Quercetin suppresses pyroptosis in mouse fibroblasts by inhibiting the NLRP3/caspase-1/GSDMD pathway

doi:10.12122/j.issn.1673-4254.2024.10.05

Abstract

Abstract:

Objective To investigate whether quercetin inhibits pyroptosis of mouse fibroblast NIH-3T3 cells by regulating the NLRP3/caspase-1/GSDMD signaling pathway. Methods NIH-3T3 cells were treated with quercetin or MCC950 (a specific inhibitor of NLRP3) before stimulation with lipopolysaccharide (LPS) and ATP to induce cell pyroptosis. The optimal quercetin concentration and duration were screened using the CCK-8assay after testing various concentrations and times. Morphological changes of the treated cells was observed, and the levels of IL-18 and IL-1β in the cell culture supernatant were detected with ELISA; the protein expressions of NLRP3, cleaved caspase-1, and GSDMD-N and the mRNA levels of NLRP3, caspase-1 and GSDMD were detected using Western blotting and qRT-PCR. The changes in cell pyroptosis were examined with TUNEL staining and LDH release assay. Results The CCK-8 assay indicated that 24-hour treatment with 20 μmol/L quercetin yielded the most favorable results. LPS and ATP stimulation of NIH-3T3 cells induced obvious swelling, cell membrane rupture and leakage of cell contents, significantly increased IL-18 and IL-1β levels, and enhanced protein expressions of NLRP3, cleaved caspase-1 and GSDMD-N and mRNA levels of NLRP3, caspase-1 and GSDMD. LPS and ATP stimulation also caused a significant increment of TUNEL-positive cell counts and LDH release in NIH-3T3 cells. Treatment with quercetin or MCC950 significantly reduced cell pyroptosis induced by LPS and ATP, lowered the concentrations of IL-18 and IL-1β, decreased the expression levels of NLRP3, caspase-1/cleaved caspase-1, GSDMD/GSDMD-N, and reduced the number of TUNEL-positive cells and LDH release. Conclusion Quercetin suppresses pyroptosis of mouse fibroblasts stimulated with LPS and ATP and reduces secretion of inflammatory cytokines by inhibiting the NLRP3/caspase-1/GSDMD pathway.

Key words: quercetin, pyroptosis, NLRP3, fibroblasts

Ping SHU, Mengke YUAN, Ke YANG, Weizhi HE, Li LIU. Quercetin suppresses pyroptosis in mouse fibroblasts by inhibiting the NLRP3/caspase-1/GSDMD pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1874-1880.

Figures/Tables 7

Fig.1 Effect of quercetin on viability of NIH-3T3 cells. *P<0.05,**P<0.01, ***P<0.001 vs 0 μmol/L.

Fig.2 Morphological changes of NIN-3T3 cells with different treatments (Original magnification: ×200). A: Control group. B: Model group. C: Quercetin group. D: MCC950 group.

Fig.3 Concentrations of IL-18 and IL-1β in the culture supernatant in each group.****P<0.0001 vs Control group; ####P<0.0001 vs Model group.

Fig.4 Comparison of mRNA expressions of NLRP3, caspase-1 and GSDMD mRNA among the groups. ****P<0.0001 vs Control group; ####P<0.0001 vs Model group.

Fig.5 Comparison of protein expressions of NLRP3, cleaved caspase-1 and GSDMD-N among the groups. ***P<0.001, ****P<0.0001 vs Control group; ##P<0.01, ###P<0.001, ####P<0.0001 vs Model group.

Fig.6 TUNEL staining of cells in each group (×200). A: Control group. B: Model group. C: Quercetin group. D: MCC950 group.

Fig.7 Comparison of LDH release among the groups. ****P<0.0001 vs Control group; ####P<0.0001 vs Model group.

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