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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-1αm RNA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 m RNA、下游信号分子Hes1 m RNA和HIF-1αm RNA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-1αm RNA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-1αm RNA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。
Objective To investigate the effect of tanshinone Ⅱ A preconditioning on malondialdehyde (MDA), apoptosis and expression of Notch1, Hes1 and HIF-1αmRNA in myocardial cells of neonatal rats after ischemia / reperfusion. To elucidate the effects of tanshinone ⅡA pretreatment on the activation of Notch signaling pathway Preventive and therapeutic effects of ⅡA on the prevention and treatment of ischemia / reperfusion (I / R). Methods Primary cultured neonatal rat cardiomyocytes were cultured and the cardiomyocyte I / R model was established. The rats were divided into control group, I / R model group (incubated with normal medium for 1 h, then simulated ischemia for 2 h, normal DMEM for 4 h), DAPT group Then simulated ischemia 2 h, normal DMEM reperfusion 4 h) and tanshinone IIA treatment group (first incubated with tanshinone IIA in DMEM for 1 h, then simulated ischemia 2 h, normal DMEM reperfusion 4 h). The level of MDA was measured by thiobarbituric acid method, the apoptosis was detected by phosphatidylserine ectopic analysis, and the Notch1 m RNA which reflects the activation of Notch signaling pathway and the downstream signaling molecules Hes1 m RNA and HIF-1α mRNA expression. Results Compared with the control group, the MDA level in neonatal rat model of I / R increased (P <0.01), the rate of early apoptosis increased, the expression of Notch1, Hes1 and HIF-1αmRNA increased (P <0.05, P < 0.01, P <0.01). Compared with the I / R model group, the MDA level in cardiomyocytes of DAPT group and Tanshinone ⅡA group decreased (P <0.01), the apoptosis rate of cardiomyocytes decreased, the expression of Notch1, Hes1 and HIF-1αmRNA decreased (P < 0.01). Conclusion Tanshinone Ⅱ A plays a protective role in I / R injury of neonatal rat cardiomyocytes. Its possible molecular mechanism is to inhibit Notch signaling pathway activation and regulate apoptosis.