Study On The Role And PI3K/Akt/mTOR Signal Pathway Of Autophagy In Piglet Hippocampal Nerve Cells Injury Induced By Deoxynivalenol

Deoxynivalenol(DON),a type B trichothecenes which are produced by Fusarium infesting that invades cereals such as,wheat and corn.Due to its high exposure level and high incidence of contamination in the food chain,it has become one of the most important problems globally especially in agricultural fields,and posing a serious threat to the health of human and animals.Recently,many researchers have found that,DON exposure can cause apoptosis and necrosis in vitro.However,whether autophagy is involved in DON induced neurotoxicity as well as the role of autophagy has not been reported.Hippocampal neurons are rigorous in the central nervous system and are most susceptible to damage by external factors,therefore,piglet hippocampal nerve cells(PHNCs)were selected as an object of in vitro study.PHNCs were treated with DON at different concentrations(0,125,250,500,1 000 and 2 000 ng/mL)for 24 h.The morphological changes of cells were observed by inverted microscopy and the survival rate of cells was detected by CCK-8 kit.The effect of DON on neurotransmitters and oxidative damage was detected by ELISA kit.The ultrastructure of cells and the number of auto-phagosomes,and autophagic vacuoles and autophagosomes,were observed by transmission electron microscopy.The rate of apoptosis,was detected by flow cytometry;GFP-LC3 plasmid transfection,while immunofluorescence were used to detect LC3 aggregation rate.The mRNA levels of autophagy and PI3K/Akt/mTOR pathway-related genes were determined by qRT-PCR,and the expression levels of related proteins were determined by western blot(WB).The autophagy inhibitor chloroquine(CQ)and autophagy activator rapamycin(RAP)were added to investigate the effect of autophagy on DON-induced PHNCs injury and the relationship between autophagy and apoptosis.The results are as follows:1.Effects of DON on cell morphology,oxidative damage and apoptotic rate:The PHNCs of the control group were full and dense,and the swellings were obvious and intertwined into a network,with full fusiform shape.The density of cells in the treatment group was gradually reduced,and their arrangement becomes thin,and the cells were obviously damaged as well as wrinkled.Compared with the control group,with the increasing concentration of DON,the survival rate of treated group was significantly decreased(P<0.01),DON also promotes the secretion of 5-HT,DA,ACH and GABA,and inhibits the secretion of NE,DON also increased the lipid peroxidation index MDA contents,and decreased the activity of CAT,GSH-Px,SOD,T-AOC and NO contents,showing significant difference(P<0.05)or extremely significant difference(P<0.01).Compared with the control group,the apoptotic rate of treatment group increased significantly with the increasing concentration of DON(P<0.01).2.Effects of DON on autophagy of PHNCs:Transmission electron microscopy(TEM)showed that,the cytoplasm of control group was homogeneous,with increasing number of cytoplasmic vacuoles in the DON-treated group and the autophagosomes,autophagic vacuoles and late autophagic lysosome structures were clearly appeared.GFP-LC3 transfection shows that the GFP-LC3 protein in the control group was diffusely distributed.When DON concentration was increased,the dot distribution of GFP-LC3 protein began to appear,and the dot distribution of green fluorescent protein becomes most obvious when DON concentration reached 1 000 ng/mL.Immunofluorescence detection showed that there was weak red fluorescence and LC3 did not form dot-like aggregation in the control group.In the DON-treated group,the spot phenomenon of LC3 was caused by different concentration of DON.The number of cells accumulating in LC3 was in highest level when DON was 1 000 ng/mL.The expression of LC3-?protein in the cells increased first and then decreased.At 1 000 ng/mL,the contents of LC3-II was the highest,and the difference was significant(P<0.05)or extremely significant(P<0.01).3.Autophagy and Cell Damage:Compared with DON treatment group,rapamycin endorsed autophagy and significantly increased cell viability(P<0.05),while chloroquine inhibited autophagy and significantly decreased cell viability(P<0.05).4.PI3K/Akt/mTOR pathway and autophagy:During comparison with the control group,the expression levels of PI3K,Akt,mTOR,P70S6K gene and phosphorylated protein generally decreased with the increasing concentration of DON,and showed significant difference(P<0.05)or highly significant(P<0.01),indicating that PI3K/Akt/mTOR signaling pathway plays a negative regulatory role in DON-induced autophagy of PHNCs.5.Cell Autophagy and Apoptosis:Compared with the control group,the apoptosis rate of each treatment group was significant(P<0.05)or extremely significant(P<0.01)increased.Compared with 1 OOOng/mL DON treatment group,the apoptotic rate of 1 000 ng/mL DON+RAP treatment group decreased significantly(P<0.01),and the apoptotic rate of 1 000 ng/mL DON+CQ treatment group increased significantly(P<0.01).In conclusion,DON exerts certain toxic effect on PHNCs,and induced apoptosis and autophagy.When inhibits autophagy,it can reduced cell activity,while induced autophagy can enhanced cell activity.The autophagy related genes and proteins LC3,PI3K,Akt,mTOR and P70S6K are involved in the regulation of DON-induced autophagy in PHNCs.At the same time,when autophagy was inhibited,apoptotic rate increased,and when autophagy was induced,it decreased the apoptotic rate,which indicated that autophagy plays a protective role in DON-induced PHNCs injury.