Hydrogen Sulfide Ameliorated Homocysteine-Induced Endothelial Dysfunction:Mechanism Of Endoplasmic Reticulum Stress-Mediated Autophagy

Part I Hydrogen sulfide improves endothelial dysfunction in hyperhomocysteinemia rats through inhibiting endoplasmic reticulum stress and autophagyObjectives:Homocysteine（Hcy）is a sulfur-containing amino acid obtained by methionine（Met）through methyl group metabolism.Research showed that Hcy could induce endothelial dysfunction with related mechanism of stimulating endoplasmic reticulum stress（ERS）and promoting autophagy.Hydrogen sulfide（H₂S）as the third gas signal molecule after nitric oxide（NO）and carbon monoxide（CO）,has numerous cardiovascular protective effects such as anti-inflammation,anti-oxidation and anti-apoptosis effect.In this part,we use renal arteries of the hyperhomocysteinemia（HHcy）rats to explore whether exogenous administration of H₂S donor sodium hydrosulfide（Na HS）could improve endothelial dysfunction induced by HHcy,and whether its mechanism is related to the inhibition of ERS and autophagy.Methods:1.Experimental protocols:Six-week-old male Wistar rats weighing 160±10g were selected for the experiment.The experimental protocols are as follows:（1）Control group,HHcy group,HHcy+Na HS group,Na HS group;（2）Control group,HHcy group,HHcy+Na HS group,TM group,HHcy+Na HS+TM group,Na HS+TM group,Na HS group;（3）Control group,HHcy group,HHcy+Na HS group,Rapamycin group,HHcy+Na HS+Rapamycin group,Na HS+Rapamycin group,Na HS group;（4）Control group,HHcy group,HHcy+Na HS group,HHcy+4-PBA group,4-PBA group;（5）Control group,HHcy group,HHcy+Na HS group,HHcy+STF-083010 group,STF-083010 group;（6）Control group,HHcy group,HHcy+Na HS group,HHcy+SP600125 group,SP600125 group;（7）Control group,HHcy group,HHcy+Na HS group,HHcy+CQ group,CQ group.The rats were fed with 2%methionine diet to set up HHcy models.Rats in HHcy+Na HS group were fed with 2%methionine diet and intraperitoneally injected with Na HS 56μmol/kg/d simultaneously.Normal food was added in Control and Na HS group.The rats were injected intraperitoneally with Na HS 56μmol/kg/d in Na HS group and normal saline in Control.All groups were kept for 8 weeks.2.Blood was obtained from inferior vena cava and centrifuged to collect plasma to determine the plasma concentration of Hcy.3.Renal arteries were taken to determine the endothelium-dependent vasodilation and endothelium-independent vasodilation.In Control group,the renal arteries were incubated with ERS inducer Tunicamycin（TM,10μmol/L）and autophagy inducer Rapamycin（100nmol/L）for 1h.In HHcy group,the renal arteries were incubated with ERS inhibitor 4-Phenylbutyric acid（4-PBA,5mmol/L）,IRE1αinhibitor STF-083010（100μmol/L）,JNK inhibitor SP600125（10μmol/L）and autophagy inhibitor Chloroquine（CQ,50μmol/L）for 1h.The renal arteries in HHcy+Na HS group and Na HS group were respectively incubated with TM（10μmol/L）and Rapamycin（100nmol/L）respectively for1h.4.Western blot was used to determine the protein expression of cystathionine-γ-lyase（CSE）,ERS or autophagy related proteins in rat renal arteries.Result:1.The plasma concentration of Hcy in HHcy group significantly increased compared with Control group,which indicated that the model of HHcy rats were set up successful.Exogenous administration of Na HS reduced the level of Hcy in HHcy rats.2.The acetylcholine（ACh）-induced endothelium-dependent vasodilation in HHcy group was significantly decreased.Na HS administration could improve the ACh-induced vasodilation.There was no significant difference between groups in sodium nitroprusside（SNP）-mediated endothelium-independent vasodilation.TM and Rapamycin incubation impaired endothelium-dependent vasodilation,while incubation of renal arteries with ERS,JNK,and autophagy inhibitors increased endothelial vasodilation in HHcy rats.3.The protein expression of p-IER1α,p-PERK,ATF6,GRP78,p-JNK,Beclin1,LC3BII/LC3BI were up-regulated in HHcy group,CSE,P62 protein expression was significantly down-regulated.Na HS exogenous administration down-regulated p-IER1α,p-PERK,ATF6,GRP78,p-JNK,Beclin1,LC3BII/LC3BI protein expression and up-regulated CSE and P62 protein expression.Conclusion:Exogenous administration of Na HS improves endothelium-dependent vasodilatation in renal arteries of HHcy rats and the mechanism may be related to the inhibition of ERS and autophagy.Part II Hydrogen sulfide protects Hcy-induced HUVECs damage by inhibiting IRE1α-JNK-autophagy pathwayObjective:The first part of the study showed that Hcy could improve endothelial function by inducing ERS and autophagy.Previous studies showed that inositol requiring enzyme 1α（IRE1α）can induce autophagy by upregulating c-Jun N-terminal kinase（JNK）expression.The IRE1α-JNK signaling pathway is a key link in the regulation of cell fate after ERS.Whether IRE1α-JNK autophagy is involved in Hcy induced endothelial injury has not been reported.In this part,human umbilical vein endothelial cells（HUVECs）were used to incubate Hcy in vitro to explore whether exogenous administration of Na HS could protect endothelial cell injury induced by Hcy by inhibiting IRE1α-JNK-autophagy pathway.Methods:1.Experimental group:HUVECs were given treatment and grouped as follows:（1）Control group,Hcy group,Hcy+Na HS group,Na HS group;（2）Control group,Hcy group,Hcy+STF-083010 group,STF-083010group;（3）Control group,Hcy group,Hcy+SP600125 group,SP600125 group;（4）Control group,Hcy group,Hcy+CQ group,CQ group.2.HUVECs activities were determined by CCK-8.3.The protein expression of CSE,ERS and autophagy related proteins in HUVECs were determined by Western blot.Result:1.Hcy incubation could reduce the bioactivity of HUVECs,and Na HS exogenous administration improves the Hcy-induced injury of HUVECs.2.Incubation of Hcy by HUVECs in vitro could significantly increase p-IER1α,p-PERK,ATF6,GRP78,p-JNK,Beclin1,LC3BII/LC3BI proteins expression,CSE and P62 protein expression were down-regulated.Na HS administration down-regulated protein expression of p-IER1α,p-PERK,ATF6,GRP78,p-JNK,Beclin1,LC3BII/LC3BI and up-regulated protein expression of CSE and P62.Incubation of IER1αinhibitor,STF-083010 and ERS inhibitor,4-PBA,the expression of p-IER1α,ATF6,GRP78,p-JNK,Beclin1 and LC3BII/LC3BI proteins in HUVECs was down regulated,while P62 protein was up regulated.After incubation of SP600125 down regulated the expression of p-JNK,Beclin1 and LC3BII/LC3BI,while P62 protein was up-regulated.Beclin1 and LC3BII/LC3BI protein expression were down-regulated and P62was up-regulated with CQ incubation.Conclusion:Exogenous administration of Na HS ameliorates Hcy-induced HUVECs injury by inhibiting the IRE1α-JNK-autophagy pathway.