Mechanism Of S.boulardii Preventing DON Induced Apoptosis Of Porcine Macrophage

Nowadays much attention has been paid to the mycotoxin in the contaminated food production and feed industry. Trichothecene mycotoxins ingestion leads to in the clinical symptoms with diarrhea, vomiting, severe bleeding,shock and even death,which is harm to human and animal health. Deoxynivalenol,also called vomiting toxins,is primarily produced by the plant pathogen Fusarium graminearum and Fusarium culmoru m. Because of its high stability and most prevalent in crops like wheat, oat or barley,its toxicity is studied extensively,although it has low toxicity.The DON-induced toxicity of immune cells has made certain progress by scholars domestic and overseas,but about how to intervene and reduce the risks of chronic poisoning may still need to be addressed.Probiotics which acts as a new immune regulator,plays an important role in improving the intestinal nutrition and regulating the body’s immune system.S.boulardii is the only yeast which palys a probiotic role.It has a very good curative effect for biological toxins-induced diarrhea. But whether S.boulardii has intervention to the immune cells apoptosis induced by DON and the function of the possible molecular mechanism have not been researched,This study aims to investigate the effect of different concentration of S.boulardii worked on the porcine alveolar macrophages(PAM) 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.At the target of p38 MAPK –their common pathway,we detected its downstream inflammatory cytokines secretion and the p38 protein expression.Thus the possible molecular mechanism of S.boulardii intervention effect could be illustrated.Moreover in order to further explore what kind of elements play a role in yeast,because of the yeast cell wall is the direct induction point contacting with the outside world,this study discussed the S.boulardii cell wall influences to the m RNA expression of inflammatory factors. The main results were as follows:1.The results of MTT experiment and Annexin V- FITC/PI showed that DON acted on PAM was time and dose dependent,and the higher concentration of toxin could obviously increase the cell inhibition rate and apoptosis rate.Utilizing ELISA detected inflammatory cytokines IL-6,TNF-α and IL-lβ,when the concentration of DON was 4μM,the concentrations presented decrease after rising first,it showed a significant rise at 4 hour.The amount of inflammatory factor m RNA expression was on the rise with the elevated concentration of DON,it had a significant different(P<0.01) at 8μM.Preliminary we determined the DON apoptosis model worked at 4 h,8μM, for subsequent S.boulardii intervention effect research.2. In the premise of DON had no toxic damage to S.boulardii,they worked together on the PAM. Using flow cytometry(FCM) detect apoptosis we found that with the increase of S.boulardii concentration, the apoptosis rate of PAM reduced.At the same time it inhibited the concentrations of inflammatory cytokines IL-6,TNF-α and IL-lβ,which increased by DON.The higher concentration(5 ×107 /m L) has a significant inhibiting effect(P<0.05).For apoptosis regulation genes Bcl-2/Bax and Bcl-xl/Bax, the medium dose of S.boulardii significantly raised the ratios compared with DON toxin treatment group(P<0.01). It urged the cells developing a antiapoptotic effect. DON obviously up-regulated p38 m RNA expression. Medium dose of S.boulardii(5 ×105 / m L) very significantly decreased(P<0.01) p38 m RNA expression. As same as the function of inhibitor SB203580.The results of Western blot which tested p38 protein expression levels showed that S.boulardii could inhibit p38 protein’s phosphorylation and block p38 MAPK pathway activation.3.Along with the higher concentration of yeast cell wall,the amount of m RNA expression of inflammatory factor increased. Inflammatory cytokines IL-6 and TNF-α m RNA expression of middle and high dose group(10ppm and 20ppm) were higher than those of DON treatment group. The yeast cell wall group only had a certain inhibition for IL-lβ.In a conclusion,the S.boulardii could reduce the expression of p38,and suppressed DON activating the p38 MAPK pathway and reduced the expression of downstream inflammatory cytokines.At the same time,it raised the expression of antiapoptotic factors to inhibit apoptosis.To a certain extent the yeast cell wall activated the PAM immune response,the active ingredient with inhibition function in yeast still needs to be further explored.