MicroRNA-223 Inhibits Soybean Glycinin- and ß-Conglycinin-Induced Apoptosis of IPEC-J2 Cells by Targeting NLRP-3 in the IEL/IPEC-J2 Co-culture System.

The apoptosis of intestinal porcine epithelial cells induced by soybean antigen protein allergy is one of the most important mechanisms responsible for enteritis. MicroRNAs (miRNAs) affect the cellular and physiological functions of all multicellular organisms. We hypothesize that microRNA-223 inhibits soybean glycinin- and ß-conglycinin-induced apoptosis of intestinal porcine enterocytes (IPEC-J2) by targeting the NLR family pyrin domain containing 3 (NLRP-3). Using the intestinal interepithelial lymphocyte (IEL)/IPEC-J2 co-culture system as an in vitro model, we investigate the role of microRNA-223 in the regulation of soybean glycinin- and ß-conglycinin-induced apoptosis. In co-cultured IEL/IPEC-J2 cells incubated with glycinin or ß-conglycinin, microRNA-223 decreased NLRP-3, ASC, caspase-1, caspase-3, FAS, BCL-2, and APAF-1 expressions in IPEC-J2 cells; decreased cytokine and cyclooxygenase-2 levels; significantly increased cell activity; and inhibited apoptosis. These data supported a novel antiallergic mechanism to mitigate the sensitization of soybean antigenic protein, which involves the upregulation of microRNA-223-targeting NLRP-3.

Soybean glycinin and ß-conglycinin damage the intestinal barrier by triggering oxidative stress and inflammatory response in weaned piglets.

PURPOSE: Soybean glycinin (11S) and ß-conglycinin (7S) are major antigenic proteins in soybean and can induce a variety of allergic reactions in the young animals. This study aimed to investigate the effect of 7S and 11S allergens on the intestine of piglets. METHODS: Thirty healthy 21-day-old weaned "Duroc × Long White × Yorkshire" piglets were randomly divided into three groups fed with the basic diet, the 7S supplemented basic diet, or the 11S supplemented basic diet for 1 week. Allergy markers, intestinal permeability, oxidative stress, and inflammatory reactions were detected, and we observed different sections of intestinal tissue. The expressions of genes and proteins related to NOD-like receptor thermal protein domain associated protein 3 (NLRP-3) signaling pathway were detected by IHC, RT-qPCR, and WB. RESULTS: Severe diarrhea and decreased growth rate were observed in the 7S and 11S groups. Typical allergy markers include IgE production and significant elevations of histamine and 5-hydroxytryptamine (5-HT). More aggressive intestinal inflammation and barrier dysfunction were observed in the experimental weaned piglets. In addition, 7S and 11S supplementation increased the levels of 8-hydroxy-2 deoxyguanosine (8-OHdG) and nitrotyrosine, triggering oxidative stress. Furthermore, higher expression levels of NLRP-3 inflammasome ASC, caspase-1, IL-1ß, and IL-18 were observed in the duodenum, jejunum, and ileum. CONCLUSION: We confirmed that 7S and 11S damaged the intestinal barrier of weaned piglets and may be associated with the onset of oxidative stress and inflammatory response. However, the molecular mechanism underlying these reactions deserves further study.

11S Glycinin Up-Regulated NLRP-3-Induced Pyroptosis by Triggering Reactive Oxygen Species in Porcine Intestinal Epithelial Cells.

11S glycinin is a major soybean antigenic protein, which induces human and animal allergies. It has been reported to induce intestinal porcine epithelial (IPEC-J2) cell apoptosis, but the role of pyroptosis in 11S glycinin allergies remains unknown. In this study, IPEC-J2 cells were used as an in vitro physiological model to explore the mechanism of 11S glycinin-induced pyroptosis. The cells were incubated with 0, 1, 5, and 10 mg·ml-1 11S glycinin for 24 h. Our results revealed that 11S glycinin significantly inhibited cell proliferation, induced DNA damage, generated active oxygen, decreased mitochondrial membrane potential, and increased the NOD-like receptor protein 3 (NLRP-3) expression of IPEC-J2 cells in a dose-dependent manner. Further, IPEC-J2 cells were transfected with designed sh-NLRP-3 lentivirus to silence NLRP-3. The results showed that 11S glycinin up-regulated the silenced NLRP-3 gene and increased the expression levels of apoptosis-related spot-like protein (ASC), caspase-1, the cleaved gasdermin D, and interleukin-1ß. The IPEC-J2 cells showed pyrolysis morphology. Moreover, we revealed that N-acetyl-L-cysteine can significantly inhibit the production of reactive oxygen species and reduce the expression levels of NLRP-3 and the cleaved gasdermin D. Taken together, 11S glycinin up-regulated NLRP-3-induced pyroptosis by triggering reactive oxygen species in IPEC-J2 cells.

Astragalus polysaccharide enhances the immune function of RAW264.7 macrophages via the NF-κB p65/MAPK signaling pathway.

The aim of the present study was to investigate the immunoregulatory effects of Astragalus polysaccharide (APS) on RAW264.7 cells. The production of cytokines by RAW264.7 cells was analyzed using ELISA, while cell viability and optimal concentration of APS were assessed using the Cell Counting Kit-8 assay. In addition, the mRNA levels of IL-6, inducible nitric oxide synthase (iNOS) and TNF-α were determined by reverse transcription-quantitative PCR analysis. The levels of co-stimulatory molecules and cell cycle distribution were assessed by flow cytometry. Electrophoretic mobility shift assay was used to determine the effects of APS on p65 expression. Compared with controls, APS enhanced the production of NO, the gene expression of TNF-α, IL-6 and iNOS and the protein levels of phosphorylated p65, p38, Jun N-terminal kinase and extracellular signal regulated kinase in RAW264.7 cells, whereas these effects of APS were alleviated by pyrrolidine dithiocarbamate. The results of the present study indicated that the immunoregulatory effects of APS are mediated, at least in part, via the activation of the NF-κB p65/MAPK signaling pathway.

Chicken serum uric acid level is regulated by glucose transporter 9.

OBJECTIVE: Glucose transporter 9 (GLUT9) is a uric acid transporter that is associated with uric absorption in mice and humans; but it is unknown whether GLUT9 involves in chicken uric acid regulation. This experiment aimed to investigate the chicken GLUT9 expression and serum uric acid (SUA) level. METHODS: Sixty chickens were divided into 4 groups (n = 15): a control group (NC); a sulfonamide-treated group (SD) supplemented with sulfamonomethoxine sodium via drinking water (8 mg/L); a fishmeal group (FM) supplemented with 16% fishmeal in diet; and a uric acid-injection group (IU), where uric acid (250 mg/kg) was intraperitoneally injected once a day. The serum was collected weekly to detect the SUA level. Liver, kidney, jejunum, and ileum tissues were collected to detect the GLUT9 mRNA and protein expression. RESULTS: The results showed in the SD and IU groups, the SUA level increased and GLUT9 expression increased in the liver, but decreased in the kidney, jejunum, and ileum. In the FM group, the SUA level decreased slightly and GLUT9 expression increased in the kidney, but decreased in the liver, jejunum, and ileum. Correlation analysis revealed that liver GLUT9 expression correlated positively, and renal GLUT9 expression correlated negatively with the SUA level. CONCLUSION: These results demonstrate that there may be a feedback regulation of GLUT9 in the chicken liver and kidney to maintain the SUA balance; however, the underlying mechanism needs to be investigated in future studies.

Mechanism of deoxynivalenol-induced neurotoxicity in weaned piglets is linked to lipid peroxidation, dampened neurotransmitter levels, and interference with calcium signaling.

Deoxynivalenol（DON） has broad toxicity in livestock, but we know little about its neurotoxic mechanisms. We investigated DON neurotoxicity in the cerebral cortex, cerebellum, and hippocampus of "Duroc × Landrace × Yokshire" piglets. Control piglets were fed a basal diet, while those in low- and high-treatment groups were fed diets with 1.3 mg/kg and 2.2 mg/kg DON, respectively. After a 60 d trial, scanning electron microscopy revealed the destruction of hippocampal cell ultrastructure. As DON concentrations increased, oxidative damage also increased in the cerebral cortex, cerebellum, and hippocampus. Norepinephrine and 5-hydroxytryptamine concentrations tended to increase, whereas dopamine and γ-aminobutyric acid concentrations decreased. We also observed an increase in calcium concentration, relative mRNA expression of calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII phosphorylation. However, calmodulin (CaM) mRNA and protein content decreased. Overall, our results suggest that DON acts through the Ca2+/CaM/CaMKII signaling pathway to influence cerebral lipid peroxidation and neurotransmitter levels.

ß-Conglycinin-Induced Intestinal Porcine Epithelial Cell Damage via the Nuclear Factor κB/Mitogen-Activated Protein Kinase Signaling Pathway.

Soybean allergy is a serious health risk to humans and animals; ß-conglycinin is the primary antigenic protein in soybean. Intestinal porcine epithelial (IPEC-J2) cells were used as an in vitro physiological model of the intestinal epithelium to study the effects of different concentrations of soybean antigen protein ß-conglycinin to identify the involved signaling pathways. The cells were divided into eight groups and either untreated or treated with different concentrations of ß-conglycinin, pyrrolidine dithiocarbamate (PDTC), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), SP600125, and SB202190 either alone or in combination. The cells were incubated with 1, 5, and 10 mg·mL-1 ß-conglycinin or 5 mg·mL-1 ß-conglycinin and 1 µmol·L-1 nuclear factor κB (NF-κB) inhibitor (PDTC), inducible nitric oxide synthase inhibitor (l-NAME), c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and p38 inhibitor (SB202190) for 24 h, separately; controls were left untreated. The mRNA, protein, and phosphorylation levels of NF-κB, p38, and JNK were higher in the treated groups than in the control group. ß-Conglycinin decreased tight junction distribution, destroyed the cytoskeleton of IPEC-J2 cells, and caused cell death. After the addition of the inhibitors, ß-conglycinin-induced IPEC-J2 cell damage was significantly reduced. ß-Conglycinin caused damage to IPEC-J2 cells via the mitogen-activated protein kinase/NF-κB signaling pathway. The results of this study are crucial for exploring the mechanisms underlying allergic reactions caused by soybean antigen proteins.

Uric acid transporters BCRP and MRP4 involved in chickens uric acid excretion.

BACKGROUND: Breast cancer resistance protein (BCRP) and multidrug resistance protein 4 (MRP4) are involved in uric acid excretion in humans and mice. Despite evidence suggesting that renal proximal tubular epithelial cells participate in uric acid excretion in chickens, the roles of BCRP and MRP4 therein remain unclear. This study evaluated the relationship between BCRP and MRP4 expression and renal function in chickens. RESULTS: Sixty laying hens were randomly divided into four treatment groups: a control group (NC) fed a basal diet; a sulfonamide-treated group (SD) fed the basal diet and supplemented with sulfamonomethoxine sodium via drinking water (8 mg/L); a fish meal group (FM) fed the basal diet supplemented with 16% fishmeal; and a uric acid injection group (IU) fed the basal diet and intraperitoneally injected with uric acid (250 mg/kg body weight). The results showed that serum uric acid, creatinine, and blood urea nitrogen levels were significantly higher in the SD and IU, but not FM, than in the NC groups. Renal tubular epithelial cells in the SD and IU groups were damaged. Liver BCRP and MRP4 mRNA and protein levels were significantly decreased in the SD and IU groups, but slightly increased in the FM group. In the SD group, BCRP and MRP4 were significantly increased in the ileum and slightly increased in the kidney. In the FM group, BCRP and MRP4 were significantly increased in the kidney and slightly increased in the ileum. In the IU group, BCRP and MRP4 were significantly increased in the kidney and ileum. BCRP and MRP4 expression in the jejunum was not affected by the treatments. CONCLUSION: Together, these results demonstrate that BCRP and MRP4 are involved in renal and intestinal uric acid excretion in chickens and that BCRP is positively related to MRP4 expression. Further, impairment of renal function results in an increase in serum uric acid as well as a compensatory increase in BCRP and MRP4 in the ileum; however, under normal renal function, renal BCRP and MRP4 are the main regulators of uric acid excretion.

Effects of 7S and 11S on the intestine of weaned piglets after injection and oral administration of soybean antigen protein.

Soybeans are used increasingly in food products because of their health benefits. In this study, we investigated the effect of soybean antigen protein on weaned piglet intestine. Seventy piglets were randomly divided into seven groups with 10 piglets each. At 7 and 14 days of age, groups A-C were injected with saline, and D-G were intramuscularly injected with or orally administered 7S or 11S. Groups B-G were artificially sensitized by dietary 7S or 11S. At 27 days, the small intestinal tissues were collected to determine levels of histamine, sIgA protein, and IgA mRNA. Histamine in B-G was significantly decreased in the duodenum and ileum. Moreover, sIgA expression was higher in all groups than in A, with B/C>D-G and F/G>D/E; the trend in IgA expression was similar. Collectively, these results indicated that soybean antigen protein-immunizing agents decrease sIgA and IgA levels. Additionally, the effect of injection immunization occurred prior to that of oral immunization.

Soybean Glycinin- and ß-Conglycinin-Induced Intestinal Damage in Piglets via the p38/JNK/NF-κB Signaling Pathway.

ß-Conglycinin (7S) and glycinin (11S) are known to induce a variety of hypersensitivity reactions involving the skin, intestinal tract, and respiratory tract. The present study aimed to identify the mechanism underlying the development of allergy to soybean antigen proteins, using piglets as an animal model. Weaned "Duroc × Landrace × Yorkshire" piglets were fed a diet supplemented with 7S or 11S to investigate the signaling pathway involved in intestinal damage in piglets. Results showed that serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and caspase-3 levels were significantly higher in 7S- and 11S-fed piglets compared to those in suckling or weaned ones. mRNA, protein, and phosphorylation levels of nuclear factor-kappa B (NF-κB), p38, and Jun N-terminal kinase (JNK) were higher in 7S- and 11S-fed piglets than in suckling and weaned ones. Overall, our results indicate that 7S and 11S damaged the intestinal function in piglets through their impact on NF-κB, JNK, and p38 expression.