益气养阴活血中药减轻大鼠离体心脏缺血再灌注损伤作用研究

Cardioprotective effects of Yiqi Yangyin Huoxue Injection against ischemia-reperfusion injury in isolated hearts of rats

目的研究益气养阴活血中药(参麦注射液与丹参注射液联合应用,YYH)通过激活PI3K-Akt与ERK1/2信号通路,抑制m PTP开放,减轻心肌缺血再灌注(I/R)损伤,发挥心肌保护作用。方法应用大鼠离体心脏I/R损伤模型,灌流2.5、5、10μL/m LYYH及PI3K特异性抑制剂LY294002,检测心脏功能指标、灌流液肌酸激酶(CK)、乳酸脱氢酶(LDH)活性、TTC染色观察心肌梗死面积、透射电镜观察心肌超微结构、Western blotting检测相关蛋白表达。YYH预处理离体心脏后进行I/R,分离心肌线粒体,以m PTP特异性抑制剂环孢素A(Cs A)作对照,检测线粒体通透性转换孔(m PTP)开放情况;药物与线粒体孵育后检测m PTP开放。结果 YYH 10μL/m L预处理能改善离体心脏功能指标,减少心肌梗死面积,减轻心肌组织超微结构损伤,5、10μL/m L降低心脏灌流液中CK、LDH活性;其改善心功能指标、减少心肌梗死面积的作用被LY294002部分或完全抵消;YYH处理后心肌组织p-Akt、p-ERK1/2、p-GSK-3β蛋白表达上调,LY294002阻断了YYH诱导的p-Akt、p-GSK-3β蛋白表达上调;在外源性钙刺激下,YYH预处理组m PTP比模型组更容易开放;YYH、参麦注射液能够直接抑制m PTP开放。结论 YYH预处理通过激活PI3K-Akt与ERK1/2信号通路,抑制m PTP开放,减轻心肌I/R损伤,发挥心肌保护作用;其抑制m PTP开放的作用机制与Cs A不完全相同。

Objective To clarify the cardioprotective effects of Yiqi Yangyin Huoxue（YYH） Injection exert against ischemia-reperfusion（I/R） injury, and the mechanisms involving activation of specific survival signals reperfusion injury salvage kinase（PI3K-Akt pathway and ERK1/2 pathway）, hence inhibiting mitochondrial permeability transition pore opening（mPTP）. Methods A Langendorff model of myocardial ischemia-reperfusion injury was employed. The rats were randomly divided into seven groups： control group, I/R group, YYH 2.5, 5, and 10 μL/mL groups, LY294002 ＋ YYH 10 μL/mL group, and LY294002 group. The cardiac parameters of left ventricular developed pressure（LVDP）, maximal rise/fall of left ventricular pressure（± dp/dtmax）, and rate-pressure product（RPP） were monitored. Creatine kinase（CK） and lactate dehydrogenase（LDH） released from effluents were measured. Infarct size was estimated by TTC staining. Transmission electron microscopy（TEM） was performed to assess morphological difference between cardiac mitochondrial isolated I/R rats and YYH pretreated rats. Western blotting was used to determine some protein expression. MPTP opening in mitochondria isolated fromLangendorff rat hearts pretreated with YYH was measured. Incubation of isolated cardiac mitochondria with YYH and mPTP opening was measured. Results YYH 10 μL/mL can significantly improve ventricular function, ameliorate the level of myocardial tissue lesions. YYH（5 and 10 μL/mL） reduced CK and LDH release. And these protections were accompanied by a significant increase in p-PKB/Akt, p-ERK1/2, and p-GSK-3β（Ser-9）. LY294002 abolished the protective effects of YYH on cardiac function and infarct size and inhibited YYH-induced phosphorylation of PKB/Akt and GSK-3β. MPTP opening in mitochondria isolated from YYH-pretreated occurred more readily invitro than I/R mitochondria. YYH（2.5, 5, 10, and 20 μL/mL）, Shenmai injection（2.5, 5, 10, and 20μL/mL） resulted in a significant inhibition of Ca2＋-induced mitochondrial swelling. Conclusion YYH produces direct cardioprotective actions. Inhibition of mPTP is a key event by which it mediates myocardial protection against I/R injury. And this effects involve activation of PI3K-Akt and ERK1/2 signal pathway. The mechanism of its action is different from that of CsA.