地黄多糖调控环状RNA 0010729/miR-326通路对海马神经元缺氧复氧损伤的影响

Effects of Rehmanniae Radix polysaccharides on hypoxia-reoxygenation-induced hippocampal neurons injury by regulating circ_0010729/miR-326 pathway

目的探讨地黄多糖对海马神经元缺氧复氧损伤的影响及其保护机制是否与调控环状RNA 0010729(circ_0010729)和微小RNA-326(microRNA-326,miR-326)表达有关。方法体外培养大鼠海马神经元建立缺氧复氧损伤模型;将大鼠海马神经元分为对照组、模型组、模型+地黄多糖低剂量组、模型+地黄多糖中剂量组、模型+地黄多糖高剂量组、模型+小干扰RNA阴性对照(Small interfering RNA negative control, si-NC)组、模型+si-circ_0010729组、模型+地黄多糖高剂量+空载质粒(Empty plasmid, pcDNA)组、模型+地黄多糖高剂量+pcDNA-circ_0010729组;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(Malonaldehyde, MDA)水平和超氧化物歧化酶(Superoxide dismutase, SOD)活性;实时定量聚合酶链式反应(Polymerase chain reaction, PCR)检测circ_0010729,miR-326表达水平;荧光素酶报告实验确定circ_0010729和miR-326靶向关系。结果与对照组比较,模型组细胞凋亡率、MDA水平、circ_0010729表达水平升高(P<0.05),SOD活性、miR-326表达水平降低(P<0.05);与模型组比较,模型+地黄多糖低剂量组、模型+地黄多糖中剂量组、模型+地黄多糖高剂量组细胞凋亡率、MDA水平、circ_0010729表达水平降低(P<0.05),SOD活性、miR-326表达水平升高(P<0.05);与模型+si-NC组比较,模型+si-circ_0010729组细胞凋亡率、MDA水平降低(P<0.05),SOD活性升高(P<0.05);与模型+地黄多糖高剂量+pcDNA组比较,模型+地黄多糖高剂量+pcDNA-circ_0010729组细胞凋亡率、MDA水平升高(P<0.05),SOD活性降低(P<0.05)。结论地黄多糖能够有效抑制缺氧复氧诱导的大鼠海马神经元凋亡和氧化损伤,其机制可能与抑制circ_0010729/miR-326通路有关。

Objective To investigate the effects of Rehmanniae Radix polysaccharides on the hypoxia-reoxygenation induced hippocampal neurons injury, and further to study whether its protective mechanism is related to the regulation of circular RNA 0010729(circ_0010729) and miR-326. Methods Rat hippocampalneuronal cells were cultured in vitro to establish hypoxia-reoxygenation injury model. The rat hippocampal neuron cells were divided into control group, model group, model+Rehmanniae Radix polysaccharides low-dose group, model+Rehmanniae Radix polysaccharides middle-dose group, model+Rehmanniae Radix polysaccharides high-dose group, model+si-NC group, model+si-circ_0010729 group, model+Rehmanniae Radix polysaccharides high dose+pcDNA group, model+Rehmanniae Radix polysaccharides high dose+pcDNA-circ_0010729 group. Flow cytometry was applied to detect cell apoptosis;Commercial kit was used to detect malondialdehyde(MDA) content and superoxide dismutase(SOD) activity;real-time quantitative PCR was selected to calculated the expression of circ_0010729 and miR-326. Luciferase reporter experiment was used to confirm the targeting relationship between circ_0010729 and miR-326. Results Compared with the control group, the apoptosis rate, MDA content, and expression of circ_0010729 in the model group were increased(P<0.05), while SOD activity and miR-326 expression were decreased(P<0.05). Compared with the model group, the apoptosis rate, MDA content, circ_0010729 expression in the model+Rehmanniae Radix polysaccharides low dose group, model+Rehmanniae Radix polysaccharides medium dose group, model+Rehmanniae Radix polysaccharides high dose group were decreased(P<0.05), SOD activity and miR-326 expression were increased(P<0.05). Compared with the model+si-NC group, the apoptosis rate and MDA content of the model+si-circ_0010729 group were decreased(P<0.05), and the SOD activity was increased(P<0.05). Compared with the model+Rehmanniae Radix polysaccharides high dose+pcDNA group, the apoptosis rate and MDA content of the model+Rehmanniae Radix polysaccharides high dose+pcDNA-circ_0010729 group were increased(P<0.05), and SOD activity was decreased(P<0.05). Conclusion Rehmanniae Radix polysaccharides can effectively inhibit the apoptosis and oxidative damage of rat hippocampal neurons induced by hypoxia and reoxygenation, and its mechanism may be related to the inhibition of circ_0010729/miR-326 pathway.