漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究

doi:10.11958/20240619

天津医药 ›› 2024, Vol. 52 ›› Issue (10): 1025-1030.doi: 10.11958/20240619

漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究

廖忠¹(), 廖伟健¹, 赖国利¹, 文茵², 苏志威², 曾举浩², 丁洪光³

1 龙南市第一人民医院急诊科（邮编341700）
2 南方医科大学附属广东省人民医院（广东省医学科学院）重症医学科
3 南方医科大学附属广东省人民医院（广东省医学科学院）急诊科

收稿日期:2024-05-17 修回日期:2024-06-11 出版日期:2024-10-15 发布日期:2024-10-14
作者简介:廖忠(1986)，男，副主任医师，主要从事脓毒症相关脏器损伤的机制研究。E-mail：liaozhong_ln@163.com
基金资助:
国家自然科学基金青年科学基金项目(82002074);江西省卫生健康委科技计划项目(202312169);广东省医学科学技术研究基金项目(A2022506)

Study on the effect of fisetin on alleviating cognitive impairment after sepsis by inhibiting the activation of microglial NLPR3 inflammasome

LIAO Zhong¹(), LIAO Weijian¹, LAI Guoli¹, WEN Yin², SU Zhiwei², ZENG Juhao², DING Hongguang³

1 Department of Emergency, Longnan First People’s Hospital, Longnan 341700, China
2 Department of Critical Care Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University
3 Department of Emergency, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University

Received:2024-05-17 Revised:2024-06-11 Published:2024-10-15 Online:2024-10-14

廖忠, 廖伟健, 赖国利, 文茵, 苏志威, 曾举浩, 丁洪光. 漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究[J]. 天津医药, 2024, 52(10): 1025-1030.

LIAO Zhong, LIAO Weijian, LAI Guoli, WEN Yin, SU Zhiwei, ZENG Juhao, DING Hongguang. Study on the effect of fisetin on alleviating cognitive impairment after sepsis by inhibiting the activation of microglial NLPR3 inflammasome[J]. Tianjin Medical Journal, 2024, 52(10): 1025-1030.

摘要/Abstract

摘要：

目的探讨漆黄素抑制小胶质细胞NOD样受体家族蛋白3（NLRP3）炎症小体活化缓解脓毒症后认知功能损害的机制。方法选用C57BL/6小鼠，用盲肠结扎穿孔法构建脓毒症模型。分组包括：假手术组、脓毒症组、脓毒症+胱天蛋白酶（caspase）-1敲除组（脓毒症+Cas-1^-/-组）、脓毒症+漆黄素组。Morris水迷宫评估小鼠的认知功能。Western blot和免疫荧光双染检测脑组织和小胶质细胞NLRP3炎症小体相关蛋白caspase-1、GSDMD蛋白N端片段（GSDMD-N）、白细胞介素（IL）-1β和IL-18，线粒体自噬相关蛋白Pink1、Parkin和微管相关蛋白轻链3（LC3）-Ⅱ的表达。用伊文思蓝检测血脑屏障的通透性。结果与假手术组相比，脓毒症组caspase-1、GSDMD-N、IL-1β和IL-18的表达水平显著升高（P<0.05）；脓毒症+Cas-1^-/-组caspase-1、GSDMD-N、IL-1β和IL-18的表达水平明显低于脓毒症组（P<0.05）。脓毒症+漆黄素组Pink1、Parkin和LC3-Ⅱ的表达水平明显高于脓毒症组（P<0.05），caspase-1、GSDMD-N、IL-1β和IL-18的表达水平明显低于脓毒症组（P<0.05）。与脓毒症组相比，脓毒症+漆黄素组伊文思蓝渗漏减少、逃逸潜伏期缩短和穿越平台次数增加（均P<0.05）。结论漆黄素可能通过Pink1/Parkin通路激活线粒体自噬，抑制小胶质细胞NLRP3炎症小体活化，进而缓解脓毒症后中枢炎症反应和认知功能损害。

关键词: 脓毒症, 认知功能障碍, 线粒体自噬, NLR家族，热蛋白结构域包含蛋白3, 漆黄素

Abstract:

Objective To investigate the mechanism of fisetin inhibiting the activation of microglia NOD-like receptor family protein 3 (NLRP3) inflammasome in microglia and alleviating cognitive impairment after sepsis. Methods C57BL/6 mice were used to establish the sepsis model by cecal ligation and puncture. Mice were divided into four groups: the sham group, the sepsis group, the sepsis+caspase-1 knockout group (sepsis+Cas-1^-/- group) and the sepsis+fisetin group. Evans blue was used to detect the permeability of blood-brain barrier (BBB). Morris water maze was used to evaluate the cognitive function of mice. Western blot assay and immunofluorescence double staining were used to detect the expression of NLRP3 inflammasome-related proteins including caspase-1, N-terminal fragment of the GSDMD (GSDMD-N), interleukin (IL)-1β, IL-18 and mitophagy-related proteins (Pink1, Parkin and LC3-Ⅱ) in brain tissue and microglia. Results Compared with the sham group, expression levels of caspase-1, GSDMD-N, IL-1β and IL-18 were significantly increased in the sepsis group (P<0.05). Compared with the sepsis group, expression levels of caspase-1, GSDMD-N, IL-1β and IL-18 were significantly decreased in the sepsis+Cas-1^-/- group (P<0.05). The expression levels of Pink1, Parkin and LC3-Ⅱ were significantly higher in the sepsis+fisetin group than those of the sepsis group (P<0.05), and expression levels of caspase-1, GSDMD-N, IL-1β and IL-18 were significantly lower (P<0.05). After fisetin intervention, the permeability of BBB was decreased and the cognitive impairment (decreased escape latency and increased frequencies of crossing the platform) was alleviated in the sepsis+fisetin group compared with those of the sepsis group (P<0.05). Conclusion Fisetin may alleviate central inflammation and cognitive impairment after sepsis by inhibiting the activation of microglial NLRP3 inflammasome through activating mitophagy.

Key words: sepsis, cognitive dysfunction, mitophagy, NLR family, pyrin domain-containing 3 protein, fisetin

中图分类号:

R515.3

图/表 11

表1 各组Morris水迷宫逃逸潜伏期结果比较（n=14，s，$\bar{x}±s$）

Tab.1 Comparison of escape latency in Morris water maze test between the three groups

组别	第2天	第3天	第4天	第5天
假手术组	74.6±14.5	63.6±13.9	39.4±8.3	25.6±4.0
脓毒症组	99.4±22.1^a	93.6±22.9^a	84.6±24.1^a	71.0±20.8^a
脓毒症+漆黄素组	77.0±26.2^b	72.8±24.6^b	60.8±22.4^b	41.5±19.3^b
F_组间/F_时间/F_交互	48.934^＊＊/30.180^＊＊/1.166

图1 Caspase-1敲除抑制脓毒症诱导的NLRP3炎症小体活化（Western blot） A：假手术组；B：脓毒症组；C：脓毒症+Cas-1-/-组。

Fig.1 Caspase-1 knockout inhibited sepsis-induced NLRP3 inflammasome activation (Western blot assay)

表2 各组caspase-1、GSDMD-N、IL-1β和IL-18水平比较（n=4，$\bar{x}±s$）

Tab.2 Comparison of caspase-1, GSDMD-N, IL-1β and IL-18 levels between the three groups

组别	caspase-1	GSDMD-N	IL-1β	IL-18
假手术组	0.16±0.06	0.14±0.08	0.06±0.02	0.09±0.03
脓毒症组	0.83±0.15^a	0.74±0.11^a	0.40±0.09^a	0.49±0.08^a
脓毒症+Cas-1^-/-组	0.15±0.08^b	0.17±0.11^b	0.09±0.03^b	0.11±0.04^b
F	58.805^＊＊	45.953^＊＊	51.213^＊＊	70.471^＊＊

图2 Caspase-1敲除抑制脓毒症诱导的小胶质细胞NLRP3炎症小体活化（免疫荧光染色，标尺=20 μm）

Fig.2 Caspase-1 knockout inhibited sepsis-induced microglial NLRP3 inflammasome activation (Immunofluorescent staining，Scale bar=20 μm)

图3 漆黄素降低脓毒症后BBB通透性

Fig.3 Fisetin reduced BBB permeability after sepsis

表3 各组伊文思蓝、Pink1、Parkin和LC3-Ⅱ水平比较（n=4，$\bar{x}±s$）

Tab.3 Comparison of Evans blue, Pink1, Parkin and LC3-II levels between three groups

组别	伊文思蓝/ （μg/mg）	Pink1	Parkin	LC3-Ⅱ
假手术组	88.69±26.51	0.06±0.01	0.05±0.02	0.21±0.05
脓毒症组	385.72±69.32^a	0.44±0.10^a	0.45±0.10^a	0.73±0.12^a
脓毒症+ 漆黄素组	142.62±42.69^b	0.79±0.11^b	0.87±0.15^b	1.26±0.17^b
F	40.985^＊＊	70.086^＊＊	63.330^＊＊	70.757^＊＊

图4 漆黄素促进脓毒症后线粒体自噬（Western blot） A：假手术组；B：脓毒症组；C：脓毒症+漆黄素组。

Fig.4 Fisetin promoted mitophagy after sepsis (Western blot assay)

图5 漆黄素促进脓毒症后小胶质细胞的线粒体自噬（免疫荧光染色，标尺=20 μm）

Fig.5 Fisetin promoted mitophagy of microglia after sepsis (Immunofluorescent staining, Scale bar=20 μm)

图6 漆黄素抑制脓毒症诱导的NLRP3炎症小体活化（Western blot） A：假手术组；B：脓毒症组；C：脓毒症+漆黄素组。

Fig.6 Fisetin inhibited sepsis-induced NLRP3 inflammasome activation (Western blot assay)

表4 各组caspase-1、GSDMD-N、IL-1β和IL-18水平比较（n=4，$\bar{x}±s$）

Tab.4 Comparison of caspase-1, GSDMD-N, IL-1β and IL-18 levels between three groups

组别	caspase-1	GSDMD-N	IL-1β	IL-18
假手术组	0.13±0.01	0.09±0.03	0.08±0.03	0.04±0.01
脓毒症组	1.26±0.15^a	1.14±0.12^a	0.73±0.12^a	0.65±0.15^a
脓毒症+漆黄素组	0.24±0.05^b	0.37±0.11^b	0.20±0.09^b	0.18±0.11^b
F	185.669^＊＊	137.843^＊＊	57.420^＊＊	32.885^＊＊

图7 漆黄素抑制脓毒症诱导的小胶质细胞NLRP3炎症小体活化（免疫荧光染色，标尺=20 μm）

Fig.7 Fisetin inhibited sepsis-induced microglial NLRP3 inflammasome activation (Immunofluorescent staining，Scale bar=20 μm)

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漆黄素抑制小胶质细胞NLRP3炎症小体活化缓解脓毒症后认知功能损害的研究

Study on the effect of fisetin on alleviating cognitive impairment after sepsis by inhibiting the activation of microglial NLPR3 inflammasome

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