Effects Of Ochratoxin A On PAMs Immune Response And Intervention Effect Of Mannan Oligosaccharide And Mechanism

Ochratoxins is a mycotoxin produced by Aspergillus and Penicillium.Ochratoxin A(OTA)is contaminating in a wide variety of foods and feeds such as grains and meats.It is one of the most harmful,and most closely related to human and animal health in nature.OTA has a variety of toxic effects,including nephrotoxicity,intestinal toxicity,immunotoxicity and teratogenic carcinogenicity.It is shown that OTA could cause immunosuppression in almost all studies in vivo.However,the vast majority of results in vitro showed that mycotoxins caused immunostimulation.The reason of results of studies in vitro are contrary to studies in vivo is unclear.It was found that the time of OTA exposure in vivo is much longer than that of OTA incubation in vitro,so we suspect that OTA treatment time may be the cause of different immunotoxicities.Therefore,we propose that different OTA treatment times may lead to two different immunotoxic effects of immunosuppression and immunostimulation,this study aimed to explore the underlying reason and mechanism of the these effects.Mannan oligosaccharide(MOS),also known as mannan oligosaccharides,derived from the cell walls of yeast,is a potent immune-modulator.MOS has two-way regulation of immune effects,and also can be physically adsorbed or directly combined with mycotoxins to eliminate the harmful effects of toxins on the body.Therefore,MOS was added to study its intervention effect on OTA-induced immunosuppression and further explored the underlying mechanisms.Experiment 1:Effect of OTA on immunotoxicity of pams and the mechanism of induced immunosuppressionIn this study,3D4/21 cells(PAMs)were used as study models in vitro to evaluate the effect of toxins.Different concentrations of OTA were used to treat PAMs for 24h,48h,and 72h,respectively.Cell activity was detected by MTT and LDH methods.To exclude the effects of cytotoxicity on immune function of PAMs,the concentration of 1.0 μg/mL was chosen for subsequent research.Subsequently,we used 1μg/ml OTA to treat PAMs for 24h,48h,and 72h,respectively,detecting the mRNA and protein expression levels of pro-inflammatory cytokines and anti-inflammatory cytokines,macrophage migration and phagocytosis,phagocyte polarization.The results showed that OTA treatment for 24 h promoted the expression of pro-inflammatory cytokines,enhanced the migration and phagocytosis of macrophages,and the macrophages were polarized toward M1.While long-time exposure of OTA significantly increased the expression of anti-inflammatory cytokines,and decreased the expression of pro-inflammatory cytokines.The migration and phagocytosis of macrophages were decreased,the number of M2 macrophages finally occupied the dominant position in total cells.The above results indicated that long-time instead of short-time exposure of OTA induced immunosuppression.In order to study the mechanism of OTA-induced immunosuppression,we detected the expression of autophagy-related proteins,the result showed that long-time exposure of OTA suppressed autophagy.Further research found that autophagy activator rapamycin(RAPA),could reverse OTA-induced immunosuppression.By detecting of Aktl and Akt2 proteins,it was found that OTA suppressed autophagy by upregulating the Aktl signaling pathway.Experiment 2:The protective effect and mechanism of mannan oligosaccharide on OTA-induced immunosuppressionIn this study,we first treated PAMs with different concentrations of mannose oligosaccharides(MOS),detected cell viability by MTT method,the concentration of 0.25 mg/ml,0.5 mg/ml,0.75 mg/l,and 1 mg/ml was chosen for subsequent research.Adding different concentrations of MOS to interfere with the OTA-induced immunosuppressive effect.The results shown that MOS decreased anti-inflammatory cytokines expression,increased pro-inflammatory cytokines expression,enhanced macrophage migration and phagocytosis.The results showed that the intervention effect was the best after the MOS concentration reached 0.75 mg/ml.The next experiment will focus on the mechanism of intervention of MOS in OTA-induced immunosuppression.MOS alleviated the autophagy suppressed by OTA,but MOS did not have a significant effect on Akt1.The study found that JNK in the MAPK signal pathway was activated by MOS.Further research showed that the JNK inhibitor also inhibited autophagy levels.JNK inhibitors significantly blocked the intervention of MOS on OTA-induced immunosuppression by increasing the expression levels of pro-inflammatory cytokines,reducing anti-inflammatory cytokines,enhancing macrophage migration and phagocytosis.It is concluded that MOS promotes autophagy by activating JNK phosphorylation,and thus played a role in intervening OTA-induced immunosuppression.