基于NF-κB信号通路探究丁香酚包合物对心肌缺血再灌注损伤大鼠保护作用

Protective effect of eugenol inclusion complex on myocardial ischemia-reperfusion injury rats based on NF-κB signaling pathway

目的:基于核转录因子κB(NF-κB)信号通路探究丁香酚包合物对心肌缺血再灌注损伤(MIRI)大鼠的保护作用。方法:70只SD大鼠随机分为假手术组,模型组,淀粉基质组(0.96 g/kg),丁香酚包合物低、中、高剂量组(0.24、0.48、0.96 g/kg),复方丹参滴丸组(85 mg/kg),每组10只。连续给药10 d,结扎左前降支冠状动脉30 min,再灌注24 h建立MIRI模型,采用生物机能实验系统检测生物电信号的方法测定大鼠的心电信号;采用全自动生化分析仪检测大鼠血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)和谷草转氨酶(AST)的活力;采用试剂盒法测定大鼠心肌组织中丙二醛(MDA)和超氧化物歧化酶(SOD)的活力;ELISA法测肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)含量;采用RT-qPCR法检测大鼠心肌组织中NF-κB、IκB、Bcl-2、Bax mRNA表达水平;采用Western Blot法检测大鼠缺血部位心肌组织中NF-κB、IκB、p-IκB、Bcl-2、Bax蛋白表达水平。结果:与模型组比较,各给药组可不同程度降低LDH、CK、AST、MDA、TNF-α、IL-6水平(P<0.01,P<0.05),升高SOD、CK-MB水平(P<0.01);下调NF-κB、Bax mRNA表达水平(P<0.01),上调IκB、Bcl-2 mRNA表达水平(P<0.01);降低核NF-κB/总NF-κB、p-IκB/IκB蛋白表达水平(P<0.01),升高Bcl-2/Bax蛋白表达水平(P<0.05,P<0.01);而淀粉基质组各指标差异均无统计学意义。结论:丁香酚包合物可通过抑制NF-κB核转位,下调p-IκB/IκB,上调Bcl-2/Bax比值,抑制NF-κB信号通路的活化,降低炎症介质表达及自由基产生,达到抗MIRI的作用。

Objective:To explore the protective effect of eugenol inclusion compound on myocardial ischemiareperfusion injury(MIRI)in rats based on nuclear factor-κB(NF-κB)signaling pathway.Methods:A total of 70 SD rats were randomly divided into sham operation group,model group,starch matrix group(0.96 g/kg),eugenol inclusion complex low,medium and high dose groups(0.24,0.48,0.96 g/kg)and Compound Danshen Dripping Pills group(85 mg/kg),10 rats in each group.The rats in each group were given medicine continuously for 10 days.The electrocardiographic signals of rats were measured by the method of bioelectrical signals,and the levels of lactate dehydrogenase(LDH),creatine kinase(CK),creatine kinase isozymes(CK-MB)and aspartate(AST)in serum of rats were detected by automatic biochemical analyzer;the activities of malondialdehyde(MDA)and superoxide dismutase(SOD)in rat myocardium were measured by kit method.The contents of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA),and the mRNA expression levels of NF-κB,IκB,Bcl-2 and Bax in rat myocardium were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot was used to detect the protein expression levels of NF-κB,IκB,p-IκB,Bcl-2 and Bax in the ischemic myocardium.Results:Compared with the model group,the levels of LDH,CK,AST,MDA,TNF-αand IL-6 in the all groups with medication were decreased(P<0.01,P<0.05),SOD and CK-MB levels were increased(P<0.01),NF-κB and Bax mRNA expression levels were down-regulated(P<0.01),IκB and Bcl-2 mRNA expression levels were up-regulated(P<0.01).The expression of nucleus NF-κB/total NF-κB and p-IκB/IκB protein was decreased(P<0.01),and the expression of Bcl-2/Bax protein was increased(P<0.05,P<0.01),but there was no significant difference between the starch matrix group and the model group.Conclusion:Eugenol inclusion complex can inhibit the nuclear translocation of NF-κB,down regulate p-IκB/IκB,up regulate the ratio of Bcl-2/Bax,inhibit the activation of NF-κB signaling pathway,reduce the expression of inflammatory mediators and the production of free radicals.