Avitinib suppresses NLRP3 inflammasome activation and ameliorates septic shock in mice

doi:10.12122/j.issn.1673-4254.2025.08.14

Abstract

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

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.

Figures/Tables 5

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.

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.

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.

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.

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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