Interference Of DON-induced Oxidative Injury Of Porcine Intestinal Epithelia Cells By Yeast Cell Wall Extract And Its Mechanism

Deoxynivalenol(DON),also known as vomitoxin(VT),is a serious and widespread trichothecene toxin contaminating cereals and a mainly toxic secondary metabolism produced by Fusarium,having a complex toxic effects on humans and animals.Studies have shown that ROS-mediated oxidative stress induced by DON is condidered as one of the mechanisms leading to cell death and apoptosis.Yeast cell wall extract(YCWE),a kind of natural substance that resists oxidation and inflammation,enhances immunity,promotes growth and improves the intestinal environment of animals,is composed of dextran,glycoprotein and mannan.At present,YCWE has been used in many livestock breeding researchs,but there has not been any in-depth study on toxin intervention.In this experiment,pig intestinal epithelial cells(IPEC-J2)were used as a cell model to explore the mechanism of toxic effect of DON on IPEC-J2 cells and the protective effect of YCWE on the cells.1.Studies on the cytotoxicity of DON and YCWE intervention in its toxicity:Using IPEC-J2 as the research object,construct intestinal epithelial cell injury model to explore the toxic effect of DON and investigate the YCWE intervention in the cytotoxicity of DON of low concentration(1?mol/L).The effects of DON and YCWE on cell viability,cell morphology,antioxidation and anti-inflammatory were evaluated.The results are as follows:1)Intestinal epithelial cell viability measured by MTS:DON significantly reduces cell viability in a dose-and time-dependent manner;the cytotoxicity of DON decreased after 12-36 hours of YCWE addition.2)Morphology of the cells observed by inverted microscope:the morphology of the cells was destroyed after 1 ?mol/L DON treatment at different times.After 36 h,the cells floating and not stuck tightly to wall were observed,while after co-treatment with YCWE and DON for 12,24 and 36 h,YCWE significantly inhibited the damage of DON on the morphology of IPEC-J2 cells and maintained the normal morphology of the cells.3)Inflammatory factors in cell culture fluid detected by ELISA:inflammatory factors IL-6,IL-8,IL-1? and TNF-? increased at 12 h and 24 h after treatment with 1 ?mol/L DON.Compared with the DON group,the content of each inflammatory factor changed in different degrees.4)Oxidation index:After treatment with 1 ?mol/L DON for 12 h and 24 h,the levels of MDA and ROS were significantly increased,and the GSH content was decreased.The laser confocal microscopy analysis showed that the ROS content in the cells treated with DON for 24 h also showed a significant increase,and both CPDT and YCWE could significantly inhibit the production of ROS.Intracellular MDA and ROS were downregulated,and GSH was upregulated after the treatment of IPEC-J2 cells damaged by DON with YCWE.The above results indicated that 1 ?mol/L DON significantly reduced cell viability,destroyed cell morphology,and induced oxidative stress and inflammation;YCWE reversed the cytotoxicity of DON at this concentration and maintained the normal morphology of the cells,and had anti-inflammatory and anti-oxidation effects.2.The molecular mechanism of DON-induced cell apoptosis and autophagy and YCWE intervention in the cytotoxicity DON:Since oxidative stress is an important factor in cell apoptosis and autophagy,this study investigated the effects of DON concentration and time on autophagy and established a model of DON-induced autophagy.The effects of DON on autophagy and apoptosis and meanwhile the effects of YCWE on autophagy and apoptosis induced by 1 ?mol/L DON were investigated.The results as follows:1)DON activated autophagy and upregulated the expression of the apoptotic protein Bax,but with the increase of concentration and time,the degree of autophagy was decreased.After co-treatment with YCWE and 1 ?mol/L DON,YCWE significantly inhibited DON-induced autophagy and apoptosis.2)Using 3-MA and Rap as inhibitors and activators of autophagy-related pathways,respectively,to investigate whether autophagy is involved in the toxicity of DON to cells and whether YCWE has a protective effect on cells by regulating autophagy.? The results of cell viability showed that both 3-MA and YCWE significantly increased the viability of DON-damaged cells,and Rap significantly decreased cell viability.? The results of oxidation index showed that both 3-MA and YCWE significantly inhibited DON-induced upregulation of MDA and ROS,and inhibited the downregulation of GSH.Rap significantly upregulated ROS and MDA,and downregulated GSH.?Western blot analysis of autophagy and apoptotic protein expression levels showed that 3-MA and YCWE significantly reversed the autophagy induction of DON and downregulated apoptosis induced by DON.The results indicated that DON induced cell autophagy and apoptosis,and DON may promote apoptosis by inducing autophagy.YCWE may inhibit apoptosis by downregulating DON-induced autophagy.3.Role of ROS-PI3K-AKT-mTOR pathway in autophagy induced by DON of low concentration and YCWE-regulated autophagy:ROS negatively regulates PI3K-AKT-mTOR signaling pathway due to oxidative stress and autophagy induced by DON.However,whether ROS-PI3K-AKT-mTOR signaling pathway is directly involved in regulating autophagy induced by DON and in DON-induced cell injury intervened by YCWE is not known to us.Therefore,this study was carried out with IPEC-J2 as the research object.1 ?mol/L DON was used alone or used with anti-oxidation pathway activator(CPDT),PI3K activator(740 YP),PI3K inhibitor(LY294002)to treat the cells for 12 h to detect IPEC-J2 cell viability,autophagy and the expression level of proteins associated with PI3K-AKT-mTOR signaling pathway to explore DON-induced autophagy involved with oxidative stress and PI3K-AKT-mTOR signaling pathway repectively,and to explore how oxidative stress induced by DON regulates PI3K-AKT-mTOR signaling pathway,thus further demonstrating molecular mechanism of DON cytotoxicity and YCWE intervention on DON cytotoxicity.The results are as follows:1)The results of cell viability showed that CPDT,YCWE and 740Y-P all significantly increased cell viability,while LY294002 significantly decreased cell viability.2)The results of DON-induced autophagy involved with oxidative stress showed that CPDT significantly decreased the autophagy-associated protein LC3-?,and the P62 protein expression level increased significantly.3)The results of DON-induced autophagy involved with PI3K-AKT-mTOR signaling pathway showed that DON significantly decreased both P-AKT/AKT and P-mTOR/mTOR.LY294002 significantly upregulated LC3-?,downregulated the expression of P62 protein,and P-AKT/AKT and P-mTOR/mTOR were downregulated;the respective result of 740 Y-P and LY294002 showed opposite trends.4)The results of that oxidative stress induced by DON regulates PI3K-AKT-mTOR signaling pathway showed that DON significantly decreased both P-AKT/AKT and P-mTOR/mTOR and CPDT significantly increased both P-AKT/AKT and P-mTOR/mTOR.5)The results of DON-induced autophagy of YCWE intervention involved with ROS-PI3K-AKT-mTOR pathway showed that DON significantly decreased both P-AKT/AKT and P-mTOR/mTOR and YCWE significantly increased both P-AKT/AKT and P-mTOR/mTOR.In summary,the oxidative stress induced by 1 ?mol/L DON may induce autophagy through regulating PI3K-AKT-mTOR signaling pathway,making apoptotic signaling pathway activated,thus exhibiting cytotoxicity,while YCWE inhibits autophagy by the activation of PI3K-AKT-mTOR signaling pathway due to antioxidation,thereby protecting cels.