柯里拉京抑制LPS和ATP激活的NLRP3炎症小体和巨噬细胞焦亡

Corilagin inhibits the NLRP3 inflammasome activation and macrophage pyroptosis induced by LPS and ATP

目的探讨柯里拉京对细菌脂多糖(LPS)联合腺嘌呤核苷三磷酸(ATP)诱导的巨噬细胞内NLRP3炎症小体活化和细胞焦亡的调控作用和机制。方法采用CCK8试剂检测不同浓度柯里拉京对J774A.1细胞活力的影响;碘化丙啶(PI)染色和乳酸脱氢酶(LDH)释放检测柯里拉京对LPS+ATP诱导的细胞死亡的影响。Western blot法检测细胞上清液中炎症小体活化标志物caspase-1 p20(Mr20000)和成熟IL-1β(Mr17000)的表达水平,以及检测细胞内NLRP3、ASC、caspase-1、IL-1β前体(pro-IL-1β)和焦亡执行蛋白GSDMD的表达。ELISA检测细胞培养上清液中IL-1β的水平。活性氧(ROS)荧光探针H2DCFDA染色观察柯里拉京对ROS产生的影响。结果柯里拉京浓度小于40μmol·L-1时对细胞活性影响较小。柯里拉京减少LPS+ATP刺激的细胞内PI阳性细胞的比例和LDH的释放。柯里拉京抑制LPS+ATP刺激的巨噬细胞内GSDMD蛋白N末端(GSDMD-NT)的表达,以及减少细胞释放caspase-1 p20和成熟IL-1β到培养上清。柯里拉京抑制细胞分泌炎症因子IL-1β和减少ROS的产生。然而ROS的诱导剂鱼藤酮能拮抗柯里拉京抑制NLRP3炎症小体活化和细胞焦亡的作用。结论柯里拉京抑制LPS+ATP诱导的巨噬细胞内NLRP3炎症小体活化和细胞焦亡依赖于抗氧化作用。

This study was designed to investigate the effects of corilagin on inflammasome activation and pyroptosis.J774A.1 cells were stimulated with LPS plus ATP to induce NLRP3 inflammasome activation and macrophage pyroptosis.CCK8 kit was used to examine the cytotoxicity of corilagin on J774A.1 cell;propidium iodide(PI)staining and lactate dehydrogenase(LDH)release were employed to observe cell death upon LPS plus ATP stimulation;Western blot was applied to detect the expression of inflammasome activation markers caspase-1 p20(Mr 20000)and mature IL-1β(Mr 17000)in cell supernatants,and intracellular NLRP3,ASC,caspase-1,precursor(pro)-IL-1βand pyroptosis executive protein GSDMD in cell lysis buffer.ELISA was used to detect the level of IL-1βin cell culture supernatant.Mouse IL1βELISA kit was applied to determine IL-1βrelease in cell supernatant;H2DCFDA staining was used to observe the effect of corilagin on ROS production.The CCK8 assay data showed that the corilagin less than 40μmol·L-1 displayed little effect on cell viability.Corilagin reduced the proportion of PI-positive cells and LDH release in LPS+ATP-stimulated cells;corilagin inhibited the expression of GSDMD-NT,as well as caspase-1 p20 and mature IL-1βproduction in the supernatant of LPS+ATP-stimulated macrophages.ELISA results showed that corilagin restrained IL-1βrelease.However,ROS inducer rotenone abrogated the effects of corilagin on NLRP3 inflammasome activation and pyroptosis.In summary,corilagin suppresses LPS+ATP-induced NLRP3 inflammasome activation and pyroptosis in macrophages through antioxidant effects.