Isolation Of Deoxynovalenol-degradation Microorganisms And Its Relieving Effects On Porcine Jejunal Epithelia Cell Injury Induced By Deoxynivalenol

Deoxynivalenol(DON),also known as vomitoxin,is one of the mycotoxins most frequently encountering in cereal-based foods throughout the world.Acute exposure to high doses of DON results in toxic effects on humans and animals.Various innate defense mechanisms can be applied to reduce the risk of invasive pathogen invasion,and the IPEC-J2 cell line,a non-transformed intestinal cell line originally derived from jejunal epithelia is an important model to study zoonosis.DON is the first known toxin to promote inflammation and immune regulation of intestinal epithelial cells.The aim of this research was to isolate microorganisms to degrade DON,construct DON-IPEC-J2 model,and study the effect of isolated microorganisms on DON degradation for relieving DON negative effect on IPEC-J2 cells proliferation.There were two parts of the experiments as follows.(1)At first,samples(mouldy corns,water,soil and faeces)were inoculated on a marine mineral culture with DON as the sole carbon source by an enrichment culture procedure to enrich DON-degrading strains.The enriched strains were isolated and purified on LB medium plates,and then detected for their ability to degrade DON using enzyme-linked immuno sorbent assay(ELISA).The result showed that a strain named as YIV capable of metabolizing deoxynivalenol was obtained from goat manure samples,which removed 48.97% deoxynivalenol after incubation for 48 h.On the basis of morphological,physiological,and phylogenetic analysis,strain YIV was classified as a bacterium belonging to Bacillus cereus group.Secondly,several strains of microbes kept in our laboratory were also used to test their degradation ability of DON.The result showed that Saccharomyces cerevisiae decreased 42.49% DON after 48 h incubation.(2)Study on the effect of Bacillus cereus or Saccharomyces cerevisiae co-incubated with DON on IPEC-J2 cell proliferation,lactate dehydrogenase(LDH)yield,DON degradation rate and the m RNA expression of IL-6,IL-8,IL-10,TJP,Occludin,as well as the cell apoptosis.The effects of DON,Bacillus cereus or Saccharomyces cerevisiae on the metabolic activity of cells were measured by MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)assay.The results showed that cells proliferation inhibition showed a DON dose-time dependent effect.The IPEC-J2 cells viability was reduced from 16.19%(P<0.05)to 50.94%(P<0.05)when DON concentrations increased from 0.15 μg/m L to 19.2 μg/m L after 24 h incubation.It also reported that the cells viability decreased by4.75%(P>0.05)and 17.73%,10.99%,17.88%,21.89%(P<0.05)when DON concentrations changed among 0.075,0.15,0.3,0.6,1.2 μg/m L after incubation for 8 h,and the LDH yield was increased by 7.14%(P>0.05)and 146.43%,160.71%,182.14%,167.86%(P<0.05),indicating that there was a severe damage to cell cytomembrane.Saccharomyces cerevisiae added at 1:1 ratio of cells might promote cell proliferation after 8 h incubation(P>0.05),whereas Bacillus cereus significantly decreased cell proliferation by 80.65%(P<0.05).For Saccharomyces cerevisiae experiment,there were three groups such as the control group,DON group(0.075,0.15,0.3,0.6,1.2 μg/m L)and Saccharomyces cerevisiae + DON group(DON levels were the same as above)at a ratio of 1:1 of cells,which was incubated for 8 h.The results showed that cell proliferation was increased by 44.83% with Saccharomyces cerevisiae addition,compared with the control group.In Saccharomyces cerevisiae+DON group,Saccharomyces cerevisiae addition could increase cell proliferation(P>0.05),decrease LDH yield by 26.67%(P>0.05)and 66.67%,69.86%,72.15%,68.00%(P<0.05),and degrade DON by 57.14%,46.15%,42.31%,26.67%,13.51%(P>0.05).The expression levels of cytokines IL-6,IL-8,IL-10,TJP,Occludin were up-regulated with DON concentrations increasing.Saccharomyces cerevisiae+DON(1.2 μg/m L)significantly up-regulated IL-6,IL-8,IL-10 m RNA expression levels compared with individual DON addition(1.2 μg/m L)(P<0.05).To study the effect of Saccharomyces cerevisiae and DON on cell apoptosis,four groups were assigned to the control,DON(DON 1.2 μg/m L),Saccharomyces cerevisiae and Saccharomyces cerevisiae+DON(1.2 μg/m L)groups.Annexin V-FITC/PI apoptosis kit was used to detect cell apoptosis by flow cytometry after incubating for 8 h.The results showed that apoptosis cells at later or earlier stage in DON group were increased by 4.53 folds and 2.23 folds(P<0.05),respectively,compared with the control group.Compared with the control group,there was no significant difference for the viable cells and apoptosis cells at later and earlier stage(P>0.05)in the Saccharomyces cerevisiae group.The early and late apoptotic cells were decreased by 44.78% and 46.37%(P<0.05),and the number of viable cells were significantly increased by 2.35% in the Saccharomyces cerevisiae+DON group,compared with the DON group(P<0.05).In conclusion,Saccharomyces cerevisiae could degrade DON,had certain ameliorative effect on DON interference with IPEC-J2 cells,and protected the integrity of the cell membrane.