灯盏花素通过转录因子cAMP反应元件结合蛋白磷酸化促进细胞自噬保护心肌细胞缺血-再灌注损伤的机制研究

Mechanism of scutellarin promoting autophagy via phosphorylation of transcription factor cAMP response element binding protein to protect myocardial cells from ischemia-reperfusion injury

背景心肌缺血-再灌注(ischemia-reperfusion injury,I/R)损伤是心肌梗死血流再灌注后出现的常见临床问题,自噬在心肌I/R损伤过程中的作用机制尚不完全明确。目的研究灯盏花素在保护心肌缺血再灌注损伤过程中通过转录因子cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)促进自噬减轻细胞损伤的机制。方法在50μmol/L灯盏花素为最佳应答浓度的基础上进行实验。实验分为正常对照(Vehicle)组、I/R组、灯盏花素处理(Scu+I/R)组和CREB抑制并灯盏花素处理(KG501+Scu+I/R)组。通过糖氧剥夺培养方式建立小鼠心肌细胞I/R损伤模型,MTT法测定细胞活力;生化法测定超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、乳酸脱氢酶(lactate dehydrogenase,LDH)和三磷酸腺苷(adenosine triphosphate,ATP)水平;RT-PCR测定线粒体内膜融合蛋白1(optic atrophy 1,Opa1)和线粒体动力相关蛋白1(dynamin-related protein 1,Drp1)表达;Western blot测定LC3、CREB和磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response element binding protein,p-CREB)表达。结果MTT测定发现:相比于其他浓度,50μmol/L灯盏花素可显著增加细胞活力,使I/R损伤心肌细胞SOD表达增加(P<0.01),MDA表达减少(P<0.01),LDH表达减少(P<0.01),ATP表达增加(P<0.01),LC3Ⅱ/Ⅰ比值增加(P<0.05),Drp1 mRNA表达减少(P<0.01),Opa1 mRNA表达增加(P<0.01),p-CREB/CREB比值增加(P<0.01)。CREB抑制剂(KG-501)可显著降低灯盏花素处理组的ATP、Opa1 mRNA表达(P均<0.01),使LC3Ⅱ/Ⅰ比值降低(P<0.05),细胞活力降低(P<0.01)。结论灯盏花素能够促进缺血心肌细胞自噬,缓解氧化损伤,提高心肌细胞活力。在保护心肌缺血-再灌注损伤过程中,灯盏花素可能通过CREB通路调节自噬作用。

Background Myocardial ischemia-reperfusion(I/R)injury is a common clinical problem after myocardial infarction reperfusion.However,the mechanism of autophagy in myocardial I/R process is not fully understood.Objective To investigate the mechanism of scutellarin in protecting myocardial ischemia reperfusion injury by promoting autophagy through the transcription factor cyclic adenylate response element binding protein(CREB).Methods Corresponding research was carried out on the basis of50μmol/L Scu as the optimal response concentration.The experimental groups were divided into normal control(vehicle)group,ischemia-reperfusion injury(I/R)group,Scu intervention(Scu+I/R)group and CREB inhibition combined with Scu intervention(KG501+Scu+I/R)group.The mouse-derived cardiomyocyte I/R model was established by glucose-oxygen deprivation culture,and MTT method was used to measure cell viability;biochemical method was used to measure SOD,MDA,LDH and ATP levels;RT-PCR was used to measure Opa1 and Drp1 expression;Western blots were used to measure LC3,CREB and p-CREB expression.Results MTT assay found that compared with other concentrations,50μmol/L of scutellarin could significantly increase cell viability,resulting in the increase of SOD expression in I/R injured cardiomyocytes(P<0.01),ATP production(P<0.01)and LC3Ⅱ/Ⅰratio(P<0.05),Opa1(P<0.01)mRNA expression and the p-CREB/CREB ratio,while the decrease of MDA expression(P<0.01),LDH expression(P<0.01),and Drp1(P<0.01)mRNA expression.CREB inhibitor(KG-501)could significantly reduce the expression of ATP and the ratio of p-CREB/CREB in the SCU treatment group(P<0.01),and reduce the ratio of LC3Ⅱ/Ⅰ(P<0.05).Conclusion Scu can promote autophagy of ischemic cardiomyocytes,relieve oxidative damage,and improve the vitality of cardiomyocytes.In the process of protecting myocardial ischemia-reperfusion injury,Scu may regulate autophagy through the CREB pathway.