山柰酚抑制NLRP3炎症小体活化和细胞焦亡提高脓毒症小鼠生存率

Kaempferol inhibits NLRP3 inflammasome activation and pyroptosis to improve survival of sepsis mouse

目的本研究以脂多糖(LPS)刺激的小鼠骨髓来源巨噬细胞(BMDMs)建立细胞炎症模型,探讨山柰酚对三磷酸腺苷(ATP)诱导的炎症小体活化、细胞焦亡的影响及其作用机制,以及对脓毒症小鼠生存率的影响。方法提取6~7周龄C57BL/6雌性小鼠原代骨髓细胞并诱导其分化为骨髓来源的巨噬细胞,根据实验设计分为空白对照组、LPS处理组、单纯药物处理组、LPS+ATP(阳性模型组)、阳性模型加药组(3个药物浓度),通过LPS+ATP处理诱导NLRP3炎症小体的活化;采用蛋白质印迹法检测caspase-1、IL-1β、gasdermin D(GSDMD)等蛋白的活化水平;采用细胞形珠阵列(CBA)检测细胞培养上清液中IL-1β的水平;碘化丙锭(PI)染色法检测细胞死亡/细胞焦亡;加入蛋白激酶A(PKA)抑制剂H89后再次检测以上炎症小体活化与焦亡的指标。盲肠结扎穿孔术(CLP)建立脓毒症小鼠模型。结果山柰酚能够抑制LPS+ATP处理后巨噬细胞中caspase-1p20和成熟IL-1β释放至细胞培养上清中;同时,山柰酚也能明显抑制巨噬细胞中具有活性的GSDMD-NT的形成以及细胞焦亡。另外,H89预处理可逆转山柰酚对LPS+ATP诱导的caspase-1p20和成熟IL-1β的释放的抑制作用,并且具有明显逆转山柰酚对ATP诱导细胞焦亡的抑制作用。通过盲肠结扎穿孔术建立脓毒症小鼠模型,山柰酚灌胃处理能够有效地延缓感染后小鼠的死亡。结论山柰酚可能通过促进PKA活性而抑制NLRP3炎症小体活化与细胞焦亡,从而提高脓毒症小鼠的生存率。

Objective To explore effects of kaempferol on ATP-induced NLRP3 inflammasome activation and pyroptosis in lipopolysaccharide(LPS)-primed murine bone marrow-derived macrophages and the underlying mechanism,and regulation of survival of sepsis mouse.Method The primary bone marrow cells of 6-7 week old C57BL/6 female mice were extracted and induced to differentiate into bone marrowderived macrophages.According to the experimental design,they were divided into blank control group,LPS treatment group,drug treatment group,LPS+ATP group(positive model group),positive model dosing group(three drug concentrations).The NLRP3 inflammasome was activated by treating with LPS plus ATP.The protein levels of caspase-1,IL-1βand GSDMD were detected by Western blotting.Soluble IL-1βin the culture supernatants was measured by CBA assay.Cell death/pyroptosis was analyzed by propidium iodide(PI)staining.After adding protein kinase A(PKA)inhibitor H89,the above indicators of inflammasome activation and pyroptosis were detected again.Cecal ligation puncture(CLP)was used to establish septicemia mouse model.Result Kaempferol inhibited caspase-1 activation and mature IL-1βsecretion in the LPS-primed BMDMs upon ATP treatment.Kaempferol also suppressed GSDMD-NT formation and pyroptosis in macrophages.In addition,H89 significantly reversed kaempferol-mediated suppression of ATP-induced caspase-1 activation,mature IL-1βsecretion and pyroptosis.We also observed that kaempferol could effectively delay the death of mice after infection.Conclusion Kaempferol probably suppressed NLRP3 inflammasome activation and pyroptosis through promoting PKA signaling in macrophages to improve the survival rate of sepsis mice.