2，6-二甲氧基-1，4-苯醌通过抑制NLRP3炎症小体活化缓解小鼠的感染性休克

doi:10.12122/j.issn.1673-4254.2024.06.02

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1024-1032.doi: 10.12122/j.issn.1673-4254.2024.06.02

• • 上一篇

2，6-二甲氧基-1，4-苯醌通过抑制NLRP3炎症小体活化缓解小鼠的感染性休克

张玮¹^,²(), 邓蒙蒙¹, 曾尧¹^,², 刘辰菲¹, 尚菲菲², 许文豪¹, 蒋昊轶², 王凤超¹, 杨燕青¹^,²()

^1.蚌埠医科大学第一附属医院检验科，安徽蚌埠 233004
^2.蚌埠医科大学慢性疾病免疫学基础与临床安徽省重点实验室，安徽蚌埠 233030

收稿日期:2024-02-25 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 杨燕青 E-mail:zw13085009617@163.com;yyqing@mail.ustc.edu.cn
作者简介:张玮，在读硕士研究生，E-mail: zw13085009617@163.com
基金资助:
国家自然科学基金(82071775);安徽省自然科学基金(2108085QH349);慢性疾病免疫学基础与临床安徽省重点实验室开放课题基金(AHIAI2022K01);安徽省科研编制计划优秀青年科研项目(2022AH030140);蚌埠医学院2023年度研究生科研创新计划项目(Byycx23070);2022年安徽省大学生创新创业训练计划项目(S202210367134)

2,6-dimethoxy-1,4-benzoquinone alleviates septic shock in mice by inhibiting NLRP3 inflammasome activation

Wei ZHANG¹^,²(), Mengmeng DENG¹, Yao ZENG¹^,², Chenfei LIU¹, Feifei SHANG², Wenhao XU¹, Haoyi JIANG², Fengchao WANG¹, Yanqing YANG¹^,²()

^1.Clinical Laboratory, First Affiliated Hospital of Bengbu Medical University, Bengbu 233004, China
^2.Anhui Provincial Key Laboratory of Immunology in Chronic Diseases, Bengbu Medical University, Bengbu 233030, China

Received:2024-02-25 Online:2024-06-20 Published:2024-07-01
Contact: Yanqing YANG E-mail:zw13085009617@163.com;yyqing@mail.ustc.edu.cn
Supported by:
National Science Foundation of China(82071775)

摘要/Abstract

摘要：

目的探究发酵小麦胚芽提取物主要活性成分2，6-二甲氧基-1，4-苯醌（DMQ）抑制NLRP3炎症小体活化并缓解小鼠感染性休克的作用机制。方法细胞学水平：在BMDM细胞中，经脂多糖（LPS）预处理、DMQ干预后，利用尼日利亚菌素（Nigericin）、ATP、尿酸钠结晶（MSU）分别活化经典NLRP3炎症小体以及胞内转染LPS活化非经典NLRP3炎症小体。利用聚脱氧腺苷酸（Poly A：T）活化AIM2炎症小体。在THP-1细胞中，利用Nigericin活化经典NLRP3炎症小体，通过Western blotting和ELISA方法测定NLRP3炎症小体活化产物的表达水平。蛋白分子水平：利用免疫共沉淀探究DMQ阻断NLRP3炎症小体活化的具体机制。动物水平：将8周龄雄性C57BL/6J小鼠随机分为空白对照组、感染性休克LPS组、DMQ 20 mg/kg治疗组、DMQ 40 mg/kg治疗组，6只/组，待DMQ治疗组小鼠预注射相应浓度DMQ后，对照组小鼠注射无菌PBS，其余3组小鼠腹腔注射相同剂量LPS，利用ELISA检测DMQ干预对LPS诱导的小鼠感染性休克模型中血清和腹腔灌洗液中TNF-α和IL-1β分泌水平的影响。DMQ预处理后注射LPS观察记录小鼠36 h内的生存状态并绘制生存曲线。结果 DMQ可有效抑制小鼠BMDM细胞和人THP-1细胞中经典NLRP3炎症小体活化（P<0.05），在小鼠BMDM细胞中对非经典NLRP3炎症小体活化也起到有效抑制作用（P<0.05），DMQ对AIM2炎症小体活化无影响（P>0.05）。进一步实验结果揭示DMQ可阻断ASC和NLRP3之间的相互作用。DMQ治疗组可显著降低小鼠血清和腹腔液中IL-1β的分泌水平（P<0.05）并延长小鼠生存时间（P<0.05）。结论发酵小麦胚芽提取物主要活性成分DMQ通过阻断ASC和NLRP3之间的相互作用有效抑制NLRP3炎症小体活化并缓解LPS诱导的小鼠感染性休克。

关键词: 2，6-二甲氧基-1，4-苯醌, NLRP3炎症小体, 感染性休克, 发酵小麦胚芽提取物

Abstract:

Objective To investigate the mechanism of 2,6-dimethoxy-1,4-benzoquinone (DMQ), an active ingredients in fermented wheat germ extract, for inhibiting NLRP3 inflammasome activation and alleviating septic shock in mice. Methods Cultured murine bone marrow-derived macrophages (BMDM) stimulated with lipopolysaccharide (LPS) were treated with DMQ, followed by treatment with Nigericin, ATP, and MSU for activating the canonical NLRP3 inflammasome; the non-canonical NLRP3 inflammasome was activated by intracellular transfection of LPS, and AIM2 inflammasome was activated using Poly A:T. In human monocytic THP-1 cells, the effect of Nigericin on inflammasome activation products was examined using Western blotting and ELISA. Co-immunoprecipitation was performed to explore the mechanism of DMQ-induced blocking of NLRP3 inflammasome activation. In a male C57BL/6J mouse model of LPS-induced septic shock treated with 20 and 40 mg/kg DMQ, the levels of IL-1β and TNF-α in the serum and peritoneal lavage fluid were determined using ELISA, and the survival time of the mice within 36 h was observed. Results Treatment with DMQ effectively inhibited LPS-induced activation of canonical NLRP3 inflammasome in mouse BMDM and human THP-1 cells and also inhibited non-canonical NLRP3 inflammasome activation in mouse BMDM, but produced no significant effect on AIM2 inflammasome activation. DMQ significantly blocked the binding between ASC and NLRP3. In the mouse models of septic shock, DMQ treatment significantly reduced the levels of IL-1β in the serum and peritoneal fluid and obviously prolonged survival time of the mice. Conclusion DMQ can effectively block ASC-NLRP3 interaction to inhibit NLRP3 inflammasome activation and alleviate LPS-induced septic shock in mice.

Key words: 2,6-dimethoxy-1,4-benzoquinone, NLRP3 inflammasome, septic shock, fermented wheat germ extract

张玮, 邓蒙蒙, 曾尧, 刘辰菲, 尚菲菲, 许文豪, 蒋昊轶, 王凤超, 杨燕青. 2，6-二甲氧基-1，4-苯醌通过抑制NLRP3炎症小体活化缓解小鼠的感染性休克[J]. 南方医科大学学报, 2024, 44(6): 1024-1032.

Wei ZHANG, Mengmeng DENG, Yao ZENG, Chenfei LIU, Feifei SHANG, Wenhao XU, Haoyi JIANG, Fengchao WANG, Yanqing YANG. 2,6-dimethoxy-1,4-benzoquinone alleviates septic shock in mice by inhibiting NLRP3 inflammasome activation[J]. Journal of Southern Medical University, 2024, 44(6): 1024-1032.

图/表 5

图1 DMQ抑制Nigericin诱导的NLRP3炎症小体活化

Fig.1 DMQ suppresses Nigericin-induced NLRP3 inflammasome activation in BMDM and THP-1 cells. A: Western blotting of cleaved IL-1β and caspase-1 (p20) in the culture supernatants, pro-IL-1β, Pro-caspase-1 (Pro-casp1) and β-actin in the cell lysate of BMDM cells. B-D: ELISA of IL-1β, TNF-α and IL-6 in the culture supernatant, respectively. E: Western blotting of cleaved caspase-1 (p20) in the culture supernatant and pro-caspase-1 (Pro-casp1) and β-actin in the cell lysate of THP-1 cells. F: ELISA of IL-1β in the culture supernatant of THP-1 cells. *P<0.05, **P<0.01, ***P<0.001.

图2 DMQ抑制ATP, MSU诱导的NLRP3炎症小体活化

Fig.2 DMQ inhibits NLRP3 inflammasome activation in BMDM induced by ATP and MSU. A: Western blotting of IL-1β and p20 in the supernatant and Pro-IL-1β, Pro-casp1 and β-actin in cell lysates of BMDM induced by ATP. B: ELISA of IL-1β in the culture supernatant of BMDM induced by ATP. C: ELISA of IL-1β in the culture supernatant of BMDM induced by MSU. D: Western blotting of IL-1β and p20 in the supernatant and Pro-IL-1β, Pro-casp1 and β-actin in cell lysates of BMDM induced by MSU. *P<0.05, **P<0.01, ***P<0.001.

图4 DMQ对抑制AIM2炎症小体活化无影响

Fig.4 DMQ does not inhibit AIM2 inflammasome activation in BMDM. A: Western blotting of IL-1β and p20 in the supernatant and Pro-IL-1β, Pro-casp1 and β-actin in cell lysates of BMDM. B-E:Quantitative analysis of the expressions of IL-1β (B), p20 (C), pro-IL-1β (D), and Pro-casp1 (E) in BMDM. F: ELISA of IL-1β in the culture supernatant of BMDM. *P<0.05, **P<0.01, ***P<0.001.

图5 DMQ阻断ASC和NLRP3之间的结合

Fig.5 DMQ blocks ASC-NLRP3 interaction. A: Western blotting of NLRP3 and NEK7 in immunoprecipitation (IP) and in cell lysates (Input) of BMDM. B, C: Quantitative analysis of the expressions of NLRP3 (B) and NEK7 (C) in BMDM. D: Western blotting of NLRP3 and ASC in IP and in cell lysates (Input) of BMDM. E, F: Quantitative analysis of the expressions of NLRP3 (E) and ASC (F) in BMDM. ***P<0.001.

图6 DMQ缓解LPS诱导的小鼠感染性休克

Fig.6 DMQ alleviates LPS-induced septic shock in mice. A, B: Serum leveld of IL-1β (A) and TNF-α (B) determined with ELISA. C, D: Levels of IL-1β (C) and TNF-α (D) in peritoneal lavage fluid determined with ELISA. E: Survive curve of the mice within 36 h after LPS injection. n=6, *P<0.05, **P<0.01. ***P<0.001.

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2，6-二甲氧基-1，4-苯醌通过抑制NLRP3炎症小体活化缓解小鼠的感染性休克

2,6-dimethoxy-1,4-benzoquinone alleviates septic shock in mice by inhibiting NLRP3 inflammasome activation

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