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 Ca2+水平,Western blotting 和 qPCR 法检测 Kim-1、OPN、NLRP3、Caspase-1、IL-1β、IL-18 蛋白及其mRNA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、72.99 μg·mL-1;与空白组相比,GP处理后HK-2细胞PI阳性率、ROS、Ca2+水平显著增加,MMP显著下降,Kim-1、OPN、IL-1β、IL-18、NLRP3、Caspase-1 蛋白及 mRNA 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.
Abstract
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-iβ and 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 Ca2+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 72.99 μg·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,Ca2+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＜0.0l),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.
NETL
NSTL
万方数据