Study On Toxic Effect Of Deoxynivalenol In Hippocampal Nerve Cells Of Piglet

Deoxynivalenol?DON?,is also named vomitoxin?VT?,mainly produced by Fusarium graminearum and Fusarium culmorum,producing varieties of mycotoxicoses in agricultural products,animal husbandry and human health,can cause neurotoxicity,cytotoxicity,immunotoxicity,carcinogenesis,teratogenesis and mutagenesis,etc.To study the toxic effects and mechanisms of apoptosis induced by DON exposure through the MAPKs signal pathway,hippocampal nerves cell of piglet?PHNCs?was cultured in vitro.After cells were treated with different concentrations of DON?0,125,250,500,1000,and 2000 ng/mL?for 24 h,changes in cellular growth and morphology were observed by an inverted microscopy and a transmission electron microscopy,the situation nuclear chromatin of nucleus was observed after dyeing with Hoechst 33258;cellular viability and the leakage rate of lactic dehydrogenase?LDH?were determined by cell counting kit-8 and LDH assay kit,respectively.5-HT content was determined by ELISA kit;Ca²⁺,CaM content and CaM mRNA expression levels were determined by calcium detection kits and real-time quantitative polymerase chain reaction?qRT-PCR?,respectively.Cellular apoptosis rates were measured using an annexin V-FITC/PI apoptosis detection kit.The mRNA levels of apoptosis-related genes were determined by qRT-PCR and the expression levels of MAPKs pathway proteins were determined by western blotting?WB?.The results were as follows:1.Effect of DON on PHNCs morphology:?i?In control group,cells presented irregular radial,densely packed;While the number of PHNCs in treatment groups were generally decreased with the increasing DON concentrations.Furthermore,cell bulge disappeared,cell volume was reduced,and cells became round.?ii?Cells in the control group showed regular nuclei,normal organelles,regular chromatin distribution,and clear nucleoli.Cells treated with DON showed typical ultrastructure characteristics of apoptotic cells as concentrations increased,including nuclear shrinking,disappearance of nucleoli,chromatin gathering to the nuclear membrane,organelles damage,and vacuolar degeneration.At concentration upon 2000 ng/mL,nuclear membrane disappeared,organelles damaged seriously,and the apoptotic bodies appeared.?iii?The cell nuclei of control group showed homogeneous pale blue,with the concentrations of DON increased,the cell nuclei bright degree and numbers increased.2.Effect of DON on PHNCs viability and LDH release:The viabilities of PHNCs were high significantly decreased with the concentrations of DON increased,compared with the control group?P<0.01?.LDH leakage rate increased with DON concentrations increased,a highly significant increase in LDH release was detected at concentrations upon 500 ng/mL of DON compared to control group?P<0.01?.3.Effect of DON on PHNCs 5-HT content and intracellular calcium homeostasis:The 5-HT content of PHNCs were high significantly increased with the concentrations of DON increased,compared with the control group?P<0.01?.The Ca²⁺,CaM content of PHNCs and CaM mRNA expression levels were high significantly increased when DON concentration upon 250 ng/mL,compared with the control group?P<0.01?.4.Effect of DON on PHNCs apoptosis rate:Compared with the control group,the apoptosis rates of PHNCs in treatment groups were significantly increased?P<0.01?,and the death rate was no obvious change,while the death rate of 2000ng/mL DON treatment group was increased.5.Effect of DON on MAPKs pathway related proteins:The total protein expression levels of JNK,ERK and p38 decreased gradually with DON concentration increased,compared with 1000 ng/mL group,the protein expression levels of JNK at DON?1000 ng/mL?+SP600125,ERK at DON?1000 ng/mL?+U0126 and p38 at DON?1000 ng/mL?+SB202190 groups were increased,respectively.The phosphorylation levels of JNK and p38 were increased with increasing DON concentration,however,the protein expression level of p-ERK was decreased with increasing DON concentration;Compared with 1000 ng/mL group,the protein expression levels of p-JNK at DON?1000 ng/mL?+SP600125,p-ERK at DON?1000ng/mL?+U0126 and p-p38 at DON?1000 ng/mL?+SB202190 groups were decreased,respectively.The ratios of p-JNK/JNK and p-p38/p38 were highly significant increased?P<0.01?,the ratio of p-ERK/ERK was highly significant decreased?P<0.01?.And compared with 1000 ng/mL group,the ratios of p-JNK/JNK,p-ERK/ERK or p-p38/p38 at DON?1000 ng/mL?+SP600125,DON?1000 ng/mL?+U0126 or DON?1000 ng/mL?+SB202190 groups were highly significant decreased?P<0.01?,respectively.6.Effect of DON on expression of apoptosis-related genes:Treatment with increasing concentrations of DON resulted in significant induction of Bax mRNA expression,Bcl-2 mRNA expression levels decreased.The Bax/Bcl-2 ratio highly significant increased DON concentration reached 250 ng/mL?P<0.01?.Compared with the 1000 ng/mL group,the Bax/Bcl-2 ratio were high significantly decreased in PHNCs treated with DON?1000 ng/mL?+SP600125 and DON?1000 ng/mL?+SB202190.However,the Bax/Bcl-2 ratio was high significantly increased in PHNCs treated with DON?1000 ng/mL?+U0126.In conclusion,DON expose caused obvious toxic effects in hippocampal nerve cells of piglet,led to cellular damage and apoptosis.Besides,DON induced apoptosis via MAPKs signaling pathway.