Effect Of Soybean 7S/11S Globulin Allergy On Intestinal Mucosal Barrier In Weaned Piglets

The purpose of this study was to evaluate the effects of soybean 7S or 11S globulin added to the diet on growth performance,intestinal morphology,intestinal flora,intestinal mucosal barrier and TLR4/My D88/NF-κB signaling pathway in weaned piglets,and to investigate the mechanism of damage to the intestinal mucosal barrier by soybean globulin-induced hypersensitivity response.Thirty 21-day-old“Duroc×Landrace×Yorkshire”ternary cross piglets were randomly divided into three groups of 10 piglets each,namely group C,group S7 and group S11.The piglets in group C were fed basal diet,in group S7 fed basal diet supplemented with soybean 7S globulin,and in group S11 fed basal diet supplemented with soybean 11S globulin.The experiment lasted for 7 days.Sera were collected from each group of weaned piglets.Allergy indexes,intestinal mucosal integrity indexes and downstream cytokines of TLR4/My D88/NF-κB signaling pathway were detected by ELISA.Intestinal villi and crypts were observed by HE staining;the number of goblet cells were observed by PAS staining;tight junction protein was detected by IHC;TLR4/My D88/NF-κB signaling pathway related genes and proteins were detected by RT-q PCR and WB.The cecum contents were collected to extract sample genomic DNA for 16S r RNA high-throughput sequencing on Illumina Mi Seq platform.Results:（1）Compared with group C,groups S7 and S11 showed a highly significant decrease inmean daily weight gain（P<0.01）,a highly significant increase in feed-to-weight ratio（P<0.01）.（2）Compared with group C,serum interleukin E,interleukin G₁,5-HT,and histamine levels in the group S7 were highly significant higher（P<0.01）,and serum interleukin E,interleukin G₁,and histamine levels were significantly higher（P<0.05）,and 5-HT levels were highly significant higher（P<0.01）in the group S11.5-HT levels were significantly lower in group S11 than in the group S7（P<0.05）.（3）The total number of valid 16S rRNA sequences obtained from the three groups was540,869,containing 861 OTUs.Theα-diversity indices（Shannon,Simpson,Ace,Chao）varied significantly（P<0.05）in the groups S7 and S11,and the Simpson index was significantly higher（P<0.05）in the group C than in the S11 group,and in all three indices except the Simpson index,the group S7 was higher（P>0.05）and significantly higher（P<0.05）than the group S11（P<0.05）,while the group S11 was lower than the group C（P>0.05）and significantly lower than the group S7（P<0.05）.（4）At the phylum level,the dominant bacteria were mainly Firmicutes,Bacteroidota and Actinobacteriota;at the genus level,the dominant bacteria in each group were mainly Lactobacillus,Prevotella,Collinsella,Subdoligranulum,Blautia,Faecalibacterium and Alloprevotella.Compared with group C,the group S11 showed a significant increase（P<0.05）in Actinobacteriota and Collinsella,a highly significant increase（P<0.01）in Subdoligranulum,a highly significant decrease（P<0.01）in Bacteroidota and Prevotella and a significant decrease（P<0.05）in Lactobacillus.（5）Compared to group C,the small intestinal villi were highly significantly reduced in groups S7 and S11（P<0.01）,the crypt depth was highly significantly thickened（P<0.01）,and the villi to crypt ratio was highly significantly reduced（P<0.01）.The number of goblet cells was significantly reduced（P<0.01）,and the level of MUC2 was significantly reduced（P<0.05）.（6）Compared to group C,the intestinal mucosal integrity indexes DAO and D-LA were significantly increased（P<0.05）and LPS was highly significantly increased（P<0.01）in groups S7 and S11.（7）Compared to group C,Claudin-1,Occludin and ZO-1 were highly significantly decreased（P<0.01）in groups S7 and S11.（8）Compared with group C,TLR4,NF-κBp65,MyD88,IL-1β,IL-6 and TNF-αwere significantly increased（P<0.05）or extremely significantly（P<0.01）in groups S7 and S11.In summary,intestinal hypersensitivity in weaned piglets due to soybean 7S/11S globulin reduced growth performance,affected intestinal microbial composition,and caused damage to the intestinal mucosal barrier,reduced the relative abundance of beneficial bacteria such as Bacteroidota,Lactobacillus and Prevotella,and increased the relative abundance of harmful bacteria such as Actinobacteriota,Collinsella and Subdoligranulum.The elevation of harmful bacteria caused an increase in bacterial LPS,which promoted the activation of TLR4/My D88/NF-κB signaling pathway,and aggravated the degree of damage to the intestinal mucosal barrier.