过表达TREM2对癫痫大鼠海马组织炎症及神经元凋亡的影响

Effect of overexpression of TREM2 on inflammation and neuron apoptosis in hippocampus of epileptic rats

目的探讨过表达髓样细胞触发受体2(triggering receptor expressed on myeloid cells 2,TREM2)对癫痫大鼠海马组织炎症及神经元凋亡的影响。方法选择清洁级SD雄性大鼠40只,随机分为4组,对照组、模型组、阴性对照组和TREM2过表达组,每组10只。实时荧光定量PCR法检测各组大鼠脑组织TREM2的表达水平;苏木精-伊红检测各组大鼠脑海马组织病理形态学变化;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测各组大鼠脑海马神经元凋亡水平;酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)检测各组大鼠脑组织肿瘤坏死因子α(tumor necrosis factorα,TNFα)、白细胞介素1β(interleukin 1β,IL-1β)含量;蛋白印迹试验和实时荧光定量PCR检测各组大鼠脑组织Toll样受体4(Toll like receptor 4,TLR4)、髓样分化因子(myeloid differentiation factor 88,MyD88)蛋白及mRNA表达水平。结果与对照组相比,模型组大鼠脑组织TREM2 mRNA表达水平显著降低(P<0.05);转染TREM2基因过表达慢病毒载体后,大鼠脑组织TREM2 mRNA表达水平显著升高(P<0.05)。与对照组相比,模型组和阴性对照组大鼠脑海马神经元核发生变形,核仁消失,胞浆着色浅,核固缩,且大鼠脑海马组织神经元凋亡细胞数、脑组织TNF-α、IL-1β含量和脑组织TLR4、MyD88 mRNA和蛋白表达水平均显著升高(均P<0.05);TREM2过表达后改善核固缩程度,细胞形态恢复正常、胞膜清楚、核仁清晰可见;与阴性对照组相比,TREM2过表达组大鼠脑海马组织神经元凋亡数、脑组织TNF-α、IL-1β含量和脑组织TLR4、MyD88 mRNA和蛋白表达水平均减少(均P<0.05)。结论过表达TREM2基因可抑制脑海马组织神经元凋亡水平及炎症水平,其作用机制可能与TREM2基因抑制TLR4/MyD88信号通路的活化有关。

Objective To investigate the effect of overexpression of triggering receptor expressed on myeloid cells 2(TREM2)on hippocampal inflammation and neuronal apoptosis in epileptic rats.Methods 40 SD male rats were randomly divided into 4 groups:control group,model group,negative control group and TREM2 overexpression group.The expression of TREM2 in brain tissue of rats in each group was detected by real-time quantitative PCR.Hematoxylin-eosin staining was used to detect the pathomorphological changes of hippocampus in each group.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)was used to detect the apoptosis level of hippocampal neurons in each group.The content of tumor necrosis factorα(TNF-α)and interleukin 1β(IL-1β)in brain tissue of rats in each group was detected by enzyme linked immunosorbent assay(ELISA).Western blot and real-time quantitative PCR were used to detect the expression levels of Toll like receptor 4(TLR4),myeloid differentiation factor 88(MyD88)protein and mRNA in the brain of rats in each group.Results Compared with the control group,the expression level of TREM2 mRNA in the model group was significantly lower(P<0.05).After transfection of TREM2 gene into lentivirus vector,the expression level of TREM2 mRNA in brain tissue of rats increased significantly(P<0.05).Compared with the control group,the nucleus of hippocampus neurons in the model group and the negative control group were deformed,the nucleolus disappeared,the cytoplasm was light colored,and the nucleus was pyknosis.The number of apoptotic cells,the content of TNF-α,IL-1βand the expression level of TLR4,MyD88 mRNA and protein were significantly increased(^(all)P<0.05).After overexpression of TREM2,the degree of nuclear pyknosis was improved,the cell morphology was returned to normal,the membrane was clear and nucleolus was clearly visible.Compared with the negative control group,the number of neuron apoptosis,TNF-α,IL-1β,TLR4,MyD88 mRNA and protein expression in the hippocampus of the rats in the TREM2 overexpression group decreased(^(all)P<0.05).Conclusion Overexpression of TREM2 gene can inhibit apoptosis and inflammation of hippocampal neurons,and its mechanism may be related to the inhibition of activation of TLR4/MyD88 signaling pathway by TREM2 gene.