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

关键词:  NLRP3通路  京尼平  HK-2细胞  细胞毒性  NLRP3抑制剂

Key words:  NLRP3 pathway  genipin  HK-2 cells  cytotoxicity  NLRP3 inhibitor

摘要:

目的 基于Nod样受体蛋白3(NLRP3)通路探讨京尼平(GP)对人源肾小管上皮细胞(HK-2)毒性作用。方法 CCK8法筛选京尼平作用于HK-2细胞的给药剂量及作用时间；设置空白组、京尼平低、中、高剂量组(50、100、200 μg/ml),蛋白免疫印迹(western-blot)法检测肾损伤标志物(Kim-1)、骨桥蛋白(OPN)及NLRP3通路蛋白NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)、白介素1β(IL-1β)、白介素18(IL-18)表达情况；设置空白组、京尼平组(100 μg/ml)、NLRP3抑制剂格列苯脲(GLY)组(400 ng/ml)、京尼平+格列苯脲组,采用Hoechst/PI染色法检测HK-2细胞凋亡情况；采用二乙酸-2",7"-二氯荧光素(DCFH-DA)法检测HK-2细胞中活性氧(ROS)水平；高内涵成像技术检测线粒体膜电位(MMP)和钙离子(Ca₂₊⁾水平；western-blot法检测Kim-1、OPN、 NLRP3、IL-1β、IL-18、Capase-1蛋白表达水平；QPCR检测Kim-1、OPN、NLRP3、Caspase-1、IL-1β、IL-18 mRNA表达水平。结果 京尼平对HK-2细胞半数抑制浓度(IC50)值在12h、24h、48h分别为433.00、110.50、72.99 μg/ml；与空白组相比,京尼平中、高剂量组Kim、OPN、NLRP3、IL-1β、IL-18、Caspase-1表达水平显著升高(P＜0.05,P＜0.01)；与空白组相比,京尼平组PI阳性率、ROS、Ca₂₊^显著增加,MMP显著下降,Caspase-1蛋白水平,Kim-1、OPN、IL-1β、IL-18、 NLRP3蛋白及mRNA水平均显著升高(P＜0.05,P＜0.01)；与京尼平组相比,京尼平+格列苯脲组PI阳性率、ROS、Ca₂₊^显著降低,MMP显著升高,Caspase-1蛋白水平,Kim-1、OPN、IL-1β、IL-18、 NLRP3蛋白及mRNA水平均显著降低(P＜0.05,P＜0.01)。结论 京尼平通过激活NLRP3通路诱导HK-2细胞损伤,抑制NLRP3通路可减轻京尼平诱导的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 dose and duration of genipin on HK-2 cells were screened by CCK8. After setting the control, low to high dose groups (50, 100, 200 μg/ml) of genipin, The proteins expression of Kim-1, OPN and NLRP3 pathway including NLRP3, Caspase-1, IL-1β and IL-18 was detected by western-blot. After setting control group, genipin group (100 μg/ml), glyburide group (400 ng/ml), glyburide combined with genipin group , apoptosis were detected by Hoechst/PI staining, and the ROS level in HK-2 cells was detected by DCFH-DA. High-content imaging techniques to detect mitochondrial membrane potential and Ca_²⁺ levels; Western-blot method to detect Kim-1, OPN, NLRP3, Caspase-1, IL-1β, IL-18 protein expression levels; QPCR detected Kim-1, OPN, NLRP3, IL-1β, IL-18 mRNA expression levels. Results The IC₅₀ values of genipin for HK-2 cells were 433.00, 110.50 and 72.99 μg/ml at 12h, 24h and 48h, respectively. Compared with the control group, the protein expression levels of Kim, OPN, NLRP3, IL-1β, IL-18 and Caspase-1 in the medium and high dose groups of genipin were significantly increased (P<0.05, P<0.01); compared with the control group, the positive rate of PI, ROS, Ca₂₊^{, Caspase-1 protein level,} the expression levels of protein and mRNA of Kim-1, OPN, IL-1β, IL-18 and NLRP3were significantly increased, and MMP was significantly decreased (P<0.05, P<0.01), compared with the genipin group, in the genipin + glyburide group, the positive rate of PI, ROS, Ca₂₊ ^{and the levels of Caspase-1 protein,} the expression levels of protein and mRNA of Kim-1, OPN, IL-1β, IL-18, NLRP3 were significantly decreased, and MMP were significantly increased (P<0.05, P<0.01). Conclusion Genipin has cytotoxicity on HK-2 cells via NLRP3 pathway, and inhibiting NLRP3 can improve the inflammation caused by genipin induced cytotoxicity on HK-2 cells.