附子-干姜对大鼠急性心肌缺血的抗炎、抗氧化、抗凋亡保护作用及机制研究

Protective Effects and Mechanism of Fuzi(附子)-Ganjiang(干姜)Against Acute Myocardial Ischemia in Rats

目的:探讨附子-干姜对异丙肾上腺素(isoproterenol,ISO)引起大鼠急性心肌缺血(acute myocardial ischemia,AMI)损伤的心肌保护作用和研究机制。方法:60只雄性SD大鼠随机分为6组:正常对照组、模型对照组、附子干姜1.4、2.8、5.6 g/kg组及复方丹参滴丸0.09 g/kg组,各组灌胃相应药物或生理盐水,在给药第4 d,除正常对照组外,其余各组大鼠皮下注射异丙肾上腺素(85 mg/kg),连续给药和造模给药4 d,建立AMI模型。测定谷草转氨酶(AST)、乳酸脱氢酶(LDH)、肌钙蛋白(cTnI)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)的活力或含量;监测心电图;HE染色观察心肌病理变化;TUNEL评估大鼠心肌细胞凋亡以及Western blot法检测Bcl-2相关X蛋白(BAX)、B细胞淋巴瘤-2基因(Bcl-2)、Bcl-2/BAX、半胱氨酸天冬氨酸蛋白水解酶-9(Caspase-9)蛋白表达。结果:与正常对照组相比,模型对照组大鼠血清中的AST、LDH、cTnI、MDA、TNF-α和IL-6的活力或含量显著升高(P<0.01),GSH-Px活力显著减弱(P<0.01);心肌细胞凋亡率显著升高(P<0.01);HE染色观察大鼠心肌损伤明显,心肌纤维排列紊乱甚至断裂,大量炎性细胞浸润;Bcl-2蛋白表达显著下调(P<0.01),BAX、Caspase-9蛋白表达显著上调(P<0.01);与模型对照组相比,附子-干姜1.4、2.8、5.6 g/kg组AST、LDH和cTnI、MDA的活力或含量明显降低(P<0.05或P<0.01);GSH-Px的活力明显升高(P<0.05或P<0.01);心肌细胞凋亡率明显降低(P<0.01);心肌组织炎症反应减轻,损伤病变明显改善;Bcl-2蛋白表达明显上调(P<0.05或P<0.01),BAX和Caspase-9蛋白表达显著下调(P<0.01);附子-干姜5.6 g/kg组TNF-α含量明显降低。结论:附子-干姜对ISO诱导的大鼠急性心肌缺血模型具有保护作用,其作用机制可能与抗炎、抗氧化、改善心肌细胞酶、改善细胞凋亡蛋白、抑制细胞凋亡相关。

Objective:To investigate the myocardial protective effects and mechanism of Fuzi(附子)-Ganjiang(干姜)against isoproterenol(ISO)-induced acute myocardial ischemia(AMI)injury in rats.Methods:Sixty male SD rats were randomly divided into a normal control group,a model control group,low-,medium-,and high-dose Fuzi-Ganjiang groups(1.4 g/kg,2.8 g/kg,and 5.6 g/kg),and a positive drug-Fufang Danshen(复方丹参)Dripping Pills,0.09 g/kg group.All rats were administered with corresponding drugs or normal saline.On day 4 of administration,the AMI model was induced by subcutaneous injection of ISO(85 mg/kg)for 4 days in rats except for those in the normal control group.The content and activities of biochemical indexes as aspartate aminotransferase(AST),lactate dehydrogenase(LDH),cardiac-specific troponin I(cTnI),malondialdehyde(MDA),glutathione peroxidase(GSH-Px),tumor necrosis factor(TNF)-α,and interleukin 6(IL-6)were determined.ECG monitoring was carried out.HE staining was used to observe the pathological changes in the myocardium.Cardiomyocyte apoptosis in rats was assessed by TUNEL.Western blot was used to detect the protein expression of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),Bcl-2/Bax,and cysteinyl aspartate-specific protease 9(Caspase-9).Results:Compared with the normal control group,the model control group showed increased serum levels and potentiated activities of AST,LDH,cTnI,MDA,TNF-α,and IL-6(P<0.01),blunted activity of GSH-Px(P<0.01),increased apoptosis rate of cardiomyocytes(P<0.01),manifest myocardial injury as revealed by HE staining,disordered myocardial fiber arrangement or even broken,infiltration of massive inflammatory cells,down-regulated protein expression of Bcl-2(P<0.01),and up-regulated protein expression of Bax and Caspase-9(P<0.01).Compared with the model control group,the Fuzi-Ganjiang groups showed reduced levels and blunted activities of AST,LDH,cTnI,and MDA(P<0.05 or P<0.01),potentiated activity of GSH-Px(P<0.05 or P<0.01),decreased apoptosis rate of cardiomyocytes(P<0.01),relieved inflammatory response of cardiomyocytes,improved lesions,up-regulated protein expression of Bcl-2(P<0.05 or P<0.01),and down-regulated protein expression of Bax and Caspase-9(P<0.01).TNF-αcontent was significantly decreased in the high-dose Fuzi-Ganjiang group.Conclusion:The herbal pair Fuzi-Ganjiang has a protective effect against ISO-induced AMI in rats,and its mechanism may be related to the anti-inflammation,anti-oxidation,the improvement of cardiomyocyte enzymes and apoptosis proteins,and the inhibition of apoptosis.