电针通过mTOR1自噬途径调节改善胰岛素抵抗认知障碍大鼠的学习记忆功能

EA Improving Learning Memory Function via mTOR1 Autophagy Pathway Modulation in IR Rats with Cognitive Impairment

目的:从哺乳动物雷帕霉素靶蛋白1(mTOR1)自噬途径探讨电针治疗胰岛素抵抗(IR)大鼠认知障碍的可能机制。方法:45只Wistar大鼠采用随机数字表法选取10只进行普通饲料喂养命名为正常组,其余35只给予高脂饲料喂养8周,造模结束后检测各组大鼠FINS、FPG,计算IRI,采用Morris水迷宫实验剔除不合格认知功能损害模型,将其余胰岛素抵抗认知障碍大鼠随机分为模型组、假电针组和电针组,电针组电针刺激“百会”“足三里(后)”“中脘”“关元”“丰隆”5穴,“百会穴”向后方平刺2~5 mm,不接电针;假电针组进行针刺后连接电针但不接通电源,每周3次,共8周。干预结束,水迷宫检测大鼠行为学改变;Western blot法检测大鼠下丘脑可溶性淀粉样蛋白(Aβ_(1-42))、海马磷酸化tau蛋白(p-Tau)、S100钙结合蛋白B重组蛋白(S100b)、哺乳动物雷帕霉素靶蛋白1(mTOR1)、WIPI2、自噬相关基因B(Atg13)及Beclin1蛋白的表达;免疫组织化学检测下丘脑阳性表达并计算Aβ_(1-42)与p-Tau平均光密度。结果:与正常组比较,模型组大鼠逃避潜伏期延长,差异具有统计学意义(P<0.01),目标停留象限时间缩短,差异具有统计学意义(P<0.01),穿越平台次数显著减少,差异具有统计学意义(P<0.01),Aβ_(1-42)、p-Tau、S100b及mTOR1蛋白表达水平显著增加,差异具有统计学意义(P<0.01),Beclin1、WIPI2及Atg13蛋白含量相对降低,差异具有统计学意义(P<0.01),模型组大鼠Aβ_(1-42)、p-Tau的阳性表达明显增多,平均光密度明显增高,差异具有统计学意义(P<0.01);与假电针组、模型组比较,电针组大鼠逃避潜伏期显著缩短,差异具有统计学意义(P<0.05或P<0.01),目标停留象限时间增加,差异具有统计学意义(P<0.01),穿越平台次数显著增加,差异具有统计学意义(P<0.01),Aβ_(1-42)、p-Tau、S100b及mTOR1的蛋白表达量降低,差异具有统计学意义(P<0.05或P<0.01),Beclin1、WIPI2及Atg13蛋白表达水平出现了不同程度的增加,差异具有统计学意义(P<0.01),电针组Aβ_(1-42)、p-Tau的阳性表达出现不同程度减少,平均光密度明显降低,差异具有统计学意义(P<0.05或P<0.01)。结论:电针可以有效改善胰岛素抵抗认知障碍大鼠下丘脑区的Aβ_(1-42)、p-Tau及S100b表达水平,降低下丘脑区Aβ_(1-42)与p-Tau阳性表达,其作用机制与电针抑制mTOR1通路激活自噬有关。

Objective:To explore the possible mechanisms of EA intervention in insulin resistance(IR)rats with cognitive impairment from the mTOR1 autophagy pathway.Methods:45 Wistar rats were randomly divided into the normal group(n=10)and the high-fat group(n=35).The rats in the normal group were fed with normal fodder,and the rats in the high-fat group were fed with high-fat fodder.After modeling,FINS and FPG were measured,IRI was calculated,and Morris water maze test was applied to screen out the unsuccessful modeling.The successful models were randomly divided into the model group,the sham EA group and the EA Group.The EA group was treated with electro-needling Baihui(DU20),Zusanli(ST36),Zhongwan(RN12),Guanyuan(RN4)and Fenglong(ST40);DU20 was punctured 2-5 mm in the posterior direction without EA.The sham EA group was treated with acupuncture connected with EA but without electricity,3 times a week for 8 weeks.At the end of intervention,the behavior of rats was detected by water maze test,and the protein expressions of Aβ_(1-42),p-Tau,S100b,mTOR1,WIPI2,Atg13 and Beclin1 in hypothalamus were detected by Western blot.The expressions of Aβ_(1-42)and p-Tau in hypothalamus were detected by immunohistochemistry,and the average optical densities(AODs)of Aβ_(1-42)and p-Tau were calculated.Results:Compared with those in the normal group,the escape latency was prolonged(P<0.01);the time of staying in the target quadrant was shortened(P<0.01);the times of crossing the platform were significantly decreased(P<0.01);the protein expressions of Aβ_(1-42),p-Tau,S100b,mTOR1 were significantly increased(P<0.01);the protein contents of Beclin1,WIPI2 and Atg13 were relatively decreased(P<0.01);the positive expressions of Aβ_(1-42)and p-Tau were significantly increased and the AODs were significantly increased in the model group(P<0.01).Compared with those in the sham EA group and the model group,the escape latency was significantly shortened(P<0.05,P<0.01);the time of staying in the target quadrant was increased(P<0.01);the number of crossing the platform was increased significantly(P<0.01);the protein expressions of Aβ_(1-42),p-Tau,S100b and mTOR1 were decreased(P<0.05,P<0.01);the protein expressions of Beclin1,WIPI2 and Atg13 were increased in different degrees(P<0.01);the positive expressions of Aβ_(1-42)and p-Tau were decreased in different degrees,and the AODs were decreased significantly in the EA group(P<0.05,P<0.01).Conclusion:EA can effectively improve the levels of Aβ_(1-42),p-Tau and S100b in hypothalamus of IR rats with cognitive impairment,and its mechanism may be related to the inhibition of mTOR1 pathway and activation of autophagy.