小鼠围术期神经认知障碍时海马miR-3065-5p与IGF-1/PI3K/Akt信号通路的关系

Relationship between hippocampal miR-3065-5p and IGF-1/PI3K/Akt signaling pathway in a mouse model of perioperative neurocognitive disorder

目的评价小鼠围术期神经认知障碍(PND)时海马miR-3065-5p与胰岛素样生长因子-1/磷脂酰肌醇3-激酶/丝氨酸-苏氨酸蛋白激酶(IGF-1/PI3K/Akt)信号通路的关系。方法清洁级健康雄性C75BL/6小鼠80只,12~14周龄,体质量20~30 g,采用随机数字表法分为4组(n=20):对照组(C组)、PND组、miR-3065-5p激动剂组(Ag组)和miR-3065-5p激动剂阴性对照组(Ag-NC组)。采用吸入1.5%异氟烷麻醉下胫骨骨折髓内固定术制备小鼠PND模型。于术前2 d,Ag组侧脑室注射miR-3065-5p agomir 2μl,Ag-NC组注射miR-3065-5p agomir阴性对照2μl。于术后7 d时行Morris水迷宫实验和旷场实验,测试结束后麻醉处死取海马组织,采用qRT-PCR法检测miR-3065-5p、IGF-1 mRNA和Bcl-2 mRNA的表达水平,采用Western blot法检测IGF-1、磷酸化Akt(p-Akt)、磷酸化糖原合成酶激酶3β(p-GSK3β)和Bcl-2的表达水平。结果4组旷场实验各指标比较差异无统计学意义(P>0.05)。与C组比较,其余3组术后逃避潜伏期延长,目标象限停留时间百分比降低,穿越原平台位置次数减少,海马组织miR-3065-5p表达上调,IGF-1 mRNA、Bcl-2 mRNA、IGF-1、p-Akt、p-GSK3β和Bcl-2表达下调(P<0.05);与PND组和Ag-NC组比较,Ag组术后逃避潜伏期延长,目标象限停留时间百分比降低,穿越原平台位置次数减少,miR-3065-5p表达上调,IGF-1 mRNA和Bcl-2 mRNA、IGF-1、p-Akt、p-GSK3β和Bcl-2表达下调(P<0.05)。结论海马miR-3065-5p表达上调可抑制IGF-1/PI3K/Akt信号通路激活,可能是小鼠PND发生机制之一。

Objective To evaluate the relationship between hippocampal miR-3065-5p and insulin-like growth factor-1/phosphatidylinositol 3-kinase/protein kinase B(IGF-1/PI3K/Akt)signaling pathway in a mouse model of perioperative neurocognitive disorder(PND).Methods Eighty clean-grade healthy male C75BL/6 mice,aged 12-14 weeks,weighing 20-30 g,were divided them into 4 groups(n=20 each)using the random number table method:control group(C group),PND group,miR-3065-5p agonist group(Ag group)and miR-3065-5p agonist negative control group(Ag-NC group).PND model was prepared by internal fixation of tibial fracture under anesthesia with 1.5%isoflurane.Two days before developing the model,miR-3065-5p agomir 2μl was injected into the lateral ventricle in Ag group,miR-3065-5p agomir negative control 2μl was injected into the lateral ventricle in Ag-NC group.Morris water maze test and open field test were performed at 7 days after surgery.The mice were sacrificed after the end of test,and hippocampal tissues were obtained for determination of the expression of miR-3065-5p,IGF-1 mRNA and Bcl-2 mRNA(by quantitative real-time polymerase chain reaction)and expression of IGF-1,phosphorylated Akt(p-Akt),phosphorylated glycogen synthase kinase-3β(p-GSK3β)and Bcl-2(by Western blot).Results There was no significant difference in each parameter in the open field test among the four groups(P>0.05).Compared with group C,the postoperative escape latency was significantly prolonged,the percentage of time of stay at the target quadrant was decreased,the number of crossing the original platform was reduced,the expression of miR-3065-5p was up-regulated,and the expression of IGF-1 mRNA,Bcl-2 mRNA,IGF-1,p-Akt,p-GSK3βand Bcl-2 was down-regulated in the other three groups(P<0.05).Compared with PND group and Ag-NC group,the postoperative escape latency was significantly prolonged,the percentage of time of stay at the target quadrant was decreased,the number of crossing the original platform was reduced,the expression of miR-3065-5p was up-regulated,and the expression of IGF-1 mRNA,Bcl-2 mRNA,IGF-1,p-Akt,p-GSK3βand Bcl-2 was down-regulated in Ag group(P<0.05).Conclusions Up-regulation of miR-3065-5p can inhibit the activation of IGF-1/PI3K/Akt signaling pathway,which might be one of the mechanisms of PND developed in mice.