摘要:目的:观察电针对衰老小鼠体力及肝脏AMP活化的蛋白激酶(AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、unc-51样自噬激活激酶1(ULK1)、Atg5、Atg7、Atg13和Beclin1表达的影响,探索其通过激活AMPK/mTOR/ULK1通路延缓衰老的机制。方法:将30周龄雄性SAMP8小鼠随机分为模型组、雷帕霉素组、电针组、电针+抑制剂组,每组6只,以同周龄抗快速衰老SAMR1小鼠6只作为对照组。雷帕霉素组予以腹腔注射诱导自噬药物雷帕霉素(2 mg·kg(-1)·d(-1)·d(-1));电针组予以电针双侧"太冲""肾俞",每次15 min;电针+抑制剂组电针治疗前予以腹腔注射自噬抑制剂3-甲基腺嘌呤(1.5 mg·kg(-1));电针组予以电针双侧"太冲""肾俞",每次15 min;电针+抑制剂组电针治疗前予以腹腔注射自噬抑制剂3-甲基腺嘌呤(1.5 mg·kg(-1)·d(-1)·d(-1))。各组均每周干预6 d,连续2周。采用力竭游泳实验观察小鼠体力变化;HE染色法观察小鼠肝脏病理形态变化;Western blot法检测小鼠肝脏AMPK/mTOR/ULK1信号通路相关蛋白表达;实时荧光定量PCR法检测小鼠肝脏自噬相关基因表达水平;免疫组织化学法观察小鼠肝脏血红素加氧酶1(HO-1)表达;羟胺法检测小鼠肝脏超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果:与对照组比较,模型组小鼠力竭游泳时间明显缩短(P<0.01);肝脏AMPK、磷酸化(p)-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性均明显下降(P<0.01);肝脏mTOR、p-mTOR蛋白表达及MDA含量升高(P<0.01)。与模型组比较,雷帕霉素组、电针组、电针+抑制剂组力竭游泳时间延长(P<0.01,P<0.05);AMPK、p-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、 ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性上升(P<0.01,P<0.05);肝脏mTOR、p-mTOR蛋白表达及MDA含量下降(P<0.01,P<0.05)。与雷帕霉素组和电针组比较,电针+抑制剂组小鼠力竭游泳时间缩短(P<0.01),肝脏AMPK、p-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、 ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性均明显下降(P<0.01);肝脏mTOR、p-mTOR蛋白表达及MDA含量明显升高(P<0.01)。模型组小鼠肝细胞排列散乱,细胞呈空泡样改变;雷帕霉素组、电针组肝细胞排列较整齐,细胞空泡减少;电针+抑制剂组肝组织病理形态改善不明显。结论:电针可通过调节自噬相关信号通路AMPK/mTOR/ULK1促进衰老小鼠肝脏自噬的发生,进而抑制其过度的氧化应激,可在一定程度上延缓机体衰老的过程。Objective To observe the effect of electroacupuncture(EA) on physical strength and expression levels of hepatic AMP-activated protein kinase(AMPK), mammalian target of rapamycin(mTOR), unc-51 like autophagy activating kinase 1(ULK1) proteins and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNAs in aging(senescence accelerated mouse/prone 8, SAMP8)mice, so as to explore its mechanism underlying delaying aging by activating AMPK/mTOR/ULK1 signaling pathway. Methods Twenty-four male SAMP8 mice were randomly divided into model group, rapamycin(autophagy inducer) group, EA group and EA+autophagy inhibitor(EA+inhibitor) group, with 6 mice in each group, and 6 homologous anti-rapid aging male(SAMR1) mice in the same age were used as the control group. Mice of the rapamycin group received intraperitoneal injection of rapamycin solution(2 mg·kg(-1))。各组均每周干预6 d,连续2周。采用力竭游泳实验观察小鼠体力变化;HE染色法观察小鼠肝脏病理形态变化;Western blot法检测小鼠肝脏AMPK/mTOR/ULK1信号通路相关蛋白表达;实时荧光定量PCR法检测小鼠肝脏自噬相关基因表达水平;免疫组织化学法观察小鼠肝脏血红素加氧酶1(HO-1)表达;羟胺法检测小鼠肝脏超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果:与对照组比较,模型组小鼠力竭游泳时间明显缩短(P<0.01);肝脏AMPK、磷酸化(p)-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性均明显下降(P<0.01);肝脏mTOR、p-mTOR蛋白表达及MDA含量升高(P<0.01)。与模型组比较,雷帕霉素组、电针组、电针+抑制剂组力竭游泳时间延长(P<0.01,P<0.05);AMPK、p-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、 ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性上升(P<0.01,P<0.05);肝脏mTOR、p-mTOR蛋白表达及MDA含量下降(P<0.01,P<0.05)。与雷帕霉素组和电针组比较,电针+抑制剂组小鼠力竭游泳时间缩短(P<0.01),肝脏AMPK、p-AMPK、ULK1、p-ULK1蛋白及Atg5、Atg7、Atg13、Beclin1、 ULK1 mRNA表达,肝脏HO-1阳性表达及SOD活性均明显下降(P<0.01);肝脏mTOR、p-mTOR蛋白表达及MDA含量明显升高(P<0.01)。模型组小鼠肝细胞排列散乱,细胞呈空泡样改变;雷帕霉素组、电针组肝细胞排列较整齐,细胞空泡减少;电针+抑制剂组肝组织病理形态改善不明显。结论:电针可通过调节自噬相关信号通路AMPK/mTOR/ULK1促进衰老小鼠肝脏自噬的发生,进而抑制其过度的氧化应激,可在一定程度上延缓机体衰老的过程。
摘要:目的:观察胰高血糖样肽-1受体(GLP-1R)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)蛋白通路在电针治疗帕金森病(PD)小鼠中的作用,探讨电针治疗PD的中脑黑质相关机制。方法:将48只SPF级C57BL/6雄性小鼠随机分为正常组、模型组、电针组、抑制剂组,每组12只。采用鱼藤酮连续灌胃4周制备PD小鼠模型。电针组予电针"风府""太冲""足三里",30 min/次,1次/d,连续干预2周。抑制剂组予二肽基肽酶4抑制剂利格列汀(10 mg·kg(-1)·d(-1)·d(-1))连续灌胃2周。观察各组小鼠行为学评分变化,用酶联免疫吸附测定法检测血清和黑质中酪氨酸羟化酶(TH)水平,用Western blot法检测小鼠中脑黑质中GLP-1R、磷酸化(p)-PI3K、p-Akt蛋白表达水平。结果:与正常组比较,模型组小鼠行为学评分显著升高(P<0.01),血清和黑质中TH水平均显著降低(P<0.01),黑质中GLP-1R、p-PI3K、p-Akt蛋白表达水平均显著降低(P<0.01)。治疗后与模型组比较,电针组和抑制剂组小鼠行为学评分均显著下降(P<0.01),血清和黑质中TH水平均显著升高(P<0.01),黑质中GLP-1R、p-PI3K、p-Akt蛋白表达水平均显著升高(P<0.01)。与抑制剂组比较,电针组血清和黑质中TH水平降低(P<0.01,P<0.05)。结论:电针"风府""太冲""足三里"可能通过上调GLP-1R/PI3K/Akt蛋白通路的活性,提高血清和黑质中的TH水平,从而改善鱼藤酮诱导的PD小鼠行为学表现。Objective To observe the effect of electroacupuncture(EA) on glucagon-like peptide-1 receptor(GLP-1 R)/phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt) protein pathway in the substantia nigra of mice with Parkinson's di-sease(PD),so as to explore its underlying mechanisms in treatment of PD. Methods Forty-eight C57 BL/6 male mice were randomly divided into normal, model, EA and inhibitor groups, with 12 mice in each group. PD mouse model was established by intragastrical administration of rotenone for 4 weeks. In the EA group, EA was applied at “Fengfu”(GV16), “Taichong”(LR3) and“Zusanli”(ST36) for 30 min, once daily, for 2 weeks. The mice of the inhibitor group received gavage of dipeptidyl peptidase-4 inhibitor ligliptin(10 mg·kg(-1))连续灌胃2周。观察各组小鼠行为学评分变化,用酶联免疫吸附测定法检测血清和黑质中酪氨酸羟化酶(TH)水平,用Western blot法检测小鼠中脑黑质中GLP-1R、磷酸化(p)-PI3K、p-Akt蛋白表达水平。结果:与正常组比较,模型组小鼠行为学评分显著升高(P<0.01),血清和黑质中TH水平均显著降低(P<0.01),黑质中GLP-1R、p-PI3K、p-Akt蛋白表达水平均显著降低(P<0.01)。治疗后与模型组比较,电针组和抑制剂组小鼠行为学评分均显著下降(P<0.01),血清和黑质中TH水平均显著升高(P<0.01),黑质中GLP-1R、p-PI3K、p-Akt蛋白表达水平均显著升高(P<0.01)。与抑制剂组比较,电针组血清和黑质中TH水平降低(P<0.01,P<0.05)。结论:电针"风府""太冲""足三里"可能通过上调GLP-1R/PI3K/Akt蛋白通路的活性,提高血清和黑质中的TH水平,从而改善鱼藤酮诱导的PD小鼠行为学表现。
摘要:金元医家窦汉卿为透穴刺法初创者已为学界共识,本文从《窦太师针经》入手梳理元明清时期多个针灸文本的透穴内容,探讨透穴刺法的体例、应用腧穴及基本要素、主治病症特点等,尝试从技术哲学角度辨析其与透刺法之关系。发现《窦太师针经》28例"透、向"体例之透穴刺法的规范和数量足以为后世模范,为元明清透穴刺法的源头文献,此后针灸文本基本沿用此体例和内容并各有发展,如《扁鹊神应针灸玉龙经》《针灸大成》《针方六集》等版本之《玉龙歌》,而《针方六集》37例居各本之首,《循经考穴编》31例记有窦氏、杨氏、凌氏三位医家之透穴特色,凌氏在窦氏一针透穴基础上发展出两针或多针相向透穴刺法。透穴刺法有针刺分寸(深度)、角度、方向、起点穴(施透穴)、终点穴(被透穴)5项基本要素,《窦太师针经》透穴刺法大多主治局部病症,肘膝以下部分腧穴兼治远端病症。透穴刺法是一种以腧穴为特定意向结构的特殊透刺法,与透刺法二者不可混同一语。It has been the consensus of the academic community that the doctor DOU Han-qing in Jin-Yuan Dynasty was the pioneer of point to point penetration acupuncture method.The contents of several texts in Yuan-Ming-Qing Dynasties were combed on the basis of Dou Taishi Zhenjing(《窦太师针经》).The paradigm, the acupoints, basic elements and the characteristics of the main treatment of diseases were discussed, and the relationship between the point to point penetration acupuncture method with the penetration acupuncture method has been tried to distinguish from the perspective of technical philosophy. It was found that the specifications and quantity of the 28 “penetration and to” cases in Dou Taishi Zhenjing(《窦太师针经》)were sufficiently exemplary for future generations, and were the source documents of Yuan-Ming-Qing's texts, which basically followed its specifications and content and developed.For example, the Yulong Poetries(《玉龙歌》)in the books of Bianque Shenying Zhenjiu Yulong Jing(《扁鹊神应针灸玉龙经》),Zhenjiu Dacheng(《针灸大成》),Zhenfang Liuji(《针方六集》).The 37 cases in Zhenfang Liuji(《针方六集》) were the most in several texts.The 31 cases in Xunjing Kaoxue Bian(《循经考穴编》)had the characteristics of DOU Han-qing, YANG Ji-zhou and LING Yun, who developed the opposite direction penetration method with two or more needles on DOU Han-qing's one needle.There were five basic elements in the point to point penetration acupuncture method: the needle pri-cking inch(depth), the needle pricking angle, the needle pricking direction, the starting acupoint, the end acupoint.This method in Dou Taishi Zhenjing(《窦太师针经》)mostly treated local diseases and symptoms, some acupoints below the elbows and knees also treated far-end diseases.The point to point penetration acupuncture method has a specific intention structure, which cannot be confused with the penetration acupuncture method.