艾灸督脉对APP/PS1双转基因小鼠mTOR/TFEB通路介导的自噬溶酶体功能及lncRNA H19表达的影响

Effect of moxibustion on autophagy lysosome function mediated by mTOR/TFEB pathway and lncRNA H19 expression in APP/PS1 double transgenic mice

目的:观察艾灸督脉穴对APP/PS1双转基因小鼠自噬溶酶体功能及长链非编码RNA H19(lncRNA H19)的影响,探讨艾灸治疗阿尔茨海默病(AD)的作用机制。方法:6月龄健康雄性APP/PS1双转基因小鼠随机分为模型组、艾灸组、艾灸+抑制剂组和雷帕霉素组,每组13只,同时选取13只6月龄健康雄性C57BL/6J小鼠作为对照组。艾灸组予隔附子饼灸“百会”及悬灸“大椎”“风府”,15 min/d;艾灸+抑制剂组在艾灸组基础上给予腹腔注射3-甲基腺嘌呤1.5 mg·kg^(-1)·d^(-1);雷帕霉素组仅腹腔注射雷帕霉素2 mg·kg^(-1)·d^(-1)。以上各组均每日干预1次,共2周。以Morris水迷宫实验检测干预前后小鼠学习记忆能力;透射电镜观察小鼠海马组织自噬体形成;免疫组织化学法检测小鼠海马区β-淀粉样蛋白(Aβ_(1-42))蛋白表达;荧光定量PCR法检测小鼠海马组织lncRNA H19、哺乳动物雷帕霉素靶蛋白(mTOR)、转录因子EB(TFEB)、溶酶体水解酶Cathepsin D、溶酶体关联膜蛋白1(LAMP1)mRNA的表达;Western blot法检测小鼠海马组织mTOR、TFEB、Cathepsin D、LAMP1、自噬标志物微管相关蛋白1轻链3B(LC3B)-Ⅱ/LC3B-Ⅰ、p62蛋白表达。结果:治疗前,与正常组比较,模型组、艾灸组、艾灸+抑制剂组和雷帕霉素组逃避潜伏期延长(P<0.05)。治疗后,与正常组比较,模型组小鼠逃避潜伏期延长(P<0.05);海马神经元细胞质内自噬泡减少,自噬溶酶体结构破坏;Aβ_(1-42)蛋白表达、lncRNA H19表达、mTOR mRNA和蛋白表达、p62蛋白表达均升高(P<0.05),TFEB、Cathepsin D、LAMP1 mRNA和蛋白表达及LC3B-Ⅱ/LC3B-Ⅰ均降低(P<0.05)。与模型组和艾灸+抑制剂组比较,艾灸组和雷帕霉素组逃避潜伏期缩短(P<0.05);海马神经元细胞质内自噬泡较多,自噬溶酶体结构清晰完整;Aβ_(1-42)蛋白表达、lncRNA H19表达、mTOR mRNA和蛋白表达、p62蛋白表达均降低(P<0.05),TFEB、Cathepsin D、LAMP1 mRNA和蛋白表达及LC3B-Ⅱ/LC3B-Ⅰ均升高(P<0.05)。结论:艾灸督脉可能通过下调lncRNA H19表达抑制mTOR/TFEB通路,改善AD小鼠自噬溶酶体功能,恢复自噬流,促进细胞自噬清除脑内Aβ,进而改善认知功能。

Objective To observe the effect of moxibustion(Moxi)at acupoints of Governor Vessel on autophagy lysosomal function and lncRNA H19 in amyloid precursor protein/presenilin 1(APP/PS1)double transgenic Alzheimer’s disease(AD)mice,so as to explore its underlying mechanisms in relieving AD.Methods Fifty two male APP/PS1 double transgenic AD mice were randomly divided into model,Moxi,Moxi+inhibitor and medication(rapamycin)groups,with 13 mice in each group.Other 13 male C57 BL/6 J mice of the same age were selected as the control group.The mice of the Moxi group received aconite cake-separated Moxi stimulation at“Baihui”(GV20),“Dazhui”(GV14)and“Fengfu”(GV16),for 15 min,those of the Moxi+inhibitor group received intraperitoneal injection of 3-methyladenine(an inhibitor of PI3 K for suppressing autophagy)1.5 mg·kg^(-1)·d^(-1)on the basis of Moxi,and those of the medication group received intraperitoneal injection of rapamycin 2 mg·kg^(-1)·d^(-1).The treatment was conducted once daily for 2 weeks.The mouse’s learning-memory ability was detected by Morris water maze tests.The hippocampus tissue was sampled for observing the formation of autophagy by using transmission electron microscope,detecting the expression of Aβ_(1-42)protein with immunohistochemical staining,and for detecting the expression levels of long noncoding RNA H19(lncRNA H19),mammalian target of rapamycin kinase(mTOR),nuclear transcription factor EB(TFEB),Cathepsin D and lysosome associated membrane protein-1(LAMP1)genes and proteins as well as microtubule associated protein 1 light chain 3 B(LC3 B)-Ⅱ/LC3 B-Ⅰand autophagy protein p62 protein by quantitative real-time PCR and Western blot,respectively.Results In contrast to the control group,the model group had an evident increase in the escape latency of Morris water maze test,and in the expression levels of Aβ_(1-42)protein,lncRNA H19 mRNA,mTOR mRNA and protein,and p62 protein(P<0.05),and a significant decrease in the expression levels of TFEB,Cathepsin D,LAMP1 mRNAs and proteins and LC3 B-Ⅱ/LC3 B-Ⅰ(P<0.05).After the treatment and relevant to the model and Moxi+inhibitor groups,both the Moxi and medication groups had an obvious down-regulation in the levels of latency of Morris water maze,expression levels of Aβ_(1-42)protein,lncRNA H19 mRNA,mTOR mRNA and protein,and p62 protein(P<0.05),and a significant up-regulation in the levels of TFEB,Cathepsin D,LAMP1 mRNAs and proteins and LC3 B-Ⅱ/LC3 B-Ⅰ(P<0.05).Conclusion Moxi at acupoints of Governor Vessel can improve cognitive function of AD mice,which may be associated with its functions in inhibiting mTOR/TFEB pathway by down-regulating the expression of lncRNA H19,improving autophagy lysosomal function,promoting autophagy and clearing away Aβin the hippocampus.