【摘要】 目的 基于腺苷酸活化蛋白激酶(AMPK)介导的炎症损伤探讨电针联合运动预处理对心肌缺血再灌注损伤(MIRI)大鼠心肌的保护作用。方法 采用随机数字表法将60只雄性SD大鼠分为对照组(Con组)、模型组(I/R组)、模型+电针联合运动预处理组(I/R+EA+EP组)、模型+AMPK抑制剂组(I/R+Compound C组)、模型+电针联合运动预处理+AMPK抑制剂组(I/R+EA+EP+Compound C组),每组12只。各预处理组依据组别先接受3周电针联合运动干预,随后采用Langendorff离体灌流装置对除Con组外的其他4组大鼠构建MIRI模型。I/R+EA+EP+Compound C组与I/R+Compound C组在造模前1～2 h腹腔注射多索吗啡(Compound C)。采用Med Lab生物信号采集处理系统检测大鼠心功能相关指标;苏木精-伊红染色(HE染色)观察心肌组织的病理改变;酶联免疫吸附试验(ELISA)检测各组大鼠心肌组织中的肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)水平;透射电子显微镜观察大鼠心肌细胞超微结构变化;Western blot法检测心肌组织中AMPK、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、沉默信息调节因子2同源蛋白1(SIRT1)、核转录因子κB(NF-κB)、磷酸化核转录因子κB(p-NF-κB)的表达情况。结果 缺血前,各组大鼠冠脉流量(CF)、心率(HR)、左心室发展压(LVDP)、左心室压力最大上升速率(+dp/dtmax)、左心室压力最大下降速率(-dp/dtmax)水平比较,差异均无统计学意义(P>0.05)。再灌注120 min后,I/R组大鼠各项心功能指标水平均低于Con组,差异均有统计学意义(P<0.05);I/R+EA+EP组大鼠各项心功能指标水平均高于I/R组,差异均有统计学意义(P<0.05);I/R+Compound C组各项心功能指标水平均低于I/R组和I/R+EA+EP组,差异均有统计学意义(P<0.05);I/R+EA+EP+Compound C组大鼠各项心功能指标水平均低于Con组和I/R+EA+EP组,HR和-dp/dtmax水平高于I/R+Compound C组,差异均有统计学意义(P<0.05)。HE染色检测结果显示,I/R+EA+EP组大鼠心肌病理损伤程度较I/R组减轻;I/R+EA+EP+Compound C组大鼠心肌病理损伤程度较I/R+Compound C组减轻。ELISA检测结果显示,I/R+EA+EP组大鼠心肌组织中的TNF-α和IL-6表达水平低于I/R组,I/R+EA+EP+Compound C组TNF-α和IL-6表达水平低于I/R+Compound C组,差异均有统计学意义(P<0.05)。透射电子显微镜检测结果显示,I/R+EA+EP组大鼠心肌细胞超微结构损伤程度较I/R组减轻;I/R+EA+EP+Compound C组大鼠心肌细胞超微结构损伤程度较I/R+Compound C组减轻。Western blot检测结果显示,I/R+EA+EP组大鼠心肌组织的p-AMPK/AMPK比值和SIRT1蛋白相对表达量较I/R组明显上调,p-NF-κB/NF-κB比值较I/R组明显下调(P<0.05);I/R+EA+EP+Compound C组大鼠心肌组织的p-AMPK/AMPK比值、SIRT1蛋白相对表达量较I/R+Compound C组明显上调,p-NF-κB/NF-κB比值较I/R+Compound C组下调(P<0.05)。结论 电针联合运动预处理能够显著减轻MIRI大鼠心肌组织的炎症损伤,其作用机制可能与调控AMPK/SIRT1/NF-κB信号通路有关。更多还原

【Abstract】 Objective To investigate the protective effects of electroacupuncture(EA) combined with exercise preconditioning(EP) on myocardial ischemia-reperfusion injury(MIRI) in rats, focusing on the role of AMP-activated protein kinase(AMPK)-mediated inflammatory damage. Methods Sixty male Sprague-Dawley rats were randomly assigned to five groups(n=12 each) using a random number table: Control(Con), I/R model(I/R), I/R+EA+EP, I/R+Compound C(AMPK inhibitor), and I/R+EA+EP+Compound C. Preconditioning groups received three weeks of EA combined with exercise according to their group assignment. Except for the Con group, all rats then underwent MIRI via Langendorff isolated heart perfusion. Compound C(dosoxmorphin) was administered intraperitoneally for 1-2 h before modeling in the I/R+Compound C and I/R+EA+EP+Compound C groups. Cardiac function indices were recorded using a Med Lab bio-signal acquisition system. Myocardial pathology was examined by hematoxylin-eosin staining. Levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in heart tissue were measured by ELISA. Transmission electron microscopy was used to assess ultrastructural changes in cardiomyocytes. Western blotting quantified myocardial expression of AMPK, phosphorylated AMPK(p-AMPK), silent information regulator 1(SIRT1), nuclear factor-κB(NF-κB), and phosphorylated NF-κB(p-NF-κB). Results Before ischemia, there were no significant differences in coronary flow(CF), heart rate(HR), left ventricular developed pressure(LVDP), maximum rate of increase of left ventricular pressure(+dp/dtmax) and maximum rate of decrease of left ventricular pressure(-dp/dtmax)(P>0.05). After 120 min of reperfusion,the levels of all cardiac function parameters in the I/R group were significantly lower than those in the Con group(P<0.05);the levels of all cardiac function parameters in the I/R+EA+EP group were significantly higher than those in the I/R group(P<0.05); the levels of all cardiac function parameters in the I/R+Compound C group were significantly lower than those in the I/R and I/R+EA+EP groups(P<0.05). The levels of all cardiac function parameters in the I/R+EA+EP+Compound C group were significantly lower than those in the Con group and I/R+EA+EP group(P<0.05). The levels of HR and-dp/dtmax in the I/R+EA+EP+Compound C group were significantly higher than those in the I/R+Compound C group(P<0.05). HE staining showed that the degree of myocardial pathological injury in the I/R+EA+EP group was alleviated compared with the I/R group, and the degree of myocardial pathological injury in the I/R+EA+EP+Compound C group was alleviated compared with the I/R+Compound C group. The results of ELISA showed that the expression levels of TNF-α and IL-6 in the myocardial tissue of rats in the I/R+EA+EP group were lower than those in the I/R group, and the expression levels of TNF-α and IL-6 in the I/R+EA+EP+Compound C group were lower than those in the I/R + Compound C group, and the differences were statistically significant(P<0.05). Transmission electron microscopy showed that the ultrastructural damage of myocardial cells in the I/R+EA+EP group was alleviated compared with the I/R group, and the ultrastructural damage of myocardial cells in the I/R+EA+EP+Compound C group was alleviated compared with I/R+Compound C group. Western blot results showed that the p-AMPK/AMPK ratio and the relative expression of SIRT1 protein in the myocardial tissue of rats in the I/R+EA+EP group were significantly up-regulated and the p-NF-κB/NF-κB ratio was significantly down-regulated compared with those in the I/R group(P<0.05); the p-AMPK/AMPK ratio and the relative expression of SIRT1 protein in the myocardial tissue of rats in the I/R+EA+EP+Compound C group were significantly up-regulated compared with those in the I/R+Compound C group, and the p-NF-κB/NF-κB ratio was down-regulated compared with that in the I/R+Compound C group(P<0.05). Conclusion Electroacupuncture combined with exercise preconditioning markedly attenuates inflammatory injury in myocardium of rats with MIRI, likely via modulation of the AMPK/SIRT1/NF-κB signaling pathway.更多还原