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

doi:10.12122/j.issn.1673-4254.2024.06.02

Abstract

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

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.

Figures/Tables 5

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.

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.

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.

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.

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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