Effect Of Wheat Arabinoxylo-oligosaccharides On Secretion Of LPS-induced Inflammatory Cytokines In Porcine Alveolar Macrophage

China is a big country in wheat production,but the by-products of wheat processing have not been fully utilized and the phenomenon of resource waste is serious.In recent years,the international research on wheat processing by-products is becoming more and more in-depth,and the products are widely used in various industries.Wheat arabinoxylans(AX)is abundant in the by-product of wheat processing,which is the main component of non-starch polysaccharides(NSP)in wheat diet.It has been found that the addition of AX to wheat diet can effectively eliminate its anti-nutritional effect,so that AX can play a prebiotic role in animals.A large number of studies have shown that the utilization mechanism of wheat arabinoxylan and its degradation product Arabinoxylo-oligosaccharides(AXOS)is different.In recent years,some scholars have carried out studies on the extraction and preparation of AXOS,and found that AXOS can improve the immune function of mice,but the mechanism is still unclear.The use of wheat AXOS as a feed additive has not been reported,and its effect on the immune function of pigs remains to be studied.Macrophages play a very important role in the body,with great plasticity.They can differentiate into different populations and play different roles according to different environmental stimuli.After being stimulated,macrophages secrete a large amount of inflammatory cytokines to clear the inflammation of the body.However,excessive secretion of inflammatory cytokines can also cause damage to the body itself.Therefore,it is very important to promote the secretion of inflammatory cytokines in a limited way.In this study,we use LPS-induced porcine alveolar macrophages as the premise to investigate the effect of wheat arabidopsis oligosaccharide in the secretion of inflammatory factors of LPS-induced porcine alveolar macrophages and its mechanism.Experiment 1 Effects of AXOS in wheat on LPS-induced macrophage inflammatory cytokines and NF-κB gene expressionThe purpose of this study was to investigate the effect of wheat AXOS in the secretion of inflammatory factors of LPS-induced porcine alveolar macrophages and its possible mechanism.In this study,LPS was used to construct the inflammatory model of porcine alveolar macrophages in vitro by means of cell culture in order to study the effect of wheat arabidopsis oligosaccharide on macrophage inflammatory factors and NF-κB gene expression.The experiment was divided into 3 groups with 3 replicates in each group,respectively:1)control group,without treatment;2)LPS group,0.1 μg/ml LPS stimulation;3)LPS+AXOS group,0.1 μg/ml LPS stimulation+different concentrations of AXOS(31.25,62.5,125,250 μg/ml)were treated for 2h,4h,and 6h,respectively.The results showed that:1)compared with the LPS group,the concentration of wheat AXOS was 31.25 μg/ml and 125 μg/ml for 4 h,and the concentration of 62.5 μg/ml for 6 h significantly decreased the content of IL-6(P<0.05).2)compared with the LPS group,the concentrations of 250 μg/ml were treated for 2 hours,125 μg/ml and 250 μg/ml for 4 hours,and the TNF-α content was significantly decreased after 6 hours of treatment(P<0.05).3)IL-4 content in LPS+AXOS group decreased significantly compared with that in LPS group(P<0.05).4)in addition to 31.25 μg/ml concentration for 2 hours,each concentration was treated for 4 hours,31.25 μg/ml and 62.5 μg/ml concentration for 6 hours significantly decreased the content of IL-10(P<0.05).5)mRNA expression of TNF-αin the LPS+AXOS group was significantly lower than that in the LPS group after 2 hours of treatment(P<0.05).6)mRNA expression levels of IL-10 and TNF-αdecreased significantly in LPS+AXOS group after 4 hours of treatment(P<0.05).7)mRNA expression of IL-10 decreased significantly after 6 h treatment(P<0.05).Compared with LPS group,NF-κB p65 expression in LPS+AXOS group was significantly increased(P<0.05).The results suggested that AXOS could regulate the secretion of some inflammatory factors in macrophages,and the mRNA expression of NF-κB increased significantly after AXOS treatment,suggesting that it might play a role through NF-κB.Experiment 2 Effect of NF-κB inhibition on AXOS in wheat on immune function of porcine alveolar macrophagesThe purpose of this study was to investigate the role of NF-κB signaling pathway in the regulation of LPS-induced porcine alveolar macrophage’s inflammatory factor secretion by wheat AXOS,and to explore the molecular mechanism by which NF-κB signaling pathway is involved in the control of macrophage inflammatory factor secretion by wheat AXOS.In this study,in vitro cell culture was used to establish an inhibitory model using the NF-κB pathway inhibitor(bay11-7082)to study the effect of NF-κB on AXOS’s regulation of macrophage inflammatory factor secretion in wheat.The experiment was divided into 3 groups with 3 replicates in each group,respectively:1)control group,without treatment;2)LPS group,0.1 μg/ml LPS stimulation;3)NF-κB inhibition group,NF-κB inhibitor pretreatment for 1 hour+0.1μg/ml LPS stimulation+different concentrations(31.25,62.5,125,25 μg/ml)AXOS treatment for 2h,4h,6h,respectively.The results showed that:1)after inhibiting the NF-κB signaling pathway,the secretion levels of IL-6 and TNF-α in LPS+AXOS group showed no significant change compared with LPS(P>,0.05).2)after inhibiting the NF-κB signaling pathway,compared with the LPS group,IL-4 secretion increased significantly at 2 h after LPS+AXOS treatment except 250 μg/ml concentration(P<0.05),but there was no significant change in IL-4 secretion at 4 h and 6 h after LPS+AXOS treatment(P>0.05).3)compared with the LPS group,the secretion of IL-10 increased significantly in the LPS+AXOS group at 31.25 μg/ml concentration for 2 h and 4 h,62.5 μg/ml concentration for 2 h and 4 h,and 125 μg/ml concentration for 2 h(P<0.05).4)mRNA expression levels of cytokines the results showed that mRNA expression levels of IL-6,IL-8,IL-10 and TNF-α in the LPS+AXOS group were not significantly changed after inhibition of the NF-κB signaling pathway compared with that in the LPS+AXOS group(P>0.05).4)protein level results showed that NF-κB p65 protein expression in LPS+AXOS treatment group was significantly lower than that in LPS group(P<0.05),and the expression level in the inhibitory group was significantly lower than that in LPS group(P<0.05).The results suggested that AXOS could regulate the partial inflammatory factor secretion of macrophages through NF-κB.