Effects Of Exogenous Hydrogen Sulfide On Cell Calcification During Phagocytic Uptake Of Lipid By Macrophage

Objective:Non-osseous tissue occurres mineral deposition, mainly calcium deposition. Vascular calcification is a sort of them, classified into medial calcification and intimal calcification. Intimal calcification is related with atherosclerosis, which the early involvement of cells is mainly macrophages and mast cells. Hydrogen sulfide play an important role in cardiovascular physiology and pathology. We used sodium hydrosulfide (Sodium hydrosulfide, NaHS) as an exogenous donor of hydrogen sulfide and Preparated the calcified model of macrophages withβ-glycerophosphate (β-sodium glycerophosphate,β-GP) and oxidized low density lipoprotein (Oxidized low density lipoprotein, ox-LDL) to investigate the effect of hydrogen sulfide on cell calcification during the process of phagocytic uptake of lipid by macrophage and other factors which may affect this process. Therefore, The results we may provide experimental clues for the further study on intimal calcification.Methods:Experiment 1: To prepare the calcified model of cells: RAW264.7 cells were incubated with 40mg/mL ox-LDL and 5mmol/Lβ-GP for three days. The calcium deposition is detected by Von kasso staining and the content of calcium is estimated by atomic absorption spectrophotometry.Experiment 2: the effect of hydrogen sulfide on calcification:1.RAW264.7 were incubated with ox-LDL,β-GP and different concentrations of NaHS (0, 25, 50, 100, 200μmol/L) for 6 days. Then we detected calcium content, calcium deposition by atomic absorption spectrophotometry and aperture scintillometers and determined the expression of OPN mRNA and protein by RT-PCR and Western blot, respectively. Raw264.7 were cultured in six different kinds of medium.2.RAW264.7 were cultured with ox-LDL,β-GP and the best concentration of NaHS at different time (0,2,4,6days). Calcium content , calcium deposition, and the expression of OPN mRNA and protein which were determined by atomic absorption spectrophotometry, aperture scintillometers, RT-PCR and Western blotting ,respectively.Experiment 3: Influencing factors:1.Oxygenation: We detected the level of ROS of cells, which were cultured with ox-LDL,β-GP and best NaHS concentration for 4 days . There were three group of them:①calcification: cells only were incubated with ox-LDl andβ-GP.②NaHS:cells were incubated with ox-LDL,β-GP and 100μmol/L NaHS.③calcification +NaHS+H2O2 : cells were incubated with ox-LDL,β-GP, 100μmol/L NaHS and 400μmol/L H2O2.2.Inflammation factor: Adding inflammation factor to medium, we research its function which may affect the role of H2S. The groups were devided as fllows:①calcification: cells were incubated with ox-LDL andβ-GP.②NaHS: cells were incubated with ox-LDL andβ-GP and 100μmol/L NaHS.③NaHS +IL-6: cells were incubated with ox-LDL andβ-GP and 100μmol/L NaHs and10ng/mL IL -6.Results:1.Compared with the control group, macrophages incubated with ox-LDL andβ-glicerophosplale observably showed a large number of black particle by Von kasso staining. Calcium content increased 6 times(p <0.05), whereas cultured with ox-LDL orβ-glycerophosphate alone, after a longer incubation time, only a small population of black cell granules exsited. The result indicated that ox-LDL andβ-GP can cause cell calcification in short time.2.Compared with calcification group, the longer incubation time and the higher the concentration of NaHS decreased the content of cell calcium and deposition , and a the expression of OPN mRNA and protein, especially at level of 100μmol/L NaHS for 4 days. compared with 0mmol/L group, The content and deposition of cell calcium and the expression of OPN mRNA and protein were decreased by 6.5 times, 7.2 times, 5times and 4.8 times, separately, (p <0.05). After treated with H2O2 and IL-6, the function of NaHS aggravating cell calcification was inhibited.Conclusions:1. The calcified model of macrophage was prepared successly byβ-GP and ox-LDL joint application2. Hydrogen sulfide can relieved cell calcification in a time and concentration dependent way during phagocytic uptake of lipid by macrophage3. IL-6 and ROS can restairn the inhibited effect of NaHS on cell calcification...