H_(2)S拮抗HUVEC衰老与促进SIRT1巯基化和减少FOXO1乙酰化有关

The antagonist role of H_(2)S against senescence of HUVEC may involve activation of SIRT1 S-sulfhydrylation and reduction of FOXO1 acetylation

[目的]探讨沉默信息调节因子1(SIRT1)/叉头转录因子O1(FOXO1)在H_(2)S拮抗H_(2)O_(2)诱导内皮细胞衰老过程中的作用。[方法]建立内皮细胞衰老模型,通过衰老相关β-半乳糖苷酶(SA-β-gal)染色在光学显微镜下观察到的蓝染细胞数(即衰老细胞)计算阳性细胞率。采用Western blot检测细胞P21、P53、纤溶酶原激活物抑制剂1(PAI-1)、叉头转录因子O1(FOXO1)、乙酰化FOXO1(ac-FOXO1)、锰超氧化物歧化酶(MnSOD)及过氧化氢酶的蛋白表达水平,采用生物素转换法测定S-巯基化SIRT1的表达,采用活性氧(ROS)检测定量评估细胞内ROS水平。[结果]经100μmol/L H_(2)O_(2)处理可显著提高SA-β-gal染色阳性细胞率和P21、P53、PAI-1的蛋白表达,提示衰老细胞模型成功建立,而100μmol/L NaHS可明显拮抗这一作用,SA-β-gal染色阳性细胞数明显下降(P<0.01),P21、P53、PAI-1的蛋白表达显著降低(P<0.01)。与对照组相比,H_(2)O_(2)组SIRT1、FOXO1、ac-FOXO1、MnSOD及过氧化氢酶的蛋白表达显著降低(P<0.05或P<0.01),ac-FOXO1/FOXO1比值显著增加(P<0.01),ROS水平明显升高(P<0.01)。与H_(2)O_(2)组相比,NaHS+H_(2)O_(2)组SIRT1、S-巯基化SIRT1、FOXO1、ac-FOXO1、MnSOD及过氧化氢酶的蛋白表达显著升高((P<0.05或P<0.01),ac-FOXO1/FOXO1比值显著下降(P<0.01),ROS水平明显降低(P<0.05)。[结论] H_(2)S可拮抗H_(2)O_(2)诱导的HUVEC衰老,其机制与促进SIRT1巯基化和减少FOXO1乙酰化有关。

Aim To explore the role of silent information regulator 1(SIRT1)/forkhead transcription factor O1(FOXO1)in H_(2)S antagonism of H_(2)O_(2)-induced endothelial cell senescence.Methods The positive cell rate was calculated by the number of blue-stained cells(senescent cells)observed under the light microscope by senescence-associatedβ-gal(SA-β-gal)staining.Western blot was used to detect the expression levels of P21,P53,plasminogen activator inhibitor-1(PAI-1),FOXO1,acetylated FOXO1(ac-FOXO1),manganese superoxide dismutase(MnSOD)and catalase proteins,and biotin-switch assay was used to measure the expression of S-sulfhydration of SIRT1,and ROS detection was used to quantitatively evaluate intracellular reactive oxygen species(ROS)level.Results Treatment with 100μmol/L H_(2)O_(2) significantly increased the SA-β-gal staining-positive cell rate and the expression of P21,P53 and PAI-1 proteins,suggesting that the senescent cell model was successfully established,whereas 100μmol/L NaHS significantly antagonized this effect,and the number of SA-β-gal staining-positive cells decreased significantly(P<0.01),and the expression of P21,P53 and PAI-1 proteins significantly reduced(P<0.01).Compared with the control group,the expression of SIRT1,FOXO1,ac-FOXO1,MnSOD and catalase proteins in the H_(2)O_(2) group significantly decreased(P<0.05 or P<0.01),the ac-FOXO1/FOXO1 ratio significantly increased(P<0.01),and the ROS level significantly increased(P<0.01).Compared with H_(2)O_(2) group,the expression of SIRT1,S-sulfhydration of SIRT1,FOXO1,ac-FOXO1,MnSOD and catalase proteins in the NaHS+H_(2)O_(2) group was significantly increased(P<0.05 or P<0.01),and the ac-FOXO1/FOXO1 ratio was significantly decreased(P<0.01),while the ROS level was significantly reduced(P<0.01).Conclusion H_(2)S can antagonize H_(2)O_(2)-induced senescence of HUVEC by a mechanism related to promoting SIRT1 sulfhydrylation and reducing FOXO1 acetylation.