氧化型低密度脂蛋白诱导人巨噬细胞焦亡的体外研究

Study on oxidized low density lipoprotein induced caspase-1 mediated pyroptosis in vitro

目的:探索性地研究一种新的细胞死亡方式——细胞焦亡是否为氧化型低密度脂蛋白(ox-LDL)诱导的人巨噬细胞死亡的途径。方法:分离、培养人单核巨噬细胞,给予非特异性caspase阻断剂或特异性caspase-1阻断剂,与ox-LDL(100μg/ml)共培养48 h,检测细胞焦亡指标。结果:与对照组比较,ox-LDL可诱导巨噬细胞caspas-1活性明显增加(P<0.001)、使巨噬细胞LDH流出增加(P<0.01)、Cacein AM/EthDⅢ染色死亡细胞明显增加;TUNEL染色表明,与PBS对照组比较,ox-LDL可诱导巨噬细胞TUNEL阳性细胞增加(P<0.05);ELISA表明,与PBS对照组比较,ox-LDL可促进巨噬细胞分泌白细胞介素(IL)-1β(P<0.001)及IL-18(P<0.001)水平明显增高。与非特异性caspas-1阻断剂相比较,特异性caspas-1,阻断剂能够显著抑制ox-LDL诱导的LDH流出(P<0.01)、Cacein AM/EthDⅢ染色死亡细胞、TUNEL阳性细胞比例(P<0.05)及IL-1β(P<0.001)和IL-18(P<0.001)分泌。结论:ox-LDL可诱导巨噬细胞发生焦亡,可能参与动脉粥样硬化不稳定斑块的发生。

Objective：To investigate whether pyroptosis occurs in ox-LDL induced human macrophage.Method：Human macrophages were cultured with ox-LDL（100 μg/ml） for 48 hours in the absence/presence of pan caspase inhibitor or specific caspase-1 inhibitor.After incubation,western blot,caspase-1 activity,LDH release,Calcein AM/KthDⅢ staining and ELISA was used to measure pyroptosis.Result：Compared with unprimed control cells,ox-LDL induced significantly activated caspsae-1 expression（P〈0.001）,elevated LDH release（P〈0.01）and dead cells,increased TUNEL positive cells（P〈0.05） and rised cytokines production of IL-1β（P〈0.001）and IL-18（P〈0.001）.Moreover,compared with pan caspase inhibitor,specific caspase-1 could significantly block ox-LDL induced LDH release（P〈0.01）,dead cells,TUNEL positive cells（P〈0.05） and IL-lβ（P〈0.001）and IL-18（P〈0.001） production.Conclusion;Our study here identified a novel cell death,pyroptosis in ox-LDL induced human macrophage.