Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigated whether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitro via activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaic acid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flow cytometry, respectively. Release of HMGB1, TNFα or IL-6 was analyzed by ELISA. Oxidative stress was evaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac) by Western blot and the association between PP2Ac and α4 by co-immunoprecipitation. Activation of the NF-κB signaling pathway was assessed by detecting Ser32 phosphorylation level of IκBα as well as nuclear entry of p65 protein by Western blot. Activation of the GSK3β/MCL1 signaling pathway was assessed by detecting Ser9 phosphorylation level of GSK3β and protein level of MCL1. We found Metformin pre-treatment attenuated human and rat cardiomyocytes apoptosis, HMGB1, TNFα and IL-6 release and ROS production that were induced by high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metformin reduced the upregulation of PP2Ac pY307 and the PP2Ac-α4 association, which was not affected by okadaic acid treatment. Metformin pre-treatment reduced NF-κB activation in human and rat cardiomyocytes apoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaic acid treatment. GSK3β/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition. In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human and rat cardiomyocytes in vitro in a PP2A dependent manner.

中文翻译：

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高葡萄糖诱导的心肌细胞凋亡、ROS 产生和促炎反应通过 PP2A 激活被二甲双胍治疗减弱

二甲双胍已被证明可以改善糖尿病心肌病。在本研究中，我们研究了二甲双胍是否会通过 PP2A 的激活来减少体外高糖刺激诱导的心肌细胞凋亡。原代人和大鼠心肌细胞受到高葡萄糖刺激。冈田酸用于抑制 PP2A 活性。分别使用 CCK-8 和流式细胞术评估细胞活力和细胞凋亡。通过ELISA分析HMGB1、TNFα或IL-6的释放。通过测量细胞 ROS 和线粒体超氧化物水平来评估氧化应激。通过丝氨酸/苏氨酸磷酸酶测定系统或通过蛋白质印迹分析 PP2A 催化结构域 (PP2Ac) 的 Y307 磷酸化水平以及通过免疫共沉淀分析 PP2Ac 和 α4 之间的关联来评估 PP2A 活性。通过检测 IκBα 的 Ser32 磷酸化水平以及通过蛋白质印迹检测 p65 蛋白的核进入来评估 NF-κB 信号通路的激活。GSK3β/MCL1 信号通路的激活通过检测 GSK3β 的 Ser9 磷酸化水平和 MCL1 的蛋白质水平来评估。我们发现二甲双胍预处理减弱了高糖刺激诱导的人和大鼠心肌细胞凋亡、HMGB1、TNFα和IL-6的释放以及ROS的产生，而冈田酸处理可以阻断二甲双胍的这些作用。二甲双胍降低了 PP2Ac pY307 和 PP2Ac-α4 关联的上调，这不受冈田酸处理的影响。二甲双胍预处理降低了由高糖刺激引起的人和大鼠心肌细胞凋亡中的 NF-κB 活化，冈田酸处理可以阻断二甲双胍的这种作用。GSK3β/MCL1 不是二甲双胍激活 PP2A 诱导的心肌细胞死亡抑制的一部分。总之，二甲双胍在体外以 PP2A 依赖性方式减少原代人和大鼠心肌细胞的细胞凋亡、ROS 产生和炎症反应。