Effects Of Hydrogen Sulfide On The Endothelial Dysfunction Of Essential Hypertension And The Underlying Mechanism

Part ? Administration of hydrogen sulfide improves renal artery endothelial dysfunction in spontaneously hypertensive rats by inhibiting NLRP3 / IL-1? pathwayObjective: Primary hypertension is associated with endothelial dysfunction,which may be related to oxidative stress and immune system disorder.NLRP3 is activated in oxidative stress state,while its downstream proinflammatory cytokine IL-1? can directly damage the vascular endothelial function.Hydrogen Sulfide?H₂S?is the third gas signaling molecules found after carbon monoxide?CO?and nitric oxide?NO?,which has the effect of improving endothelial dysfunction,but the mechanism is unclear.The aim of this study is to investigate the effects of H₂S on the NLRP3 / IL-1? pathway and endothelial function in vivo and in vitro.Methods:1 Experiment groupIn vivo experiment: Rats were randomly divided into four groups : Na SH group,Wistar-Kyoto rats?WKY?,WKY + sodium hydrosulfide?Na SH?,spontaneously hypertensive rats?SHR?,SHR + Na SH.The animals in Na SH groups were given Na SH 100umol/kg intraperitoneally daily from 8 weeks old to the end of 24 weeks old.In vitro experiment: 24-week-old experimental animals were selected and rats were divided into five groups,namely WKY group,SHR group,SHR + H₂S?100?mol/L?group,SHR + LPS?10?g/ml?group and SHR + LPS + H₂S group.2 The systolic blood pressure?SBP?of the tail arteries of each group was measured using the rat tail arterial blood pressure measurement system,which was measured from 8 weeks to 24 weeks every two weeks.3 Animals were sacrificed at 24 weeks old,and the seconary branches of the kidney arterioles were taken for experiment.The endothelium-dependent relaxation function and endothelium-dependent contraction function were measured.After 16 hours of incubation in vitro,the endothelium-dependent relaxation and endothelium-dependent contraction function were also measured.4 The expressions of CSE,Nrf2,NOX4,P67 phox,SOD,CAT,and IL-1? pathway protein NLRP3,Caspase-1 and IL-1? in renal arterioles were determined by Western Blot.5 Plasma levels of H₂S were measured by liquid chromatography-mass spectrometry?HPLC-MS?,the content of malondialdehyde?MDA?was determined by chemical method and IL-1? was determined by ELISA.Results:1 SBP in SHR group was significantly higher than that in WKY group at the end of treatment,and the blood pressure of SHR was significantly decreased after administration of Na SH,which was statistically significant.While administration of Na SH had no significant effect on the SBP of WKY.2 SHR plasma H₂S and CSE level were significantly lower than the WKY group.Na SH supplemented the lack of endogenous hydrogen sulfide in hypertensive animals,up-regulated the expression of CSE and increased H₂S level.3 The endothelium-dependent relaxation response induced by acetylcholine?Ach?in the SHR group was significantly lower than that in the WKY group,while the endothelium-dependent contraction was significantly enhanced.Exogenous administration of Na SH significantly improved endothelium-dependent vasodilatation and inhibited excessive endotheliumdependent contractile response.In vitro incubation of H₂S significantly improved endothelium-dependent vasodilatation and inhibited endotheliumdependent systolic function in SHR rats,but its protection against LPS-induced endothelial dysfunction was abolished.4 The expression of NOX4 and P67 phox in the renal arteries of SHR increased,while the expression of antioxidant protein Nrf2,SOD and CAT decreased.Chronic administration of Na SH could significantly down-regulate the expression of oxidized protein and up-regulate the expression of antioxidant protein.In addition,chronic administration of Na SH reduced the level of malondialdehyde?MDA?in SHR plasma,which was an oxidative stress marker.After LPS activated the NLRP3 pathway in vitro,this trend of anti-oxidation of H₂S was eliminated.5 The expression of NLRP3 / caspase-1 / IL-1? pathway protein in SHR renal artery was significantly increased,and chronic administration of Na HS could down-regulate the expression of NLRP3 pathway.In addition,chronic administration of Na SH reduced the level of IL-1? in SHR plasma.In vivo incubation experiment used LPS to over-activated NLRP3 pathway in SHR,and results showed that the above-mentioned protein didn't increase significantly.However,afterwards when given Na SH,its down-regulation of inflammatory protein disappeared.Conclusion: Exogenous administration of H₂S can enhance SHR endothelium-dependent relaxation,inhibit its excessive endotheliumdependent contraction function,thereby reducing SHR blood pressure.The role of H₂S in improving endothelial function may be achieved by inhibiting the NLRP3 inflammatory pathway and blocking its interaction with oxidative stress.Part ? Hydrogen sulfide exerts endothelial protective effect by up-regulating Nrf2 protein and enhancing antioxidant capacityObjective: Nuclear factor erythroid-2-related factor 2?Nrf2?belongs to the nuclear transcription factor family.It binds to the molecular chaperone Keap-1 under normal condition.When the cells are oxidatively or chemically activated,the Nrf2 and Keap1 are isolated and Nrf2 transfers into the nucleus,activating the antioxidative genes after its binding to the antioxidant response element?ARE?.The aim of this part is to test whether H₂S can exert endothelial protective effect by up-regulating the expression of Nrf2 and enhancing the antioxidant capacity in human umbilical vein endothelial cells?HUVECs?.Methods:1 The effects of different concentration of H₂S?0?mol / L,50?mol / L,100?mol / L,200?mol / L?on the activity of HUVECs were measured by CCK-8 method under the stimulation of 1?M /L Ang?.2 Nrf2 gene in HUVECs was knocked down with lentivirus and divided into six groups:1)Negative control?NC?group2)NC+ Angiotensin ??Ang??1?mol / L group3)NC+ Ang? 1?mol / L + H₂S 100?mol / L group4)Nrf2 knockdown?Small interferce RNA,si RNA?group5)Si RNA + Ang ? 1?mol / L group6)Si RNA + Ang? 1?mol / L + H₂S 100?mol / L groupDHE staining was used to measure the level of reactive oxygen species?ROS?.The expression of Nrf2,SOD,CAT,NOX4,P67 phox,NLRP3,Caspase-1 and IL-1? were measured by western blot.Results:1 Ang ? can significantly reduce the activity of HUVEC,while H₂S can increase the biological activity of HUVECs in a dose-dependent manner.2 DHE staining found that Ang ? can significantly increase the level of ROS,whereas H₂S can significantly reduce the level of ROS.ROS level significantly increased after knockout of Nrf2,and the effect of H₂S on redcing ROS was blocked.3 The expression of NOX4,p67 phox in NC+ Ang?,Si RNA group and Si RNA + Ang? group increased,while the expression of antioxidant protein Nrf2 SOD and CAT decreased.In the NC group giving H₂S can reverse the above changes,whereas in the Nrf2 knock-out groups this reverse effect of H₂S was eliminated.4 Compared with NC group,the expression of NLRP3,Caspase-1 and IL-1? in NC+ Ang?group and three Si RNA knockout groups increased.In NC+H₂S group the expression of the above protein was down-regulated,while afer knockout of Nrf2,this down-regulating effect of H₂S disappeared.Conclusion: H₂S enhances the expression of Nrf2,increases the expression of antioxidant proteins,reduces oxidative stress,inhibits the downstream inflammatory cytokine expression and enhances endothelial cell bioactivity to exert endothelial cell protective and antihypertensive effects.