二十碳五烯酸通过调节Nrf2通路抑制脂多糖诱导肾小球系膜细胞焦亡  

Eicosapentaenoic acid inhibited lipopolysaccharide-induced mesangial cell pyroptosis by regulating Nrf2 pathway

作　　者：李洁[1] 冯烨 陆庆雪 梁君梦 LI Jie;FENG Ye;LU Qingxue;LIANG Junmeng(Department of Rheumatology,Minzu Hospital of Guangxi Zhuang Autonomous Region,Guangxi Nanning 530001,China)

机构地区：[1]广西壮族自治区民族医院风湿免疫科,广西南宁530001

出　　处：《江苏大学学报（医学版）》2024年第4期277-282,共6页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:以核因子E2相关因子2(nuclear factor E2 related factor 2,Nrf2)蛋白为切入点,基于Nrf2/NLRP3/GSDMD信号通路探究二十碳五烯酸(eicosapentaenoic acid,EPA)对脂多糖(lipopolysaccharide,LPS)诱导肾小球系膜细胞焦亡的调控作用,结合Nrf2激动剂(4-OI)、核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-bound oligomerized domain-like receptor protein 3,NLRP3)受体抑制剂(MCC950)探究EPA调控焦亡信号通路的机制。方法:LPS体外诱导肾小球系膜细胞(HBZY-1)焦亡模型,并根据不同药物处理分别设置空白对照组、LPS组、LPS+EPA组、LPS+EPA+4-OI组和LPS+EPA+MCC950组,其中LPS浓度为10μg/mL,EPA、4-OI和MCC950的浓度均为200μmol/L,按分组将药物同时加入培养板中处理48 h,利用CCK-8法、乳酸脱氢酶(LDH)释放实验、TUNEL染色检测细胞损伤,ELISA检测细胞上清液中炎症因子IL-1β、IL-18释放量,蛋白免疫印迹法检测Nrf2、NLRP3、Caspase-1、GSDMD蛋白表达水平。结果:LPS与EPA共处理HBZY-1细胞的细胞活力实验表明,10μg/mL LPS刺激48 h显著降低了细胞活力(P<0.05),但20~200μmol/L EPA对其有明显的保护作用(P<0.05)。采用200μmol/L EPA进行后续实验发现,EPA可明显保护LPS造成的细胞损伤(P<0.01),减少炎性因子(P<0.01)、LDH的释放(P<0.01)和DNA的断裂并抑制焦亡信号通路的激活,促进Nrf2蛋白的表达(P<0.01)。结合4-OI、MCC950共处理发现4-OI可以促进EPA对焦亡的抑制作用(P<0.05),减少相关蛋白的表达以及因子的释放(P<0.05),并增强EPA对细胞的保护(P<0.01),而MCC950对EPA发挥的作用无显著影响。结论:EPA通过Nrf2抑制LPS诱导的HBZY-1细胞焦亡信号通路各分子的表达,发挥细胞保护作用。Objective:To explore the regulatory effect of eicosapentaenoic acid(EPA)on lipopolysaccharide(LPS)-induced HBZY-1 cell pyroptosis based on the Nrf2/NLRP3/GSDMD signaling pathway.Additionally,nuclear factor E2 related factor 2(Nrf2)agonist(4-OI)and nucleotide-bound oligomerized domain-like receptors(NLRP3)receptor inhibitor(MCC950)were chosen to explore the regulating mechanism of EPA on Nrf2.Methods:According to different drug treatments strategy,HBZY-1 cells were divided into five groups:control group,LPS group,LPS+EPA group,LPS+EPA+4-OI group,and LPS+EPA+MCC950 group,in which 10μg/mL LPS was applied to induce the cell pyroptosis in vitro,and the concentration of EPA,4-OI,MCC950 was all 200μmol/L,the drugs were added to the culture plate and treated for 48 h.In addition,CCK-8 assay,LDH release test,and TUNEL staining were performed to detect cell damage.The release of inflammatory factors IL-1βand IL-18 in the cell supernatant were detected by ELISA kits,protein levels of Nrf2,NLRP3,Caspase-1 and GSDMD were detected by Western Blotting.Results:Cell viability assay showed that 10μg/mL LPS stimulation for 48 hours significantly reduced the cell viability(P<0.05),but 20-200μmol/L EPA had significantly protective effects(P<0.05).Moreover,200μmol/L EPA significantly attenuated the cell damage caused by LPS(P<0.01),reduced the release of inflammatory factors(P<0.01),LDH release(P<0.01)and DNA fragmentation,inhibited the activation of the pyroptosis,and promoted the expression of Nrf2 protein(P<0.01).In addition,co-treatment with 4-OI and MCC950 found that 4-OI can promote the inhibitory effect of EPA on pyroptosis(P<0.05),reduce the expression of related proteins and the release of IL-1βand IL-18(P<0.05),and enhance the protection effect of EPA(P<0.01),while MCC950 has no significant on the effect of EPA.Conclusion:EPA inhibits the expression of various molecules in the pyroptosis of HBZY-1 cells induced by LPS through Nrf2,and exerts a significant cytoprotective effect.

关 键 词：二十碳五烯酸 焦亡 细胞损伤 核因子E2相关因子2(Nrf2) 

分 类 号：R692.6[医药卫生—泌尿科学]