丹参酮ⅡA对缺血再灌注损伤的乳鼠心肌细胞Notch信号通路的影响

Effect of Tanshinone ⅡA on Notch Signal Pathway in the Neonatal Rat Cardiac Myocytes of Ischemic/Reperfusion Injury

关键词:  丹参酮ⅡA  Notch信号通路  缺血再灌注损伤  心肌细胞

Key words:  tanshinone ⅡA  Notch signal pathway  ischemic/reperfusion injury  cardiomyocytes

摘要:

目的 研究丹参酮ⅡA预处理对缺血再灌注后乳鼠心肌细胞丙二醛(MDA)水平、细胞凋亡及细胞内Notch1、Hes1及HIF-1α mRNA表达的影响，从Notch信号通路活化水平阐明丹参酮ⅡA预处理防治缺血再灌注损伤(ischemia/ reperfusion，I/R)的分子机制。方法 培养乳鼠原代心肌细胞，建立心肌细胞I/R模型。并分为对照组、I/R模型组(先用正常培养液孵育1 h，然后模拟缺血2 h，正常DMEM再灌注4 h)、DAPT处理组(先用含DAPT的DMEM中孵育1 h，然后模拟缺血2 h，正常DMEM再灌注4 h)和丹参酮ⅡA处理组(先用含丹参酮IIA的DMEM中孵育1 h，然后模拟缺血2 h，正常DMEM再灌注4 h)。应用硫代巴比妥酸法测定MDA水平，用磷脂酰丝氨酸外翻分析法检测细胞凋亡，并用荧光定量PCR技术检测反映Notch信号通路活化水平的Notch信号通路受体Notch1 mRNA、下游信号分子Hes1 mRNA和HIF-1α mRNA表达。结果 与对照组比较，乳鼠I/R模型组心肌细胞MDA水平升高(P<0.01)，细胞早期凋亡率增加，Notch1、Hes1、HIF-1α mRNA表达水平增加(P<0.05，P<0.01，P<0.01)。与I/R模型组比较，DAPT处理组和丹参酮ⅡA处理组心肌细胞MDA水平降低(P<0.01)，心肌细胞早期凋亡率降低，Notch1、Hes1、HIF-1α mRNA表达水平下降(P均<0.01)。结论 丹参酮ⅡA在乳鼠心肌细胞I/R损伤中起到心肌保护作用，其可能的分子机制为抑制Notch信号通路的活化及调节细胞凋亡。

Abstract:

OBJECTIVE To illuminate the mechanism of tanshinone ⅡA on the prevention and treatment of myocardial ischemic/reperfusion(I/R) injury from the perspective of Notch signal pathway, the effect of tanshinone ⅡA on the level of MDA, cell apoptosis and the expression of Notch1, Hes1 and HIF-1α mRNA in the neonatal rat cardiac myocytes of I/R injury were studied. METHODS Culture the primary neonatal rat myocardiocytes, and establish a model of I/R injury according to previous reports. Four groups were settled, including control group, I/R model group, DAPT- treated group and tanshinone ⅡA-treated group. Myocardiocytes of I/R model group were incubated in DMEM medium for 1 h, then simulate ischemia for 2 h, after that DMEM medium were utilized for reperfusion for 4 h. DAPT-treated group were incubated with DMEM within DAPT for 1 h, then treated with the I/R injury procedure. Cells in tanshinone ⅡA-treated group were pretreated in DMEM medium with tanshinone ⅡA for 1 h, and simulate ischemia then reperfusion with DMEM. Production of MDA in myocardiocytes was tested by colorable reaction. Cell apoptosis was observed through Annexin V-PI staining. The activation of Notch signaling pathway and level of HIF-1α mRNA was detected by Real-time PCR. RESULTS Compared with control group, MDA levels in the myocardial cell of I/R model group rised (P<0.01), early cell apoptosis rate increased and the expression of Notch1, Hes1, HIF-1α mRNA were upregualted (P<0.05, P<0.01, P<0.01). Compared with I/R model group, MDA levels reduced(P<0.01), early apoptosis rate of myocardial cell decreased, the expression of Notch1, Hes1, and HIF-1α mRNA were downregulated(P<0.01) in the myocardial cell of DAPT-treated group and tanshinone ⅡA-treated group. CONCLUSION Chinese medicine monomer tanshinone ⅡA may protect neonatal rat cardiomyocytes from I/R injury through inactivate Notch pathway and regulating and cell apoptosis.