Objective: To investigate the mechanism by which the small molecule compound WP1130 inhibits NLRP3 inflammasome activation and alleviates septic shock.
Methods: Mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells were pre-treated with WP1130 before stimulation with different NLRP3 inflammasome agonists (Nigericin, ATP, MSU and intracellular LPS transfection), and AIM2 inflammasomes were activated with poly A: T. The levels of caspase-1 and IL-1β in the cell culture supernatant were determined using Western blotting and ELISA, and mitochondrial damage in the cells was observed using confocal microscopy. In the animal experiment, male C57BL/6 mice were randomized into blank control group, septic shock group (LPS group) and WP1130 treatment group (WP1130+LPS group), and the levels of IL-1β and TNF-α in the serum and peritoneal cavity were detected using ELISA.
Results: In murine BMDM and human THP-1 cells, WP1130 significantly inhibited NLRP3 agonists-induced caspase-1 and IL-1β secretion in a dose-dependent manner (P < 0.05) but did not obviously affect the secretion of such inflammatory factors as IL-6 and TNF-α that were not associated with inflammasomes (P>0.05). Treatment with WP1130 did not significantly affect poly A: T-induced activation of AIM2 inflammasomes (P>0.05) or induce obvious changes in mitochondrial damage, an upstream signal of NLRP3 inflammasome activation. In the mouse model of LPS-induced septic shock, WP1130 treatment significantly reduced the level of IL-1β (P < 0.05) without obviously affecting TNF-α level either in the serum or in the peritoneal cavity (P>0.05).
Conclusion: WP1130 specifically inhibits NLRP3 inflammasome activation to alleviate LPS-induced septic shock in mice.
目的: 探究小分子化合物WP1130抑制NLRP3炎症小体活化并缓解感染性休克的作用机制。
方法: 细胞生物学水平：WP1130预处理小鼠骨髓来源巨噬细胞（BMDM）或人THP-1细胞后，利用多种NLRP3炎症小体活化剂（Nigericin、ATP、MSU、胞内LPS转染）活化NLRP3炎症小体，使用poly A: T活化AIM2炎症小体，通过Western blot和ELISA检测细胞培养上清中caspase-1和IL-1β的分泌水平，确定WP1130对NLRP3炎症小体的抑制效果及其特异性。利用激光共聚焦显微镜观察Nigericin诱导的线粒体损伤情况，探究WP1130是否影响NLRP3炎症小体组装活化的上游信号。动物水平：将SPF级雄性C57BL/6小鼠随机分为3组，即空白对照组（Control组）、感染性休克组（LPS组）、WP1130治疗组（WP1130+LPS组），ELISA检测小鼠血清和腹腔液中IL-1β、TNF-α的分泌水平。
结果: WP1130可剂量依赖性的抑制多种激动剂诱导的NLRP3炎症小体活化（P < 0.05），但对非炎症小体相关炎性因子IL-6、TNF-α的分泌无显著影响（P>0.05）。此外，WP1130对AIM2炎症小体的活化无显著影响（P>0.05）。机制研究表明，WP1130并不影响NLRP3炎症小体组装活化的上游信号线粒体损伤。动物实验结果表明，相较感染性休克组（LPS组），WP1130治疗组（WP1130+LPS组）小鼠血清和腹腔液中IL-1β的水平显著降低（P < 0.05），而TNF-α的分泌水平无统计学差异（P>0.05）。
结论: WP1130能够特异性抑制NLRP3炎症小体活化并缓解LPS诱导的小鼠感染性休克。
Keywords: NLRP3 inflammasomes; WP1130; inflammasome-driven diseases; septic shock.