Protective Effect Of Proanthocyanidin B2 On T-2 Toxin Induced Damage In TM3 Cells

Objective: To explore the protective effect and its molecular mechanism of procyanidins B2(PB2)on mouse Leydig cell line(TM3)injury induced by T-2 toxin,which provide an important experiment basis and theoretical reference for the use of PB2 as an antidote to mycotoxins.Methods:In this study,TM3 cells were used as the experimental cell model.Different concentrations of PB2(20 ?M,100 ?M,and 500 ?M)were used to co-treat cultured cells with 100 nM T-2 toxin,and water-soluble vitamin E Trolox was used as a positive control.A series of experiments were performed to investigate the antagonistic effects of PB2 on T-2 toxin induced apoptosis of TM3 cells through oxidative stress.(1)Protective effects of PB2 on oxidative damage induced by T-2 toxin in TM3 cells: TM3 cells were treated with PB2 and T-2 toxins for 24 h,MTT assay was used to detect the cell viability.Activities of SOD,GSH-PX,CAT and contents of GSH and MDA were detected,and the level of ROS was assayed by flow cytometry.The protein expressions of ASK1?JNK and P38 were analysis by Western Blot.(2)Antagonistic effect of PB2 on mitochondrial damage induced by T-2 toxin in TM3 cells: Mitochondrial contents and mitochondrial membrane potentials were detected by flow cytometry,and the activities of mitochondrial respiratory chain complex enzyme I-V and CS,and content of ATP were detected,respectively.(3)The protective effects of PB2 on apoptosis induced by T-2 toxin in TM3 cells: The protein expressions of CyPD,Bak,Bax,Bcl-2,Bcl-xl,Caspase-3 and Caspase-9 were detected by Western Blot.Contents of cyt c in mitochondria and cytoplasm were detected,and apoptosis of TM3 cells were detected by flow cytometry.Results:(1)T-2 toxin decreased the activities of SOD and GSH-PX,and contents of GSH,increased the contents of MDA and ROS,and enhanced protein expression of ASK1,JNK,and P38,which lead to a decrease in cell viability.While TM3 cells were treated with T-2 toxin combined with PB2,the cell viability and the levels of SOD,GSH-PX and GSH were increased,and the contents of MDA and ROS were reduced.Meanwhile protein expression of ASK1,JNK and P38 were decreased.(2)T-2 toxin reduced the amounts of mitochondria and mitochondrial membrane potential,meanwhile,decreased the activities of respiratory chain complex enzyme I-V and CS,ATP production.When TM3 cells were treated with T-2 toxin combined with PB2,mitochondrial contents,mitochondrial membrane potential,activities of respiratory chain complex enzyme I-V and CS,the amounts of ATP in cells were increased,these indicate mitochondrial function was recovered.(3)PB2 treatment down-regulated protein expressions of CyPD,Bak,Bax,Caspase-3 and Caspase-9,the ratio of Bax/Bcl-2,attenuated delivering of cyt c from mitochondria to cytoplasm,and decreased cellular apoptosis by T-2 toxin,and up-regulated protein expression of Bcl-2 and Bcl-xl.These protein are related to mitochondrial apoptosis signal pathway.Conclusions: These findings reveal a protective capability of PB2 against T-2 toxin-induced cell apoptosis by reducing oxidative stress via mitochondrial mediated signal pathway.