毛蕊花苷对心肌缺血再灌注大鼠Notch信号通路及Sirt3的影响

Influences of verbascoside on Notch signaling pathway and Sirt3 in rats with myocardial ischemia reperfusion

目的探究毛蕊花苷对心肌缺血再灌注(MI/R)大鼠Notch信号通路及沉默信息调节因子3(Sirt3)的影响。方法用结扎左冠状动脉前降支30 min,再灌注2 h建立MI/R模型。按照体质量将SD大鼠随机分为5组:假手术组、模型组、阳性对照组和毛蕊花苷低、高剂量组,每组12只。毛蕊花苷低、高剂量组分别腹腔注射毛蕊花苷10和20 mg·kg^(-1),阳性对照组腹腔注射5.4 mg·kg^(-1)曲美他嗪,假手术组和模型组分别注射等量的0.9%NaCl。再灌注开始时进行给药,共1次,假手术组分离左冠状动脉前降支,不结扎。用2,3,5-氯化三苯基四氮唑染色法检测大鼠心肌梗死面积,用TUNEL染色法检测心肌细胞凋亡指数,用试剂盒检测血浆中乳酸脱氢酶(LDH)含量及心肌组织丙二醛(MDA)、超氧化物歧化酶(SOD)含量,用流式细胞仪检测心肌组织内活性氧ROS的水平,用蛋白质印迹法检测心肌组织中Notch1、发状分裂相关增强子-l(Hes-1)和Sirt3蛋白的表达水平。结果假手术组、模型组、阳性对照组、毛蕊花苷高剂量组大鼠的心肌梗死面积百分比分别为0、(35.32±3.67)%、(11.49±1.01)%和(14.61±1.53)%;心肌细胞凋亡指数分别为(0.35±0.02)%、(36.23±3.06)%、(14.08±1.24)%和(13.26±1.86)%;MDA含量分别为(6.02±1.05)、(17.24±2.13)、(7.15±1.21)和(7.89±1.32)μmol·mg^(-1);SOD含量分别为(2.69±0.12)、(1.03±0.02)、(2.43±0.12)和(2.35±0.11)U·mg^(-1);Notch1蛋白表达水平分别为0.22±0.02、0.83±0.07、0.35±0.03和0.39±0.04;Sirt3蛋白表达水平分别为0.97±0.10、0.54±0.05、0.85±0.09和0.81±0.08。模型组的上述指标与假手术组比较,差异均有统计学意义(均P<0.05);模型组的上述指标与毛蕊花苷高剂量组比较,差异均有统计学意义(均P<0.05)。结论毛蕊花苷能够调节Notch信号通路及Sirt3的表达,减轻MI/R大鼠心肌组织氧化应激反应,缓解心肌组织损伤。

Objective To explore the influences of verbascoside on Notch signaling pathway and silent information regulator 3(Sirt3)in rats with myocardial ischemia/reperfusion(MI/R).Methods MI/R model was established by ligating the left anterior descending coronary artery for30 minutes and reperfusion for 2 h.According to body weight,SD rats were randomly divided into five groups:Sham operation group,model group,positive control group,experimental-L group,and experimental-H group,with 12 rats in each group.Experimental-L group and experimental-H group were injected intraperitoneally with 10 and 20 mg·kg^(-1)of verbascoside,respectively;the positive control group was injected with trimetazidine 5.4 mg·kg^(-1),and the sham operation group and the model group were injected with the same amount of normal saline.Dosing was administered at the start of reperfusion,once in total.In the sham operation group,the left anterior descending coronary artery was isolated without ligation.The size of myocardial infarction in rats was detected by 2,3,5-triphenyl tetrazolium chloride(TTC)staining;cardiomyocyte apoptosis index was detected by TUNEL staining;the kits were used to detect the content of lactate dehydrogenase(LDH)in plasma and the content of malondialdehyde(MDA)and superoxide dismutase(SOD)in myocardial tissue;flow cytometry was used to detect the level of reactive oxygen species(ROS)in myocardial tissue;Western blot was used to detect the protein expression levels of Notch1,hairy division-related enhancer-1(Hes-1)and Sirt3 in myocardial tissue.Results The percentage of myocardial infarction area in the sham operation group,model group,positive control group and experimental-H group were 0,(35.32±3.67)%,(11.49±1.01)%and(14.61±1.53)%,respectively;the apoptosis index of cardiomyocytes in these four groups were(0.35±0.02)%,(36.23±3.06)%,(14.08±1.24)%and(13.26±1.86)%,respectively;the MDA content of these groups were(6.02±1.05),(17.24±2.13),(7.15±1.21)and(7.89±1.32)μmol·mg^(-1),respectively;the SOD content of these four groups were(2.69±0.12),(1.03±0.02),(2.43±0.12)and(2.35±0.11)U·mg^(-1),respectively;the expression level of Notch1 protein in these four groups were 0.22±0.02,0.83±0.07,0.35±0.03and 0.39±0.04,respectively;the expression level of Sirt3 protein in these four groups were 0.97±0.10,0.54±0.05,0.85±0.09 and 0.81±0.08,respectively;there were statistically differences between the model group and the sham operation group in the above indexes(all P<0.05);there were statistically differences between the model group and the experimental-H group(all P<0.05).Conclusion Verbascoside can regulate the Notch signaling pathway and Sirt3 expression,reduce the oxidative stress response of myocardial tissue in MI/R rats,and alleviate myocardial tissue damage.