Mechanism Of Astragalus Polysaccharide On Intestinal Immune Function In Canines

The aim of this experiment was to study the effect of astragalus polysaccharide on the serum immunoglobulin and cytokine levels of canines,the expression of intestinal secretory IgA,TLR4,TLR7,My D88,TRAF-6 and AP-1 proteins,and the composition of cecum microbial community,to explore its mechanism on the intestinal immune function of canines.Eighteen Beagles were randomly divided into the following three groups,with six canines in each group,which were control group,low dosage group(basal diet +1% astragalus polysaccharide),and high dosage group(basic diet + 2% astragalus polysaccharide).The experiment lasted for 14 days.Blood samples were collected at 0,7,and 14 days.On day 14,all canines were sacrificed and dissected for sample collection.The duodenum,middle jejunum,and ileum were collected and stored in 4%paraformaldehyde and liquid nitrogen,and the contents of the cecum were collected aseptically and stored in liquid nitrogen.The serum immunoglobulin and cytokine levels were detected by ELISA,and the secreted IgA,TLR4,TLR7,MyD88,TRAF-6,and AP-1 proteins in the intestine of canines were detected by immunohistochemistry,real-time PCR and Western-Blotting.High throughput sequencing was performed to detect changes in the cecum microflora.The results were as follows:(1)ELISA results showed that,compared with the control group,the serum immunoglobulins(IgA,IgG and IgM)and interferon gamma levels of canines in the astragalus polysaccharide groups were significantly or extremely significantly increased(P<0.05 or P<0.01),the serum interleukin-2,interleukin-4 and interleukin-6 levels of canines in low dosage group were significantly or extremely significantly increased(P<0.05 or P<0.01)and were extremely significant increased(P<0.01)in high dosage group There was no significant difference between the low dosage and high dosage groups(P > 0.05).(2)The results of immunohistochemistry,real-time PCR and Western-Blotting showed that,compared with the control group,the density mean,protein expression and mRNA relative expression of secretory IgA in the canine duodenum,jejunum and ileum of low dosage group were significantly increased(P<0.05),and were significantly or extremely significantly increased(P<0.05 or P <0.01)in high dosage group;the density mean,protein expression and mRNA relative expression of TLR4,TLR7,MyD88,TRAF-6 and AP-1 in canine duodenum of low dosage group were significantly increased(P<0.05),and were significantly or extremely significantly increased in the high dosage group(P<0.05 or P<0.01).There was no significant difference between the low dosage and high dosage groups(P> 0.05).(3)High-throughput sequencing results showed that 885,399 tags were obtained from9 samples in the canine cecum of the three groups.The total number of OTU was 1710,559 in the control group,573 in the low dosage group,and 578 in the high dosage group.The representative contribution rate of principle component 1(PC1)to total microbes was 48.44%,the contribution rate of principle component 2(PC2)was 22.13%.The composition of microbial community in the canine cecum of astragalus polysaccharide groups were changed,in which the main features were the increase in the number of Actinobacteria and Firmicutes bacteria and the decrease in the number of Fusobacteria and Proteobacteria,the increase in the number of Actinobacteria and Bacilli and the decrease in the number of Fusobacteria and ?-proteobacteria,the increase in the number of Bifidobacteriaceae,Prevotellaceae and Lactobacillaceae and the decrease in the number of Fusobacteriaceae,and the increase in the number of Prevotella and Lactobacillus and the decrease in the number of Fusobacterium.The experiment results showed that the astragalus polysaccharide can increase the serum immunoglobulin and cytokine levels,the expression of intestinal secretory IgA and toll-like receptor signaling pathway-related proteins,change the composition of the microbial community,increase the beneficial flora in the cecum,reduce the proportion of opportunistic pathogens,and enhance canine immunity.