硫化氢对巨噬细胞吞噬脂质过程中钙化的影响及其机制

Effect of hydrogen sulfide on calcification during macrophage phagocytosis of lipids and its mechanisms

摘要:

目的]探讨硫化氢对巨噬细胞钙化的影响及其潜在分子机制。 [方法]采用油红O染色观察细胞内脂质蓄积情况,von Kossa染色和原子吸收光谱法分别对单核巨噬细胞钙化模型的钙盐沉积和细胞内钙含量进行形态学分析及定量分析。利用Western blot和RT-PCR检测硫化氢不同剂量和不同处理时间下骨桥蛋白(OPN)的mRNA和蛋白表达。同时,利用Western blot检测早期生长反应因子1(EGR1)和内质网应激相关标志物C/EBP同源蛋白(CHOP)和葡萄糖调节蛋白78(GRP78)的表达变化,活性氧荧光探针检测评估活性氧水平,并结合von Kossa染色和钙离子荧光探针检测评估硫化氢对巨噬细胞钙化的影响。通过干扰EGR1表达和使用内质网应激抑制剂4-苯基丁酸(4-PBA)探讨硫化氢影响巨噬细胞钙化的分子机制。 [结果]与氧化型低密度脂蛋白(ox-LDL)组相比,β-甘油磷酸钠(β-GP)＋40 g/L ox-LDL组细胞内脂质蓄积明显增加,硫化氢显著抑制巨噬细胞钙化,且呈剂量和时间依赖性。与β-GP＋ox-LDL组相比,100 μmol/L NaHS处理4天效果最为显著,细胞内钙含量降低66%(P＜0.01),细胞间钙盐沉积减少71%(P＜0.01),OPN mRNA和蛋白表达分别降低50%和48%(P＜0.05)。硫化氢处理使EGR1的表达上调了21%,CHOP和GRP78的表达分别下调了58%和59%(P＜0.01)。内质网应激抑制剂4-PBA使OPN表达下调73%(P＜0.01),而干扰EGR1表达则完全抵消了硫化氢对OPN表达及钙盐沉积的抑制作用(P＜0.01)。 [结论]硫化氢通过上调EGR1表达抑制内质网应激,从而显著抑制巨噬细胞钙化。

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.