SIRT3 Mediates the Antioxidant Effect of Hydrogen Sulfide in Endothelial Cells

Abstract

Aim: Oxidative stress is a key contributor to endothelial dysfunction and associated cardiovascular pathogenesis. Hydrogen sulfide (H2S) is an antioxidant gasotransmitter that protects endothelial cells against oxidative stress. Sirtuin3 (SIRT3), which belongs to the silent information regulator 2 (SIR2) family, is an important deacetylase under oxidative stress. H2S is able to regulate the activity of several sirtuins. The present study aims to investigate the role of SIRT3 in the antioxidant effect of H2S in endothelial cells.

Results: Cultured EA.hy926 endothelial cells were exposed to hydrogen peroxide (H2O2) as a model of oxidative stress-induced cell injury. GYY4137, a slow-releasing H2S donor, improved cell viability, reduced oxidative stress and apoptosis, and improved mitochondrial function following H2O2 treatment. H2S reversed the stimulation of MAPK phosphorylation, downregulation of SIRT3 mRNA and reduction of the superoxide dismutase 2 and isocitrate dehydrogenase 2 expression which were induced by H2O2. H2S also increased activator protein 1 (AP-1) binding activity with SIRT3 promoter and this effect was absent in the presence of the specific AP-1 inhibitor, SR11302 or curcumin. Paraquat administration to mice induced a defected endothelium-dependent aortic vasodilatation and increased oxidative stress in both mouse aorta and small mesenteric artery, which were alleviated by GYY4137 treatment. This vasoprotective effect of H2S was absent in SIRT3 knockout mice.

Innovation: The present results highlight a novel role for SIRT3 in the protective effect of H2S against oxidant damage in the endothelium both in vitro and in vivo.

Conclusion: H2S enhances AP-1 binding activity with the SIRT3 promoter, thereby upregulating SIRT3 expression and ultimately reducing oxidant-provoked vascular endothelial dysfunction. Antioxid. Redox Signal. 24, 329-343.

FIG. 1.

Protective effect of H₂S on H₂O₂-induced cell injury in endothelial cells. EA.hy926 endothelial cells were pretreated with GYY4137 (12.5, 25, 50, and 100 μM) for 4 h before H₂O₂ (250 μM, 4 h). (A) Cell viability in EA.hy926 endothelial cells was measured by the CCK-8 kit. (B) Lactate dehydrogenase (LDH) release in cell culture medium was assayed by a commercial LDH kit. (C) Shape of EA.hy926 cells was detected with a light microscope. (D) Cells were stained with Hoechst 33342 and the images were taken under a fluorescence microscope, white arrows indicate the apoptotic cells. (E, F) Cells were stained with Annexin V/PI and apoptotic rates were analyzed by flow cytometry. **p < 0.01 versus control; ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group, n = 5–7. H₂O₂, hydrogen peroxide; LDH, lactate dehydrogenase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Effect of H₂S on ROS accumulation, SOD activity, mitochondrial function, and NO synthesis in endothelial cells with H₂O₂ treatment. EA.hy926 endothelial cells were pretreated with GYY4137 (25–100 μM) for 4 h before H₂O₂ (250 μM, 4 h). (A, B) Cellular ROS production was detected by DHE and DCFH-DA staining. (C, D) Total SOD and Mn-SOD (SOD2) enzyme activities were assessed using an SOD kit. (E) NO production was measured with an NO sensor. (F) The bioenergetic profiles of EA.hy926 cells were detected by a Seahorse Extracellular Flux Analyzer, oxygen consumption rate (OCR) in cells treated with oligomycin (2 μg/ml), FCCP (2 μM), and antimycin A and rotenone (rot and AA, respectively, 4 μM). (G) Basal respiration, ATP generation, maximal respiratory, and respiratory reserve capacity are shown. (H) Mitochondrial permeability transition (ΔΨm) was determined by JC-1 staining. *p < 0.05, **P < 0.01, ***p < 0.001 versus control, ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group, n = 3–8. ROS, reactive oxygen species; SOD, superoxide dismutase; NO, nitric oxide; DHE, dihydroethidium; DCFH-DA: dichlorodihydrofluorescein diacetate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Effect of H₂S on MAPK signal pathway and caspase-3. EA.hy926 endothelial cells were pretreated with GYY4137 (50 μM) for 4 h before H₂O₂ (250 μM, 4 h). (A–C) Representative Western blots and quantification of p-p38 MAPK, p-ERK, and p-JNK protein expression. (D) Representative examples of Western blots and quantification of cleaved capase-3 protein expression. *p < 0.05, **p < 0.01 versus control, ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group, n = 4–6. MAPK, mitogen-activated protein kinase.

FIG. 4.

Role of SIRT3 in H₂S-mediated protection of endothelial cell injury by H₂O₂. (A, B) EA.hy926 endothelial cells were pretreated with GYY4137 (50 μM) for 4 h before H₂O₂ (250 μM, 4 h). Quantification of SIRT family (SIRT1 to SIRT7) mRNA expression was determined by real-time PCR. *p < 0.05 versus control, ^#p < 0.05 versus the H₂O₂-treated group, n = 6–7. (C) Representative examples of Western blots and quantification of SIRT3 protein. *p < 0.05 versus control, ^##p < 0.01 versus the H₂O₂-treated group, n = 5. (D) EA.hy926 endothelial cells were transfected with SIRT3-specific siRNA (SIRT3siRNA) or a nonspecific control siRNA (CTLsiRNA). Transfection efficiency was assessed by immunofluorescence. (E, F) Representative examples of Western blots and quantification of SIRT3 after transfection. **p < 0.01 versus CTLsiRNA, n = 6. Transfected with CTLsiRNA or SIRT3siRNA for 24 h, EA.hy926 endothelial cells were exposed to H₂O₂ (250 μM, 4 h) after pretreatment with GYY4137 (50 μM) for 4 h, (G, H) ROS in endothelial cells was examined by DHE and DCFH-DA staining, and (I) apoptosis in endothelial cells was detected with Hoechst 33342 staining, white arrows indicate the apoptotic cells. (J, K) Cells were stained with Annexin V/PI and apoptotic rates were analyzed by flow cytometry. **p < 0.01 versus CTLsiRNA transfection, ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group with CTLsiRNA transfection, ^&&p < 0.01 versus SIRT3siRNA transfection, n = 5. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Effect of H₂S on the SIRT3-regulated SOD2, IDH2, and MAPK signaling pathways in endothelial cells. Transfected with CTLsiRNA or SIRT3siRNA for 24 h, EA.hy926 endothelial cells were exposed to H₂O₂ (250 μM, 4 h) after pretreatment with GYY4137 (50 μM) for 4 h. (A, B) Representative Western blots and quantification of SOD2 and IDH2 protein expression. (C–F) Representative Western blots and quantification of p-p38 MAPK, p-ERK, and p-JNK protein expression. *p < 0.05, ***p < 0.001 versus CTLsiRNA transfection, ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group with CTLsiRNA transfection, ^&p < 0.05, ^&&p < 0.01 versus SIRT3siRNA transfection, n = 3–4. IDH, isocitrate dehydrogenase; NS, no statistical significance.

FIG. 6.

Effect of H₂S on SIRT3 gene transcript activity. (A) EA.hy926 endothelial cells were transfected with SIRT3 promoter (−491/+146) luciferase fusion plasmid and pRL-TK plasmids. Twenty-four hours later, cells were pretreated with GYY4137 (50 μM) for 4 h before H₂O₂ (250 μM, 4 h). SIRT3 promoter luciferase activity was determined using a dual-luciferase reporter assay system. (B) Cells were transfected with plasmids contain the SIRT3 promoter region up to −491, −242, and −161, respectively. The 3′ end of the promoter of all of these constructs corresponds to +146. Transfected cells were treated and promoter luciferase activities were measured as described above. **p < 0.01 versus control, ^#p < 0.05, ^##p < 0.01 versus the H₂O₂-treated group, n = 5–7. (C) Cells were pretreated with GYY4137 (50 μM) for 4 h before H₂O₂ (250 μM, 4 h). Chromatin fragments for ChIP assays were immunoprecipitated with anti-AP-1 antibody. Precipitated DNA was amplified by real-time PCR with primers spanning the SIRT3 promoter region. IgG as a negative control. **p < 0.01 versus control, ^##p < 0.01 versus the H₂O₂-treated group, n = 4. (D, E) Cells were transfected with SIRT3 promoter (−491/+146) luciferase fusion plasmid. Twenty-four hours later, cells were treated with specific AP-1 inhibitor, SR11302 (1 μM) or curcumin (20 μM), for 4 h. Then, GYY4137 (50 μM) was pretreated for 4 h before H₂O₂, and then luciferase activities were determined. ^##p < 0.01 versus the H₂O₂-treated group, n = 4. AP-1, activator protein 1; NS, no statistical significance.

FIG. 7.

Effects of H₂S on vasorelaxation in SIRT3 KO mice. (A) WT or SIRT3 KO mice were injected with either paraquat (50 mg/kg, i.p) or saline 1 h before either GYY4137 (133 μM/kg, ip) or saline. Mice were killed and aortic segments were removed 24 h thereafter. Endothelium-dependent vasorelaxation to acetylcholine of precontracted aortic sections was assessed. **p < 0.01 versus the saline-treated group of the same genotype, ^##p < 0.01 versus the paraquat-treated group of the same genotype, n = 6. (B) DHE staining of aorta for superoxide production (n = 4). (C, D) Above aortic segments were preincubated with tempol (1 mM) for 30 min, then endothelium-dependent vasorelaxation to acetylcholine of precontracted aortic sections was assessed. **p < 0.01 versus the saline-treated group of the same genotype, ^##p < 0.01 versus the paraquat-treated group of the same genotype, ^&p < 0.05 versus paraquat- and GYY4137-treated groups of the same genotype, n = 6. (E, F) Vasorelaxation to SNP of precontracted aortic sections was assessed. (G, H) Endothelium-dependent vasorelaxation in small mesenteric artery to acetylcholine of precontracted aortic sections was assessed. **p < 0.01 versus the saline-treated group of the same genotype, ^##p < 0.01 versus only the paraquat-treated group of the same genotype, n = 4–6. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars