Study On The Regulatory Mechanism Of Phenylethyl Isothiocyanate Inducing Apoptosis Of IPEC-J2 Cells Based On Mitochondrial Signaling Pathway

Objective:The aim of this study was to investigate the toxic effect of phenethyl isothiocyanate（PEITC）on porcine jejunal epithelial cells（IPEC-J2）,and to preliminarily explore the possible mechanism of PEITC induced apoptosis in IPEC-J2 cells.Methods:In this study,IPEC-J2 cell were treated with PEITC at different concentrations（3μM,9μM and 27μM）for 24 h,and the following experiments were performed:（1）The effects of PEITC on oxidative damage of IPEC-J2 cells:Cell proliferation rate was measured by MTT assay.The activities of SOD,GSH-Px and CAT,the contents of GSH,MDA and H₂O₂ in cells and the activity of LDH in culture supernatants were measured.The levels of ROS and Ca²⁺were measured by flow cytometry.（2）The effects of PEITC on mitochondrial damage in IPEC-J2 cells:The changes of mitochondrial membrane potential were detected by flow cytometry.ATP level in mitochondria was measured by kits instruction.（3）The effects of PEITC on apoptosis of IPEC-J2 cells:The apoptotic rate of IPEC-J2cells was detected by flow cytometry.Western Blot was used to analyzed the expression of mitochondrial apoptosis-related proteins Bax,Bcl-2,Caspase-3,Caspase-9 and PPAR-1.The content of Cyt c in mitochondria and cytoplasm were assessed.IPEC-J2 cells were treated with Caspase-9 inhibitor for 30 min,then treated with different concentrations of PEITC（3μM,9μM and 27μM）for 24 h.The apoptotic rate of IPEC-J2 cells was detected by flow cytometry.（4）The effects of PEITC on IPEC-J2 cell cycle:Flow cytometry was used to detect the changes of IPEC-J2 cell cycle;Western Blot was used to detect the expression of cell cycle-related proteins p53,Cyclin A2 and Cdc25c.Result:（1）With the increase of PEITC concentration,IPEC-J2 cell proliferation rate decreased in a dose-dependent manner.The levels of MDA,ROS,Ca²⁺,LDH and the activities of SOD,CAT were increased in a dose-dependent manner.The activity of GSH-Px and the level of GSH were decreased in a dose-dependent manner.（2）With the increase of PEITC concentration,mitochondrial membrane potential of IPEC-J2 cells suppressed in a dose-dependent manner,and the level of ATP was decreased in a dose-dependent manner.（3）PEITC increased the proteins ratio of Cleave Caspsese-3/Pro Caspase-3,Cleave Caspsese-9/Pro Caspase-9,Bax/Bcl-2 and Cleave PARP-1/Pro PARP-1,and released Cyt c from the mitochondria to the cytoplasm.With the increase of PEITC concentration,the apoptosis rate of IPEC-J2 was increased in a dose-dependent manner.After pretreatment with Caspase-9 inhibitor,the apoptosis rate of IPEC-J2 was significantly decreased.（4）With the increase of PEITC concentration,the ratio of G₀/G₁ phase decreased in a dose-dependent manner,while ratio of S and G₂/M phases increased in a dose-dependent manner.The expression of cell cycle proteins was detected by Western Blot.The results showed that PEITC up-regulated the expression of p53 protein and down-regulated the expression of Cyclin A2 and Cdc25c protein.Conclusion:PEITC has a certain toxic effect on IPEC-J2 cells.Its toxic mechanism is to induce oxidative stress in IPEC-J2 cells,thereby damaging mitochondrial function,leading to cell cycle arrest in S phase and G₂/M phase,and ultimately inducing apoptosis of IPEC-J2cells.