水晶兰苷调控巨噬细胞极化减少THP-1源性泡沫细胞形成

Monotropein inhibits THP-1-derived foam cell formation by regulating macrophage polarization

目的:探讨水晶兰苷(MON)对巨噬细胞极化和THP-1源性泡沫细胞形成的影响。方法:THP-1细胞与100 ng/mL佛波酯(PMA)共孵育48 h诱导M0巨噬细胞,采用流式细胞术进行鉴定;采用不同浓度的氧化低密度脂蛋白(ox-LDL)(0μg/mL、25μg/mL、50μg/mL、100μg/mL、150μg/mL)、不同的ox-LDL干预时间(0 h、3 h、6 h、12 h、24 h)处理巨噬细胞以诱导泡沫细胞;采用ox-LDL或MON干预巨噬细胞,CCK-8检测细胞活力;设置分组:control组、ox-LDL组和ox-LDL+MON组,采用油红O染色观察各组细胞脂质吞噬情况,采用western blotting、荧光定量PCR(RT-qPCR)评估巨噬细胞极化表现及其与铁死亡相关信号通路的结果。结果:ox-LDL呈浓度和时间依赖性增加iNOS、TNF-α蛋白表达;ox-LDL能显著降低细胞活力,200μmol/L MON可显著恢复细胞活力,单纯MON浓度高达1000μmol/L时,细胞活力下降;经ox-LDL诱导可观察到细胞内大量明显红色脂滴,加入MON处理,细胞内的脂滴可见明显减少;与对照组比较,ox-LDL诱导的巨噬细胞CD86 mRNA、iNOS蛋白表达水平升高,CD206 mRNA、Arg-1、SLC7A11、GPX4蛋白表达水平降低,而经过MON干预后,CD86 mRNA、iNOS蛋白表达水平降低,Arg-1、SLC7A11、GPX4蛋白表达水平升高,差异均有统计学意义(均P<0.05)。结论:MON通过减少巨噬细胞M1极化,促进M2极化,从而抑制ox-LDL诱导的泡沫细胞形成、增强细胞活力,这可能与抑制铁死亡相关。

Objective:To investigate the effect of monotropein(MON)on macrophage polarization and the formation of THP-1 derived foam cells.Methods:THP-1 cells were incubated with 100 ng/mL phorbol 12-myristate 13-acetate(PMA)for 48 hours to induce M0 macrophages,which were identified by flow cytometry.Foam cells were induced by treating macrophages with different concentrations of oxidized low-density lipoprotein(ox-LDL)(0μg/mL,25μg/mL,50μg/mL,100μg/mL,150μg/mL)for different ox-LDL intervention times(0 h,3 h,6 h,12 h,24 h).Macrophages were intervened with ox-LDL or MON,and cell viability was assessed using CCK-8.The experimental groups were divided into control group,ox-LDL group,and ox-LDL+MON group.Oil Red O staining was employed to observe the lipid engulfment of cells in each group.Western blotting and reverse transcription-quantitative PCR(RT-qPCR)were utilized to evaluate the polarization of macrophages and the outcomes of signaling pathways associated with ferroptosis.Results:The ox-LDL increased iNOS and TNF-αprotein expression in a concentration-and time-dependent manner.The ox-LDL significantly reduced cell viability,which could significantly be restored by 200μmol/L MON,but would be decreased when the concentration was up to 1000μmol/L MON.A large number of obvious ox-LDL-induced intracellular lipid droplets were observed in the cells,and the amount of lipid droplets in the cells were markedly reduced with MON treatment.Compared with the control group,ox-LDL-induced macrophages showed increased CD86 mRNA and iNOS protein expression levels and decreased CD206 mRNA,Arg-1,SLC7A11,and GPX4 protein expression levels,while MON intervention decreased CD86 mRNA and iNOS protein expression levels and increased Arg-1,SLC7A11,and GPX4 protein expression levels(all P<0.05).Conclusion:MON inhibits ox-LDL-induced foam cell formation and enhances cell viability by reducing M1 polarization and promoting M2 polarization in macrophages,which may be associated with inhibition of ferroptosis.