Effects And Mechanism Of Hydrogen Sulfide On Vascular Smooth Muscle Cells Endocytosis Oxidized Low Density Lipoprotein

Objective To explore the efects and mechanisms of Hydrogen Sulfide on lipidendocytosis in vascular smooth muscle cells and provide a new compelling evidence forprevention of the atherosclerosis.Methods Vascular smooth muscle cells（VSMCs） were isolated from abdominal aortaof healthy SD rats which were cultured in10%FBS Deulbecco’s Modified EagleMedium（DMEM） at37℃in a humidified atmosphere containing5%CO2. Cells crawl outat3to5days, and could receive the first passage at7to9days. VSMCs used were thosethat have underpassed3to4passages. According to the different detection methods, cellswere used as follows:①Cells for MTT assay were plated in96-well culture plates. Everywell contains200μl cell suspension（cell density:5×104ml）.②The experimental cells usedfor Oil Red O staining and Immunofluorescence staining, were plated in24-well cultureplates with a piece of glass placed horizontally at the bottom of each well. Every wellcontains1ml cell suspension （cell density:1×105/ml）.③.The VSMCs of each group,which were collected to detect the protein expression of CD36and LOX-1by western blot,were culutred in a6well culture plates. Every well contains2ml cell suspension（celldensity:1×106ml）.The groups were divided as follows:1.MTT assay: the control group; NaHS（25、50、100、200、500、1000μmol/L） group2. Oil Red O staining and expression of CD36、LOX-1:the control group; ox-LDL（80μg/ml）group; ox-LDL（80μg/ml）+NaHS（50μmol/L） group;ox-LDL（80μg/ml）+PPG（3mmol/L） group;3. ox-LDL endocytosis assays: DiI-oxLDL（10μg/ml）group; DiI-oxLDL（10μg/ml）+NaHS（50μmol/L） group; DiI-oxLDL（10μg/ml） +PPG（3mmol/L） group;4. Western blot analysis: the control group; ox-LDL（80μg/ml）group; ox-LDL（80μg/ml）+NaHS（25、50、100、200μmol/L）group.Results1. MTT assay results showed that treatment with high concentrations（500-1000μmol/L）of NaHS resulted in a significant reduction in cell viability and relatively loweoncentrations （25-200μmol/L） didn’t affect cell viability compared with untreated controlgroup.2.The original aim of this study was to establish whether NaHS （donor of H2S） acts atthe lipid accumulation in vascular smooth muscle cells. The result of the Oil Red Ostaining indicated that treating with ox-LDL alone caused intracellular lipid accumulationin VSMC compared with the control group. This ox-LDL-induced lipid accumulation wassignificantly exacerbated by an additional treatment of PPG （a CSE inhibitor）, butsuppressed by NaHS treatment.3.We investigated whether decreased endocytosis of ox-LDL could account forinhibiting lipid accumulation on treatment with NaHS. Ox-LDL endocytosis in VSMC wasstudied with confocal microscopy. We found that H2S significantly blunted ox-LDLendocytosis in VSMC, while PPG treatment markedly increased the ox-LDL-induced lipidendocytosis,the results correspond to Oil Red O staining.4.To elucidate the molecular mechanism, we examined the effects of NaHS on theexpressions of CD36and LOX-1with confocal microscopy. We show here that ox-LDLsignificantly downregulated CD36and LOX-1expressions, While H2S exerted oppositeeffects, PPG up-regulated the expressions remarkably.5.To see whether CD36and LOX-1are responsible for the endocytosis of ox-LDL inVSMC, we used Western blot to analyze the protein expressions of CD36and LOX-1. Thisresult showed that, ox-LDL remarkably induced scavenger receptor CD36、LOX-1proteinexpressions which were abolished by H2S in a dose-dependent manner.Conclusion:1. H₂S suppressed lipid accumulation by blunting ox-LDL endocytosis significantly inVSMC, while PPG treatment exerted opposite effects. 2. H₂S could inhibit the lipid accumulation via down-regulation expressions of CD36and LOX-1in VSMC.