miR-133b-3p对大鼠脑缺血/再灌注损伤的影响及其作用机制

Effect and mechanism of miR-133b-3p oncerebral ischemia/reperfusion injury in rats

目的:探讨miR-133b-3p对脑缺血/再灌注(I/R)损伤大鼠的影响。方法:将36只雄性SD大鼠随机分为6组,每组6只,分别为假手术组、I/R组、I/R+miR-133b-3p激动剂(agomir)组、I/R+miR-133b-3p抑制剂(antagomir)组、I/R+agomir阴性对照(NC)组、I/R+antagomir NC组。采用实时荧光定量PCR(qPCR)法检测大鼠脑组织miR-133b-3p表达水平,苏木精-伊红(HE)染色观察大鼠脑组织病理形态变化,原位末端标记(TUNEL)染色检测神经元细胞凋亡,利用DIANA数据库筛选miR-133b-3p的靶基因,通过生物信息学方法对靶基因的上、下游完整调控机制进行分析。结果:与假手术组比较,I/R组大鼠神经行为学评分显著升高(P<0.01),脑组织miR-133b-3p表达水平明显降低(P<0.05);与相应NC组比较,I/R+agomir组行为学评分显著升高,miR-133b-3p表达明显升高,而I/R+antagomir组miR-133b-3p表达显著降低(均P<0.01)。与I/R组比较,I/R+agomir组神经元损伤严重,神经元细胞萎缩,核深染且形状不规则,细胞凋亡率明显升高(P<0.01);相反,I/R+antagomir组神经元损伤少,神经元细胞核染适中且均匀,神经元凋亡率明显降低(P<0.01)。KEGG通路富集分析发现,靶基因主要作用于自噬信号通路和MAPK信号通路等,上游转录因子和激酶富集结果显示miR-133b-3p主要与UBTF和MAPK1等因子相关。结论:miR-133b-3p可能通过调控自噬通路和MAPK信号通路加重大鼠脑I/R损伤,抑制miR-133b-3p表达对大鼠神经元有保护作用。

Objective:To investigate the effect of miR-133b-3p on cerebral ischemia/reperfusion(I/R)injury in rats Methods:A total of 36 male SD rats were randomly divided into 6 groups:sham-operation group I/R group,I/R+miR-133b-3p agonist(agomir)group,I/R+miR-133b-3p inhibitor(antagomir)group,I/R+agomir negative control(NC)group,I/R+antagomir NC group,with 6 rats in each group.The expression of miR-133b-3p in the brain of rats in each group was detected by RT-qPCR method.The pathomorphology of brain tissues was observed by hematoxylin-eosin(HE)staining.Theneuronal apoptosis was determined by TdT-mediated dUTP Nick-End Labeling(TUNEL)staining.The target genes of miR-133b-3p regulation were screened by DIANA database,and the complete regulation mechanism of upstream and downstream of target genes was analyzed by bioinformatics method.Results:Compared with the sham-operationgroup,the neurobehavioral score was increased,and the expression level of miR-133b-3p in the I/R group was decreased(P<0.05).Compared with NC group,the behavioral score and the miR-133b-3p expression in I/R+agomir group wereincreased,while themiR-133b-3p expressionin I/R+agomir group wasdecreased(P<0.01).In I/R+agomir group,neuronal damage was increased,neuronal cells were atrophied,nuclei were deeply stained and irregular,and the apoptotic rate of cells was increased(P<0.01).On the contrary,there was less neuronal damagein I/R+antagomir group,the neuronal nuclei were moderately stained and evenly stained,and the neuronal apoptosis rate was significantly reduced(P<0.01).KEGG pathway enrichment analysis showed that target genes mainly acted on autophagy signal pathway and MAPK signal pathway.Upstream transcription factors and kinase enrichment results showed that miR-133b-3p was mainly related to factors such as UBTF and MAPK1.Conclusion:MiR-133b-3p may aggravate brain I/R injury in rats by regulating autophagy pathway and MAPK signaling pathway,and inhibition of miR-133b-3p expression has a protective effect on rat neurons.