Study On The Cytotoxic Mechanism Of Tannic Acid On IPEC-J2 Cells Based On Mitochondrial Signaling Pathway

Objective: Tannic acid(TA)is the main disadvantage of clinical utilization of non-grain feed,which widely distribute in plant.Understanding toxic mechanism of tannic acid on animals has important sense to prevention tannin acid poisoning,reduction antinutritional factors and clinical development of tannic acid.To study the toxic dose and toxicity mechanism of TA on IPEC-J2 cells,and to provide theoretical basis and experimental evidence for clinic utilizition of TA.Method: After treatment with 2.5,10 and 40?M of TA for 24 h,we determined the effects on cells apoptosis and cell cycle in IPEC-J2 cells,(1)MTT used for detecting the cells viability.The activity of SOD,GSH-PX,and CAT,and the content of MDA and GSH were determined by commercial kit.The content of ROS was detected by fluorescent.(2)The content of 8-OHdG was detected by ELISA.(3)The mitochondrial membrane potential was deteced by fluorescent.The content of ATP was determined by kit.(4)The espression of Bax,Bcl-2,casapse-3,caspase-9,PARP and cytochrome c were detected by western blot.The apoptosis cells was determined by flow cytometry.(5)The espression of ATM,P53,CDK2 and Cyclin A2 were detected by western blot.The cell cycle distribution was determined by flow cytometry.Result:(1)TA inhibited the proliferation of IPEC-J2 cells,decresed the activities of GSH-PX,SOD and CAT,inrcresed the contents of ROS and MDA,and decreased the content of GSH.(2)TA induced the production of 8-OHdG in a dose-dependent manner.(3)TA induced the dysfunction of mitochondrial and decreased the content of ATP.(4)TA increased the expression of Bax,cleaved-caspase-9,cleaved-caspase-3 and cleaved PARP,while decrased the expression of Bcl-2.TA induced the apoptosis via mitochondrial pathway,however it was attenuated by Z-LEDH-FMK which is an inhibitor of caspase-9.(5)TA increased the expression of p-ATM.P53,while decresed expression of p-CDK2 and Cyclin A2.TA imduced the S phase arrest in IPEC-J2 cells in a dose-dependent manner.Conclusion: At the concentration in our experience,TA induced the accumulation of ROS,oxidative stress and dysfunction of mitochondrial by decreasing the activity of anti-oxidative enzyme and increasing the lipid peroxide.On the one hand,TA induced the apoptosis via mitochondrial pathway,on the other hand,TA caused S phase arrest in IPEC-J2 cells via DNA damage.