连梅芪物汤调控AMPK/TrkA/TRPM7信号通路减轻糖尿病心脏自主神经病变  

Lianmei Qiwu Decoction relieves diabetic cardiac autonomic neuropathy by regulating AMPK/TrkA/TRPM7 signaling pathway

作　　者：孙雪梅[1,2] 季海刚 高昕[1] 王新东[1] SUN Xue-mei;JI Hai-gang;GAO Xin;WANG Xin-dong(The Third School of Clinical Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Changzhou Hospital of Traditional Chinese Medicine,Changzhou 213004,China)

机构地区：[1]南京中医药大学第三临床医学院,江苏南京210028 [2]常州市中医医院,江苏常州213004

出　　处：《中国中药杂志》2023年第7期1739-1750,共12页China Journal of Chinese Materia Medica

基　　金：江苏省中医局科技项目(YB201922);全国中医药创新骨干人才项目(国家中医药管理局办人教2019-91);全国名老中医药专家传承工作室建设项目(国中医药人教函[2022]75号);江苏省名老中医专家董其美传承工作室建设项目(苏中医科教[2019]10号);国家自然科学基金项目(82274469)。

摘　　要：以高脂饮食诱导的糖尿病大鼠模型,探讨连梅芪物汤(Lianmei Qiwu Decoction,LMQWD)改善糖尿病心脏自主神经重构的作用,以腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)/肌钙蛋白受体激酶A(tropomyosin receptor kinase A,TrkA)/瞬时性受体电位通道M7(transient receptor potential melastatin 7,TRPM7)通路为切入点探讨其可能的作用机制。将糖尿病大鼠随机分为糖尿病模型(model)组、连梅芪物汤(LMQWD)组、AMPK激动剂(AMPK agonist)组、空载TRPM7腺病毒(TRPM7-N)组、过表达TRPM7腺病毒(TRPM7)组、连梅芪物汤+空载TRPM7腺病毒(LMQWD+TRPM7-N)组、连梅芪物汤+过表达TRPM7腺病毒(LMQWD+TRPM7)组、TRPM7通道抑制剂(TRPM7 inhibitor)组。分组给药4周后,行程序电刺激(program electrical stimulation,PES)检测大鼠心律失常易感性;采集大鼠心肌和神经节标本,通过苏木素-伊红(hematoxylin-eosin,HE)染色和马松(Masson)染色观察心肌细胞结构和心肌组织纤维化情况;运用免疫组化、免疫荧光、实时定量逆转录聚合酶链反应(real-time quantitative polymerase chain reaction,RT-PCR)及蛋白免疫印迹(Western blot)检测心肌组织及神经节组织中TRPM7、酪氨酸羟化酶(tyrosine hydroxylase,TH)、乙酰胆碱转移酶(choline acetyltransferase,ChAT)、生长相关蛋白-43(growth associated protein-43,GAP-43)、神经生长因子(nerve growth factor,NGF)、p-AMPK/AMPK等基因和相关神经标志物的分布与表达。结果显示,LMQWD可显著降低糖尿病大鼠的心律失常易感性,并减轻糖尿病状态下心肌组织的纤维化,降低心肌及神经节中TH、ChAT、GAP-43水平,升高NGF水平,抑制TRPM7表达,并上调p-AMPK/AMPK、p-TrkA/TrkA水平。该研究表明,LMQWD可以减轻糖尿病状态下的心脏自主神经重构,其机制与激活AMPK、进一步磷酸化TrkA而抑制TRPM7表达相关。This study investigated the effect of Lianmei Qiwu Decoction(LMQWD)on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient recept or potential melastatin 7(TRPM7)signaling pathway.The diabetic rats were randomly divided into a model group,an LMQWD group,an AMPK agonist group,an unloaded TRPM7 adenovirus group(TRPM7-N),an overexpressed TRPM7 adenovirus group(TRPM7),an LMQWD+unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N),an LMQWD+overexpressed TRPM7 adenovirus group(LMQWD+TRPM7),and a TRPM7 channel inhibitor group(TRPM7 inhibitor).After four weeks of treatment,programmed electrical stimulation(PES)was employed to detect the arrhythmia susceptibility of rats.The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE)staining and Masson staining.The immunohistochemistry,immunofluorescence,real-time quantitative polymerase chain reaction(RT-PCR),and Western blot were adopted to detect the distribution and expression of TRPM7,tyrosine hydroxylase(TH),choline acetyltransferase(ChAT),growth-associated protein(GAP-43),nerve growth factor(NGF),AMPK/p-AMPK,and other genes and related neural markers.The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues,decrease the levels of TH,ChAT,and GAP-43 in the myocardium and ganglion,increase NGF,inhibit the expression of TRPM7,and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels.This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state,and its mechanism was associated with the activation of AMPK,further phosphorylation of TrkA,and inhibition of TRPM7 expression.

关 键 词：糖尿病心脏自主神经病变 连梅芪物汤 AMPK TRKA TRPM7 

分 类 号：R285.5[医药卫生—中药学]