Effect Of Soybean Antigen Protein On Serum Cytokines And Intestinal Allergization In Weaned Piglets

The allergization model was established by adding different levels of purified β-conglycinin (7S) and Glycinin (11S) to diets of weaned piglets. Cytokines, intestinal mucosal morphology, expression of tight junctions protein Claudin-1 and its genes expression levels were studied by the methods of ELISA, paraffin sections, immunohistochemistry and real-time fluorescence quantitative PCR (qRT-PCR), respectively, in order to explore the allergization of 7S and 11S in weaned piglets.Seventy crossbred weaned piglets (DurocxLandracex Yorkshire),24 days old, were selected and allotted to seven groups randomly, ten piglets in each goup. All of the piglets were fed with milk replacer at the age of 17 day, weighed and weaned at day 24, then started to feed with experimental diets. The piglets in the control group (A) were fed with basic diet without soybean antigen protein. The piglets in Group B, Group C, and Group D were fed with the basic diet with 7S at 500 μg/kg·W,2000 μg/kg·W, and 5000 μg/kg·W, respectively. The piglets in Group E, Group F, and Group G were fed with the basic diet with 11S at 500 ug/kg-W,2000 μg/kg·W, and 5000 μg/kg·W, respectively. Blood samples were collected from the anterior vena cava at days 26,28 and 30 for measuring cytokines (IL-2, IL-4, IL-6, IL-8, and INF-y) in sera. At the age of 31 day, five piglets of each group were slaughtered. Segments of duodenum, mid-jejunum and ileum were collected immediately after slaughtered and divided into two parts. One part was fixed in 4% paraformaldehyde for paraffin section and immunohistochemistry examination of intestinal epithelial tight junction protein Claudin-1, another part was frozen rapidly in liquid nitrogen for detecting the mRNA expression levels of tight junction protein Claudin-1 in the intestinal epithelial cells by qRT-PCR.The results were as follows:1. The levels of serum IL-2, IL-4, IL-6 were significantly increased (P<0.05 or P<0.01) by adding 7S with the doses of 2000 μg/kg·W,5000 μg/kg·W and 11S with the doses of 5000 ug/kg-W, while the levels of serum INF-y were significantly decreased (P<0.05 or P<0.01). The level of serum IL-8 was significantly increased (P<0.05) by adding both 7S and 11S with the doses of 5000 μg/kg·W. No significant effects were observed in the levels of serum IL-2, IL-4, IL-6, IL-8 and IFN-y by adding 7S and 11S with the doses of 500 μg/kg·W.2. The structure of mucosa in small intestine could be changed by adding 7S and 11S, which showed villous atrophy and crypt hyperplasia. Villus height on duodenum and jejunum were decreased significantly by adding 7S and 11S with the doses of 2000 μg/kg-W,5000 μg/kg-W. Villus height on ileum were decreased significantly by adding 7S and 11S with a doses of 5000 μg/kg·W. Crypt depth of duodenum, jejunum and ileum were increased significantly by adding 7S with the doses of 2000 μg/kg-W,5000 μg/kg-W and 11S with the dose of 5000 μg/kg·W (P<0.05 or P<0.01).3. The expression of TJ protein Claudin-1 of duodenum, jejunum and ileum were reduced by adding 7S and 11S. The expression levels of claudin-1 in jejunum and ileum were decreased significantly by adding 7S with the doses of 2000 μg/kg-W,5000 μg/kg·W and 11S with the dose of 5000 μg/kg-W (P<0.05 or P<0.01). The expression levels of Claudin-1 of ileum were decreased significantly by adding 7S and 11S with the doses of 5000 μg/kg-W (P<0.01).4. The relative expression of intestinal TJ protein Claudin-1 mRNA were reduced by adding 7S and 11S in piglets. The relative expression of intestinal TJ protein Claudin-1 mRNA in duodenum were reduced significantly by adding 7S and 11S with the doses of 2000 μg/kg-W, and 5000 μg/kg-W (P<0.05 or P<0.01). The relative expression of intestinal TJ protein Claudin-1 mRNA on proximal jejunum were reduced significantly by adding 7S and 11S with the doses of 500 μg/kg-W,2000 μg/kg-W, and 5000 μg/kg-W (P<0.05 or P<0.01). The relative expression of intestinal TJ protein Claudin-1 mRNA in middle jejunum, distal jejunum and ileum were reduced significantly by adding 7S and 11S with the doses of 5000 μg/kg-W.The experiment indicated that 7S and 11S could increase the levels of IL-2, IL-4, IL-6 and IL-8 in serum and decrease the level of INF-y significantly. Villus height of each intestinal segment were decreased, crypt depth were increased, and the expression of intestinal mucosa TJ protein Claudin-1 and its mRNA were decreased by adding soybean protein antigens.