Effect Of Selenium-Chitosan Anti-ZEA Induced Toxicity Of Porcine In Vivo And Vitro

Zealenone（ZEA）is one of the most extensive mycotoxins in the world that contaminates crops.ZEA had a variety of toxic effects on humans and animals and could cause severe economic losses to the livestock industry.ZEA had caused serious harm to humans and livestock,and its methods and technologies for detoxification have become hot research topics.Chitosan-Se playing the dual effects of chitosan and organic selenium,which could promote animal growth and development,improve disease resistance,and exert antioxidant and so on.This study investigated the chitosan-Se against toxicity of ZEA on pigs via the JNK / SAPK pathway through in vivo and in vitro experiments.The purpose was to elucidate the relationship between chitosan –Se antagonizing the toxicity of ZEA on pigs and the JNK / SAPK signaling pathway,and to provide a theoretical basis for reducing or even eliminating the harm of ZEA.The main contents are as followings:1.Research the effects of chitosan-Se anti-ZEA on the cell viability,morphological ultrastructure and cell cycle of porcine endometrial epithelial cells（PEECs）.The cell viability were detected induced by ZEA by using CCK8;The cell cycle,cell ultrastructure and activities of Lactate dehydrogenase（LDH）were detected by using flow cytometry,electron microscope and ELISA.The results showed that the viability of PEECs decreased with the increase of ZEA concentration and the prolongation of action time.ZEA concentration of 40 μg / m L and maintain 24 h was selected for subsequent research.Microscopy and scanning transmission electron microscopy showed that PEECs in group C and 3μmol / L Se + ZEA group were rich in cytoplasm,obvious nucleoli,and nuclear membrane intact;in ZEA group,the cell membrane was ruptured,the nucleus was condensed,no obvious nucleoli and chromatin was condensed close to the nucleus.Compared with the ZEA group,the G1% of PEECs of ZEA+3 Se group was decreased（P<0.05）.3μmol / L Se could significantly reduce the increase of LDH activity caused by ZEA（P <0.01）.These results suggested that ZEA inhibited the proliferation of PEECs cells in a dose-dependent manner;chitosan-Se could alleviate the G1 phase arrest induced by ZEA in PEECs;chitosan-Se could anti-ZEA cause PEECs cell membrane rupture,nuclear shrinkage and mitochondrial swelling and so on;Chitosan-Se can reduce the LDH activity of PEECs induced by ZEA.2.Research the effects of chitosan-Se anti-ZEA on reactive oxygen species（ROS）,mitochondrial membrane potential（MMP）and apoptosis in PEECs.The study was divided into 5 groups: C group,ZEA group,0.5Se + ZEA group,1.5Se + ZEA group and 3Se + ZEA group;flow cytometry was used to detect ROS,MMP and apoptosis in PEECs.The results showed that the chitosan-Se could reduce the increase of the ROS level and apoptosis rate exposed to ZEA（P <0.01 or P <0.05）;chitosan-Se could improve the ZEA-induced decrease of JC-1（%）（p < 0.01）.These results suggested that chitosan-Se could anti-ZEA reduce ROS levels in PEECs;chitosan-Se could increase the MMP reduction of PEECs induced by ZEA;chitosan-Se protects PEECs from apoptosis caused by ZEA by reduce the ROS level.3.Research the effect of chitosan-Se against ZEA on Caspase-3 and Caspase-9 of PEECs via SAPK/JNK pathway.Protein and m RNA expression of Caspase-3 and Caspase-9 were detected by q PCR and western bloting,and p-JNK/JNK,p-ASK1/ASK1,p-c-Jun/c-Jun,p-p53/p53,p-MEK4/MEK4 of SAPK/JNK signal pathway were detected by western bloting.The results showed that chitosan-Se could decreased the pro-apoptotic related m RNA and protein caspase-3 and caspase-9,shown no significantly differences compared with control group（P > 0.05）;chitosan-Se could significantly reduce the ZEA-induced increased of p-JNK/JNK,p-ASK1/ASK1 and p-c-Jun/c-Jun（p < 0.01）,Se-CTS could significantly improve the ZEA-induced decreased of p-p53/p53 and p-MEK4/MEK4（p < 0.01）.These results confirmed that chitosan-Se could reduce the increase m RNA and protein expression of caspase-3 and caspase-9induced by ZEA in PEECs;chitosan-Se could reduce p-c-Jun/c-Jun,p-ASK1/ASK1 and p-JNK/JNK protein ratio,increase the p-MKK4/MKK4 and p-p53/p53 protein ratio of PEECs with ZEA,chitosan-Se could inhibit PEECs apoptosis induced by ZEA through modulating the JNK/SAPK signaling pathway.4.Research the effects of chitosan-Se anti-ZEA on the growth performance,reproductive toxicity,blood biochemical indexes,3α-HSD and 3β-HSD activity of weaned piglets.The experiment was divided into 4 groups,C group was fed basal diets;ZEA group was basal diets supplemented with 2.0 mg/kg ZEA;ZEA-Se group was basal diets supplemented with2.0 mg/kg ZEA and 0.3 mg/kg selenium as chitosan selenium;Se group was fed basal diets supplemented with 0.3 mg/kg selenium as chitosan selenium to test growth performance,reproductive toxicity,blood biochemical indexes,3α-HSD and 3β-HSD activity.The results showed that there was no significant difference in growth performance and viscera index between each group in weaned piglets（P> 0.05）;chitosan-Se could reduce the increase the vaginal length and width,ovarian index and uterine index induced by ZEA（P <0.01 or P <0.05）;ZEA-Se group could significantly reduce serum ALT,AST,liver 3α-HSD and 3β-HSD activity of ZEA group in piglets（P<0.01 or P <0.05）.These results confirmed that chitosan-Se had no effect on growth performance and viscera index of ZEA;chitosan-Se could anti-ZEA the reduced vaginal length and width,uterus and ovarian index of ZEA-reduced;Chitosan-Se could reduce the increase AST,ALT,liver 3α-HSD and 3β-HSD activities induced by ZEA,reduce the liver toxicity ZEA-caused on weaned piglets.5.Research the effects of chitosan-Se anti-ZEA on antioxidant of blood,liver and kidney and immune function in weaned piglets.Test group and animal treatment are the same as expriment 4.T-AOC,T-SOD,GSH-Px,and MDA,serum E2,IL-2,and immunoglobulin levels were measured by ELISA.The results showed that chitosan-Se could increased the decrease blood,liver and kidney T-AOC,T-SOD,and GSH-Px activities with ZEA（P <0.05 or P <0.01）,chitosan-Se could reduced the increase MDA content（P <0.05）caused by ZEA;the serum E2 content,the swine fever antibody level and the Ig G level in the ZEA-Se group were significantly higher than ZEA group（P<0.01 or P<0.05）.These results confirmed that chitosan-Se could alleviate the oxidative damage of weaned piglets induced by ZEA,to improve the antioxidant function of blood,liver and kidney;chitosan-Se could anti-ZEA reduce E2 in weaned piglets;chitosan-Se could increase Ig G,Ig A and swine fever antibodies in weaned piglets,to alleviate the immune suppression of piglets caused by ZEA.