Phenyl Isothiocyanate Induces Cytotoxicity And Apoptosis Of Porcine Kidney Cells Through The Endoplasmic Reticulum Stress Pathway.Introduction

Objective:To observe the toxic effects of phenylethyl isothiocyanate(PEITC)on pig kidney cells(PK-15)and preliminarily explore the mechanisms of endoplasmic reticulum stress in apoptosis of PK-15 cells induced by PEITC.This study provides theory basis for the utilization of protein resources of rapeseed meal,and have important implications for further understanding the toxicity of PEITC.Methods:In this experiment,PK-15 cells were taken as the research objects,PK-15 cells were treated with PEITC(2.5,5,10μM)for 24 hours,and the following experiments were conducted in this study:(1)Cytotoxicity:After treated with PEITC for 24 hours,the activity of PK-15 cells was measured by MTT method,and the cell morphology was observed under inverted microscope;(2)Oxidative damage:After PEITC treated cells for 24 hours,the content of reactive oxygen species(ROS)was detected by fluorescence,the activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),catalase(CAT)and the content of glutathione(GSH)and malondialdehyde(MDA)were detected by detection kit;(3)Apoptosis:the intracellular calcium level,cell cycle and apoptosis rate of PK-15cells were detected by flow cytometry;the expression of caspase family proteins(caspase 3,9,7,12)and Bcl-2 family proteins(Bcl-2 and Bax)were detected by Western blot;(4)Endoplasmic reticulum(ER)stress pathway:Western blot was used to detect the expression of ER stress-related proteins(CHOP,GRP-78,GRP-94,ATF-6,PERK,IRE1-α),and the effect of ER stress blocker 4-PBA on the expression of CHOP,GRP78 and apoptosis rate of PK-15 cells.Result:(1)PEITC inhibited the activity of PK-15 cells in a dose-dependent manner after 24hours,and had an effect on the cell morphology(2)PEITC increased the content of MDA and LDH and decreased the activity of SOD,CAT,GSH-Px and GSH in a dose-dependent manner.(3)PEITC increased the expression levels of apoptotic proteins caspase 3,9,7,12,Bax,CHOP,GRP-78,GRP-94,ATF-6、PERK,IRE1-αand decreased the expression level of Bcl-2,(4)Compared with PEITC stimulation group(10μM),after 4-PBA treatment on PK-15 cells for 2 hours,the expression levels of CHOP and GRP78 protein and the apoptosis rate decreased in 4-PBA group.Conclusion:A certain dose of PEITC has some toxic effects on PK-15 cells,which can reduce the activity of PK-15 cells.Through the induction of ROS and Ca2~+accumulation in cells,resulting in ER stress and cell cycle was blocked in S and G2/M phase,thus eventually triggering apoptosis.