针刺内关加公孙穴调控循环外泌体circ_0001439S表达对冠状动脉粥样硬化大鼠Sclt1、ET-1和eNOS表达影响机制研究  

Mechanism of Acupuncture at Neiguan(PC6)and Gongsun(SP4)on the Expression of Sclt1,ET-1 and eNOS in Rats with Coronary Atherosclerosis via Regulating the Expression of Circulating Exosomal circ_0001439S

作　　者：梁冰雪 王丹 马翔 樊文彬 LIANG Bingxue;WANG Dan;MA Xiang;FAN Wenbin(Chongqing College of Traditional Chinese Medicine,Chongqing 401331,China)

机构地区：[1]重庆中医药学院,重庆401331

出　　处：《辽宁中医药大学学报》2025年第3期165-175,共11页Journal of Liaoning University of Traditional Chinese Medicine

基　　金：重庆市教委科学技术资助项目(KJQN202315121)。

摘　　要：目的探讨针刺内关+公孙穴对冠状动脉粥样硬化大鼠钠离子通道和网格蛋白连接蛋白1(sodium channel and clathrin linker 1 gene,Sclt1)、内皮素-1(endothelin-1,ET-1)、血管内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)表达的影响以及其对循环外泌体环状RNA(circRNAs)0001439S(circ_0001439S)表达的调控机制。方法提取并鉴定健康对照组受试者和冠状动脉粥样硬化心脏病组患者血清中的外泌体;通过芯片分析以及实时荧光定量反转录聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)验证外泌体中差异表达的circRNAs。通过高脂饲料持续喂养大鼠8周构建动物模型,以针刺内关+公孙穴进行干预,以circ_0001439S模拟物(circ_0001439S-mimic,mimic)进行功能挽救实验。脱氧核苷酸转移酶介导的脱氧三磷酸鸟苷原位末端标记(terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling,TUNEL)检测大鼠冠状动脉组织中的细胞凋亡率,罗丹明123(Rhodamine 123,Rh123)染色、二氢乙锭(dihydroethidium,DHE)染色以及油红O染色检测大鼠冠状动脉组织中的线粒体膜电位、活性氧(reactive oxidative species,ROS)水平以及脂质沉积,免疫组化法检测初级纤毛标志物抗ADP核糖基化因子相似蛋白-13B(antiADP-ribosylation factor-like protein 13B,ARL13B)和纤毛内转运蛋白88(intraflagellar transport 88,IFT88)的表达;免疫荧光法检测乙酰辅酶A羧化酶(acetyl-coA carboxylase,ACC)和固醇调节元件结合蛋白(sterol regulatory element binding proteins,SREBP)的表达;蛋白质免疫印记(Western blot)法检测Sclt1、ET-1和eNOS的表达。结果经电镜以及Western blot鉴定后确为外泌体;经芯片分析以及qRT-PCR验证显示circ_0001439S在疾病中的表达明显升高;针灸内关+公孙穴能明显降低细胞凋亡率、ROS水平以及脂质沉积,升高冠状动脉组织中的线粒体膜电位以及冠状动脉组织中ARL13B和IFT88的表Objective To observe the effect of acupuncture at Neiguan(PC6)+Gongsun(SP4)on the expression of sodium channel and clathrin linker 1 gene(Sclt1),endothelin 1(ET-1)and endothelial nitric oxide synthase(eNOS)in rats with coronary atherosclerosis and its regulatory mechanism on the expression of circulating exocrine circ_0001439S.Methods The exosomes in serum of control group and patients with coronary atherosclerotic heart disease were extracted and identified,and the differentially expressed circRNAs in exosomes was verified by chip analysis and real-time quantitative polymerase chain reaction(RT-qPCR).High-fat diet for 8 weeks was used to establish animal model.Aacupuncture at Neiguan(PC6)+Gongsun(SP4)was used for intervention,and functional salvage experiment was carried out with circ_0001439S-mimic(mimic)for functional rescue experiments.Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling(TUNEL)was used to detect the cell apoptosis rate in rat coronary artery tissue.Rhodamine 123(Rh123)staining,dihydroethidium(DHE)staining and oil red O staining were used to detect the mitochondrial membrane potential,reactive oxidative species(ROS)level and lipid deposition in rat coronary artery tissue.Immunohistochemistry was used to detect the expression of anti-ADP-ribosylation factor-like protein 13B(ARL13B)and intraflagellar transport 88(IFT88).Immunofluorescence was used to detect the expression of acetyl-coA carboxylase(ACC)and sterol regulatory element binding proteins(SREBP).Western blot was used to detect the expression of Sclt1,ET-1 and eNOS.Results The circulating exosomal was identified by electron microscope and Western blot.Chip analysis and qRT-PCR verification showed that the expression of circ_0001439S in the disease was significantly increased.Acupuncture at Neiguan(PC6)+Gongsun(SP4)could significantly reduce the apoptosis rate,ROS level and lipid deposition,and increase the mitochondrial membrane potential in coronary artery and the expression of ARL13B and IFT88 in c

关 键 词：针刺内关+公孙穴 冠状动脉粥样硬化 循环外泌体circ_0001439S 钠离子通道和网格蛋白连接蛋白1 内皮素-1 血管内皮型一氧化氮合酶 

分 类 号：R245.31[医药卫生—针灸推拿学]