Effects Of Endogenous Sulfureted Hydrogen On Calcification And BMP Expression During Macrophages Lipid Loading

Objective:Heterotopic calcification, namely, the mineral deposition occurred in the nonosteonal tissue, mainly refers to the deposition of calcium salt, and one kind, the calcification of vascular tissue, is classified into tunica medial calcification, and tunica intimal calcification. The tunica intimal calcification is closely relevant to artherosclerosis, and its early involvements primarily include macrophages and clasmatoblast. The solfurated hydrogen possesses important physiological action and pathological action of cardiovascular. With L-cysteine as the endogenous donor of solfurated hydrogen, based on the model of calcium salt deposition in macrophages which is guided byβ-GP and oxLDL, this research attempts to study the effects of endogenous sulfureted hydrogen on calcification during macrophages lipid loading and the factors behind them, providing some experimental evidence for further exploration of its effects on the tunica intimal calcification.Methods:Expriment 1: Establish the model of the calcification: Incubate RAW264.7 with 40mg/ml oxLDL and 5mmol/Lβ-GP for three days; obseve calcium deposition by von Kossa staining and determine cumulation content by atomic absorption spectrophotometry.Expriment 2: Effect on the cell calcification of endogenous sulfureted hydrogen with different concentration:1.Control group: cell incubated in RPMI -1640 with 10% tetal caif serum .2.Calfication and 0mmol/L L-lysine groups:cell incubated with oxLDL,β-GP and 0mmol/L L-lysine.3. Calfication and 50mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 50mmol/L L-lysine; calfication and 100mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 100mmol/L L-lysine; calfication and 200mmol/L L-lysine groups:cell incubarted with oxLDL,β-GP and 200mmol/L L-lysine.Expriment 3: Effect of endogenous sulfureted hydrogen on the cell calcification in different periods of time:1. Control group: cell incubated in RPMI -1640 with 10% tetal calf serum.2. 0d :cell incubated with oxLDL,β-GP and bestL-lysine contration for 0 day; 2d: cell incubated with cell were incubated with oxLDL,β-GP and best L-lysine contration for 2 days; 4d: cell incubated with oxLDL,β-GP and best L-lysine contration for 4 days; 6d: cell incubated with oxLDL,β-GP and best L-lysine contration for 6 days.Expriment 4: Effects of PPG on cell calcification:RAW264.7 was incubated with oxLD,β-GP and PPG at different concentrations(0,2,4,8mmol/L) for 4 days.then we detected calcium content, calcium deposition and OPN mRNA and protein eapression were determined by RT-PCR and Westen blot, respectively. Raw264.7 were divided into five groups as followings:1) control group:cell incubated in RPMI -1640 with 10% tetal caif serum . 2) calfication and 0mmol/L PPG group:cell incubarted with oxLDL,β-GP and 0mmol/L PPG. 3) calfication and 2 mmol/L PPG group:cell incubarted with oxLDL,β-GP and 2mmol/L PPG. 4) calfication and 4mmol/L PPG group:cell incubated with oxLDL,β-GP and5 mmol/LPPG. 5) calfication and 8mmol/L PPG group:cell incubarted with oxLDL,β-GP and 8mmol/L PPG.Results:Compared with the control group, macrophages incubated with oxLDl andβ-glicerophosplale evidently showed a numbers of black particles, oxLDl andβ-glicerophosplale caused recrease the content of calium by 210%, and they also can recrease BMP mRNA and protein expression by 180% and 160% respectively. Compared with the calcification group, calcification macrophages incubated with L-cysteine macrophages showed less black particles,and also the contend of the calium. BMP mRNA and protein expression decreased too.expecially at the 50mmol/L level. L-cysteine caused a deceased BMP mRNA and protein expression by 161% and 220% respectively compared with calcification cell. Calcification cell incubated with PPG showed the opposite results compared with the cell incubated with L-cysteine..Conclusion:1.Endogenous sulfureted hydrogen can decrease the calcium content and calcium deposition during the macrophages lipid loading.2.Endogenous sulfureted hydrogen can down-regulate BMP expression during the macrophages lipid loading.