Mechanism Of MTOR/AKT/Ca²⁺Signaling Pathway In TM3 Cell Apoptosis Induced By T-2 Toxin

Objective:The purpose of this study was to investigate the mechanism of m TOR/AKT/Ca²⁺signaling pathway in T-2 toxin-induced apoptosis of TM3 cell.Methods:In this study,TM3 cells were used as the research object,and treated with T-2toxin（100n M）for different time（oh,12h,24h,48h）,the following experiments were performed:（1）The effects of T-2 toxin on TM3 cell viability and apoptosis:TM3 cells were treated with T-2 toxin（100n M）at different times（0h,12h,24h,and 48h）and cell viability was detected by MTT assay.The apoptosis rate of TM3 cells was measured by flow cytometry,and the expression of caspase-3 protein in TM3 cells was detected by Western Blot.（2）The effects of T-2 toxin on m TOR/AKT/Ca²⁺signaling pathway:TM3 cells were treated with T-2 toxin（100n M）for different time（0h,12h,24h,48h）,and the expressions of m TOR,p-m TOR ser2481,p-m TOR ser2448,AKT and p-AKT ser473 proteins in m TOR/AKT pathway were detected by Western Blot.The Ca²⁺sensitive dye Fura2-AM was used for staining,and the changes in intracellular Ca²⁺concentration in TM3 cells were measured by fluorescence microscope and Fluorescent enzyme labeling instrument.（3）The mechanism of m TOR/AKT/Ca²⁺signaling pathway in T-2 toxin induced TM3cell apoptosis:TM3 cells were pretreated with BAPTA-AM（calcium chelators）for 30min,and treated with T-2 toxin for 24 hours,the concentration of intracellular free Ca²⁺was detected by Fluorescent enzyme labeling instrument,the apoptosis rate of TM3 cells was detected by flow cytometry,and the expression of caspase-3 protein in the cells was detected by Western Blot.TM3 cells were pretreated with m TOR activator MHY1485 for 30min,and treated with T-2 toxin for 24 hours,the expression of proteins m TOR,p-m TOR ser2481,p-m TOR ser2448,AKT and p-AKT ser473 were detected by Western Blot,and the concentration of free Ca²⁺in cells was detected by Fluorescent enzyme labeling instrument,the apoptosis rate of TM3 cells was detected by flow cytometry.Result:（1）With the increase of the time of T-2 toxin treatment,the TM3 cell viability decreased in a time-dependent manner.The expression of Cleaved-caspase-3 increased in time-dependent manner.The apoptosis rate increased in time dependence.（2）With the increase of the time of T-2 toxin treatment,the ratio of p-m TOR ser2481/m TOR,p-m TOR ser2448/m TOR,p-AKT ser473/AKT was significantly reduced,which inhibited the activation of m TOR/AKT signaling pathway.The intracellular Ca²⁺concentration in TM3 cells were increased in a time-dependent manner.（3）After treated with BAPTA-AM significantly reduced the increase in Ca²⁺concentration in TM3 cells caused by T-2 toxin,and significantly reduced the increase in Cleaved-caspase-3 expression caused by T-2 toxin,as well as the increase in apoptosis rate.After pretreated with MHY1485（m TOR activator）,the ratio of p-m TOR ser2481/m TOR,p-m TOR ser2448/m TOR,p-AKT ser473/AKT was significantly increased,alleviated the inhibition of t-2 toxin on the m TOR/AKT signaling pathway,and reduced the increase of intracellular free Ca²⁺concentration and TM3 cell apoptosis caused by T-2 toxin.Conclusion:T-2 toxin induces apoptosis by inhibiting m TOR/AKT signaling to promote Ca²⁺production in TM3 cells.