摘要: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)·d(-1);雷帕霉素组仅腹腔注射雷帕霉素2 mg·kg(-1);雷帕霉素组仅腹腔注射雷帕霉素2 mg·kg(-1)·d(-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)。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。
摘要:目的:观察针刺对惊厥性脑损伤大鼠不同时点海马神经元内质网Ca(2+)表达、细胞凋亡和Caspase-12蛋白表达的影响,探讨针刺维持内质网钙稳态及抑制细胞凋亡级联反应的可能作用机制。方法:SD大鼠随机分为正常组、模型组、针刺组,每组36只。采用腹腔一次性注射惊厥剂量(50 mg/kg)戊四唑造成大鼠惊厥。针刺组于惊厥后即刻针刺“百会”“大椎”30 min。各组分别于惊厥发作后2、12、48 h采用TUNEL法检测海马神经元细胞凋亡情况;采用内质网Ca(2+)表达、细胞凋亡和Caspase-12蛋白表达的影响,探讨针刺维持内质网钙稳态及抑制细胞凋亡级联反应的可能作用机制。方法:SD大鼠随机分为正常组、模型组、针刺组,每组36只。采用腹腔一次性注射惊厥剂量(50 mg/kg)戊四唑造成大鼠惊厥。针刺组于惊厥后即刻针刺“百会”“大椎”30 min。各组分别于惊厥发作后2、12、48 h采用TUNEL法检测海马神经元细胞凋亡情况;采用内质网Ca(2+)荧光探针技术检测大鼠海马神经元内质网Ca(2+)荧光探针技术检测大鼠海马神经元内质网Ca(2+)表达;采用免疫组织化学法观察各组大鼠海马组织中Caspase-12蛋白表达。结果:与正常组比较,模型组2、12、48 h海马神经元凋亡细胞数明显增加(P<0.01),海马神经元内质网Ca(2+)表达;采用免疫组织化学法观察各组大鼠海马组织中Caspase-12蛋白表达。结果:与正常组比较,模型组2、12、48 h海马神经元凋亡细胞数明显增加(P<0.01),海马神经元内质网Ca(2+)表达降低(P<0.01),Caspase-12蛋白表达升高(P<0.01);与模型组比较,针刺组在惊厥发作2、12、48 h海马神经元凋亡减轻,凋亡细胞数明显减少(P<0.05,P<0.01),海马神经元内质网Ca(2+)表达降低(P<0.01),Caspase-12蛋白表达升高(P<0.01);与模型组比较,针刺组在惊厥发作2、12、48 h海马神经元凋亡减轻,凋亡细胞数明显减少(P<0.05,P<0.01),海马神经元内质网Ca(2+)表达升高(P<0.05,P<0.01),Caspase-12蛋白表达降低(P<0.05,P<0.01)。结论:针刺有利于海马神经元内质网Ca(2+)表达升高(P<0.05,P<0.01),Caspase-12蛋白表达降低(P<0.05,P<0.01)。结论:针刺有利于海马神经元内质网Ca(2+)表达的恢复,从而维持内质网内钙稳态,避免诱发内质网应激介导的Caspase-12凋亡级联反应,减少细胞凋亡,从而治疗惊厥性脑损伤。Objective To observe the effect of acupuncture on endoplasmic reticulum calcium, apoptosis number and Caspase-12 protein expression in hippocampal neurons of convulsive rats, so as to explore its mechanisms underlying improvement of convulsion. Methods SD rats were randomly divided into normal control, model and acupuncture groups, with 36 rats in each group. Rats in the normal control group received intraperitoneal injection(i.p.) of normal saline(2 mL), and those of the other 2 groups received i.p. of pentylenetetrazole(50 mg/kg) for establishing convulsion model. Manual acupuncture stimulation was applied to “Baihui”(GV20) and “Dazhui”(GV14) for 30 min after modeling. The hippocampal tissues were taken at 2, 12 and 48 h after modeling. The endoplasmic reticulum Ca(2+)表达的恢复,从而维持内质网内钙稳态,避免诱发内质网应激介导的Caspase-12凋亡级联反应,减少细胞凋亡,从而治疗惊厥性脑损伤。