游离胆固醇激活未折叠蛋白反应诱导巨噬细胞凋亡

目的探讨游离胆固醇激活未折叠蛋白反应以及对巨噬细胞凋亡的影响。方法取小鼠腹腔巨噬细胞进行培养,然后分别以普通培养基、普通培养基加胆固醇乙酰转移酶抑制剂和乙酰低密度脂蛋白、普通培养基加胆固醇乙酰转移酶抑制剂和乙酰低密度脂蛋白再加胆固醇移动抑制剂分组孵育,Annexin-V和碘化丙锭双染色后流式细胞仪检测细胞凋亡,Western-bolt检测下游转录因子C/EBP同源蛋白表达。结果普通培养基孵育的巨噬细胞无C/EBP同源蛋白表达,只有少量细胞凋亡;而普通培养基加胆固醇乙酰转移酶抑制剂和乙酰低密度脂蛋白孵育的巨噬细胞C/EBP同源蛋白表达明显,细胞凋亡也达到21.83%±2.47%;同时使用胆固醇移动抑制剂干预后,C/EBP同源蛋白无表达,而细胞凋亡比单纯促进游离胆固醇聚集状态孵育组显著减少。结论过多的游离胆固醇聚集是诱导巨噬细胞凋亡的重要原因之一,未折叠蛋白反应参与这一过程,而特异性胆固醇移动抑制剂U18666A可以有效阻止游离胆固醇进入内质网激活未折叠蛋白反应诱导巨噬细胞凋亡。

Free Cholesterol Loading of Macrophages Induces Apoptosis Involving the Unfolded Protein Response

Aim To explore the cellular mechanisms of free cholesterol(FC)-induced macrophage apoptosis by activating unfolded protein response(UPR).Methods Macrophages were harvested from the peritonea of female C57BL6/J mice,then incubated in DMEM and 1% fetal bovine serum(FBS) medium alone,or medium containing acetyl-low density lipoprotein(ac-LDL) and compound 58035(free cholesterol-loading conditions),or medium containing ac-LDL and compound 58035 plus U18666A respectively.Western-blot was used to determine expression of C/EBP homology protein(CHOP).The apoptosis percentages of macrophages were detected with flow cytometry(FCM) after the staining of Annexin-V and propidium(PI).Results Macrophages incubated in DMEM and 1% FBS alone had no expression of CHOP and showed very little evidence of cell apoptosis.After incubated with medium containing ac-LDL and compound 58035 in our cell culture model,the expression of CHOP increased notably and about 21.83%±2.47% apoptosis of the cells had been detected.But apoptosis was diminished markedly in free cholesterol-loaded plus U18666A and also no CHOP expression had been detected.Conclusions Excess intracellular FC accumulation plays an important role in macrophage apoptosis,and UPR is involved in this process.But U18666A can block FC transport into endoplasmic reticulum(ER) to activate the CHOP and subsequent apoptosis.