槲皮素通过抑制NLRP3/Caspase-1/GSDMD信号通路抑制小鼠成纤维细胞焦亡

doi:10.12122/j.issn.1673-4254.2024.10.05

摘要/Abstract

摘要：

目的探究槲皮素通过NOD样受体热蛋白结构域相关蛋白3（NLRP3）/含半胱氨酸的天冬氨酸蛋白水解酶1（Caspase-1）/ 消皮素D（GSDMD）信号通路调控小鼠成纤维细胞（NIH-3T3）焦亡的影响及其机制。方法实验分为对照（Control）组、模型（Model）组、槲皮素（HPS）组、NLRP3特异性抑制剂（MCC950）组。CCK-8法检测槲皮素对NIH-3T3细胞活力的影响。光镜下观察细胞形态变化，ELISA检测白细胞介素（IL）-18、IL-1β含量，Western blotting和qRT-PCR检测NLRP3、活化半胱氨酸蛋白酶 1（Cleaved caspase-1）、GSDMD-N蛋白表达及NLRP3、Caspase-1、GSDMD mRNA水平。TUNEL染色及乳酸脱氢酶（LDH）释放实验检测细胞焦亡情况。结果槲皮素作用于NIH-3T3细胞最佳干预浓度和时间分别是20 μmol/L和24 h。与Control组相比，Model组细胞出现明显肿胀、破裂、细胞内容物流出，IL-18、IL-1β含量和NLRP3、Cleaved caspase-1、GSDMD-N蛋白表达及NLRP3、Caspase-1、GSDMD mRNA水平均明显升高（P<0.001，P<0.0001），TUNEL染色的阳性细胞数及LDH释放量增加（P<0.0001）。与Model组相比，HPS组和MCC950组细胞焦亡情况改善，IL-18、IL-1β含量和NLRP3、Cleaved caspase-1、GSDMD-N蛋白表达及NLRP3、Caspase-1、GSDMD mRNA水平显著降低（P<0.01，P<0.001），TUNEL染色阳性细胞数及LDH释放量明显减少（P<0.0001）。结论槲皮素可能通过抑制NLRP3/Caspase-1/GSDMD信号通路抑制成纤维细胞焦亡，下调炎症因子释放从而发挥治疗作用。

关键词: 槲皮素, 细胞焦亡, NLRP3, 成纤维细胞

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

舒萍, 袁孟珂, 杨珂, 何伟志, 刘丽. 槲皮素通过抑制NLRP3/Caspase-1/GSDMD信号通路抑制小鼠成纤维细胞焦亡[J]. 南方医科大学学报, 2024, 44(10): 1874-1880.

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.

图/表 7

图1 槲皮素对NIH-3T3细胞活力的影响

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

图2 各组细胞光镜下形态

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.

图3 各组IL-18、IL-1β含量比较

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.

图4 各组细胞NLRP3、Caspase-1、GSDMD mRNA比较

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.

图5 各组细胞NLRP3、Cleaved caspase-1、GSDMD-N蛋白比较

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.

图6 各组TUNEL染色情况

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

图7 各组LDH释放量比较

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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