Study On Effects Of Vomitoxin On Apoptosis And Function Of IPEC-J2 Cells

Vomitoxin,also known as deoxynivalenol(DON)is a class of potent immunosuppressive agents.DON easily infects with grains,corn,sweet potatoes and other commonly used crops,in addition,it pollutes the animal’s secondary metabolites,such as pork,milk and blood.Therefore,it poses threat on human and animal health especially the immune function.DON has a high immunosuppressive and cytotoxic effects,can cause immune stimulation or immunosuppression according to the dose and action time of DON.It will lead to anorexia,vomiting,fever,diarrhea,standing instability,unresponsive and other acute poisoning symptoms,severe hematopoietic system and even death when people eat food contaminated by DON.The proportion of grain in Chinese traditional diet is much higher than in west,which makes the risk of vomiting toxins more prominent.Objective:To screen the appropriate concentration and acted time of DON from the view of CCK-8 proliferation toxins,cycle and apoptosis through an established in vitro model of jejunal epithelial cell line IPEC-J2,the level of Caspase3(17)and Caspase3(32)was detected by werstern-blot,and the expression of genes related to cytokines,mitochondrial oxidative stress and free amino acid transport vector was detected by quantitative real-time PCR.Methods:1)The proliferation of CCK-8 cells in IPEC-J2 at different concentrations of DON was studied.The cells were cultured in 96-well plates for 24 hours,48 hours and 72 hours.Cell relative survival ratio was calculated by measuring the OD value of the culture medium in A low concentration of 200 ng/ml DON and high concentration of 2000 ng/ml DON2)IPCE-J2 cell cycle detection after treatment with different DON concentrations:cells cultured in culture dishes were treated with high concentration of 2000 ng/ml DON and low concentration 200 ng/ml DON for 6 hours,12 hours and 24 hours.The percentages of the cells in the PI fluorescence histogram were analyzed according to operated instructions of experimental kit.3)Apoptosis detection of IPCE-J2 cells treated with different DON concentrations:cell culture was the same as above.According to the kit operation,the flash point was collected and the apoptotic rate was calculated by flow cytometry.4)Protein expression of IPEC-J2 cells based on the target gene after DON treatment:The cells were incubated with 50 ul of RIPA lysate.Detection of protein concentration,electrophoresis,transfer membrane,closed,primary antibody incubation,secondary antibody incubation,color exposure,grayscale analysis and then data processing were conducted.5)Immunofluorescence experiments of IPEC-J2 cells on the target gene after DON treatment:Cell culture and DON treatment refered to the previous section,RNA extraction,RNA concentration determination,DNA removal reaction,reverse transcription and data processing were conducted.Results:1)DON had significant effects on the growth of IPEC-J2 cells and inhibited cell proliferation.24 hours for the termination of the time period were choosed to research the reaction of 6,12,24 hours respectively.2)Most of cells treated with 200 ng/ml DON stayed in the S phase wherever 2000 ng/ml DON stayed in the G2/M phase.3)In 6 hours,the early apoptosis rate and the total apoptosis rate increased with the increase of DON concentration.4)DON activated the most important terminal cleavage enzyme Caspase3(17)fragment during the process of apoptosis,and the significant effect time was at 6 hours.5)Cytokine-related gene expression:high concentration of 2000 ng/ml DON two genes were significantly upregulated,IL-8 and IL-1β inflammatory gene expression were significantly activated.The expression of TFAM,mt SSB,ND4 and CCOXIV increased significantly after high concentration of 2000 ng/ml DON at 12 hours and the expression of Cytc increased at 24 hours.The expression of free amino acid transport vector gene:DON inhibited the expression of free amino acid transport vector BO + AT at 12 hours,and inhibited the expression of SGLT1,ASCT2 and GIUT2 after 24 hours.Conclusion:DON has a significant effect on the growth of IPEC-J2 cells by inhibiting its proliferation,inducing apoptosis and stimulating the inflammatory response.The low concentration of 200 ng/ml DON makes cell mitosis in the S phase;high concentration of DON in the late mitosis G2/M phase.In the apoptosis of DON mainly in the 6 hours or before the time period,which correlatived with the activation of Caspase3 gene in 6 hours.The expression of TFAM,mt SSB,ND4,CCOXV and CCOXIV upregulated at 12 hours after high concentration of 2000 ng/ml DON.MtSSB expression disorder,and mtDNA gene mutation occurred such as high oxidative stress,hydrolysis deamination reaction,alkylation and mass accumulated DNA damage,which ultimately lead to a variety of related diseases.ND4 gene promoted genetic neuropathy,mitochondrial myopathy,tension disorders and other related diseases.CCOXIV gene caused cytochrome deepen and cell membrane aged.The expression of IL + and SGLT1 decreased at 12 hours after treatment with low and high concentration of DON,indicating that DON inhibited the passive absorption of carbohydrate and the transport of fructose and galactose,and the transport efficiency of glucose in small intestine decreased,resulting in metabolic disorders,and the effect of low concentration of 200 ng/ml DON is more dominatant.