基于mTORC1/4EBP1信号通路探讨银盏心脉滴丸对缺氧/复氧损伤大鼠心肌细胞线粒体功能的影响

The exploration of the effect of Yinzhan Xinmai dropping pills on mitochondrial function of anoxia/reoxygenation injured rat cardiomyocytes, based on mtorc1/4ebp1 signaling pathway

目的观察银盏心脉滴丸对大鼠心肌细胞(H9C2)mTORC1/4EBP1信号通路的影响以探讨其对线粒体功能的机制。方法制备银盏心脉滴丸含药血清。将细胞分为对照组、模型组(缺氧/复氧)、空白血清组及银盏心脉滴丸含药血清组。制备缺氧/复氧细胞损伤模型,利用激光共聚焦检测线粒体膜电位(ΔΨm),利用流式细胞仪检测活性氧(reactive oxygen species, ROS),运用Western Blot技术检测细胞雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、真核翻译起始因子4E结合蛋白(eukaryotic translation initiation factor 4E-binding proteins,4E-BP)、mTOR复合体蛋白(regulatory-associated protein of mammalian target of rapamycin,Raptor)蛋白表达,运用q-PCR方法检测mTORC1、4EBP1、Raptor的mRNA含量。结果与对照组相比,模型组细胞内绿色荧光增强,ΔΨm显著高于对照组(P<0.05);而与模型组相比,银盏心脉滴丸组红色荧光增强(P<0.05),说明银盏心脉保护ΔΨm稳定,保护线粒体膜结构;与对照组相比,模型组的ROS合成较对照组升高(P<0.05);而与模型组相比,银盏心脉滴丸组细胞内ROS产生明显降低(P<0.05);与对照组相比,模型组mTOR、Raptor基因mRNA水平下调(P<0.05),4ebp1基因mRNA水平上调,差异有统计学意义(P<0.05)。与模型组比较,银盏心脉滴丸组mTOR、Raptor基因mRNA水平上调(P<0.05),4ebp1基因mRNA水平下调(P<0.05)。结论银盏心脉滴丸改善缺氧复氧损伤的机制可能是通过激活mTORC1/4EBP1信号通路,从而抑制线粒体膜电位,改善活性氧,保护线粒体功能。

Objective To observe the effect of Yinzhan Xinmai dropping pills on the mTORC1/4EBP1 signaling pathway in rat cardiomyocytes(H9C2)and to explore its mechanism of mitochondrial function.Methods Prepare serum containing Yinzhan Xinmai dropping pills.The cells were divided into control group and model group(hypoxia/reoxygenation,blank serum group,and serum containing Yinzhan Xinmai dropping pills group The hypoxia/reoxygenation cell injury model was prepared.The mitochondrial membrane potential(ΔΨm)was detected by laser confocal microscopy.The reactive oxygen species(reactive oxygen species,ROS)were detected by flow cytometry.The expressions of mTORC1(mammalian target of rapamycin),eukaryotic translation initiation factor 4E-binding proteins,4E-BP,and Raptor(regulatory-associated protein of mammalian target of rapamycin)proteins were detected by Western Blot.The mRNA levels of mTORC1,4EBP1 and Raptor were detected by q-PCR.Results Compared with the control group,the intracellular green fluorescence of the model group was enhanced(P<0.05),andΔΨm was significantly higher than that of the control group.Compared with the model group(P<0.05),red fluorescence of serum containing Yinzhan Xinmai dropping pills group enhanced,indicating that the serum containing Yinzhan Xinmai dropping pills protectΔΨm stability,protect the mitochondrial membrane structure;compared with the model group,the ROS synthesis was higher than that of the control group(P<0.05).Compared with the control group,intracellular ROS production was significantly decreased in the Yinzhan Xinmai dropping pills group(P<0.05)was significantly decreased;Compared with the control group,the mRNA level of mTOR and Raptor were decreased(P<0.05),and the mRNA level of 4ebp1 gene was increased(P<0.05).Compared with model group,mRNA levels of mTOR and Raptor in Yinzhan Xinmai dropping pills group were increased(P<0.05)and 4ebp1 gene mRNA level decreased(P<0.05).Conclusion Yinzhan Xinmai dropping pills can improve the mechanism of hypoxia-reoxygenation injury by activating the mTORC1/4EBP1 signaling pathway,thereby inhibiting mitochondrial membrane potential,improving reactive oxygen species,and protecting mitochondrial function.