京尼平调控NLRP3通路致HK-2细胞毒性作用机制研究  

Study of the Mechanism of Genipin Induced Cytotoxicity on HK-2 Cell via NLRP3 Pathway

作　　者：石明珠 叶田香 程含笑 杨卫东 李会芳 SHI Mingzhu;YE Tianxiang;CHENG Hanxiao;YANG Weidong;LI Huifang(College of Chinese Medicine and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学中药与食品工程学院,山西晋中030619

出　　处：《中国现代应用药学》2024年第19期2599-2607,共9页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金项目(81903913);山西省中医药管理局科研课题(2022ZYYC093);山西中医药大学科技创新团队(2022TD1016)。

摘　　要：目的基于NLRP3通路探讨京尼平(genipin,GP)对人源肾小管上皮细胞(human tubular epithelial cells,HK-2)毒性作用。方法CCK8法筛选GP致HK-2细胞毒性浓度及作用时间;Western blotting检测肾损伤标志物Kim-1、OPN及NLRP3通路蛋白NLRP3、Caspase-1、IL-1β、IL-18表达;研究GP对HK-2细胞的毒性作用,并采用Hoechst/PI染色法检测HK-2细胞凋亡,DCFH-DA法检测HK-2细胞中ROS水平,高内涵成像检测线粒体膜电位(mitochondrial membrane potential,MMP)和Ca^(2+)水平,Western blotting和qPCR法检测Kim-1、OPN、NLRP3、Caspase-1、IL-1β、IL-18蛋白及其m RNA表达水平,研究NLRP3抑制剂格列苯脲对GP毒性的拮抗作用及其机制。结果GP对HK-2细胞在12、24、48 h的IC50分别为433.00、110.50、72.99μg·mL^(-1);与空白组相比,GP处理后HK-2细胞PI阳性率、ROS、Ca^(2+)水平显著增加,MMP显著下降,Kim-1、OPN、IL-1β、IL-18、NLRP3、Caspase-1蛋白及m RNA水平均显著升高(P<0.05,P<0.01);NLRP3通路抑制剂格列苯脲可以明显改善GP细胞毒性作用及相关分子变化。结论GP可能通过激活NLRP3通路诱导HK-2细胞损伤,抑制NLRP3通路可减轻GP诱导的HK-2细胞炎症损伤。OBJECTIVE To investigate the toxicity of genipin on human tubular epithelial cells(HK-2)based on NLRP3 pathway.METHODS The cytotoxic concentration and action time of genipin on HK-2 were detected by CCK8.The Western blotting was used to detect the expression of Kim-1,OPN and NLRP3 pathway protein NLRP3,Caspase-1,IL-1βand IL-18.Studied on the toxic effects of genipin on HK-2 cells,apoptosis in HK-2 cell was detected by Hoechst/PI staining,and the level of ROS in HK-2 cell was detected by DCFH-DA.High-content imaging techniques was used to detect mitochondrial membrane potential(MMP)and Ca^(2+)levels,Western blotting and qPCR methods was used to detect the expression levels of Kim-1,OPN,NLRP3,Caspase-1,IL-1β,IL-18 protein and mRNA.To study the antagonistic effect of NLRP3 inhibitor glyburide on GP toxicity and its mechanism.RESULTS The IC50 values of genipin for HK-2 cell were 433.00,110.50 and 72.99μg·mL^(-1)at 12,24 and 48 h,respectively.Compared with the blank group,after the treatment of genipin,the positive rate of PI,ROS,Ca^(2+)levels of HK-2 cell increased significantly,MMP decreased significantly,and the levels of Kim-1,OPN,IL-1β,IL-18,NLRP3,Caspase-1 protein and mRNA were significantly increased(P<0.05,P<0.01),and the NLRP3 pathway inhibitor glyburide could significantly improve the cytotoxic effects of genipin and related molecular changes.CONCLUSION Genipin might have cytotoxicity on HK-2 cells via NLRP3 pathway,and inhibiting NLRP3 pathway can improve the inflammation caused by genipin induced cytotoxicity on HK-2 cells.

关 键 词：NLRP3通路 京尼平 HK-2细胞 细胞毒性 NLRP3抑制剂 

分 类 号：R285.5[医药卫生—中药学]