To observe the effect of moxibustion at the governor vessel on lncRNA-RP4/miR-939-5p and Bnip3 in APP/PS1 double transgenic mice mediated by Wnt/β-catenin pathway, and to explore the mechanism of moxibustion in the treatment of Alzheimer's disease (AD). Sixty 6-month-old APP/PS1 mice were randomly divided into a model group, a rapamycin group, a moxibustion+ 3-methyladenine (3-MA) group and a moxibustion group, with 15 mice in each group. Fifteen C57BL/6J mice of the same age were used as the control group. The rapamycin group was given intraperitoneal injection of rapamycin (2 mg/kg). The moxibustion group was given moxibustion at "Baihui" (GV20),suspended moxibustion at "Fengfu" (GV16) and "Dazhui" (GV14) for 20 min. The moxibustion+3-MA group was injected with 1.5 mg/kg 3-MA on the basis of the moxibustion group. After 6 consecutive treatments, rest for 1 d, and lasted 2 weeks.HEK293T cells were cultured in vitro and transfected with miR-939-5p and its empty plasmid, and transfected with lncRNA-RP4 and Bnip3 wild-type and mutant. HT22 cells cultured in vitro were randomly divided into a control group and a model (Aβ 1-42) group. The lncRNA-RP4 overexpression group, the lncRNA-RP4 knockdown group, the miR-939-5p mimic group, the miR-939-5p inhibitor group, the Bnip3 overexpression group, the Bnip3 knockdown group and the corresponding empty plasmid group were set up, and transfection was performed on the basis of the model group. Morris water maze test was used to detect the learning and memory ability of mice. HE staining was used to observe the morphology of hippocampus in each group. The structure of nerve cells, the number and structure of autophagic vacuoles and autophagic lysosomes in hippocampal CA1 region of mice in each group were observed by transmission electron microscopy. The expression of Aβ 1-42 protein in hippocampus was detected by immunohistochemistry. The expression of mTOR, TFEB, P62, Wnt3 a, β-catenin, GSK-3β, lncRNA-RP4, miR-939-5 p and Bnip3 mRNA in hippocampus of mice in each group was detected by real-time fluorescence quantitative PCR. Western blot was used to detect the expression of mTOR, TFEB, P62, LC3 B-Ⅰ,LC3 B-Ⅱ, CTSB, Lamp1, V-ATPase, Wnt3a, β-catenin, GSK-3β and Bnip3 protein in hippocampus of mice in each group.Dual luciferase assay was used to verify the targeting relationships among lncRNA-RP4, miRNA-939-5p and Bnip3 in HEK293T cells. The concentration of Aβ 1-42 in HT22 cells of each group was detected by ELISA. The expression of lncRNA-RP4, miR-939-5p, Bnip3, Wnt3a, β-catenin and GSK-3β mRNA in HT22 cells of each group was detected by real-time fluorescence quantitative PCR. The expression of Bnip3, Wnt3a, β- catenin and GSK-3β protein in HT22 cells of each group was detected by Western blot. Compared with the model group, the escape latency of the rapamycin group and the moxibustion group was shortened (P<0.05), and the number of crossing the platform was increased (P<0.05).The number of hippocampal neurons was large, and a small amount of cell necrosis was observed. The cells were arranged in an orderly manner with clear boundaries. Some neurons were deformed, atrophied and irregular, and autophagic vacuoles increased. The expression of A β 1-42 protein, mTOR, P62, GSK-3β mRNA and protein, and miR-939-5p mRNA in hippocampus was decreased (P<0.05), while the expression of TFEB, Wnt3a, β-catenin, Bnip3 mRNA and protein, LC3B-Ⅰ, LC3B-Ⅱ, CTSB, Lamp1, V-ATPase protein, and lncRNA-RP4 mRNA was increased (P<0.05). Compared with the rapamycin and moxibustion groups, the escape latency of the moxibustion+3-MA group was prolonged (P<0.05), and the number of crossing the platform was decreased (P<0.05). The number of hippocampal neurons decreased slightly, the cell necrosis was more, the cell arrangement was irregular, the boundary was blurred, and a small amount of autophagic vacuoles and more deformed neurons were occasionally seen. The expression of A β 1-42 protein, mTOR, P62, GSK-3β mRNA and protein, and miR-939-5p mRNA in hippocampus increased (P<0.05), while the expression of TFEB, Wnt3a, β-catenin,Bnip3 mRNA and protein, LC3B-Ⅰ, LC3B-Ⅱ, CTSB, Lamp1, V-ATPase protein, and lncRNA-RP4 mRNA decreased (P<0.05). Dual luciferase assay confirmed that there was a targeting relationship among lncRNA-RP4, miR-939-5p and Bnip3.After the intervention of lncRNA-RP4 in vitro, compared with the model group, the expression of Aβ 1-42 protein,miR-939-5p mRNA, GSK-3 β mRNA and protein in the lncRNA-RP4 overexpression group was decreased (P<0.05), and the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a, β-catenin mRNA and protein was increased (P<0.05). The expression of Aβ 1-42 protein, miR-939-5p mRNA, GSK-3 β mRNA and protein in lncRNA-RP4 knockdown group was increased (P<0.05), while the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a and β-catenin mRNA and protein was decreased (P<0.05). Compared with the lncRNA-RP4 overexpression group, the expression of Aβ 1-42 protein, miR-939-5p mRNA, GSK-3 β mRNA and protein in the lncRNA-RP4 knockdown group was increased (P<0.05), and the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a, β-catenin mRNA and protein was decreased (P<0.05). After intervention with miR-939-5p, compared with the model group, the expression of Aβ 1-42 protein, miR-939-5p mRNA, GSK-3 β mRNA and protein in the miR-939-5p mimic group was increased (P<0.05), and the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a,β-catenin mRNA and protein was decreased (P<0.05). The expression of A β 1-42 protein, miR-939-5p mRNA, GSK-3 β mRNA and protein in miR-939-5p inhibitor group was decreased (P<0.05), while the expression of lncRNA-RP4 mRNA,Bnip3, Wnt3a and β-catenin mRNA and protein was increased (P<0.05). Compared with the miR-939-5p mimic group, the expression of Aβ 1-42 protein, miR-939-5p mRNA, GSK-3β mRNA and protein in the miR-939-5p inhibitor group was decreased (P<0.05), and the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a, β-catenin mRNA and protein was increased (P<0.05). After Bnip3 intervention, compared with the model group, the expression of Aβ 1-42 protein,miR-939-5p mRNA, GSK-3 β mRNA and protein in the Bnip3 overexpression group was decreased (P<0.05), while the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a and β-catenin mRNA and protein was increased (P<0.05). The expression of A β 1-42 protein, miR-939-5p mRNA, GSK-3 β mRNA and protein in the Bnip3 knockdown group was increased (P<0.05), while the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a and β-catenin mRNA and protein was decreased (P<0.05). Compared with the Bnip3 overexpression group, the expression of Aβ 1-42 protein, miR-939-5p mRNA,GSK-3β mRNA and protein in Bnip3 knockdown group was increased (P<0.05), and the expression of lncRNA-RP4 mRNA, Bnip3, Wnt3a, β-catenin mRNA and protein was decreased (P<0.05). Moxibustion at the governor vessel ameliorates AD cognitive deficits by activating the lncRNA-RP4/miR-939-5p/Bnip3 axis, enhancing Wnt/β-catenin pathway, restoring autophagosome-lysosome activity, promoting autophagy, accelerating A β 1-42 clearance, and improve cognitive dysfunction of AD. This study elucidates a novel epigenetic mechanism underlying moxibustion's therapeutic efficacy in AD. 目的：观察艾灸督脉介导长链非编码RNA（lncRNA-RP4）/微小RNA-939-5p（miR-939-5p）及B细胞淋巴瘤-2基因/腺病毒E1B 19 kDa相互作用蛋白3（Bnip3）调控Wnt/β-连环蛋白（β-catenin）通路对APP/PS1双转基因小鼠的影响，探究艾灸治疗阿尔茨海默病（AD）的机制。 方法：将60只6月龄APP/PS1小鼠随机分为模型组、雷帕霉素组、艾灸+3-甲基腺嘌呤（3-MA）组、艾灸组，每组15只；15只同月龄C57BL/6J小鼠作为对照组。雷帕霉素组予腹腔注射雷帕霉素（2 mg/kg），艾灸组予隔附子饼实按灸“百会”，悬灸“风府”“大椎”各20 min，艾灸+3-MA组小鼠在艾灸组基础上按1.5 mg/kg剂量注射3-MA溶液，连续干预6次后休息1 d，共干预2周。体外培养HEK293T细胞，按照转染miR-939-5p及其空载质粒，转染lncRNA-RP4及Bnip3野生型和突变型并进行分组。体外培养HT22细胞随机分为对照组、Aβ 1-42组（模型组）；设置lncRNA-RP4过表达组、lncRNA-RP4敲减组、miR-939-5p模拟物组、miR-939-5p抑制剂组、Bnip3过表达组、Bnip3敲减组及各自对应的空载质粒组，在模型组基础上进行转染。以Morris水迷宫实验检测小鼠学习记忆能力，HE染色观察各组小鼠海马组织形态，透射电镜观察各组小鼠海马CA1区神经元结构、自噬泡及自噬溶酶体数量和结构，免疫组化法检测各组小鼠海马区Aβ 1-42蛋白表达，实时荧光定量PCR法检测各组小鼠海马区哺乳动物雷帕霉素靶蛋白（mTOR）、转录因子EB（TFEB）、泛素结合蛋白P62（P62）、Wnt3a、β-catenin、糖原合酶激酶-3β（GSK-3β）、lncRNA-RP4、miR-939-5p、Bnip3 mRNA的表达，Western blot法检测各组小鼠海马区mTOR、TFEB、P62、微管相关蛋白1轻链3B-Ⅰ（LC3B-Ⅰ）、微管相关蛋白1轻链3B-Ⅱ（LC3 B-Ⅱ）、组织蛋白酶B（CTSB）、溶酶体相关膜蛋白1（Lamp1）、V型ATP酶（V-ATPase）、Wnt3a、β-catenin、GSK-3β、Bnip3蛋白表达，双荧光素酶实验验证各组HEK293T细胞中lncRNA-RP4、miR-939-5p、Bnip3之间的靶向关系，ELISA法检测各组HT22细胞Aβ 1-42蛋白浓度，实时荧光定量PCR法检测各组HT22细胞lncRNA-RP4、miR-939-5p、Bnip3、Wnt3a、β-catenin、GSK-3β mRNA表达，Western blot法检测各组HT22细胞Bnip3、Wnt3a、β-catenin、GSK-3β蛋白表达。 结果：与模型组比较，雷帕霉素组、艾灸组逃避潜伏期缩短（P<0.05），穿越平台次数增加（P<0.05）；海马神经元细胞数量较多，见少量的细胞坏死，细胞排列较有序、界线较清晰，部分神经元变形、萎缩和不规则，自噬泡增多；海马Aβ 1-42蛋白表达，mTOR、P62、GSK-3β mRNA和蛋白表达，miR-939-5p mRNA表达均降低（P<0.05），TFEB、Wnt3a、β-catenin、Bnip3 mRNA和蛋白表达、LC3B-Ⅰ、LC3B-Ⅱ、CTSB、Lamp1、V-ATPase蛋白表达，lncRNA-RP4 mRNA表达均升高（P<0.05）。与雷帕霉素组及艾灸组比较，艾灸+3-MA组逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05）；海马神经元细胞数量小幅度减少，细胞坏死较多，细胞排列较不规则、界线较模糊，偶见少量自噬泡和较多变形神经元；海马Aβ 1-42蛋白表达，mTOR、P62、GSK-3β mRNA和蛋白表达，miR-939-5p mRNA表达均升高（P<0.05）；TFEB、Wnt3a、β-catenin、Bnip3 mRNA和蛋白表达，LC3B-Ⅰ、LC3 B-Ⅱ、CTSB、Lamp1、V-ATPase蛋白表达，lncRNA-RP4 mRNA表达均降低（P<0.05）。双荧光素酶实验证实lncRNA-RP4、miR-939-5p、Bnip3之间存在靶向关系。体外实验干预lncRNA-RP4后，与模型组比较，lncRNA-RP4过表达组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均降低（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均升高（P<0.05）；lncRNA-RP4敲减组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均升高（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均降低（P<0.05）。与lncRNA-RP4过表达组比较，lncRNA-RP4敲减组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均升高（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均降低（P<0.05）。干预miR-939-5p后，与模型组比较，miR-939-5p模拟物组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均升高（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均降低（P<0.05）；miR-939-5p抑制剂组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均降低（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均升高（P<0.05）。与miR-939-5p模拟物组比较，miR-939-5p抑制剂组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均降低（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均升高（P<0.05）。干预Bnip3后，与模型组比较，Bnip3过表达组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均降低（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均升高（P<0.05）；Bnip3敲减组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达及GSK-3β mRNA和蛋白表达均升高（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均降低（P<0.05）。与Bnip3过表达组比较，Bnip3敲减组Aβ 1-42蛋白浓度、miR-939-5p mRNA表达、GSK-3β mRNA和蛋白表达均升高（P<0.05），lncRNA-RP4 mRNA表达及Bnip3、Wnt3a、β-catenin mRNA和蛋白表达均降低（P<0.05）。 结论：艾灸督脉可通过调节lncRNA-RP4/miR-939-5p/Bnip3轴激活Wnt/β-catenin通路，改善自噬溶酶体活性，促进细胞自噬，加速Aβ 1-42清除，改善AD认知功能障碍。.
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