摘要
目的探讨胆绿素(biliverdin,BV)对脂多糖诱导RAW264.7巨噬细胞NLRP3炎性体激活的影响及其作用机制。方法用不同浓度胆绿素(10、20、30μmol/L)处理小鼠RAW264.7巨噬细胞30min后再用LPS(1μg/ml)处理细胞6h,ELISA法检测培养上清液IL-β和IL-18中的分泌量。30μmol/L胆绿素处理细胞30min后再用LPS处理细胞6h,Western blot法检测胞内NLPR3、caspase-1、ASC和磷酸化IκB(p-IκB)的蛋白表达水平;NF-κB抑制剂BAY11-7082处理细胞1h后,然后给予细胞30μmol/L胆绿素孵育30min,经1μg/ml LPS孵育6h,ELISA法检测细胞培养上清液中IL-β、IL-18的表达水平。结果 (1)在受到LPS作用后,RAW264.7巨噬细胞分泌IL-β、IL-18水平显著升高;而在预先给予胆绿素后,抑制了LPS诱导的IL-β、IL-18表达,并且呈剂量依赖性(P<0.05)。(2)与空白对照组相比,LPS处理巨噬细胞6h后,NLRP3炎性体各组分蛋白和pIκB蛋白表达明显上调(P均<0.05),在同时间点,巨噬细胞在LPS处理前经过胆绿素预处理后,NLRP3炎性体各组分蛋白和p-IκB蛋白表达都显著降低(均P<0.05)。(3)与LPS组相比,BAY+胆绿素明显抑制了LPS诱导的IL-β、IL-18的表达(P<0.05),并且BAY和胆绿素的对于LPS诱导炎性反应对的联合抑制作用显著的高于BAY或者胆绿素的单独作用(P<0.05)。结论在RAW264.7巨噬细胞中,胆绿素可以通过抑制NF-κB的活性进而抑制NLRP3炎性体的形成,从而发挥抗炎作用。
Objective To investigate the effects of biliverdin on NLRP3 inflammasome pathway in LPS - induced RAW264.7 mac- rophages. Methods RAW264.7 macrophage was pre- treated with different concentrations （10,20,30μmol/L） of biliverdin（BV） for 30 minutes, then administrated with LPS（ 1 μg/ml） for 6 hours, and the expression of supernatant levels of proinflammatory cytokine IL - 1β,IL- 18 were measured respectively by ELISA. Cells were treated with LPS（ 1 μg/ml） for 6h,and pretreated with BV（30μmol/L） for 30min before LPS stimulation, the protein expression of NLPR3, caspase - 1, ASC and phosphorylated IKB were determined by western blot. Cells were preincubated with BAY11 - 7082 （an inhibitor of NF -KB） for 1 hour, followed by BV treatment and then LPS stimula- tion. Meanwhile, the levels of pro - inflammatory cytokine IL - 1β, IL - 18 in the supernatant were measured by ELISA. Results LPS significantly increased IL- 1β, IL- 18 seretion levels in the supernatant, however, pretreatment with BV suppressed the LPS -induced IL - 1β, IL - 18 in a concentration - dependent manner（ P 〈 0. 05 ）. The protein expressions of NLRP3 inflammasome and phosphorylated IKB were up - regulated significantly 6 hours after LPS stimulation compared with control group （ P 〈 O. 05） ,while downregulated by BV on the same time point（P 〈 0.05 ）. Compared with LPS group, a large reduction in IL - 1β, IL - 18 expression were observed in BAY ＋ BV ＋ LPS group（ P 〈 0.05 ）. Furthermore, these cytokines were much less in BAY ＋ BV ＋ LPS group than in BAY ＋ LPS group or BV ＋ LPS group（ P 〈 0.05 ）. Conclusion Biliverdin exerted anti - inflammatory effects by regulating NLRP3 expression partially through the NF - KB pathway in RAW264.7 macrophages.
出处
《医学研究杂志》
2017年第10期35-39,共5页
Journal of Medical Research
基金
国家自然科学基金资助项目(81171785)
黑龙江省自然科学基金资助项目(ZJY0704-02)
哈尔滨医科大学研究生创新科研项目(YJSCX2015-21HYD)
