MiR-132调节PTEN/Akt/Foxo3信号通路对细菌性脑膜炎大鼠神经元凋亡的影响

Effect of MiR-132 on neuronal apoptosis in bacterial meningitis rats by regulating the PTEN/Akt/Foxo3 signaling pathway

目的探究miR-132调节磷酸酯酶-张力蛋白同源基因(PTEN)/蛋白激酶B(AKT)/叉头蛋白3(Foxo3)信号通路对细菌性脑膜炎(BM)大鼠神经元凋亡的影响。方法将60只SD大鼠随机分为对照组(Control组)、模型组(Model组)、激活剂阴性对照组(NC agomir组)、miR-132激活剂组(miR-132 agomir组)和miR-132激活剂+AKT抑制剂MK-2206组(miR-132 agomir+MK-2206组),每组12只。经大鼠小脑延髓池注射B族溶血性链球菌(GBS)建立大鼠BM模型,采用Loeffler神经学评分评估大鼠神经功能损伤情况,采用ELISA检测大鼠脑脊液中白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)水平。将大鼠处死,取海马组织,苏木精-伊红(HE)染色观察病理损伤,TUNEL染色观察神经元凋亡情况;提取大鼠海马组织核酸,采用实时荧光定量PCR(RT-qPCR)检测miR-132水平;提取大鼠海马组织蛋白,采用Western blot检测PTEN/AKT/FOXO3信号通路和凋亡相关蛋白表达情况。结果与Control组相比,Model组大鼠神经功能评分、海马组织miR-132水平、Bcl-2蛋白表达水平、AKT及FOXO3磷酸化水平均显著降低(均P<0.05),脑脊液IL-6和TNF-α水平、海马组织神经元凋亡率、PTEN和Bax蛋白水平均显著升高(均P<0.05)。HE染色观察海马组织脑膜增厚,细胞排列疏松、紊乱,部分核固缩,组织受损严重。与Model组相比,miR-132 agomir组大鼠相关指标变化与上述相反(均P<0.05);海马组织损伤减轻,神经元细胞排列较为整齐,细胞较为完整。TUNEL染色检查大鼠海马组织神经元凋亡率,Model组较Control组显著升高(P<0.05),miR-132 agomir组较Model组和NC agomir组显著降低(P<0.05),miR-132 agomir+MK-2206组较miR-132 agomir组显著升高(P<0.05)。结论miR-132可下调PTEN表达,从而促进AKT与FOXO3的磷酸化水平,抑制BM大鼠的神经元凋亡。

Objective To investigate the effect of miR-132 on neuronal apoptosis in bacterial meningitis(BM)rats by regulating the phosphatase tensin homologous gene(PTEN)/protein kinase B(AKT)/forkhead protein 3(Foxo3)signaling pathway.Methods Sixty SD rats were randomly separated into Control group,Model group,activator negative control group(NC agomir group),miR-132 activator group(miR-132 agomir group),and miR-132 activator+AKT inhibitor MK-2206 group(miR-132 agomir+MK-2206 group),with 12 rats in each group.The injection of group B hemolytic streptococci(GBS)into the cerebellomedullary cistern of rats was used to establish a rat BM model.Loeffler neurological score was applied to evaluate the neurological impairment in rats.Enzyme linked immunosorbent assay(ELISA)was applied to detect levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in cerebrospinal fluid.Rats were euthanized and hippocampal tissue was taken.Hematoxylin-eosin(HE)staining was applied to observe pathological damage in hippocampal tissue.TUNEL staining was applied to observe neuronal apoptosis in hippocampal tissue.Extracted nucleic acid from rat hippocampal tissue and detected miR-132 levels using real-time fluorescence quantitative PCR(RT-qPCR).Extracted proteins from rat hippocampal tissue and used Western blot to detect the expression of PTEN/AKT/FOXO3 signaling pathway and apoptosis related proteins.Results Compared with the Control group,the neural function score,the level of miR-132,the expression of Bcl-2 protein,the phosphorylation levels of AKT and FOXO3 in hippocampal tissues in the Model group were obviously reduced(P<0.05),the levels of IL-6 and TNF-αin cerebrospinal fluid,the rate of neuronal apoptosis,and the expression of PTEN and Bax proteins in hippocampal tissues were obviously increased(P<0.05),the meninges of the hippocampus tissue thickened,the arrangement of cells was loose and disordered,some nuclei pyknosis,and the tissue was severely damaged.Compared with the Model group,the changes in relevant indicators in miR-132 agomir group were opposite to the above(P<0.05),the damage to the hippocampal tissue was alleviated,and the arrangement of neuronal cells was relatively neat and the cells were relatively intact.AKT inhibitor MK-2206 was able to alleviate the inhibitory effect of miR-132 on neuronal apoptosis in BM rats.Conclusion MiR-132 promotes phosphorylation levels of AKT and FOXO3,and inhibits neuronal apoptosis in BM rats by down-regulating PTEN expression.