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

Abstract

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.

Keywords

• epilepsy
• temporal lobe
• hippocampus
• apoptosis
• disease models
• animal