Mechanism Of Neurotoxicity Induced By Deoxynivalenol In Weanling Piglets

Deoxynivalenol（DON）produced by Fusarium is widely exists in agricultural products and feed.DON was confirmed as one of the most dangerous abiogenetic food contaminants.DON not only has the effects of cytotoxicity and immunotoxicity but also can induce carcinogenic,teratogenic and mutagenic.However,the neurotoxic mechanism of DON has not been fully exposed.Therefore,the further explorations of neurotoxicity of DON are urgently needed.To study the mechanism of DON’s neurotoxicity in piglets,the Fusarium graminearum was cultivated and DON concentration was measured in this experiment.Thirty healthy“Duroc×Landrace×Yokshire”weaned piglets were selected and randomly divided into 3 groups according to single factor experimental design,10 piglets in each group.Piglets in control were fed the diets without DON.Piglets in low and high concentration groups were fed the diets with 1 mg/kg and 2 mg/kg DON,respectively.The experimental trials were lasted for 60 days.At the end of the trials,5 piglets from each group were slaughtered and its brain tissues were quickly collected.The cerebral cortex,cerebellum,medulla oblongata and hippocampus were removed and preserved for further analysis.The changes in the pathological structure and ultrastructure of hippocampus were observed.The oxidation and antioxidant index as well as the concentration of neurotransmitters and Ca²⁺in the cerebral cortex,cerebellum,medulla and hippocampus were detected by enzyme linked immune sorbent assay（ELISA）method.The mRNA relative expression of CaM and CaMKⅡ was detected by quantitative real time polymerase chain reaction（qRT-PCR）.Western Blot was used to detect the protein levels of CaM,CaMKⅡ and p-CaMKⅡ.The result shows that:1.Histopathological observation of hippocampus in low DON concentration group showed that,the gap of hippocampal cells increased and the number of microglia around some neurons increased during comparison with control group.In high concentration group,the hippocampus cells became thin accompanied by some nerve fiber fracture and the neuronophagia was observed obviously.Scanning electron microscopy of hippocampal cells showed that the cell structures of control group were normal but in low concentration group it showed degeneration with uneven chromatin distribution and vacuolar degeneration of some mitochondria.In the high concentration group,hippocampal cells exhibited watery degeneration or even necrosis,indicating destruction of the cell and nuclear membrane,severe cytoplasmic swelling,swelling of the mitochondria as well as disappearance of some organelles.2.In cerebral cortex,compared with the control group,the activities of superoxide dismutase（SOD）in high DON concentration group were decreased significantly（P<0.05）while the activities of the glutathione peroxide enzymes（GSH-Px）in high and low concentration groups were significantly decreased（P<0.01）,the concentrations of Malondialdehyde（MDA）in low concentration group were significantly increased（P<0.01）and the levels of MDA and NO in high concentration group was significantly increased（P<0.01）.In cerebellum,the activities of SOD and GSH-Px in high concentration group were significantly decreased（P<0.01）,the activities of GSH-Px in low concentration group were also significantly decreased（P<0.01）while the concentrations of NO in high and low concentration groups were increased significantly（P<0.01）when compared with the control group.In medulla oblongata,the activities of SOD and GSH-Px in high concentration groups were decreased significantly（P<0.01）while the concentrations of NO in high and low concentration groups were increased significantly（P<0.01）when compared with the control group.In hippocampus,compared with the control group,the activities of SOD and GSH-Px in high concentration groups were decreased significantly（P<0.01）as well as the activity of SOD in low concentration group were also decreased significantly（P<0.01）while the concentrations of MDA and NO in high concentration group were increased significantly（P<0.01）.3.Compared with the control group,the concentration of 5-HT was significantly increased in high DON concentration group in cerebral cortex,cerebellum and hippocampus（P<0.01）,the concentrations of 5-HT in the medulla oblongata were significantly increased in the high and low concentration groups（P<0.01）.In hippocampus,compared with the control group,the concentrations of NE were increased significantly in low concentration group（P<0.05）and in high concentration group（P<0.01）.Compared with the control group,the concentrations of DA in the high concentration group was reduced significantly in cerebral cortex,cerebellum and hippocampus（P<0.01）and in the medulla oblongata（P<0.05）,the concentrations of DA in the cerebellum of the low concentration group was reduced significantly（P<0.01）.Compared with the control group,the concentrations of GABA in the high concentration group showed a significant decrease in the cerebral cortex and hippocampus（P<0.01）and in medulla oblongata（P<0.05）In cerebral cortex,cerebellum,medulla oblongata and hippocampus,the concentrations of ACH in the high and low concentration group was reduced significantly compared with the control group（P<0.01）.4.Compared with the control group,the concentration of calcium(Ca²⁺)in cerebral cortex,cerebellum,medulla oblongata and hippocampus of the high DON concentration group increased significantly（P<0.01）but the concentration of Ca²⁺in low concentration groups increased significantly in cerebellum and medulla（P<0.01）.By Comparison with the control group,the mRNA relative expression levels of CaM and CaMKⅡ of low and high concentration group were reduced significantly in cerebral cortex（P<0.01）while in the high concentration group it was reduced significantly in cerebellum（P<0.01）,the mRNA relative expression levels of CaMKⅡ in the low concentration group were reduced significantly in cerebellum（P<0.01）and the mRNA relative expression levels of CaM in the low concentration group was reduced significantly in the medulla oblongata（P<0.05）,the mRNA relative expression levels of CaMKⅡ in the low and high concentration group was reduced significantly in medulla oblongata（P<0.01）,the mRNA relative expression levels of CaM in the high concentration group was reduced significantly in hippocampus（P<0.01）,the relative expression levels of CaMKⅡ mRNA in the high concentration group was reduced significantly in hippocampus（P<0.01）.Compared with the control group,the levels of CaM protein in the high and low concentration groups were decreased significantly in cerebral cortex（P<0.05）,the levels of CaM protein in the high concentration group were reducedsignificantly in cerebellum and medulla oblongata（P<0.01）but the levels of CaM protein in the low concentration group were decreased significantly in medulla oblongata（P<0.05）while the levels of CaM protein in the high and low concentration groups were decreased significantly in hippocampus（P<0.01）.Compared with the control group,the phosphorylation levels of CaMKⅡ protein in the high and the low concentration group increased significantly in cerebral cortex and hippocampus（P<0.01）and the phosphorylation levels of CaMKⅡ protein in the high concentration group were increased significantly in cerebellum and medulla oblongata（P<0.01）.In summary,the morphological changes and damage of neuronal cells can be caused byDON exposed in brain tissue of piglets.The oxidative damage and neurotransmitter secretion disorder can be caused by DON.The calcium overload can also be leaded by DON exposed in nerve cells.The relative expression of CaM mRNA and the levels of CaM protein in brain tissue were down-regulated by DON,the relative expression of CaMKⅡ mRNA was down-regulated and the phosphorylation of CaMKⅡ protein was increased.It is suggests that the expose of DON has neurotoxic effects on the piglets.