Avitinib抑制NLRP3炎症小体活化并改善小鼠感染性休克

doi:10.12122/j.issn.1673-4254.2025.08.14

摘要/Abstract

摘要：

目的探究Avitinib抑制NLRP3炎症小体活化并缓解感染性休克的作用及其机制。方法预先给药Avitinib作用小鼠骨髓来源巨噬细胞（BMDM）、人单核细胞白血病细胞系（THP-1）、健康志愿者外周血分离提取的单个核细胞（PBMC），后加入多种NLRP3炎症小体激动剂如尼日利亚菌素（Nigericin），单钠尿酸盐（MSU）结晶，三磷酸腺苷（ATP）活化经典NLRP3炎症小体，或在细胞内转染脂多糖（LPS）活化非经典形式NLRP3炎症小体（Western blotting）检测NLRP3炎症小体活化的分泌蛋白指征及细胞焦亡情况。利用酶联免疫吸附技术（ELISA）测定细胞上清中相关炎症因子的水平。构建小鼠感染性休克模型，采取随机分组的方法，将8周龄雄性C57BL/6J小鼠分为空白对照组（Control组），感染性休克模型组（LPS组），给药组（LPS+Avitinib组），6只/组。ELISA测定各组眼球血和腹腔灌洗液中的相关炎症因子水平，并记录小鼠的存活情况，采用Kaplan-Meier法进行生存分析。结果 Avitinib在多种细胞类型中抑制NLRP3炎症小体活化，剂量依赖性地抑制IL-1β分泌和caspase-1剪切，同时抑制GSDMD介导的细胞焦亡（P<0.05），但对炎症早期预警因子白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）的分泌并无影响（P>0.05）。动物实验结果表明，在LPS诱导的感染性休克模型中，给予Avitinib干预后小鼠血清和腹腔液中IL-1β的水平降低（P<0.05），并延长小鼠生存时间（P<0.05）。结论 Avitinib可抑制NLRP3炎症小体活化并改善感染性休克。

关键词: Avitinib, NLRP3炎症小体, EGFR抑制剂, 感染性休克

Abstract:

Objective To investigate the effect of avitinib for suppressing NLRP3 inflammasome activation and alleviating septic shock and explore the underlying mechanism. Methods Mouse bone marrow-derived macrophages (BMDM), human monocytic leukemia cell line THP-1, and peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were pre-treated with avitinib, followed by activation of the canonical NLRP3 inflammasome using agonists including nigericin, monosodium urate (MSU) crystals, or adenosine triphosphate (ATP). Non-canonical NLRP3 inflammasome activation was induced via intracellular transfection of lipopolysaccharide (LPS). Western blotting was used to detect the secretory protein markers of NLRP3 inflammasome activation and assess pyroptosis, and the levels of inflammatory cytokines in cell culture supernatant were determined with ELISA. In a mouse model of LPS-induced septic shock, the effect of avitinib treatment on the levels of inflammatory cytokines in serum and peritoneal lavage fluid were examined with ELISA, and survival curves of the mice were plotted using the Kaplan-Meier method. Results Avitinib significantly inhibited NLRP3 inflammasome activation in multiple cell types, and dose-dependently reduced IL-1β secretion and caspase-1 cleavage while suppressing GSDMD-mediated pyroptosis without obviously affecting IL-6 or TNF-α levels. In the mouse models of LPS-induced septic shock, avitinib significantly lowered IL-1β levels in serum and peritoneal fluid and extended survival time of the mice. Conclusion Avitinib suppresses NLRP3 inflammasome activation and alleviates septic shock in mice.

Key words: avitinib, NLRP3 inflammasome, EGFR inhibitor, septic shock

尚菲菲, 师晓可, 曾尧, 陶循浅, 李天真, 梁艳, 杨燕青, 宋传旺. Avitinib抑制NLRP3炎症小体活化并改善小鼠感染性休克[J]. 南方医科大学学报, 2025, 45(8): 1697-1705.

Feifei SHANG, Xiaoke SHI, Yao ZENG, Xunqian TAO, Tianzhen LI, Yan LIANG, Yanqin YANG, Chuanwang SONG. Avitinib suppresses NLRP3 inflammasome activation and ameliorates septic shock in mice[J]. Journal of Southern Medical University, 2025, 45(8): 1697-1705.

图/表 5

图1 Avitinib抑制鼠BMDM细胞Nigericin诱导的经典NLRP3炎症小体活化与焦亡

Fig.1 Avitinib inhibits nigericin-induced activation of the canonical NLRP3 inflammasome in mouse bone marrow-derived macrophages (BMDMs). A: Western blotting for detecting cleaved IL-1β and caspase-1 (p20) in the culture supernatants and expressions of pro-IL-1β, pro-caspase-1 (Pro-casp1) and β-actin in the cell lysate of BMDMs. B-D: ELISA for detecting IL-1β (B), IL-6 (C), and TNF-α (D) levels in the culture supernatant. E: Western blotting of full-length and cleaved Gasdermin D (GSDMD) and β-actin in BMDM cell lysates. *P<0.05, **P<0.01, ***P<0.001 vs LPS+Nigericin group.

图2 Avitinib抑制THP-1细胞、人PBMC细胞中经典NLRP3炎症小体活化

Fig.2 Avitinib inhibits nigericin-induced activation of the canonical NLRP3 inflammasome in THP-1 cells and human peripheral blood mononuclear cells (PBMCs). A: Western blotting of cleaved caspase-1 (p20) in the culture supernatants and expressions of pro-caspase-1 (Pro-casp1) and β-actin in THP-1 cell lysate. B: ELISA of IL-1β levels in the culture supernatant of THP-1 cells. C: ELISA of IL-1β levels in the culture supernatant of PBMCs. *P<0.05, **P<0.01, ***P<0.001 vs LPS+Nigericin group.

图3 Avitinib抑制鼠BMDM细胞ATP、MSU诱导的经典NLRP3炎症小体活化

Fig.3 Avitinib inhibits ATP- and MSU-induced activation of the canonical NLRP3 inflammasome in BMDMs. A,B: Western blotting of cleaved IL-1β and caspase-1 (p20) in culture supernatants and pro-IL-1β, Pro-caspase-1 (Pro-casp1), and β-actin in cell lysates of BMDMs stimulated with ATP (A) or MSU (B). C, D: ELISA of IL-1β in the culture supernatant of BMDMs stimulated with ATP (C) or MSU (D). *P<0.05, **P<0.01, ***P<0.001 vs LPS+ATP group; ##P<0.01, ###P<0.001 vs LPS+MSU group.

图4 Avitinib抑制鼠BMDM细胞非经典NLRP3炎症小体活化

Fig.4 Avitinib inhibits activation of the non-canonical NLRP3 inflammasome in BMDMs. A: Western blotting of cleaved IL-1β and caspase-1 (p20) in the culture supernatants and pro-IL-1β, Pro-caspase-1 (Pro-casp1) and β-actin in the cell lysate of BMDM cells. B: ELISA of IL-1β in the culture supernatant. **P<0.01, ***P<0.001 vs Pam3+cLPS group.

图5 Avitinib缓解小鼠感染性休克

Fig.5 Avitinib relieves septic shock in mice. A, B: ELISA for detecting IL-1β levels in serum (A) and peritoneal lavage fluid (B). C,D: ELISA for detecting IL-6 levels in serum (C)and peritoneal lavage fluid (D). E, F: ELISA for detecting TNF‑α levels in serum (E) and peritoneal lavage fluid (F). G: Survive curves of the mice within 36 h after LPS injection. n=6. **P<0.01 vs LPS group.

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