| Vomiting toxins also known as deoxynivalenol(DON),is secondary metabolism products produced by Fusarium,as major mycotoxins of food pollution.DON is widespread in crops such as wheat,oats and barley,and is highly hazardous to human and animal because it can cause acute poisoning symptom such as vomiting,diarrhea and fever.There is also a closed relation between DON and some diseases like anemia,immune suppression,esophageal cancer,the harmfulness can't be ignored.Some certain progress about DON have been made,most of which focus on cytotoxicity,teratogenicity,mutagenicity and carcinogenicity,but about how to intervene and reduce the risks of chronic poisoning may still need to be addressed.S.boulardii is a kind of probiotic yeast,which has a very good curative effect for biological toxins-induced diarrhea.But whether S.boulardii has intervention on the porcine jejunum epit helial cells apoptosis induced by DON has not been reported in any research yet.After the oral intake of animal feed contaminated with DON,the intestinal epithelial cells become the first target site of DON for being exposed to high concentrations of toxins.This research aims to investigate the effect of different concentration of S.boulardii worked on the porcine jejunum epithelial cell line(IPEC-J2)as immune cell model with immune cell apoptosis inducted by DON.Based on this model,we explore different concentration of S.boulardii's intervention effect on cells that are damaged by DON.The main results were as follows:1.The results of detecting cell inhibition ratio by MTS experiment showed that DON acted on IPEC-J2 was time dependent,and the longer incubation time could obviously increase the cell inhibition rate and apoptosis rate.According to the data accumulated in our laboratory,we determined the DON apoptosis model worked at 4 h,8?M,for subsequent S.boulardii intervention effect research.2.To confirm whether yeast itself will cause damage to IPEC-J2 cell,we evaluate its toxicity by the cell morphological analysis observed by electron microscopy and the observation of using flow cytometry detect apoptosis.It turns out that yeast action group brings no obviously different effect on apoptosis rate(including living cells rate,early apoptosis rate,late apoptosis rate and death rate),compared with the blank control group.And morphological results add to the confirm that yeast itself cause no toxi c damage to IPEC-J2.3.Based on the results of the two experiments above,we pick out three different concentrations of yeast,namely high,middle and low(5×104?/m L?5×105?/m L?5×106?/m L),to act on IPEC-J2 cell induced by DON(8?M),and concentrate on evaluating the yeast's intervention from cell apoptosis,ultrastructural morphology of cells and the tight-junction proteins between cells and determining whether there exists a dose-response relationship.We choose the three tight junction proteins,Occludin?Claudin?ZO-1,to evaluate the S.boulardii's intervention on tight-junction proteins' injury of cells induced by DON from the change of m RNA expression and protein expression.The findings show that:(1)When the three different concentrations of yeast act on the DON toxin group,the early apoptosis rate and late apoptosis rate both decline.The early apoptosis rate of the low concentration group decline from 14.82% to 0.45%,the late apoptosis rate decline from the initial 2.85% to 0.52%,and necrotic cell rate decline from 2.85% to 0.45%.There is no dose-response relationship for no significant difference between the single dose group.(2)Comparing the m RNA's relative expression of IPEC-J2 cells' tight-junction proteins,Occludin?Claudin and ZO-1,with DON toxin group,we find that only the low concentration group(5×104/m L)up-regulate the m RNA level of tight-junction proteins,Occludin?Claudin and ZO-1m,and the middle concentration group can only up-regulate the m RNA level of Cccludin.Whereas,the high concentration group down-regulate the m RNA's relative expression of Claudin and ZO-1.(3)Western bolt's determination of protein shows that there is no distinct expression difference in ZO-1and Occludin of blank control group and S.boulardii(5 × 105/m L).DON toxin group and high-concentration group lower the expression of the two kind of protein,but the low and middle-concentration group distinctly enhance it,approaching that of the blank control group.(4)Under the 1700-times electron microscope,nucleus of cells infected by DON are in irregular shape,with its margin denting badly,the fine hair between the cells reducing,cytoplasm condensing and big bubble appearing.Nucleus of cells of low-concentration group are in regular round shape,with the cytoplasm dispersing uniformly and the injury within the cell being healed to some degree.The cell shape of the middle and high-concentration group is between the above two.In conclusion,certain concentration of S.boulardii(5×105/m L)can act as intervention on the porcine jejunum epithelial cells apoptosis induced by DON.Whereas,the molecular mechanism that make S.boulardii act like that need to be further explored. |
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