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.

References 34

1	Jännebring J, Liv P, Knuts M, et al. Impact of patient-reported salpingitis on the outcome of hysterectomy and adnexal surgery: a national register-based cohort study in Sweden[J]. Acta Obstet Gynecol Scand, 2024, 103(6): 1153-64.
2	Xijiao HU, Shuoxi LI, Yang DX, et al. Modified Gexiazhuyu Decoction alleviates chronic salpingitis p38 signaling pathway[J]. Chung I Tsa Chih Ying Wen Pan, 2022, 42(2): 213-20.
3	Zhang RN, Sun ZJ, Zhang L. Pyroptosis in inflammatory bone diseases: molecular insights and targeting strategies[J]. FASEB J, 2022, 36(12): e22670.
4	Hachim MY, Khalil BA, Elemam NM, et al. Pyroptosis: the missing puzzle among innate and adaptive immunity crosstalk[J]. J Leukoc Biol, 2020, 108(1): 323-38.
5	Coll RC, Schroder K, Pelegrín P. NLRP3 and pyroptosis blockers for treating inflammatory diseases[J]. Trends Pharmacol Sci, 2022, 43(8): 653-68.
6	Sefik E, Qu RH, Junqueira C, et al. Inflammasome activation in infected macrophages drives COVID-19 pathology[J]. Nature, 2022, 606(7914): 585-93.
7	Arrè V, Scialpi R, Centonze M, et al. The 'speck'-tacular oversight of the NLRP3-pyroptosis pathway on gastrointestinal inflammatory diseases and tumorigenesis[J]. J Biomed Sci, 2023, 30(1): 90.
8	Hsu CG, Chávez CL, Zhang CY, et al. The lipid peroxidation product 4-hydroxynonenal inhibits NLRP3 inflammasome activation and macrophage pyroptosis[J]. Cell Death Differ, 2022, 29(9): 1790-803.
9	洪金妮, 林达涛, 陈仔颖, 等. 少腹逐瘀汤对输卵管炎性不孕大鼠NLRP3炎症小体的作用研究[J]. 药物评价研究, 2022, 45(12): 2486-93.
10	刘梅, 谢星, 匡继林. 通管方对输卵管炎性不孕模型大鼠NLRP3/Caspase-1细胞焦亡通路的影响[J]. 中医药导报, 2023, 29(2): 8-13.
11	袁孟珂, 刘进哲, 穆姿辰, 等. 膈下逐瘀汤加减方通过lncRNAs/NLRP3炎症小体通路抑制慢性输卵管炎大鼠细胞焦亡[J]. 中华中医药杂志, 2024, 39(5): 2582-6.
12	刘丽, 袁孟珂, 陈婧, 等. 针药联合治疗输卵管炎性不孕症临床观察[J]. 辽宁中医药大学学报, 2020, 22(8): 4-7.
13	李杨, 刘丽. 基于网络药理学探讨膈下逐瘀汤加减方治疗慢性盆腔炎性疾病的作用机制[J]. 世界中西医结合杂志, 2021, 16(10): 1858-64.
14	王月田, 刘鑫国, 杨恿. 槲皮素调控p38 MAPK/NF-κB/NLRP3信号通路对大鼠分泌性中耳炎的作用机制[J]. 中国药理学通报, 2023, 39(4): 672-8.
15	杨昊若, 任军华, 张昱, 等. 槲皮素通过激活Keap1/Nrf2信号通路减轻脂多糖诱导的大鼠急性肾损伤[J]. 中国临床药理学杂志, 2023, 39(21): 3134-8.
16	祝珊珊, 谭博文, 秦飞, 等. 槲皮素通过PTEN/PI3K/JNK信号通路减轻小鼠RAW264.7巨噬细胞炎症[J]. 中国病理生理杂志, 2023, 39(3): 510-9.
17	Li H, Guan YL, Liang B, et al. Therapeutic potential of MCC950, a specific inhibitor of NLRP3 inflammasome[J]. Eur J Pharmacol, 2022, 928: 175091.
18	Song ZH, Gong Q, Guo JW. Pyroptosis: mechanisms and links with fibrosis[J]. Cells, 2021, 10(12): 3509.
19	Gan C, Cai QY, Tang CW, et al. Inflammasomes and pyroptosis of liver cells in liver fibrosis[J]. Front Immunol, 2022, 13: 896473.
20	Kang JY, Xu MM, Sun Y, et al. Melatonin attenuates LPS-induced pyroptosis in acute lung injury by inhibiting NLRP3-GSDMD pathway via activating Nrf2/HO-1 signaling axis[J]. Int Immunopharmacol, 2022, 109: 108782.
21	Sanz AB, Sanchez-Niño MD, Ramos AM, et al. Regulated cell death pathways in kidney disease[J]. Nat Rev Nephrol, 2023, 19(5): 281-99.
22	Dong ZP, Yang BN, Jia ME, et al. DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition[J]. Biochim Biophys Acta Mol Cell Res, 2024, 1871(4): 119712.
23	Weng XZ, Luo X, Dai XY, et al. Apigenin inhibits macrophage pyroptosis through regulation of oxidative stress and the NF-κB pathway and ameliorates atherosclerosis[J]. Phytother Res, 2023, 37(11): 5300-14.
24	Wu WJ, Wu XH, Qiu LX, et al. Quercetin influences intestinal dysbacteriosis and delays alveolar epithelial cell senescence by regulating PTEN/PI3K/AKT signaling in pulmonary fibrosis[J]. Naunyn Schmiedebergs Arch Pharmacol, 2024, 397(7): 4809-22.
25	冯桃花, 刘文康, 武宁, 等. 益肾健脾泻浊中药对慢性肾脏病妊娠大鼠肾脏NLRP3/caspase-1/IL-1β信号通路及细胞焦亡的影响[J]. 世界科学技术-中医药现代化, 2021, 23(2): 451-61.
26	邹长鹏, 郭燕, 张长旺, 等. 补中益气汤中的有效成分槲皮素通过GSTP1-JAK-STAT途径增强肺腺癌耐药细胞株A549/DDP对顺铂的敏感性[J]. 广州中医药大学学报, 2023, 40(10): 2606-14.
27	兰悦嘉, 孟宪丽, 吴嘉思. 黄芩素调控NLRP3/Caspase-1/GSDMD通路介导的焦亡减轻小鼠急性肺损伤作用 [J]. 中草药, 2023, 54(20): 6694-703.
28	Zhang HX, Li YY, Liu ZJ, et al. Quercetin effectively improves LPS-induced intestinal inflammation, pyroptosis, and disruption of the barrier function through the TLR4/NF‑κB/NLRP3 signaling pathway in vivo and in vitro [J]. Food Nutr Res, 2022, ecollection. 66.
29	Mulay SR. Multifactorial functions of the inflammasome component NLRP3 in pathogenesis of chronic kidney diseases[J]. Kidney Int, 2019, 96(1): 58-66.
30	Platnich JM, Muruve DA. NOD-like receptors and inflammasomes: a review of their canonical and non-canonical signaling pathways[J]. Arch Biochem Biophys, 2019, 670: 4-14.
31	Li Z, Guo JL, Bi LQ. Role of the NLRP3 inflammasome in autoimmune diseases[J]. Biomed Pharmacother, 2020, 130: 110542.
32	Wang K, Sun Q, Zhong X, et al. Structural mechanism for GSDMD targeting by autoprocessed caspases in pyroptosis[J]. Cell, 2020, 180(5): 941-55.e20.
33	Jorgensen I, Rayamajhi M, Miao EA. Programmed cell death as a defence against infection[J]. Nat Rev Immunol, 2017, 17(3): 151-64.
34	Luo X, Bao XY, Weng XZ, et al. The protective effect of quercetin on macrophage pyroptosis via TLR2/Myd88/NF‑κB and ROS/AMPK pathway[J]. Life Sci, 2022, 291: 120064.

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