miR-433通过JAK2/STAT3信号通路调控氧化应激干预术后认知功能障碍的实验研究

Experimental Study of miR-433 in Regulating Oxidative Stress through JAK2/STAT3 Signaling Pathway to Intervene in Postoperative Cognitive Impairment

目的:探讨miR-433对术后认知功能障碍(POCD)大鼠海马氧化应激和认知功能的影响及其作用机制。方法:将大鼠随机分为Control组、POCD组、POCD+NC mimic组、POCD+miR-433 mimic组。构建大鼠POCD模型,采用实时荧光定量聚合酶链式反应(RT-qPCR)检测miR-433在POCD大鼠海马组织中的表达;Morris水迷宫试验评价大鼠认知功能;苏木精-伊红(HE)染色检测海马组织病理学变化;原位末端凋亡(TUNEL)染色检测细胞凋亡情况;采用酶联免疫吸附(ELISA)法检测氧化应激水平;双荧光素酶报告基因系统检测miR-433和酪氨酸激酶2(JAK2)的靶向关系;免疫印迹法(Western Blot)检测海马组织Cleaved Caspase-3、Bad、Bcl-2、JAK2、p-JAK2、转录激活因子3(STAT3)、p-STAT3蛋白表达。结果:与Control组比较,POCD组大鼠海马组织miRNA-433表达降低,逃避潜伏期延长,穿越平台次数减少,POCD组神经细胞紊乱,细胞数量减少,神经元细胞凋亡率、Cleaved Caspase-3和Bad蛋白表达升高,Bcl-2蛋白表达、超氧化物歧化酶(SOD)水平降低,丙二醛(MDA)水平升高,差异均有统计学意义(P<0.05)。与POCD+NC mimic组比较,POCD+miR-433 mimic组大鼠海马组织中miRNA-433表达升高,逃避潜伏期缩短,穿越平台次数增加,海马内细胞排列较规则,坏死神经细胞数量减少,神经元细胞凋亡率、Cleaved Caspase-3和Bad蛋白表达降低,Bcl-2蛋白表达和SOD水平升高,MDA水平降低,差异均有统计学意义(P<0.05)。双荧光素酶报告基因实验结果显示:JAK2是miRNA-433的靶基因,与POCD+miR-433 mimic+Vector组比较,POCD+miR-433 mimic+pcDNA-JAK2组大鼠海马组织p-JAK2、p-STAT3、Cleaved Caspase-3、Bad蛋白及MDA水平升高,Bcl-2蛋白和SOD水平降低,差异有统计学意义(P<0.05)。结论:miR-433通过JAK2/STAT3信号通路抑制氧化应激,从而预防大鼠发生术后认知功能障碍。

Objective: To investigate the effects of miR-433 on oxidative stress and cognitive function in the hippocampus of rats with postoperative cognitive impairment(POCD) and its mechanism.Methods: The rats were randomly divided into the Control group, POCD group, POCD+NC mimic group, and POCD+miR-433 mimic group.Rat POCD model was established.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect miR-433 expression in hippocampal tissue of POCD rats, Morris water maze test was used to evaluate cognitive function in rats, hematoxylin-eosin(HE) staining was used to detect histopathological changes in hippocampus, terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) staining was used to detect apoptosis, enzyme-linked immunosorbent assay(ELISA) was used to detect oxidative stress levels, dual luciferase reporter gene system was used to detect the targeting relationship between miR-433 and tyrosine kinase 2(JAK2),Western Blot was used to detect expressions of Cleaved Caspase-3,Bad, Bcl-2,JAK2,p-JAK2,transcription activator 3(STAT3),and p-STAT3 protein in hippocampal tissue.Results: Compared with the Control group, the expression of miR-433 in the hippocampus tissue of the POCD group was significantly decreased, the escape latency was prolonged, the time spent in the target quadrant and the number of times to cross the target platform were decreased.The cells in the hippocampus were arranged more regularly, the number of necrotic nerve cells, the apoptosis rate of neuronal cells, the expression of Cleaved Caspase-3 and Bad protein, and the level of malondialdehyde(MDA) were significantly deceased, while the expression of Bcl-2 protein, and the level of superoxide dismutase(SOD) were significantly increased(P<0.05).Compared with the POCD+NC mimic group, the expression of miR-433 in the hippocampus tissue of the POCD+miR-433 mimic group was significantly increased, the escape latency was shortened, the time spent in the target quadrant and the number of times to cross the target platform were increased.The cells in the hippocampus were arranged more regularly and the number of necrotic nerve cells was significantly decreased.The apoptosis rate of neuronal cells, the expressions of Cleaved Caspase-3,Bad and Bcl-2 protein, and the level of MDA were significantly increased, while the level of SOD was significantly increased(P<0.05).The results of dual luciferase reporter gene assay showed that JAK2 was the target gene of miRNA-433.Compared with the POCD+miR-433 mimic+Vector group, the levels of p-JAK2,p-STAT3,Cleaved Caspase-3,Bad protein and MDA in hippocampal tissue of the POCD+miR-433 mimic+pcDNA-JAK2 group were increased, while the levels of Bcl-2 protein, and SOD were decreased(P<0.05).Conclusion: The results suggested that miR-433 could inhibit oxidative stress through the JAK2/STAT3 signaling pathway, thereby preventing postoperative cognitive impairment in rats.