| Soybean allergy poses a serious health risk to humans and animals;β-conglycinin and glycinin are the primary antigenic protein in soybean.Intestinal porcine epithelial cells（IPEC-J2）were used as an in vitro physiological model of the intestinal epithelium to study the effects of different concentrations ofβ-conglycinin and glycinin to identify the signaling pathways involved.Fresh untreated cells were defined as the control group and incubated with 1,5,or 10 mg·mL-1β-conglycinin/glycinin or 5 mg·mL-1β-conglycinin/glycinin and 1μmol·L-1nuclear factor-κB（NF-κB）inhibitor（PDTC）,inducible nitric oxide synthase（iNOS）inhibitor（L-NAME）,c-Jun N-terminal kinase（JNK）inhibitor（SP600125）,or p38 inhibitor（SB202190）for 24 h,the inhibitors were pretreated for 30 min.After completion of the culture,the cell culture supernatant and lysis supernatant were collected for the detection of each index.The expression of cytokines,Caspase-3/-8 and COX-2 in IPEC-J2 cells was detected by ELISA;alkaline phosphatase activity in the culture supernatant was detected using an alkaline phosphatase test kit.Cell viability was detected by CCK8 reagent.Digital images were taken with a JEM-1230 transmission electron microscopy.The expression of tight junction（TJ）proteins（occludin,claudin-1,and ZO-1）was assessed by western blot,qRT-PCR,and immunofluorescence.The cytoskeleton in IPEC-J2 cells was detected via immunofluorescence.The expression of NF-κB,iNOS,JNK,and p38 mRNA and their downstream genes was detected by qRT-PCR.Mitogen-activated protein kinase（MAPK）/NF-κB-related protein expression was detected by western blot.NF-κB p65,CREB,and AP-1 DNA-binding activities were detected using the electrophoretic mobility shift assay.The experimental results were as follows:1.The cell viability was significantly decreased after addingβ-conglycinin/glycinin（P<0.01）.However,the cell viability was considerably higher in the 5 mg·mL-1β-conglycinin/glycinin+1μmol·L-1inhibitors（PDTC,L-NAME,SP600125,and SB202190）groups than in the 5 mg·mL-1β-conglycinin group（P<0.05 or P<0.01）.2.The cytokine,caspase-3/-8,and COX-2 levels were significantly enhanced after addingβ-conglycinin/glycinin to IPEC-J2 cells（P<0.01）.However,their levels were significantly lower in the 5 mg·mL-1β-conglycinin/glycinin+1μmol·L-1the inhibitors（PDTC,L-NAME,SP600125,and SB202190）groups than in the 5mg·mL-1β-conglycinin group（P<0.05 or P<0.01）.3.The alkaline phosphatase activity increased significantly after the addition ofβ-coglycinin/glycinin（P<0.01）.The alkaline phosphatase activity was significantly lower after the addition of 1μmol·L-1with the inhibitors（PDTC,L-NAME,SP600125,and SB202190）（P<0.01）,compared with that in the 5 mg·mL-1β-conglycinin/glycinin group（P<0.05 or P<0.01）.4.In the control group,IPEC-J2 cells displayed complete nuclei with homogeneously dispersed chromatin.In the 5 mg·mL-1β-conglycinin/glycinin group,the IPEC-J2 cells exhibited mitochondrial alterations.In the 10 mg·mL-1β-conglycinin/glycinin group,small vesicles were observed near the nucleus,which was broken up into several discrete fragments.Compared with that in the 5 mg·mL-1β-conglycinin/glycinin group,the structure of IPEC-J2 cells in the 5 mg·mL-1β-conglycinin/glycinin+1μmol·L-1the inhibitors groups tended to be intact.5.Theβ-conglycinin/glycinin-treated groups showed a significant decrease in the protein and mRNA expression of claudin-1,ZO-1,and occludin（P<0.01 or P<0.05）.Compared with that in the 5 mg·mL-1β-conglycinin group,the protein and mRNA expression of claudin-1,ZO-1,and occludin in IPEC-J2 cells in the 5 mg·mL-1β-conglycinin+1μmol·L-1the inhibitors（PDTC,L-NAME,SP600125,and SB202190）groups was significantly higher（P<0.01 or P<0.05）.6.After culturing the cells withβ-conglycinin or glycinin,the immunofluorescence signal of the tight junction proteins in IPEC-J2 cells was significantly weakened,and the cell connection location was decreased.The tight junction proteins in the 5 mg·mL-1β-conglycinin/glycinin+1μmol·L-1the inhibitors（PDTC,L-NAME,SP600125,and SB202190）groups tended to be intact than those in the 5 mg·mL-1β-conglycinin/glycinin group.7.The cytoskeleton of IPEC-J2 cells in the 1 mg·mL-1β-conglycinin/glycinin group was slightly damaged,whereas that of cells in the 5 and 10 mg·mL-1β-conglycinin/glycinin groups was severely damaged.However,the addition of 5mg·mL-1β-conglycinin/glycinin with the inhibitors（PDTC,L-NAME,SP600125,and SB202190）resulted in only slight damage to the integrity of the cytoskeleton.8.The expression of NF-κB,iNOS,JNK,and p38 mRNA and their downstream genes was increased after incubation withβ-conglycinin/glycinin（P<0.01 or P<0.05）.Treatment with the inhibitors（PDTC,L-NAME）significantly inhibited theβ-conglycinin/glycinin（P<0.01 or P<0.05）.9.β-Conglycinin/glycinin treatment increased the phosphorylation of JNK,p38,ERK,NF-κB,c-Fos,c-Jun,and IKKα/β（P<0.01 or P<0.05）.However,these levels were decreased after adding 1μmol·L-1inhibitors（PDTC,L-NAME,SP600125,and SB202190）（P<0.01 or P<0.05）.10.After addingβ-conglycinin or glycinin,the apoptosis rate of IPEC-J2 cells increased significantly（P<0.01）and decreased after adding 1μmol·L-1inhibitors（PDTC,L-NAME,SP600125,and SB202190）（P<0.01）.The experiment confirmed thatβ-conglycinin and glycinin decreased the distribution of TJ proteins,destroyed the cytoskeleton in IPEC-J2 cells,and caused cell death.After the addition of the inhibitors,theβ-conglycinin/glycinin-induced IPEC-J2 cell damage was significantly reduced.β-Conglycinin and glycinin caused damage to the IPEC-J2 cells via the MAPK/NF-κB signaling pathway.The results of this study are crucial for exploring the mechanisms underlying the allergic reactions caused by soybean antigen proteins. |
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