电针预处理对心肌缺血再灌注大鼠的保护作用及FXR/SHP基因的调控作用
|
摘要
目的:探讨电针预处理对大鼠心肌缺血再灌注损伤的保护机制。方法:将SPF级雄性Wistar大鼠88只,随机分为正常组、假手术组、模型组和电针组,每组22只。正常组常规饲养7 d,不做任何处理;假手术组和模型组每日捆绑1次,每次20 min,共7 d;电针组电针"内关""足三里""关元"穴,每天1次,频率2 Hz、强度为1 mA的连续波,每次20 min,共7 d。第8天,正常组采用10%乌拉坦(10 mL/kg)腹腔注射麻醉后60 min腹主动脉取血,再取心脏组织;模型组与电针组结扎左冠状动脉前降支20 min,再灌注40 min后取样;假手术组开胸后不做任何处理,60min后取样。采用氯化硝基四氮唑蓝(NBT)染色法测定缺血再灌注损伤后大鼠心肌梗死的面积和梗死区质量,采用酶联免疫分析法检测心肌损伤标志物和炎性因子[血清乳酸脱氢酶(LDH)、肌酸激酶(CK)、血清肌钙蛋白(c Tn I)]含量,采用荧光定量PCR法检测法尼酯衍生物X受体(FXR)、小异二聚体配体(SHP)基因表达情况。结果:①与正常组比较,假手术组心肌梗死面积与梗死区质量比较差异无统计学意义(均P>0.05),模型组的梗死面积与梗死区质量均升高(均P<0.01),电针组的梗死面积与梗死区质量均低于模型组(均P<0.01)。②与正常组比较,假手术组血清LDH、CK、c Tn I各项指标比较差异无统计学意义(均P>0.05);与正常组比较,模型组血清LDH、CK和c TnI水平均升高(均P<0.05),电针组血清LDH、CK和c TnI水平均低于模型组(均P<0.05)。③模型组FXR、SHP基因表达水平明显高于正常组(P<0.05),电针组FXR、SHP基因表达水平低于模型组(均P<0.05)。结论:电针预处理能显著改善大鼠心肌缺血再灌注后的心功能,降低梗死面积,减少炎性因子,下调FXR/SHP的基因表达,其保护作用可能是基于调控FXR/SHP凋亡信号通路产生的。
Objective To explore the protective mechanism of electroacupuncture preconditioning on myocardial ischemia-reperfusion injury in rats. Methods A total of 88 SPF-grade Wistar male rats were randomized into a normal group, a sham-operation group, a model group and an electroacupuncture(EA) group, 22 rats in each one. The rats in the normal group received normal diet and no intervention was given for 7 d. The rats in the sham-operation and the model group were bound for 20 min, once a day for 7 d. In the EA group, EA, continuous wave, 2 Hz and 1 mA, was applied at "Neiguan"(PC 6), "Zusanli"(ST 36) and "Guanyuan"(CV 4) for 20 min, once a day for 7 d. In the 8 th day, the blood was collected by abdominal aortic method 60 min after intraperitoneal injection of 10% urethane anesthesia by 10 mL/kg in the normal group,and then the tissue of heart was collected. In the model group and the EA group, ligating the left anterior descending coronary artery(LAD) was adopted for 20 min and reperfusion was applied for 40 min, then the samples were collected. The rats in the sham-operation group recevied open-chest operation and no other intervention, the samples were collected in 60 min. Nitro Blue Tetrazolium Chloride monohydrate(NBT) stain was used to detected myocardial infarct size and weight, ELISA was used to measured myocardial injury markers and inflammatory factors(LDH、CK、cTnI), and the fluorescent quantitative PCR method was used to determine the expressions of FXR and SHP gene. Results ①There was no significant difference between the normal group and the sham-operation group in myocardial infarct size and weight(P>0.05).Compared with the normal group,myocardial infarct size and weight were higher in the model group(P<0.01). Compared with the model group, myocardial infarct size and weight were lower in the EA group(P<0.01). ②There was no significant difference between the normal group and the sham-operation group in serum contents of LDH、CK、cTnI(P>0.05). Compared with the normal group, the serum contents of LDH、CK、c Tn I were higher in the model group(P<0.05). Compared with the model group, the serum contents of LDH、CK、c Tn I were lower in the EA group(P<0.05). ③The expressions of FXR and SHP gene in the model group were higher than those in the normal group(P<0.05). The expressions of FXR and SHP gene in the EA group were lower than those in the model group(P<0.05). Conclusion Electroacupuncture preconditioning could significantly improve cardiac function of rats with myocardial ischemia-reperfusion, reduce infarct size and inflammatory factors, and down-regulate the expressions of FXR and SHP gene. The protective effect may generate based on regulation of FXR/SHP apoptosis signal pathway.
Objective To explore the protective mechanism of electroacupuncture preconditioning on myocardial ischemia-reperfusion injury in rats. Methods A total of 88 SPF-grade Wistar male rats were randomized into a normal group, a sham-operation group, a model group and an electroacupuncture(EA) group, 22 rats in each one. The rats in the normal group received normal diet and no intervention was given for 7 d. The rats in the sham-operation and the model group were bound for 20 min, once a day for 7 d. In the EA group, EA, continuous wave, 2 Hz and 1 mA, was applied at "Neiguan"(PC 6), "Zusanli"(ST 36) and "Guanyuan"(CV 4) for 20 min, once a day for 7 d. In the 8 th day, the blood was collected by abdominal aortic method 60 min after intraperitoneal injection of 10% urethane anesthesia by 10 mL/kg in the normal group,and then the tissue of heart was collected. In the model group and the EA group, ligating the left anterior descending coronary artery(LAD) was adopted for 20 min and reperfusion was applied for 40 min, then the samples were collected. The rats in the sham-operation group recevied open-chest operation and no other intervention, the samples were collected in 60 min. Nitro Blue Tetrazolium Chloride monohydrate(NBT) stain was used to detected myocardial infarct size and weight, ELISA was used to measured myocardial injury markers and inflammatory factors(LDH、CK、cTnI), and the fluorescent quantitative PCR method was used to determine the expressions of FXR and SHP gene. Results ①There was no significant difference between the normal group and the sham-operation group in myocardial infarct size and weight(P>0.05).Compared with the normal group,myocardial infarct size and weight were higher in the model group(P<0.01). Compared with the model group, myocardial infarct size and weight were lower in the EA group(P<0.01). ②There was no significant difference between the normal group and the sham-operation group in serum contents of LDH、CK、cTnI(P>0.05). Compared with the normal group, the serum contents of LDH、CK、c Tn I were higher in the model group(P<0.05). Compared with the model group, the serum contents of LDH、CK、c Tn I were lower in the EA group(P<0.05). ③The expressions of FXR and SHP gene in the model group were higher than those in the normal group(P<0.05). The expressions of FXR and SHP gene in the EA group were lower than those in the model group(P<0.05). Conclusion Electroacupuncture preconditioning could significantly improve cardiac function of rats with myocardial ischemia-reperfusion, reduce infarct size and inflammatory factors, and down-regulate the expressions of FXR and SHP gene. The protective effect may generate based on regulation of FXR/SHP apoptosis signal pathway.
引文
[1]FrankA,BonneyM,BonneyS,etal.Myocardialischemia reperfusion injury from basic science to clinical bedside[J]. Semin Cardiothorac Vasc Anesth, 2012, 16(3):123-132.
[2]陈福晖,刘达兴,容松,等.心肌缺血再灌注损伤发生机制的研究进展[J].安徽医药, 2017, 21(12):2145-2148.
[3]Kinnally KW, Peixoto PM, Ryu SY, et al. Is mPTP the gatekeeper for necrosis, apoptosis, or both?[J]. Biochim Biophys Acta, 2011,1813(4):616-622.
[4]Shahzad T, Kasseckert SA, Iraqi W, et al. Mechanisms involved in postconditioningprotectionofcardiomyocytesagainstacute reperfusion injury[J]. J Mol Cell Cardiol, 2013, 58:209-216.
[5]邵明璐,李洋,崔华峰,等.针刺预处理对大鼠心肌缺血再灌注氧化应激损伤的保护作用[J].中国针灸, 2017, 37(3):285.
[6]NiX,XieY,WangQ,etal.Cardioprotectiveeffectof transcutaneouselectricacupointstimulationinthepediatric cardiacpatients:arandomizedcontrolledclinicaltrial[J].Pediatr Anesth, 2012, 22(8):805-811.
[7]YangL,YangJ,WangQ,etal.Cardioprotectiveeffectsof electroacupuncturepretreatmentonpatientsundergoingheart valvereplacementsurgery:arandomizedcontrolledtrial[J].Ann Thorac Surg, 2010, 89(3):781-786.
[8]TongF,ZhangH.PulmonaryExposuretoparticulatematter(PM2.5)affects the sensitivity to myocardial ischemia/reperfusion injurythroughfarnesoid-X-receptor-inducedautophagy[J].Cell Physiol Biochem, 2018, 46(4):1493-1507.
[9]李明磊,王华,陈泽斌,等.电针内关穴对AMI大鼠自主神经与中枢P物质、一氧化氮合酶的影响[J].中华中医药杂志, 2007,22(8):543-546.
[10]洪亚群,王华,HongYQ,等.“双固一通”针法对甲亢性心肌病大鼠AngⅡ、ET及CGRP的影响[J].中国康复,2008,23(3):150-151.
[11]王华,涂乾,王亚文,等.电针“内关”穴抗心肌缺血损伤中下丘脑室旁核内阿片肽的作用[J].中国针灸,2005,25(10):720-724.
[12]汪梦霞,廖锦华,马达,等.大鼠在体心肌缺血再灌注模型的建立[J].临床医学工程, 2013, 20(5):533-535.
[13]陈松,韩永丽,吴松,等.针刺“内关”预处理对大鼠缺血再灌注心肌的保护作用及机制研究[J].华中科技大学学报(医学版),2017, 46(5):526-530.
[14]闵清,白育庭,张志,等.山楂叶总黄酮对大鼠心肌缺血再灌注损伤的心电图、一氧化氮及丙二醛变化的影响[J].中国临床药理学与治疗学, 2007, 12(7):800-803.
[15]蔡荣林,胡玲,申国明,等.电针预处理对急性心肌缺血再灌注损伤大鼠心肌组织水通道蛋白1表达及蛋白激酶C活性的影响[J].中国针灸, 2017, 37(2):157-161.
[16]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验, 1991(1):1-5.
[17]李晓泓.“逆灸”对佐剂性关节炎的影响及机制研究[D].北京:北京中医药大学, 2004.
[18]陈晨,孙忠人,吕晓琳,等.针灸预处理改善心肌缺血再灌注损伤机制的研究[J].针灸临床杂志, 2016, 32(11):102-106.
[19]钟峰,曾芳,郑晖,等.腧穴配伍拮抗作用的研究现状[J].中国针灸, 2011, 31(12):1093-1096.
[20]王华,梁凤霞.腧穴配伍研究思路和展望[J].中国针灸,2012,32(4):359-362.
[21]谢俊,王华,吴松,等.标本配穴对心肌缺血大鼠心肌沉默信息调节因子1的影响[J].中华中医药杂志,2016,31(7):2761-2764.
[22]王华,卢继东,吴松,等.电针不同腧穴对心肌缺血模型大鼠细胞凋亡及心肌细胞miRNAs表达的影响[J].中国针灸,2016,36(3):281-286.
[23]李江山,严洁,何军锋,等.电针对心肌缺血模型大鼠孤束核c-fos表达及心电图STII的影响[J].针刺研究, 2009, 34(3):171-174.
[24]叶小丰,李建国,杜朝晖,等.电针“足三里”穴对大鼠迷走神经放电的影响[J].针刺研究, 2006, 31(5):290-293.
[25]许敬.运动后大鼠血清cTnI及CK、CK-MB、LDH变化的比较研究[D].扬州:扬州大学, 2003.
[26] MakishimaM,OkamotoAY,RepaJJ,etal.Identificationofa nuclearreceptorforbileacids[J].Science,1999,284(5418):1362-1365.
[27] WangYD,ChenWD,MooreDD,etal.FXR:ametabolic regulator and cell protector[J]. Cell Res, 2008, 18(11):1087-1095.
[28] Bishop-BaileyD,WalshDT,WarnerTD.Expressionand activationofthefarnesoidXreceptorinthevasculature[J].Proc Natl Acad Scien USA, 2004, 101(10):3668-3673.
[29] PuJ,YuanA,ShanP,etal.Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes tomyocardialischaemia/reperfusioninjury[J].EurHeartJ,2013,34(24):1834-1845.
[2]陈福晖,刘达兴,容松,等.心肌缺血再灌注损伤发生机制的研究进展[J].安徽医药, 2017, 21(12):2145-2148.
[3]Kinnally KW, Peixoto PM, Ryu SY, et al. Is mPTP the gatekeeper for necrosis, apoptosis, or both?[J]. Biochim Biophys Acta, 2011,1813(4):616-622.
[4]Shahzad T, Kasseckert SA, Iraqi W, et al. Mechanisms involved in postconditioningprotectionofcardiomyocytesagainstacute reperfusion injury[J]. J Mol Cell Cardiol, 2013, 58:209-216.
[5]邵明璐,李洋,崔华峰,等.针刺预处理对大鼠心肌缺血再灌注氧化应激损伤的保护作用[J].中国针灸, 2017, 37(3):285.
[6]NiX,XieY,WangQ,etal.Cardioprotectiveeffectof transcutaneouselectricacupointstimulationinthepediatric cardiacpatients:arandomizedcontrolledclinicaltrial[J].Pediatr Anesth, 2012, 22(8):805-811.
[7]YangL,YangJ,WangQ,etal.Cardioprotectiveeffectsof electroacupuncturepretreatmentonpatientsundergoingheart valvereplacementsurgery:arandomizedcontrolledtrial[J].Ann Thorac Surg, 2010, 89(3):781-786.
[8]TongF,ZhangH.PulmonaryExposuretoparticulatematter(PM2.5)affects the sensitivity to myocardial ischemia/reperfusion injurythroughfarnesoid-X-receptor-inducedautophagy[J].Cell Physiol Biochem, 2018, 46(4):1493-1507.
[9]李明磊,王华,陈泽斌,等.电针内关穴对AMI大鼠自主神经与中枢P物质、一氧化氮合酶的影响[J].中华中医药杂志, 2007,22(8):543-546.
[10]洪亚群,王华,HongYQ,等.“双固一通”针法对甲亢性心肌病大鼠AngⅡ、ET及CGRP的影响[J].中国康复,2008,23(3):150-151.
[11]王华,涂乾,王亚文,等.电针“内关”穴抗心肌缺血损伤中下丘脑室旁核内阿片肽的作用[J].中国针灸,2005,25(10):720-724.
[12]汪梦霞,廖锦华,马达,等.大鼠在体心肌缺血再灌注模型的建立[J].临床医学工程, 2013, 20(5):533-535.
[13]陈松,韩永丽,吴松,等.针刺“内关”预处理对大鼠缺血再灌注心肌的保护作用及机制研究[J].华中科技大学学报(医学版),2017, 46(5):526-530.
[14]闵清,白育庭,张志,等.山楂叶总黄酮对大鼠心肌缺血再灌注损伤的心电图、一氧化氮及丙二醛变化的影响[J].中国临床药理学与治疗学, 2007, 12(7):800-803.
[15]蔡荣林,胡玲,申国明,等.电针预处理对急性心肌缺血再灌注损伤大鼠心肌组织水通道蛋白1表达及蛋白激酶C活性的影响[J].中国针灸, 2017, 37(2):157-161.
[16]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验, 1991(1):1-5.
[17]李晓泓.“逆灸”对佐剂性关节炎的影响及机制研究[D].北京:北京中医药大学, 2004.
[18]陈晨,孙忠人,吕晓琳,等.针灸预处理改善心肌缺血再灌注损伤机制的研究[J].针灸临床杂志, 2016, 32(11):102-106.
[19]钟峰,曾芳,郑晖,等.腧穴配伍拮抗作用的研究现状[J].中国针灸, 2011, 31(12):1093-1096.
[20]王华,梁凤霞.腧穴配伍研究思路和展望[J].中国针灸,2012,32(4):359-362.
[21]谢俊,王华,吴松,等.标本配穴对心肌缺血大鼠心肌沉默信息调节因子1的影响[J].中华中医药杂志,2016,31(7):2761-2764.
[22]王华,卢继东,吴松,等.电针不同腧穴对心肌缺血模型大鼠细胞凋亡及心肌细胞miRNAs表达的影响[J].中国针灸,2016,36(3):281-286.
[23]李江山,严洁,何军锋,等.电针对心肌缺血模型大鼠孤束核c-fos表达及心电图STII的影响[J].针刺研究, 2009, 34(3):171-174.
[24]叶小丰,李建国,杜朝晖,等.电针“足三里”穴对大鼠迷走神经放电的影响[J].针刺研究, 2006, 31(5):290-293.
[25]许敬.运动后大鼠血清cTnI及CK、CK-MB、LDH变化的比较研究[D].扬州:扬州大学, 2003.
[26] MakishimaM,OkamotoAY,RepaJJ,etal.Identificationofa nuclearreceptorforbileacids[J].Science,1999,284(5418):1362-1365.
[27] WangYD,ChenWD,MooreDD,etal.FXR:ametabolic regulator and cell protector[J]. Cell Res, 2008, 18(11):1087-1095.
[28] Bishop-BaileyD,WalshDT,WarnerTD.Expressionand activationofthefarnesoidXreceptorinthevasculature[J].Proc Natl Acad Scien USA, 2004, 101(10):3668-3673.
[29] PuJ,YuanA,ShanP,etal.Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes tomyocardialischaemia/reperfusioninjury[J].EurHeartJ,2013,34(24):1834-1845.