二甲双胍激活Sirt3信号抑制高糖诱导内皮细胞衰老的实验研究

目的: 研究二甲双胍抑制高糖诱导人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)衰老的作用与机制。方法: 传代培养HUVECs,高糖刺激HUVECs 48 h建立血管内皮细胞衰老模型,采用噻唑蓝法(MTT)检测HUVECs活力,2',7'-二氢二氯荧光素二乙酸酯(DCFH-DA)为荧光探针检测细胞内活性氧(ROS)水平,硫代巴比妥酸法检测丙二醛(MDA)含量,酶联免疫吸附实验(ELISA)检测细胞培养上清液中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的分泌量,β-半乳糖苷酶染色检测细胞衰老水平。使用Sirt3抑制剂3-TYP作用HUVECs后,蛋白免疫印迹法(Western blot)检测p53、PAI-1及Sirt3的蛋白表达水平。结果: 与正常对照组相比,高糖组中HUVECs的活力降低,细胞内ROS的水平和MDA含量增加,IL-6、TNF-α的分泌量增加,衰老阳性细胞的百分比增加,二甲双胍干预后能明显改善高糖诱导HUVECs的上述变化。3-TYP能显著逆转二甲双胍对p53、PAI-1、Sirt3蛋白表达的调控作用。结论: 二甲双胍对高糖诱导的HUVECs衰老具有明显改善作用,其作用机制与激活Sirt3密切相关。

Inhibitory effects of metformin on endothelial cell senescence induced by high glucose via activating Sirt3 signal

OBJECTIVE To explore the inhibitory effects and mechanism of metformin on human umbilical vein endothelial cells (HUVECs) senescence induced by high glucose.METHODS HUVECs was sub-cultured in medium, and then cell senescence model was successfully reproduced by exposing to high glucose for 48 h. Cell viability was detected by thiazole blue assay (MTT). The intracellular reactive oxygen species (ROS) levels was determined by using DCFH-DA probes, and thiobarbituric acid method was used to detect malondialdehyde (MDA) content. The secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant was measured by ELISA. The number of senescence cells was analyzed by β-galactosidase staining. 3-TYP used as Sirt3 inhibitor was treated on HUVECs, the protein expression levels of p53, PAI-1 and Sirt3 were determined by Western blot.RESULTS Compared with those of the control group, the cell viability was significantly decreased, the intracellular contents of ROS, MDA, IL-6 and TNF-α were markedly up-regulated, and the percentage of senescence-positive cells were increased in the high glucose group. Preincubating with metformin could ameliorate the relevant indexes. Besides, 3-TYP could significantly reverse the regulatory effects of metformin on the protein expressions of p53, PAI-1 and Sirt3.CONCLUSION Metformin can alleviate high glucose-induced HUVECs senescence, and the mechanism may involve in activating Sirt3.