摘要
目的以脂多糖(LPS)激活的腹腔巨噬细胞为炎症细胞模型,探讨糖尿病常用药物二甲双胍对胞外ATP诱导炎症小体活化并释放白细胞介素-1β(IL^(-1)β)的影响。方法C57BL/6小鼠腹腔注射30 g·L^(-1)巯基乙酸盐(thioglycollate,TG)诱导腹腔巨噬细胞大量产生;利用LPS+ATP处理激活炎症小体,采用碘化丙锭(PI)染色法检测二甲双胍对LPS+ATP诱导的腹腔巨噬细胞焦亡的影响,免疫印迹法检测该细胞胞内及上清中IL^(-1)β和caspase-1的表达水平;免疫荧光技术检测巨噬细胞P_2X_7嘌呤能ATP受体7(P_2X_7R)的亚细胞分布及荧光强度。结果单独二甲双胍不会导致LPS激活细胞发生焦亡,但二甲双胍呈剂量依赖性促进LPS+ATP诱导的细胞焦亡;免疫印迹法显示,在蛋白水平上,单独LPS(或LPS+二甲双胍)刺激不能诱导细胞释放成熟IL^(-1)β(17 ku)到细胞外;当加入ATP刺激后,成熟IL^(-1)β和活化caspase-1(10 ku)释放到胞外;而二甲双胍可以剂量依赖性地促进LPS激活、ATP刺激下腹腔巨噬细胞释放成熟IL^(-1)β和活化caspase-1的水平。结论二甲双胍增强LPS激活的腹腔巨噬细胞在ATP刺激下的炎症小体活化,提高caspase-1活化和成熟IL^(-1)β释放,促进炎症反应。
Aim To explore the influence of metformin（ a first-line drug for type 2 diabetes） on ATP-induced inflammasome activation and the release of interleukin-1β（ IL-1β） by LPS-activated peritoneal macrophages,a commonly-used inflammatory cell model.Methods Peritoneal macrophages were elicited by intraperitoneal injection of 30 g · L-1thioglycollate into C57BL/6mice.Inflammasome was activated and cell pyroptosis was induced by LPS plus ATP treatment,and the pyroptotic cells were calculated after propidium iodide（ PI） staining.The protein levels of IL-1β and caspase-1 expressed in the cells and released from them into the supernatant were evaluated by Western blot.Immunofluorescent microscopy was recruited to detect the subcellular distribution and fluorescent intensity of the purinergic P2X7receptor（ P2X7R）.Results Metformin per se did not induce pyroptosis in LPS-activated peritoneal macrophages,but it signifi-cantly and dose-dependently increased cell pyroptosis induced by ATP treatment.At protein levels,maturated IL-1β（ 17 ku） could not be released from the cells upon single LPS or LPS plus metformin stimulation;but after ATP was added,maturated IL-1β was released into the supernatants of the cells.Moreover,metformin dose-dependently increased the protein levels of both maturated IL-1β and active caspase-1 released by the LPS-activated peritoneal macrophages upon ATP stimulation.Conclusion Metformin intensifies the activation of inflammasome and increases the release of active caspase-1 and maturated IL-1β upon ATP stimulation in the LPS-activated peritoneal macrophages,which should promote inflammatory responses.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2017年第4期474-479,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81173604
81373423)
