木犀草素对膀胱癌5637细胞活力、焦亡及NLRP3-Caspase-1-焦亡素D通路的影响OA
Effects of luteolin on viability,pyroptosis,and NLRP3-Caspase-1-gasder-min D pathway of bladder cancer 5637 cells
目的:探讨木犀草素对膀胱癌5637 细胞活力、焦亡的影响及其分子机制.方法:CCK-8 法检测低、中、高剂量(25、50、100 μmol/L)木犀草素分别处理5637 细胞12、24、48 h 后细胞活力.5637 细胞分为5 组,正常对照组(PBS)、阳性对照组(5 μmol/L 焦亡诱导剂尼日利亚霉素)以及木犀草素低、中、高剂量组(25、50、100 μmol/L 木犀草素),处理24 h 后采用免疫荧光染色法检测焦亡素 D(GSDMD)蛋白的表达,ELISA 法检测细胞上清液中乳酸脱氢酶(LDH)、IL-18、IL-1β 分泌量,2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)法检测细胞内活性氧(ROS)含量,Western blot 法检测焦亡通路蛋白 NLRP3、Caspase-1、GSDMD 及炎症因子 IL-1β、IL-18 的表达.另取5637 细胞分为3 组:正常对照组、木犀草素低剂量组和焦亡抑制剂组(25 μmol/L 木犀草素+20 μmol/L Caspase-1 抑制剂 VRT-043198),处理24 h 后采用Western blot 法检测Caspase-1、GSDMD、IL-1β、IL-18 的表达.结果:随木犀草素作用时间的增加、剂量的增大,5637 细胞活力有降低的趋势(P＜0.05).与正常对照组比较,木犀草素各剂量组及阳性对照组 GSDMD 蛋白大量聚集在细胞膜上,细胞 LDH、IL-1β 和 IL-18 分泌增加,提示诱发焦亡;同时细胞内 ROS 含量增加,NLRP3、Caspase-1、GSDMD、IL-1β、IL-18 蛋白表达上调(P＜0.05).另外,与木犀草素低剂量组相比,焦亡抑制剂组 Caspase-1、GSDMD、IL-1β、IL-18 蛋白的表达下调(P＜0.05).结论:木犀草素可能通过激活 NLRP3-Caspase-1-GSDMD 通路,诱导膀胱癌细胞焦亡,抑制细胞活力.
Aim:To explore the role of luteolin in viability and pyroptosis of bladder cancer cells and its molecular mechanism.Methods:CCK-8 assay was used to detect the viability of 5637 cells after treatment with low,medium,and high doses(25,50,100 μmol/L)of luteolin for 12,24,and 48 hours.5637 cells were divided into 5 groups:normal control group(PBS),positive control group(5 μmol/L pyroptosis inducer nigericin),and low-dose(25 μmol/L),medium-dose(50 μmol/L),and high-dose(100 μmol/L)luteolin groups.After 24 hours of treatment,immunofluorescence staining was used to detect gasdermin D(GSDMD)protein expression in each group.ELISA was performed to measure the secretion levels of lactate dehydrogenase(LDH),IL-18,and IL-1β in the cell supernatant.The 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)method was used to detect intracellular reactive oxygen species(ROS)levels.Western blot was employed to determine the expression of pyroptosis pathway proteins NLRP3,Caspase-1,GSDMD,and inflammatory cytokines IL-1β and IL-18.Additionally,5637 cells were divided into 3 groups:normal control group,low-dose luteolin group,and pyroptosis in-hibitor group(25 μmol/L luteolin+20 μmol/L Caspase-1 inhibitor VRT-043198),Western blot was used to detect the ex-pression of Caspase-1,GSDMD,IL-1β,and IL-18 after 24 hours of treatment.Results:With increasing luteolin treatment time and dose,the viability of 5637 cells showed a decreasing trend(P＜0.05).Compared with the normal control group,the luteolin dose groups and the positive control group exhibited abundant GSDMD protein aggregation on the cell mem-brane,increased secretion of LDH,IL-1β,and IL-18,indicating induced pyroptosis.Meanwhile,intracellular ROS level in-creased,and the protein expression of NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 was upregulated(P＜0.05).Further-more,compared with the low-dose luteolin group,treatment with Caspase-1 inhibitor markedly decreased the protein expres-sion of Caspase-1,GSDMD,IL-1β and IL-18 in the pyroptosis inhibitor group.Conclusion:Luteolin may inhibit the viabili-ty of bladder cancer cells by inducing pyroptosis of 5637 cells through activating the NLRP3-Caspase-1-GSDMD pathway.
姚晓含;李赫阳;孙振邦;杨璨宇;王明;韩滨
郑州大学第一附属医院医学研究中心 郑州 450052郑州大学第一临床医学院 郑州 450052郑州大学第一临床医学院 郑州 450052郑州大学第一临床医学院 郑州 450052郑州大学第一附属医院医学研究中心 郑州 450052郑州大学第一附属医院放射治疗部 郑州 450052
医药卫生
木犀草素膀胱癌焦亡焦亡素D5637细胞NLRP3-Caspase-1-GSDMD通路
luteolinbladder cancerpyroptosisgasdermin D5637 cellNLRP3-Caspase-1-gasdermin D pathway
《郑州大学学报（医学版）》 2026 (3)
11-15,5
河南省创新研发专项(HNGD2024005)
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