抑制miRNA-193a-5p表达对癫痫模型大鼠海马神经元保护机制研究

Effect of inhibiting the expression of miRNA-193a-5p on hippocampal neuron protection in epileptic model rats

目的探讨抑制miRNA-193a-5p表达对癫痫大鼠海马组织神经元损伤和凋亡的保护作用及其在颞叶癫痫发病机制中的作用。方法采用氯化锂-匹罗卡品制备癫痫大鼠模型,随机分为模型组、miRNA-193a-5p抑制剂组(miRNA-193a-5p antagomir组)及其阴性对照组(antagomir-NC组)。分别于建模后第1、3和7天时记录模型大鼠单位时间内异常脑电波次数;第7天经酶联免疫吸附试验检测海马组织炎性因子[白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)]表达变化,化学比色法分析超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量,TUNEL染色检测神经元凋亡数目,荧光定量聚合酶链反应和Western blotting法分别检测miRNA-193a-5p和G蛋白耦联受体39(GPR39)mRNA,以及cleaved Caspase-3、Bax、Bcl-2和GPR39蛋白相对表达量,双荧光素酶报告实验验证miRNA-193a-5p与GPR39的靶向作用关系。结果(1)各项指标在不同处理组和各观察时间点之间的变化差异均具有统计学意义(均P=0.000)。与正常对照组相比,模型组、antagomir-NC组和miRNA-193a-5p antagomir组miRNA-193a-5p相对表达量(均P=0.000),炎性因子[IL-1β(均P<0.05)、IL-6(均P=0.000)和TNF-α(均P=0.000)]表达水平,MDA含量(均P<0.01),神经元凋亡数目(均P=0.000),cleaved Caspase-3(均P=0.000)和Bax蛋白(均P=0.000)相对表达量升高,而SOD活性(均P<0.01)、Bcl-2蛋白相对表达量(均P=0.000)、GPR39 mRNA(均P=0.000)和GPR39蛋白(均P<0.01)相对表达量降低。与模型组和antagomir-NC组相比,miRNA-193a-5p antagomir组miRNA-193a-5p相对表达量(均P=0.000),炎性因子[IL-1β(均P=0.000)、IL-6(均P=0.000)和TNF-α(均P<0.01)]表达水平,MDA含量(均P=0.000),神经元凋亡数目(均P=0.000),cleaved Caspase-3(均P=0.000)和Bax蛋白(均P=0.000)相对表达量,以及第1、3和7天脑电波异常次数减少或降低(均P<0.05),而SOD活性(均P=0.000)、Bcl-2蛋白相对表达量(均P=0.000)、GPR39 mRNA(均P=0.000)和GPR39蛋白(均P=0.000)相对表达量升高。(2)经双荧光素酶报告实验验证,miRNA-193a-5p mimic组人胚肾细胞293T(HEK293T)转染GPR39-WT后荧光素酶活性降低且低于mimic-NC组(t=16.340,P=0.000)。结论抑制miRNA-193a-5p表达可以降低癫痫大鼠海马组织氧化应激和炎症反应,抑制海马组织神经元凋亡,其机制可能与靶向上调GPR39表达有关。

Objective To investigate the effect of inhibiting the expression of miRNA-193a-5p on neuronal damage and apoptosis in hippocampus of epileptic rats,and to explore its role in the pathogenesis of temporal lobe epilepsy.Methods Epileptic rat models were prepared with lithium chlorine-pilocarpine and randomly divided into model group,miRNA-193a-5p inhibitor group(miRNA-193a-5p antagomir group)and negative control group(antagomir-NC group).The frequency of abnormal brainwaves per unit time was recorded at first,3th and 7th day after model preparation.On the 7th day,the expression of hippocampal inflammatory factors[interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)]were detected by enzyme-linked immunosorbent assay(ELISA),the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)were detected by chemical colorimetry,and the number of neuronal apoptosis was calculated by TUNEL staining.The relative expression of miRNA-193a-5p and G-protein coupled receptor 39(GPR39),and expression of cleaved Caspase-3,Bax,Bcl-2,and GPR39 proteins were detected by fluorescence quantitative polymerase chain reaction(PCR)and Western blotting,respectively.Double luciferase reporter assay confirmed the targeting relationship between miRNA-193a-5p and GPR39.Results 1)There were statistically significant differences in each index in different treatment groups and at each observation time point(P=0.000,for all).Compared with normal control group,the relative expression of miRNA-193a-5p in model group,antagomir-NC group and miRNA-193a-5p antagomir group(P=0.000,for all),the expression of inflammatory factors[IL-1β(P<0.05,for all),IL-6(P=0.000,for all)and TNF-α(P=0.000,for all)],the content of MDA(P<0.01,for all),the number of apoptosis of neurons(P=0.000,for all),the relative expression of cleaved Caspase-3(P=0.000)and Bax(P=0.000)increased.The relative expression of SOD activity(P<0.01,for all),Bcl-2 relative expression(P=0.000,for all),GPR39 mRNA(P=0.000,for all)and GPR39 protein(P<0.01,for all)were decreased.Compared with model group and antagomir-NC group,the relative expression of miRNA-193a-5p in antagomir group(P=0.000,for all),the expression of inflammatory cytokines[IL-1β(P=0.000,for all),IL-6(P=0.000,for all)and TNF-α(P<0.01,for all)],the content of MDA(P=0.000,for all),the number of neuronal apoptosis(P=0.000,for all),the relative expression of cleaved Caspase-3(P=0.000,for all)and Bax(P=0.000,for all),as well as the number of abnormal brain waves at day 1,3 and 7(P<0.05,for all)were decreased.The relative expression of SOD activity(P=0.000,for all),Bcl-2(P=0.000,for all),GPR39 mRNA(P=0.000,for all)and GPR39 protein(P=0.000,for all)increased.2)Double luciferase assay showed the luciferase activity of HEK293T cells transfected with miRNA-193a-5p mimic was decreased after transfection of GPR39-WT,and was lower than that of mimic-NC group(t=16.340,P=0.000).Conclusions Inhibiting the expression of miRNA-193a-5p can reduce the expression of oxidative stress and inflammation in the hippocampus of epileptic rats,and inhibit hippocampal neuron apoptosis.The mechanism may be related to the targeted up-regulation of GPR39 expression.