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

Protective effect and regulating effect on FXR/SHP gene of electroacupuncture preconditioning on myocardial ischemia-reperfusion injury in rats

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.