Abstract:Aim To investigate the effect of hydrogen sulfide on macrophage calcification and its underlying molecular mechanisms. Methods Oil red O staining was used to observe intracellular lipid accumulation, and von Kossa staining and atomic absorption spectroscopy were used for morphological and quantitative analysis of calcium deposition and intracellular calcium content in a mononuclear macrophage calcification model. Western blot and RT-PCR were used to detect the mRNA and protein expression of osteopontin (OPN) at different doses and treatment times of hydrogen sulfide.At the same time, Western blot was used to detect the expression changes of early growth response factor 1 (EGR1), endoplasmic reticulum stress-related markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78).Reactive oxygen species levels were evaluated by fluorescence probe staining, and the effect of hydrogen sulfide on macrophage calcification was evaluated by combining von Kossa staining and calcium ion fluorescence probe staining. The molecular mechanisms of hydrogen sulfide affecting macrophage calcification were explored by interfering with EGR1 expression and using endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA). Results Compared with oxidized low density lipoprotein (ox-LDL) group, β-glycerophosphate (β-GP)+40 g/L ox-LDL group showed a significant increase in intracellular lipid accumulation, while hydrogen sulfide significantly inhibited macrophage calcification in a concentration- and time-dependent manner. Compared with the β-GP+ox LDL group, the most significant effect was observed after incubation with 100 μ mol/L NaHS for 4 days. The hydrogen sulfide group showed a 66% decrease in intracellular calcium content (P<0.01), a 71% decrease in intercellular calcium deposition (P<0.01), and a 50% and 48% decrease in OPN mRNA and protein expression, respectively (P<0.05). Hydrogen sulfide treatment upregulated the expression of EGR1 by 21%, while downregulating the expression of CHOP and GRP78 by 58% and 59%, respectively (P<0.01). The endoplasmic reticulum stress inhibitor 4-PBA could downregulate OPN expression by 73% (P<0.01), while interfering with EGR1 expression completely counteracts the inhibitory effect of hydrogen sulfide on OPN expression and calcium deposition (P<0.01). Conclusion Hydrogen sulfide significantly inhibits macrophage calcification by upregulating EGR1 expression and suppressing endoplasmic reticulum stress.