基于PI3K/PDK1/Akt信号通路研究丹参-红花药对对寒凝血瘀型心肌缺血大鼠的保护作用及机制

Protective mechanism of Salviae Miltiorrhizae Radix et Rhizoma-Carthami Flos drug pair on myocardial ischemia rats with cold coagulation and blood stasis syndrome based on PI3K/PDK1/Akt signaling pathway

目的 研究丹参-红花药对对异丙肾上腺素(isoprenaline hydrochloride,ISO)诱导的心肌缺血大鼠的保护作用及其机制。方法 大鼠随机分为对照组、模型组、复方丹参滴丸(73 mg/kg)组和丹参-红花低、中、高剂量(4、8、16 g/kg)组,给予相应药物干预7 d。于第6天开始ig药物1 h后,大鼠于0℃的冰浴中寒冷刺激4 min,sc ISO(85 mg/kg),连续2 d。采用2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色法测定各组大鼠心肌梗死面积;采用TUNEL法检测各组大鼠心肌细胞凋亡情况;采用苏木素-伊红(HE)染色法观察各组大鼠心肌组织病理变化;采用ELISA法检测各组大鼠血浆中肌酸激酶同工酶(creatine kinase,CK)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、乳酸脱氢酶(lactate dehydrogenase,LDH)、CK-肌红蛋白(myoglobin,MB)活性及心肌肌钙蛋白-Ⅰ(cardiac troponin-Ⅰ,CTn-Ⅰ)、CTn-T、C反应蛋白(C-reaction protein,CRP)、MB、N末端利钠肽前体(N-terminal pro-brain natriuretic peptide,NT-proBNP)和B型脑利钠肽(B-brainnatriureticpeptides,BNP)水平;采用Westernblotting检测各组大鼠心肌组织中磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、磷酸化PI3K(phosphorylated PI3K,p-PI3K)、磷酸肌醇依赖性蛋白激酶1(pyruvate dehydrogenase kinase isozyme 1,PDK1)、蛋白激酶B(protein kinase B,Akt)、p-Akt、内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)、p-eNOS、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、核因子-κB p65(nuclear factor-κB p65,NF-κB p65)、p-NF-κB p65、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、细胞色素C(cytochrome C,Cyt C)、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)和Caspase-9蛋白表达。结果 丹参-红花药对显著抑制ISO诱导的心肌缺血模型大鼠的心肌梗死面积(P<0.01);改善心肌组织病理变化;显著抑制心肌细胞的凋亡(P<0.05、0.01);显著降低血浆中心肌酶活性以及心肌蛋白水平(P<0.05、0.01);显著上调心肌组织中Bcl-2、PDK1、p-eNOS、p-PI3K和pAkt蛋白表达水平(P<0.05、0.01),降低Bax、Cyt C、剪切型Caspase-3(cleaved Caspase-3)、cleaved Caspase-9和p-NF-κB p65蛋白表达水平(P<0.05、0.01)。结论 丹参-红花药对能够通过调控PI3K/PDK1/Akt信号通路,从而发挥对ISO诱导的心肌缺血的保护作用。

Objectives To study the protective effect and mechanism of Danshen(Salviae Miltiorrhizae Radix et Rhizoma)-Honghua(Carthami Flos) on myocardial ischemia rats induced by isoprenaline hydrochloride(ISO). Methods Rats were randomly divided into control group, model group, Compound Danshen Dropping Pills(73 mg/kg) group and Salviae Miltiorrhizae Radix et RhizomaCarthami Flos low-, medium-and high-dose(4, 8, 16 g/kg) groups, rats were given corresponding drug for 7 d. 1 h after ig drug on 6th day, rats were stimulated with cold in ice bath at 0 ℃ for 4 min and sc ISO(85 mg/kg) for 2 d. 2,3,5-Triphenyltetrazolium chloride(TTC) staining was used to determine the myocardial infarction area of rats in each group;TUNEL method was used to detect myocardial cell apoptosis of rats in each group;Hematoxylin-eosin(HE) staining method was used to observe the pathological changes in myocardial tissue of rats in each group;ELISA method was used to detect creatine kinase(CK), aspartate aminotransferase(AST),lactate dehydrogenase(LDH), CK-myoglobin(MB) activities and cardiac troponin-Ⅰ(CTn-Ⅰ), CTn-T, C-reaction protein(CRP), MB,N-terminal pro-brain natriuretic peptide(NT-proBNP), B-brain natriuretic peptides(BNP) levels in plasma of rats in each group;Western blotting was used to detect phosphatidylinositol 3-kinase(PI3K), phosphorylated PI3K(p-PI3K), pyruvate dehydrogenase kinase isozyme 1(PDK1), protein kinase B(Akt), p-Akt, endothelial nitric oxide synthase(eNOS), p-eNOS, B-cell lymphoma-2(Bcl-2), nuclear factor-κB p65(NF-κB p65), p-NF-κB p65, Bcl-2 associated X protein(Bax), cytochrome C(Cyt C), cysteine-aspartate protease-3(Caspase-3) and Caspase-9 protein expressions in myocardial tissue of rats in each group. Results Salviae Miltiorrhizae Radix et Rhizoma-Carthami Flos significantly inhibited the myocardial infarction area in ISO-induced myocardial ischemia model rats(P < 0.01), improved the pathological changes of myocardial tissue, significantly inhibited the apoptosis of cardiomyocytes(P < 0.05,0.01), significantly decreased myocardial enzyme activities and myocardial protein levels in plasma(P < 0.05, 0.01), significantly increased protein expression levels of Bcl-2, PDK1, p-eNOS, p-PI3K and p-Akt in myocardial tissue(P < 0.05, 0.01), decreased protein expression levels of Bax, Cyt C, cleaved Caspase-3(cleaved Caspase-3), cleaved Caspase-9 and p-NF-κB p65(P < 0.05, 0.01).Conclusion Salviae Miltiorrhizae Radix et Rhizoma-Carthami Flos drug pair can exert a protective effect on ISO-induced myocardial ischemia by regulating PI3K/PDK1/Akt signaling pathway.