【摘要】 目的：探讨Klotho蛋白对缺氧/复氧（hypoxia/reoxygenation, H/R）损伤大鼠心肌细胞的保护作用及机制研究。方法：以大鼠心肌细胞H9c2为研究对象，分为对照组、H/R组、低浓度Klotho+H/R组、高浓度Klotho+H/R组，经1μmol/L或10μmol/L Klotho预处理心肌细胞后，进行H/R以诱导细胞损伤。流式细胞仪检测各组心肌细胞凋亡率，DCFH-DA探针法检测各组心肌细胞中活性氧（reactive oxygen species, ROS）水平，试剂盒检测各组心肌细胞中超氧化物歧化酶（superoxide dismutase, SOD）活性和丙二醛（malondialdehyde, MDA）含量，JC-1法检测各组心肌细胞线粒体膜电位变化，测定各组心肌细胞中caspase-3和caspase-9活性，Western blot法检测各组心肌细胞中细胞色素C(cytochrome C, Cyt-C)、B细胞淋巴瘤2(B-cell lymphoma-2, Bcl-2)和Bcl-2关联X蛋白（Bcl-2-associated X protein, Bax）的蛋白表达水平。结果：与对照组比较，H/R组心肌细胞凋亡率增加（P<0.05）,ROS水平和MDA含量升高，SOD活性降低（P<0.05），线粒体膜电位下降（P<0.05）,caspase-3和caspase-9活性升高（P<0.05），线粒体Cyt-C和Bcl-2蛋白相对表达量下调（P<0.05），细胞质Cyt-C和Bax蛋白相对表达量上调（P<0.05）；与H/R组比较，低、高浓度Klotho+H/R组大鼠心肌细胞凋亡减少（P<0.05）,ROS水平和MDA含量降低而SOD活性升高（P<0.05），线粒体膜电位升高（P<0.05）,caspase-3和caspase-9活性降低（P<0.05），线粒体Cyt-C和Bcl-2蛋白相对表达量上调（P<0.05），且细胞质Cyt-C和Bax蛋白相对表达量下调（P<0.05）。此外，高浓度Klotho对H/R诱导的心肌细胞中各项损伤指标改善更为显著（P<0.05）。结论：Klotho蛋白能够减轻H/R诱导的大鼠心肌细胞损伤，其作用机制可能与抑制线粒体介导的凋亡有关。更多还原

【Abstract】 AIM: To investigate the protective effects of Klotho protein against hypoxia/reoxygenation(H/R)-induced damage in rat cardiomyocytes, and to elucidate its underlying mechanisms. METHODS: Rat cardiomyocyte H9c2 cells were divided into 4 groups: control, H/R, low-concentration(1 μmol/L) Klotho+H/R, and high-concentration(10 μmol/L) Klotho+H/R groups. Cells were pretreated with Klotho at specified concentrations before induction of H/R injury. Flow cytometry was used to determine cardiomyocyte apoptosis rates, while reactive oxygen species(ROS) levels were measured using the DCFH-DA probe. Additionally, superoxide dismutase(SOD) activity and malondialdehyde(MDA) content were assessed using biochemical assay kits. Mitochondrial membrane potential was evaluated using the JC-1 assay, and activity of caspase-3 and caspase-9 was quantified. Western blot analysis was conducted to measure the protein expression of cytochrome C(Cyt-C), B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax) in cardiomyocytes from each group. RESULTS: Compared with the control group, the H/R group exhibited significantly increased apoptosis rates(P<0. 05), elevated ROS levels and MDA content, decreased SOD activity(P<0. 05), reduced mitochondrial membrane potential(P<0. 05), increased caspase-3 and caspase-9 activity(P<0. 05), decreased mitochondrial CytC and Bcl-2 protein expression(P<0. 05), and increased cytoplasmic Cyt-C and Bax protein expression(P<0. 05). In comparison with the H/R group, both low-and high-concentration Klotho treatments significantly reduced cardiomyocyte apoptosis(P<0. 05), lowered ROS levels and MDA content(P<0. 05), increased SOD activity(P<0. 05), restored mitochondrial membrane potential(P<0. 05), decreased caspase-3 and caspase-9 activity(P<0. 05), increased mitochondrial Cyt-C and Bcl-2 expression(P<0. 05), and decreased cytoplasmic Cyt-C and Bax expression(P<0. 05). Notably, the high-concentration Klotho group demonstrated more pronounced protective effects(P<0. 05). CONCLUSION: Klotho protein exerts protective effects against H/R-induced cardiomyocyte injury, possibly by inhibiting mitochondria-mediated apoptosis.更多还原