Investigation On The Effect And Mechanisms Of Hydrogen Sulfide Against Apoptosis Induced By OxLDL In Human Umbilical Vein Endothelial Cells

Objective: To investigate the effects of hydrogen sulfide against oxLDL-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and the mechanisms involved .Methods: 1.HUVECs were pre-treated with NaHS at different concentrations (25μmol /L, 50μmol /L, 100μmol /L and 200μmol /L) for 24 hours or for different incubation time(6h, 12h, 24h) at the concentration of 50μmol /L NaHS, followed by incubation with oxLDL(100μg/ml) for 24 hours. The change of nuclear morphology were observed by nuclear staining with Hoechst 33258. The apoptosis of HUVECs was detected by flow cytometry (FCM) with propidium iodide (PI) staining.2. HUVECs were divided into four groups as follows: (1) Control group(CON): Cells were incubated in RPMI-1640 medium; (2) NaHS+oxLDL group: HUVECs were pre-treated with NaHS(50μmol /L) for 24 hours and then incubated with oxLDL(100μg/ml) for another 24 hours; (3) oxLDL group: HUVECs were incubated with oxLDL(100μg/ml) for 24 hours; (4) NAC(N- acetyl-L-cysteine,antioxidant)+oxLDL group: Cells were pre-treated with NAC(1 mmol/L) for 24 hours and then incubated with oxLDL(100μg/ml) for another 24 hours. To study the change of reactive oxygen species (ROS) content in HUVECs, the fluorescence intensity of Dihydrohodamine123(DHR123)-stained or rhodamine123 (Rh123)-stained(for detection of mitochondrial membrane potential) HUVECs was observed by fluorescence microscope and detected by FCM. Western Blot was used to detect protein expression of Caspase-3 and Caspase-9 , respectively, in the HUVECs. Results: 1. The antagonist effect of H2S against apoptosis induced by oxLDL in HUVECs①As shown in Hoechst 33258 staining assay, compared with the control group, the number of cells with nuclear condensation induced by oxLDL(100μg/ml) increased significantly. Pre-treatment with different concentrations (25μmol /L, 50μmol /L, 100μmol /L and 200μmol /L) of NaHS for 24 hours or with different incubation time(6, 12, 24 hours) at the comcemtration of 50μmol /L NaHS remarkably lowered the number of apoptotic cells induced by oxLDL(100μg/ml).②As shown in FCM assay, compared with the control group, the apoptotic proportion of the HUVECs in oxLDL group was significantly increased(p <0.05). But pre-treatment with different concentrations (25μmol /L, 50μmol /L, 100μmol /L and 200μmol /L) of NaHS for 24 hours caused a decreased apoptotic proportion by 80%, 85%, 89%, 92%, respectively, compared with oxLDL group (all p <0.05). The apoptotics proportion of the HUVECs pretreated with 50μmol /L NaHS for different times(6, 12, 24 hours) was lowered by 80%, 81%, 84%, respectively, in comparison with oxLDL-treated group (all p <0.05).2. The mechanisms of hydrogen sulfide against oxLDL-induced apoptosis in HUVECs①Compared with the control group, the DHR123 fluorescence in HUVECs treated with oxLDL(100μg/ml) for 24 hours were increased by 230%(p <0.05) ,which was notably inhibited by 63% and 67% decrease(both p <0.05) by pre-treatment with NaHS(50μmol/L) or NAC(1mmol/L), suggesting that H2S can inhibit the production of ROS induced by oxLDL in HUVECs.②The Rh123 fluorescence in HUVECs treated with oxLDL(100μg/ml) for 24h were decreased by 56% (p <0.05),which was remarkably inhibited by pre-treatment with NaHS(50μmol/L) or NAC(1mmol/L).Both pre-treatment caused an elevation of the MFI by 104% and 106% (both p <0.05),respectively, suggesting that H2S can inhibit the decrease of mitochondrial membrane potential (△Ψm) induced by oxLDL in HUVECs.③Compared with the control group, expression of Caspase-3 and Caspase-9 was increased by 29% and 24%(p <0.05),respectively, after 100μg/ml oxLDL treatment 51. The antagonist effect of H2S against apoptosis induced by oxLDL in HUVECs①As shown in Hoechst 33258 staining assay, compared with the control group, the number of cells with nuclear condensation induced by oxLDL(100μg/ml) increased significantly. Pre-treatment with different concentrations (25μmol /L, 50μmol /L, 100μmol /L and 200μmol /L) of NaHS for 24 hours or with different incubation time(6, 12, 24 hours) at the comcemtration of 50μmol /L NaHS remarkably lowered the number of apoptotic cells induced by oxLDL(100μg/ml).②As shown in FCM assay, compared with the control group, the apoptotic proportion of the HUVECs in oxLDL group was significantly increased(p <0.05). But pre-treatment with different concentrations (25μmol /L, 50μmol /L, 100μmol /L and 200μmol /L) of NaHS for 24 hours caused a decreased apoptotic proportion by 80%, 85%, 89%, 92%, respectively, compared with oxLDL group (all p <0.05). The apoptotics proportion of the HUVECs pretreated with 50μmol /L NaHS for different times(6, 12, 24 hours) was lowered by 80%, 81%, 84%, respectively, in comparison with oxLDL-treated group (all p <0.05). 2. The mechanisms of hydrogen sulfide against oxLDL-induced apoptosis in HUVECs①Compared with the control group, the DHR123 fluorescence in HUVECs treated with oxLDL(100μg/ml) for 24 hours were increased by 230%(p <0.05) ,which was notably inhibited by 63% and 67% decrease(both p <0.05) by pre-treatment with NaHS(50μmol/L) or NAC(1mmol/L), suggesting that H2S can inhibit the production of ROS induced by oxLDL in HUVECs.②The Rh123 fluorescence in HUVECs treated with oxLDL(100μg/ml) for 24h were decreased by 56% (p <0.05),which was remarkably inhibited by pre-treatment with NaHS(50μmol/L) or NAC(1mmol/L).Both pre-treatment caused an elevation of the MFI by 104% and 106% (both p <0.05),respectively, suggesting that H2S can inhibit the decrease of mitochondrial membrane potential (△Ψm) induced by oxLDL in HUVECs.③Compared with the control group, expression of Caspase-3 and Caspase-9 was increased by 29% and 24%(p <0.05),respectively, after 100μg/ml oxLDL treatment for 24h. But a decrease of Caspase-3 expression by 19% or 20% was observed when pretreated with 50μmol/L NaHS or 1mmol/L NAC(both p <0.05). These two pre-treatments also resulted in a lowered expression of Caspase-9 by 12% and 19%(both p <0.05),respectively,Conclusions:1.H2S can exert antagonist effect against apoptosis induced by oxLDL in HUVECs; 2.The antagonist effect of H2S against ox-LDL-induced apoptosis in HUVECs is involved in preservation of△Ψm and attenuation of the intracellular ROS generation.