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 C57BL/6J 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 PI3K 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 3B (LC3B)-Ⅱ/LC3B-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 LC3B-Ⅱ/LC3B-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 LC3B-Ⅱ/LC3B-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 Aβ1-42 in the hippocampus.
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。.
Keywords: Alzheimer’s disease; Autophagy; Hippocampus; Learning-memory ability; Long noncoding RNA H19; Moxibustion; mTOR/TFEB pathway.