Effects And Mechanism Of Hydrogen Sulfide On Endocytosis Of Ox-LDL By Macrophages

Background and ObjectiveFormation of macrophage-derived foam cells plays a critical role in the initiation and progression of atherosclerosis, oxidized low-density lipoprotein (ox-LDL) is the key element in the process. Macrophages take up oxidized lipoproteins via scavenger receptors such as CD36and scavenger receptor A (SR-A) as well as a novel lectin-like oxidized low-density lipoprotein receptor (LOX-1), which causes intracellular lipids accumulation and foam cells formation. Endogenous hydrogen sulfide (H2S) was recognized as a novel gastrotransmitter followed by NO and CO, recent evidence indicates that hydrogen sulfide (H2S) exerts an anti-atherogenic effect, but the potential mechanism is still unclear. Therefore, in the current study, ectogenic H2S donor sodium hydrosulfide (NaHS) was used to investigate the effects of H2S on macrophage-derived foam cell formation and discuss the molecular mechanism herein.Materials and Methods1. Macrophages Raw264.7were pretreated with NaHS (100μmol/L) and a H2S generating enzyme cystathionine gamma lyase (CSE) inhibitor DL-propargylglycine (PPG,3mmol/L) for one hour, and then incubated for another six hours with Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL;20μmol/L), the ox-LDL uptaking ability of Raw264.7was evaluated by Laser scanning confocal microscope, flow cytometer (FCM) as well as Live-cell imaging using Olympus Cell＾R imaging system.2. As for the investigation of mechanism in the process, Raw264.7cells were separately pretreated with NaHS of different concentration ((25,50,100,200μmol/L) for one hour and100μmol/L of different time (10、30、60】120min), and then co-cultured for another24h in the presence of ox-LDL (20μl/mL). Immunofluorescence and western blotting assay were used to detect the expression of macrophage cell-surface receptors including LOX-1, CD36as well as CD68.3. To identify the signaling pathways involved in the inhibitory effect of H2S on foam cell formation, macrophages were pretreated for one hour with the NF-κB inhibitor PDTC before the treatment of NaHS. The expression of LOX-1and CD36was detected by western blotting assay.Results1. Treating Raw264.7cells with ox-LDL alone caused intracellular lipid accumulation in macrophages; this ox-LDL-induced lipid accumulation was significantly exacerbated by an additional treatment of PPG (a CSE inhibitor) while suppressed by NaHS (H2S donor) treatment.2. The high levels of LOX-1, CD36and CD68expressions induced by ox-LDL were significantly attenuated in the presence NaHS in a dose-dependent manner, with the strongest decrease at200μmol/L, and in a time-dependent manner, with the peak at120min of LOX-1and60min of CD36. 3. The decreased expression of ox-LDL receptors LOX-1and CD36induced by NaHS (50μmol/L) was reversed by NF-κB signaling pathway inhibitor PDTC, but was facilitated by ERK1/21/2inhibitor PD98059.Conclusion1. Ectogenic and endogenic hydrogen sulfide can suppress the cellular uptake ability of ox-LDL by macrophages.2. The inhibitory effect of hydrogen sulfide in suppressing cellular ox-LDL uptake is attributed to down-regulating the expressions of cell-surface ox-LDL receptors LOX-1, CD36and CD68.3. The down-regulation of ox-LDL receptors LOX-1, CD36and CD68by hydrogen sulfide is via NF-κB signal pathway.