| Soybean allergenic proteins can induce diarrhea in piglet,but the mechanism remains largely unknown.This study was carried out to investigate the effect of conglycinin on the expression of genes and proteins associated with growth,transport channel,barrier function and autophagy in the porcine intestine by using experiments of in vivo piglets feeding and in vitro intestinal porcine epithelial cells(IPEC-1)incubation.1.Effect of conglycinin on the growth and vitality of IPEC-1 cellsThe IPEC-1 cells were divided into six treatment groups: control and five treatment groups with different concentrations of conglycinin(0.28 mg/m L,0.56 mg/m L,1.11 mg/m L,2.23 mg/m L and 4.45 mg/m L).The results showed that with the increasing concentrations of conglycinin,the growth and vitality of IPEC-1 cells was substantially reduced.Additionally,the concentration of median lethal dose(TC50)of conglycinin in IPEC-1 cells is 1.20 mg/mL.2.Effect of conglycinin on expression of genes associated with intestinal growth,proliferation,transport channel,barrier function and autophagy in IPEC-1 cellsThe IPEC-1 cells were divided into two groups: control and 1.20 mg/mL conglycinin group.The culture medium was changed every day,and total sample RNA and protein was collected from IPEC-1 cells post 3 days of incubation.The results showed that:(1)Conglycinin reduced the mRNA levels for CFTR,SGLT-1,AQP3,AQP4 and Claudin-1(P < 0.05),but increased the mRNA abundances for Bcl-2,Bcl-xl,PepT1,AQP10,Occludin and ZO-1 in IPEC-1 cells(P < 0.05).(2)Conglycinin increased the AQP4,Bcl-2,and caspase-3 protein expression in IPEC-1 cells.3.Conglycinin increased the autophagy activation of IPEC-1 cellsExp1.The transfected IPEC-1 cells by GFP-LC3 were divided into two groups: control and 1.20mg/m L conglycinin group.Similarly,the culture medium was changed every day,and slices of GFP-LC3-IPEC-1 cells were made after three days incubation.Autophagy spots were counted in slices.Exp2.IPEC-1 cells were divided into two groups: control and 1.20 mg/m L conglycinin group.Total protein of IPEC-1 cells was collected after 3 days incubation and the autophagy markers,LC3-I and LC3-II,were determined by western blot assay.Exp3.The transfected IPEC-1 cells by GFP-LC3 were divided into six groups: control group,addition 1.20mg/m L conglycinin group,addition of 8.9 nM bafilomycin group,addition 100 nM wortmannin group,addition 1.20 mg/m L conglycinin and 8.9 nM bafilomycin,and addition 1.20 mg/m L conglycinin and 100 n M wortmannin.The culture medium was changed every day,and slices of GFP-LC3-IPEC-1 cells were made after three days incubation.Exp4.IPEC-1 cells were divided into six groups: control group,addition 1.20mg/m L conglycinin group,addition of 8.9 nM bafilomycin group,addition 100 nM wortmannin group,addition 1.20 mg/m L conglycinin and 8.9 nM bafilomycin,and addition 1.20 mg/mL conglycinin and 100 nM wortmannin.The culture medium was changed every day,and the proteins LC3-I and LC3-II were determined in IPEC-1 cells.The results indicated that:(1)Conglycinin elevated the autophagy spots in GFP-LC3-IPEC-1 cells.(2)Conglycinin significantly increased the LC3-II expression and the ratio of LC3-II/LC3-I.(3)When autophagy inhibitor was added to culture medium,the autophagy spots of GFP-LC3-IPEC-1 cells were decreased(P < 0.05),whereas the ratio of LC3-II/LC3-I in IPEC-1 cells was increased.Therefore,conglycinin could induce the elevation of autophagy activation in IPEC-1 cells.4.Effect of conglycinin on the growth performance,intestinal morphology,and intestinal functions in piglets.Twelve 21-days-old piglets(duroc × landrace × landrace)were randomly assigned to two treatment groups: control group(fed with 4% casein)and treatment group(fed with 4% conglycinin).On day 8 of trial,piglets were slaughtered after taken blood,and intestinal samples(duodenum,jejunum,ileum and colon)were collected.The results showed that:(1)Diet supplemented with conglycinin reduced the average daily gain of piglet,but increased the feed conversion ratio and diarrhea.(2)Conglycinin improve the crypt depth of duodenum,jejunum and colon in piglet,whereas decreased the villus width in duodenum and villus height and villus area in ileum,indicating that conglycinin could impair intestinal morphology in piglets.(3)Conglycinin increased the mRNA levels for CFTR and MMP4 in duodenum,but decreased the mRNA levels for AQP3,MMP3,and villin.Moreover,the expression of gene Bcl-2,CFTR,PepT1,SGLT-1,NHE3,AQP3,AQP4,AQP8 and Occludin were upregulated,while the expression of AQP10 was downregulated in jejunum of piglets in conglycinin group.Additionally,the expression of PepT1 and Occludin were increased,but the expression of Bax,AQP 3,villin and MMP3 were decreased in ileum of piglets in conglycinin group.(4)Conglycinin reduced the expression of protein AQP4 in jejunum,but increased ratio of protein LC3-II / LC3-I in colon of piglets.Taken together,our results suggest that conglycinin reduces the growth performance and enterocyte growth,impairs the intestinal morphology,induces the enhancement of autophagy activation,and regulates the expression of genes and proteins associated with intestinal growth,transport channel,and barrier functions. |
PDF Full Text Request