依托咪酯调控Sirt1/FOXO1通路对心肌缺血再灌注大鼠模型的保护作用

Protective Effect of Etomidate on Myocardial Ischemia-reperfusion via Regulating Sirt1/FOXO1 Pathway in Rats

目的基于沉默信息调节因子1(Sirt1)/叉头框转录因子O亚族1(FOXO1)通路探究依托咪酯(Eto)对心肌缺血再灌注损伤(MIRI)大鼠模型的保护作用。方法将60只大鼠随机分为假手术组(Sham组)、MIRI组、依托咪酯低、高剂量组(L-Eto、H-Eto组,0.5、1 mg/kg)、通路抑制剂组(Eto+EX527组,1 mg/kg Eto+2 mg/kg EX527),每组12只。除Sham组外,其余大鼠采用手术结扎冠状动脉左前降支的方法复制大鼠MIRI模型。给药组大鼠于再灌注后2 h尾静脉注射相应剂量药物;1次/d,持续28 d。于手术前和末次给药2 h后超声心电图检查大鼠心脏功能;生化法检测血清中肌酸激酶同工酶MB(CKMB)、肌钙蛋白I(cTnI)、乳酸脱氢酶(LDH)水平和心肌组织匀浆中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;苏木精-伊红(HE)染色观察心脏组织形态变化;流式细胞术检测心肌细胞凋亡;荧光探针测定心肌中活性氧(ROS)水平;JC-1法检测线粒体膜电位(MMP)变化;Western blot检测心肌组织Sirt1/FOXO1通路和凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)的表达。结果与Sham组相比,MIRI组大鼠左心室舒张末期容积(LVEDV)、左心室收缩末期容积(LVESV)、血清CKMB、cTnI、LDH水平、心肌MDA水平、心肌细胞的凋亡率、ROS水平、心肌乙酰化FOXO1(Ac-FOXO1)、Bax、Caspase-3的表达显著升高(均P<0.05),左心室射血分数(LVEF)和左心室短轴缩短率(LVFS)、心肌SOD、GSH-Px水平、MMP、心肌Sirt1、Bcl-2的表达显著下降(均P<0.05),心肌纤维紊乱,细胞肿胀、坏死,大量炎性细胞浸润;与MIRI组相比,L-Eto、H-Eto组大鼠的LVESV、LVEDV、血清CKMB、cTnI、LDH水平、心肌MDA水平、心肌细胞的凋亡率、ROS水平、心肌Ac-FOXO1、Bax、Caspase-3的表达明显下降(均P<0.05),LVEF、LVFS、心肌SOD、GSH-Px水平、MMP、Sirt1、Bcl-2的表达明显升高(均P<0.05);心肌损伤明显减轻。EX527可逆转Eto的抗氧化活性和心肌保护作用。结论Eto对MIRI的保护作用可能与激活Sirt1,下调乙酰化FOXO1的表达,进而改善线粒体功能障碍,抑制氧化应激和细胞凋亡有关。

Objective To explore the protective effect of etomidate(Eto)on myocardial ischemia-reperfusion injury(MIRI)rat model based on silence information regulator 1(Sirt1)/forkhead box transcription factor O subgroup 1(FOXO1)pathway.Methods Sixty rats were randomly divided into sham operation group(Sham group),MIRI group,low-and high-dose Eto groups(L-Eto,H-Eto groups,0.5 and 1 mg/kg),and pathway inhibitor group(Eto+EX527 group,1 mg/kg Eto+2 mg/kg EX527),with 12 rats in each group.In addition to Sham group,the rest of the rats were used to establish MIRI model by ligating left anterior descending coronary artery.The rats in the treatment groups were injected with the corresponding dose of drugs through tail vein 2 h after reperfusion;once a day for 28 days.The cardiac function of rats was examined by ultrasonic cardiogram before operation and 2 h after the last administration;the levels of creatine kinase isoenzyme MB(CKMB),cardiac troponin I(cTnI),lactate dehydrogenase(LDH)in serum and levels of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in myocardial tissue homogenate were detected by biochemical method;the morphological changes of heart tissue were observed by HE staining;cardiomyocyte apoptosis was detected by flow cytometry;the level of reactive oxygen species(ROS)in myocardium was measured by fluorescence probe;the changes of mitochondrial membrane potential(MMP)were detected by JC-1 method;the expression of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)and Caspase-3 was detected by Western blotting.Results Compared with those in Sham group,the left ventricular end diastolic volume(LVEDV),left ventricular end systolic volume(LVESV),serum CKMB,cTnI and LDH levels,myocardial MDA level,apoptosis rate of cardiomyocytes,ROS level,acetylated-FOXO1(ac-FOXO1),Bax and Caspase-3 expression in MIRI group were significantly higher(all P<0.05),left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),myocardial SOD and GSH-Px levels,MMP,Sirt1 and Bcl-2 expression were significantly lower(all P<0.05),myocardial fibers were disordered,the cells were swollen and necrotic,a large number of inflammatory cells were infiltrated,compared with those in MIRI group,the LVESV,LVEDV,serum CKMB,cTnI and LDH levels,myocardial MDA level,apoptosis rate of cardiomyocytes,ROS level,ac-FOXO1,Bax and Caspase-3 expression in L-Eto、H-Eto groups were significantly lower(all P<0.05),the LVEF,LVFS,myocardial SOD and GSH-Px levels,MMP,Sirt1 and Bcl-2 expression were significantly higher(all P<0.05);myocardial injury was significantly reduced.EX527 can significantly reverse the antioxidant activity and myocardial protection of Eto.Conclusion The protective effect of Eto on MIRI may be related to activation of Sirt1,down-regulation of acetylated FOXO1 expression,improvement of mitochondrial dysfunction,inhibition of oxidative stress and apoptosis.