【摘要】 目的:研究微小RNA let-7a对脑出血(ICH)大鼠神经元凋亡的影响及其分子机制。方法:从48只健康SD大鼠中随机选取8只作为假手术组,将2μLⅦ型胶原酶注入其余大鼠苍白球以构建ICH模型。将造模后的大鼠随机分为模型(2μL生理盐水)组、let-7a激动剂(agomir)组、阴性对照(NC)agomir组、let-7a拮抗剂(antagomir)组和NC antagomir组,每组8只,在侧脑室注射相应药物。7 d后进行神经功能评分;HE染色观察病理损伤;RT-qPCR和Western blot检测相关蛋白表达水平;TUNEL染色检测神经元凋亡情况;利用生物信息学软件预测let-7a与丝裂原活化的蛋白激酶激酶激酶激酶3(MAP4K3)的靶向关系,并在HEK293T细胞中用双萤光素酶报告基因实验进一步验证。结果:动物实验中,let-7a过表达时,神经功能评分降低,病理损伤减轻,胶质细胞原纤维酸性蛋白(GFAP)低表达,神经元凋亡减少,cleaved caspase-3和cleaved PARP低表达(P<0. 05)。细胞实验中,MAP4K3是let-7a的靶基因之一,且两者为负向调控;let-7a过表达抑制MAP4K3/MKK4/JNK的表达。结论:let-7a通过抑制MAP4K3/MKK4/JNK信号通路,减少ICH大鼠神经元凋亡。更多还原

【Abstract】 AIM:To investigate the effect of microRNA let-7a on neuronal apoptosis in a rat model of intracerebral hemorrhage(ICH).METHODS:Forty-eight healthy SD rats were selected,8 of which served as sham group,and 2 μL of type VII collagenase was injected into globus pallidus of the remaining rats to construct ICH model.These ICH rats were randomly divided into model(2 μL normal saline) group,let-7a agomir group,NC agomir group,let-7 a antagomir group and NC antagomir group,with 8 rats in each group,and the corresponding drugs were injected into the lateral ventricle.After 7 d,the neurological function scoring was performed,and HE staining was used to observe the pathological damage.RT-qPCR and Western blot were used to detect the expression of related proteins.TUNEL staining was used to detect neuronal apoptosis.The targeting relationship between let-7a and mitogen-activated protein kinase kinase kinase kinase 3(MAP4K3) was predicted by bioinfomatic software,and further verified by dual-luciferase reporter assay.RESULTS:In animal experiments,when let-7 a was over-expressed,neurological function scores were reduced,and the pathological damage was alleviated.Glial fibrillary acidic protein(GFAP) expression was down-regulated,neuronal apoptosis was reduced,and the protein levels of cleaved caspase-3 and cleaved PARP were decreased(P<0.05).In cell assays,MAP4K3 was one of the target genes of let-7 a,and MAP4K3 was negatively regulated by let-7a.let-7a overexpression inhibited the expression of MAP4K3/MKK4/JNK.CONCLUSION:let-7a reduces neuronal apoptosis in ICH rats by inhibiting the MAP4K3/MKK4/JNK signaling pathway.更多还原