expression level of Bax protein and mRNA was significantly decreased,and the expression level of Bcl-2 protein and mRNA was up-regulated(P<0.05).Conclusion Inhibition of LMO3 gene expression can improve the cognitive dysfunction of middle and PSD rats,and its mechanism may be through reducing the level of neuron apoptosis in hippocampus.