Department of Cardiology, Yantaishan Hospital, Yantai, Shandong 264001, PR China.
Department of Diagnostics, BinZhou Medical University, Yantai, Shandong 264003, PR China.
Tissue Cell. 2022 Aug;77:101828. doi: 10.1016/j.tice.2022.101828. Epub 2022 May 20.
The regulation of GPX4 by AAR and AAR was investigated, and whether the inhibition of AAR and AAR on ferroptosis of myocardial cell is related to GPX4 was also discussed.
we constructed a rat model of myocardial ischemia and reperfusion (MIR) model and hypoxia/reoxygenation (H/R) model of H9C2 cells, and MIR rats were intraperitoneally injected with A1AR and A2bAR agonists and antagonists. TTC staining, DHE, TUNEL, western blot experiments, immunohistochemistry assay were implemented to analyze the influence of AAR and AAR on ferroptosis and potential role of GPX4. To further authenticate the result of non-specific agonists and antagonists, we transfected siRNA interference or overexpression vectors into cells. CCK8, flow cytometry and western blot were performed to evaluate cell proliferation and apoptosis, and the expression of GPX4 and ferroptosis-related proteins.
The experimental results showed that reduced expression of AAR, AAR and GPX4 was found after MIR. AAR and AAR activation by agonists increased GPX4 expression and decreased production of lipid ROS, further inhibiting apoptosis of cardiomyocytes. In addition, we also analyzed the effect of AAR and AAR on ferroptosis-related proteins. We found that expression of FIH1 protein increased and expression of ACSL4 and NOX1 proteins decreased. Consistent with results in vivo, cellular data also indicated that AAR and AAR overexpression could increase proliferation ability of H9C2, and inhibit apoptosis and ROS production, upregulate GPX4 and FIH1, and downregulate ACSL4 and NOX1.
AAR and AAR could regulate GPX4, thereby affecting ferroptosis of cardiomyocytes in a rat model of MIR.
研究 AAR 和 A2bAR 对 GPX4 的调节作用，探讨抑制 AAR 和 A2bAR 对心肌细胞铁死亡的作用是否与 GPX4 有关。
构建大鼠心肌缺血再灌注（MIR）模型和 H9C2 细胞缺氧/复氧（H/R）模型，腹腔注射 A1AR 和 A2bAR 激动剂和拮抗剂，采用 TTC 染色、DHE、TUNEL、western blot 实验、免疫组化检测分析 AAR 和 A2bAR 对铁死亡的影响及其潜在的 GPX4 作用。为进一步验证非特异性激动剂和拮抗剂的结果，我们将 siRNA 干扰或过表达载体转染细胞。CCK8、流式细胞术和 western blot 检测细胞增殖和凋亡情况以及 GPX4 和铁死亡相关蛋白的表达。
实验结果表明，MIR 后 AAR、A2bAR 和 GPX4 的表达减少。激动剂激活 AAR 和 A2bAR 增加了 GPX4 的表达，减少了脂质 ROS 的产生，进一步抑制了心肌细胞的凋亡。此外，我们还分析了 AAR 和 A2bAR 对铁死亡相关蛋白的影响。结果表明，FIH1 蛋白表达增加，ACSL4 和 NOX1 蛋白表达减少。与体内结果一致，细胞数据也表明 AAR 和 A2bAR 过表达可以增加 H9C2 的增殖能力，抑制细胞凋亡和 ROS 产生，上调 GPX4 和 FIH1，下调 ACSL4 和 NOX1。
AAR 和 A2bAR 可以调节 GPX4，从而影响 MIR 大鼠模型中心肌细胞的铁死亡。
