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