目的: 研究远隔缺血预处理(RIPC)分离液对人肾小管上皮细胞(HK-2)缺氧/复氧(H/R)损伤的影响及机制。方法: 将HK-2细胞分为时间对照组、H/R、RIPC处理组、自噬干预组。时间对照组细胞在正常条件下培养,RIPC组细胞使用RIPC分离液预处理,自噬干预组细胞在RIPC处理基础上,加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3MA),后三组细胞使用三气培养箱进行H/R构建损伤模型。模型成功后使用光学显微镜观察各组细胞状态,应用Western blot观察LC3Ⅱ/Ⅰ蛋白、Beclin1蛋白的表达水平。结果: 镜下示RIPC分离液可明显减轻H/R对HK-2细胞的损伤,加入自噬抑制剂,RIPC分离液对HK-2细胞H/R损伤的保护作用被阻断。Western blot结果显示,RIPC分离液可使HK-2细胞线粒体中的自噬特异性蛋白LC3Ⅱ/Ⅰ、Beclin1表达水平增高。Real-time PCR结果显示,与H/R组比,RIPC组LC3及Beclin1的mRNA表达量明显升高;加入自噬抑制剂,HK-2细胞线粒体中的自噬特异性蛋白LC3Ⅱ/Ⅰ、Beclin1表达降低,LC3及Beclin1的mRNA表达量同时也明显降低。结论: RIPC分离液对HK-2细胞H/R后损伤具有保护作用,作用机制可能与线粒体自噬相关。
Objective: To investigate the effect and mechanism of remote ischemic preconditioning (RIPC) separating medium on hypoxia/reoxygenation (H/R) injury of human renal tubular epithelial cells (HK-2). Methods: HK-2 cells were divided into time control group, H/R, RIPC treatment group and autophagy intervention group. Cells in the time control group were cultured under normal conditions. Cells in the RIPC group were pretreated with RIPC separating medium. Cells in the autophagy intervention group were treated with autophagy inhibitor 3-methyladenine (3MA) on the basis of RIPC treatment. Cells in the latter three groups were subjected to H/R in a three-gas incubator to construct a damage model. After the success of the model, the cell status of each group was observed by optical microscope, and the expression levels of LC3Ⅱ/Ⅰ protein and Beclin1 protein were observed by Western blot. Results: Under the microscope, RIPC separating medium could significantly reduce the damage of H/R to HK-2 cells. After adding autophagy inhibitor, the protective effect of RIPC separating medium on H/R injury of HK-2 cells was blocked. The results of Western blot showed that RIPC separating medium could increase the expression levels of autophagy-specific proteins LC3Ⅱ/Ⅰ and Beclin1 in mitochondria of HK-2 cells. The results of Real-time PCR showed that the mRNA expression of LC3 and Beclin1 in RIPC group was significantly higher than that in H/R group; the expression of autophagy-specific proteins LC3Ⅱ/Ⅰ and Beclin1 in mitochondria of HK-2 cells was decreased by adding autophagy inhibitors, and the mRNA expression of LC3 and Beclin1 was also significantly decreased. Conclusion: RIPC separating medium has a protective effect on HK-2 cells after H/R injury, and the mechanism may be related to mitophagy.
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