Hydrogen Sulfide Donor Drugs Inhibit Potential Atherosclerosis Regulating Cell Autophagy In Vitro

Objective:Human umbilical vein endothelial cells(HUVECs)and macrophages were induced by oxidized low-density lipoprotein(ox-LDL)to establish the model of atherosclerotic cells,and the effect of ox-LDLon autophagy was studied.Several references have shown that hydrogen sulfide has therapeutic effect on atherosclerosis.The aim of this study is to explore the effect of hydrogen sulfide donor to the cell autophagy and to reveal its new mechanism of anti-atherosclerosis.Methods:(1)Construction of HUVECs injury cell model and study of autophagy:we incubated HUVECs with different concentrations of ox-LDL(0?g/mL?20?g/mL?40?g/mL?8?g/mL?120?g/mL?160?g/mL)for 24 hours,Cell Counting kit-8(CCK-8)was used to detect cell growth activity,(Enzyme Linked Immunosorbent Assay)ELISA was used to detect interleukin-6(IL-6)and tumor necrosis factor(TNF-?),oil red O staining was used to detect lipid accumulation,Western Blot(WB)was used to detect the expression ofLC3 ?and p62 which were related to autophagy.Baf-A1 was used to investigate the flow and arrest of autophagy.(2)Construction of THP-1 derived foam cell model and the study of autophagy:THP-1 cells were treated with 100ng/mL PMA for 48h and then were induced to THP-1 derived macrophages(THP-M).Nextly,different concentrations of ox-LDL(0?g/mL?40?g/mL?80?g/mL?120?g/mL?160?g/mL?200?g/mL)were adopted to incubate THP-M for 24h,CCK-8was used to detect cell growth activity,cholesterol esterase method was used to detect total cholesterol(TC)and free cholesterol(Fch),oil O red staining showed lipid droplets accumulation,WB was used to detect autophagy related proteins of LC3 ? and p62,Baf-A1 was used to investigate the flow and arrest of autophagy.(3)GYY4137 was used to treat human umbilical vein endothelial cells and detect the cell growth activity and autophagy.Autophagy study of GYY4137 on model cells:HUVECs and THP-M cells were pretreated with different concentrations of GYY4137(0?mol/L?50?mol/L?100?mol/L?200?mol/L?400?mol/L)for 30 minutes,and then incubated with corresponding concentrations of ox-LDL for 24 hours.The autophagy related proteins of LC3 ? and p62 were detected by WB.Studies on autophagy and anti-inflammatory effects of hydrogen sulfide donor drugs:GYY4137(100?mol/L)and sodium hydrosulfide(NaHS)(100?mol/L)were pretreated for 30 minutes and then incubated with ox-LDL for 24 hours.Meanwhile,Baf-Al and 3-methyladenine(3-MA)were used as the control group.WB was used to detect LC3 ? and p62 and ELISA was used to detect cell secretion.Results:(1)CCK-8 results showed that the activity of HUVEC cells increased firstly and then decreased with ox-LDL.When ox-LDL was 160?g/mL,the cell growth activity of HUVEC cells decreased significantly(*p<0.05).When the ox-LDL was 200?g/mL,the cell growth activity of THP-M cells decreased significantly(*p<0.05).Meanwhile,GYY4137 had no effect on the cell growth activity for the selected concentrations(0?mol/L?50?mol/L?100?mol/L?200?mol/L?400?mol/L)(the difference was no statistically significant).(2)Injury model of Human umbilical vein endothelial cells:Elisa kit results showed that the expression of IL-6,TNF-? in HUVEC cellshadconcentration dependence on the concentration of ox-LDL(80?g/mL and 120?g/mL,statistically significant).Oil O red staining showed lipid droplets were increased with the ox-LDL increasing concentration.The results suggested that there would be cell injury when the concentration of ox-LDL is 120?g/mL.WB showed that ox-LDL could increase the expression of LC3 ?and p62 in HUVECs in a dose-dependent manner.Baf-Alcould not increase the expression ofLC3 ?(ns),but increase the expression of p62.(3)Foam cell model:cholesterol esterase assay showed that ox-LDL increased TC and Fch in a dose-dependent manner.When ox-LDL concentration was 160?g/mL,CE/TC=55.74±2.49%>50%(**p<0.01)was in accordance with the foam regulations,while oil red staining showed large number of lipid droplets.It was suggested that ox-LDL concentration of 160?g/mL can form foam cells.The results of WB showed that ox-LDL could increase the expression of LC3? in THP-M cells in a dose-dependent manner,and p62 showed a trend of decreasing first and increasing later,which was statistically significant.Baf-Al increased the expression of LC3 ?(*p<0.05),but did not increase the expression of p62.(4)GYY4137 could not cause the change of autophagy protein of HUVECs in the concentration range(0-400?mol/L).(5)Different hydrogen sulfide donors drugs(GYY4137,NaHS)100?mol/L can reduce the LC3 ?/LC3 ? ratio of HUVEC injured cells and foam cells,the expression of p62 protein(*p<0.05,**p<0.01),and the release of inflammatory factors IL-6,TNF-?,IL-1?(*p<0.05,*p<0<0.01).Meanwhile,Baf-Al can significantly increase the release of IL-6,TNF-?(**p<0.01),and 3-MA can not significantly reduce the release of TNF-?,IL-1?.Conclusion:GYY4137 and NaHS have protective effects on ox-LDL induced Human umbilical vein endothelial cell inflammatory injury model and macrophage foam cell model,and this protective effect is related to the regulation of autophagy.