■作为血管保护气体发射器家族的新成员,硫化氢(H2S)的功能类似于一氧化氮(NO)和一氧化碳(CO)。内皮细胞(EC)死亡和自噬使细胞能够应对心血管疾病的进展。然而,H2S在ECs自噬过程中的影响和潜在机制尚不完全清楚。这里,我们研究了H2S对人血管内皮细胞自噬的影响。
■将人脐静脉内皮细胞(HUVEC)暴露于不同浓度(0、50、100、200、500和1,000µmol/L)的GYY4137(H2S供体)指定时间(0、0.5、1、2、4和8小时),有或没有用自噬抑制剂3-甲基腺嘌呤(3-MA)或巴弗洛霉素A1进行预处理。用沉默蛋白1(Sirt1)过表达质粒(PIRES-Sirt1)转染HUVEC,使用Lipofectamine2000的Sirt1-siRNA或叉头盒O1(FoxO1)-siRNA。通过蛋白质印迹和荧光显微镜评估细胞自噬。免疫共沉淀测定用于测量FoxO1的乙酰化水平。采用蛋白质印迹法和免疫荧光法观察FoxO1在细胞质和细胞核中的分布。西方印迹,流式细胞仪分析,采用细胞计数试剂盒-8法评价H2S对氧化低密度脂蛋白(Ox-LDL)诱导HUVECs凋亡的影响。
■使用功能增益和损失实验,我们表明,Sirt1依赖性的FoxO1的激活,包括其核转位和去乙酰化,对介导H2S诱导的ECs自噬至关重要。此外,H2S诱导的自噬通过激活Sirt1保护ECs免受Ox-LDL诱导的细胞凋亡。
■这些结果表明,Sirt1介导的ECs自噬是H2S发挥血管保护作用的一种新机制。
UNASSIGNED: As a new member of the vasculoprotective gasotransmitter family, hydrogen sulfide (H2S) functions similar to nitric oxide (NO) and carbon monoxide (CO). Endothelial cell (EC) death and autophagy enable cells to cope with the progression of cardiovascular diseases. However, the impacts and underlying mechanisms of H2S in the autophagic process in ECs are not completely understood. Here, we investigated the effects of H2S on autophagy in human vascular ECs.
UNASSIGNED: Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations (0, 50, 100, 200, 500 and 1,000 µmol/L) GYY4137 (H2S donor) for indicated times (0, 0.5, 1, 2, 4 and 8 h), with or without pre-treatment with the autophagy inhibitor 3-methyladenine (3-MA) or bafilomycin A1. HUVECs were transfected with sirtuin 1 (Sirt1) overexpression plasmids (PIRES-Sirt1), Sirt1-siRNAs or forkhead box O1 (FoxO1)-siRNA using Lipofectamine 2000. Cell autophagy was evaluated via Western blotting and fluorescence microscopy. Co-immunoprecipitation assay was used to measure acetylation level of FoxO1. The distribution of FoxO1 in the cytoplasm and nucleus was observed using Western blotting and immunofluorescence. Western blotting, flow cytometric analysis, and cell count kit-8 assay were conducted to evaluate the effect of H2S on the oxidized low-density lipoprotein (Ox-LDL) induced apoptosis of HUVECs.
UNASSIGNED: Using both gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1, including its nuclear translocation and deacetylation, was critical for mediating H2S-induced autophagy in ECs. Furthermore, H2S-induced autophagy protected ECs from Ox-LDL-induced apoptosis by activating Sirt1.
UNASSIGNED: These results suggest that Sirt1-mediated autophagy in ECs is a novel mechanism by which H2S exerts vascular-protective actions.