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

doi:10.19871/j.cnki.xfcrbzz.2022.01.003

新发传染病电子杂志 ›› 2022, Vol. 7 ›› Issue (1): 12-16.doi: 10.19871/j.cnki.xfcrbzz.2022.01.003

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

吴森泉, 方年新, 莫伟良, 张平

东莞市人民医院呼吸与危重症医学科,广东东莞 523059

收稿日期:2021-07-29 出版日期:2022-02-28 发布日期:2022-07-07
通讯作者: 张平,Email: 1728340667@qq.com
基金资助:
1.2020年东莞市社会科技发展一般项目（202050715001774）; 2.2020年东莞市人民医院院内科研项目（K202025）

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

Wu Senquan, Fang Xinnian, Mo Weiliang, Zhang Ping

Department of Respiratory and Critical Care Medicine, Dongguan People's Hospital, Guangdong Dongguan 523059, China

Received:2021-07-29 Online:2022-02-28 Published:2022-07-07

摘要/Abstract

摘要： 目的本研究以脂多糖（LPS）刺激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BMDMs,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-1p20、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。通过盲肠结扎穿孔术（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,山柰酚对LPS+ATP诱导的caspase-1p20和成熟IL-1β的释放有抑制作用,对ATP诱导细胞焦亡也有抑制作用,而H89预处理则可逆转其抑制作用。山柰酚灌胃处理能够有效提高脓毒症模型小鼠的存活率。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存活率。

关键词: 山柰酚, 炎症小体, 细胞焦亡, 蛋白激酶A, 脓毒症

Abstract: 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 marrow-derived 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-1p20, IL-1β and GSDMD were detected by Western blotting. Soluble IL-1β in 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-1p20 activation and mature IL-1β secretion 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-1p20 activation, mature IL-1β secretion and pyroptosis. We also observed that Intragastric administration of kaempferol can effectively improve the survival rate of sepsis model mice. Conclusion Kaempferol probably suppressed NLRP3 inflammasome activation and pyroptosis through promoting PKA signaling in macrophages to improve the survival rate of sepsis mice.

Key words: Kaempferol, Inflammasome, Pyroptosis, Protein kinase A, Sepsis

吴森泉, 方年新, 莫伟良, 张平. 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[J]. 新发传染病电子杂志, 2022, 7(1): 12-16.

Wu Senquan, Fang Xinnian, Mo Weiliang, Zhang Ping. Kaempferol inhibits NLRP3 inflammasome activation and pyroptosis to improve survival of sepsis mouse[J]. Electronic Journal of Emerging Infectious Diseases, 2022, 7(1): 12-16.

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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

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