Zheng Meihua, Qiao Weili, Cui Jie, Liu Lei, Liu Hong, Wang Zhirong, Yan Changdong
Department of Physiology, Xuzhou Medical College, #209 Tongshan Road, Xuzhou, 221004, Jiangsu, China.
Mol Cell Biochem. 2014 Aug;393(1-2):59-67. doi: 10.1007/s11010-014-2046-y. Epub 2014 Apr 13.
The present study was designed to investigate the effect of hydrogen sulfide on cellular senescence of human umbilical vascular endothelial cells (HUVECs CC-2517) and its underlying mechanism. The premature senescence-like phenotype HUVECs (the fourth passage) was induced by treatment with nicotinamide (NAM, an inhibitor of SIRT1, 5 mmol/L, 12 h). Cells were cultured with sodium hydrosulfide (NaHS, 12.5, 25, 50 and 100 μmol/L) for 48 h in premature senescence-like phenotype HUVECs. The fourth passage of HUVECs was considered as young group. Senescence-associated (SA)-β-galactosidase activities were detected to evaluate cell senescence, and the expression of SA heterochromatin foci (SAHF) was visualized by DAPI DNA staining. The mRNA and protein levels of SIRT1 were detected using RT-PCR and western blotting analysis, respectively. The results showed that β-galactosidase positive cells and the formation of SAHF were markedly increased after treatment with NAM (5 mmol/L) for 12 h. We also found that NaHS (12.5 μmol/L) had no effect on the percentage of SA β-gal positive cells and the expression of SAHF, and the hallmarks decreased at the concentration of 25 and 50 μmol/L, reaching the minimum at 50 μmol/L, while the percentage of SA β-gal positive cells and the expression of SAHF increased at the concentration of 100 μmol/L. Furthermore, we found that both on protein and mRNA levels of SIRT1 in the Y+N+S50 group was significantly increased compared with that in Y+N group. In conclusion, NaHS delays senescence of HUVECs induced by NAM via upregulation of SIRT1 expression.
本研究旨在探讨硫化氢对人脐静脉内皮细胞（HUVECs CC-2517）细胞衰老的影响及其潜在机制。用烟酰胺（NAM，一种SIRT1抑制剂，5 mmol/L，12小时）处理诱导过早衰老样表型的HUVECs（第4代）。将过早衰老样表型的HUVECs用硫氢化钠（NaHS，12.5、25、50和100 μmol/L）培养48小时。第4代HUVECs作为年轻组。检测衰老相关（SA）-β-半乳糖苷酶活性以评估细胞衰老，并通过DAPI DNA染色观察SA异染色质灶（SAHF）的表达。分别使用RT-PCR和蛋白质印迹分析检测SIRT1的mRNA和蛋白质水平。结果显示，用5 mmol/L NAM处理12小时后，β-半乳糖苷酶阳性细胞和SAHF的形成明显增加。我们还发现，12.5 μmol/L的NaHS对SA β-半乳糖苷阳性细胞百分比和SAHF的表达没有影响，在25和50 μmol/L浓度时这些特征降低，在50 μmol/L时达到最低，而在100 μmol/L浓度时SA β-半乳糖苷阳性细胞百分比和SAHF的表达增加。此外，我们发现Y+N+S50组中SIRT1的蛋白质和mRNA水平均比Y+N组显著增加。总之，NaHS通过上调SIRT1表达延缓了NAM诱导的HUVECs衰老。
