Involvement of PKA signalling in anti‐inflammatory effects of chitosan oligosaccharides in IPEC‐J2 porcine epithelial cells

Weaning is characterized by intestinal inflammation, which is a big challenge in pig industry. Control of intestinal inflammation is important for improvement of growth performance and health. Therefore, the study was focused on the anti‐inflammatory activity of low‐molecular‐weight chitosan oligosaccharide (LCOS) in a porcine small intestinal epithelial cell line (IPEC‐J2). The results showed that TNF‐α, as inflammation inducer, significantly upregulated the mRNA expression of IL‐8 and MCP‐1. Afterwards, LCOS significantly attenuated mRNA expression of IL‐8 and MCP‐1 induced by TNF‐α in the cells. Mannose (MAN), as ligand of mannose receptor, had no effect on the anti‐inflammatory activity of LCOS, which suggested that mannose receptor may not involve in the anti‐inflammatory activity of LCOS in IPEC‐J2 cells. Interestingly, N‐[2‐(p‐bromocinnamylamino)ethyl]‐5‐isoquinolinesulfonamide 2HCl hydrate (H89), as PKA (protein kinase A)‐specific inhibitor, reversed the mRNA expression of IL‐8 when co‐cultured with LCOS. Furthermore, LCOS concentration dependent downregulated the mRNA expression of claudin‐1 compared with TNF‐α treatment. However, the trans‐epithelial electric resistance (TEER) was not affected by LCOS when co‐cultured with TNF‐α in 3 hr. In conclusion, LCOS have a potent anti‐inflammatory activity, and as a feed additives, may be useful for the inhibition of inflammatory process in weaning period of pigs with intestinal inflammation occurring.     

Eleutheroside B increase tight junction proteins and anti‐inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC‐J2)

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti‐inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC‐J2). The IPEC‐J2 cells were inoculated into 96‐well plates at a density of 5 × 10³ cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20 mg/ml for 48 hr. Then, 0.10 mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC‐J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin‐6 (IL‐6), interferon‐γ (INF‐γ), and tumour necrosis factor‐α (TNF‐α). The supplementation of EB in IPEC‐J2 cells increased tight junction protein expression and anti‐inflammatory cytokines, interleukin 10 (IL‐10) and transforming growth factor beta (TGF‐β). In addition, the western blotting and real‐time quantitative polymerase chain reaction (RT‐qPCR) results indicated that EB significantly (p < 0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin‐3, Occludin, and Zonula Occludins protein‐1 (ZO‐1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti‐inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC‐J2 cells.     

Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells

High oxalate consumption has been recognized as a risk factor for renal calcium oxalate stones in companion animals (dogs and cats). However, the cellular signaling involved in oxalate-induced dysfunction in renal tubular epithelial cells remains not fully elucidated. In this study, Mardin–Darby canine kidney (MDCK) cells, an epithelial cell line derived from canine kidney tubule, were tested for cell proliferation activity and barrier function after being exposed to sodium oxalate (NaOx). Further, the involvement of Wnt/β-catenin in NaOx-induced renal epithelial barrier dysfunction was evaluated. MDCK cells treated with NaOx exhibited reduction in cell proliferation and migration. Besides, NaOx exposure led to a decrease in transepithelial electrical resistance and an increase in paracellular permeability. The deleterious effects of NaOx on epithelial barrier function were related to the suppressed abundance of tight junction proteins including zonula occludens, occludin, and claudin-1. Of note, protein levels of β-catenin and phosphorylated (p)-β-catenin (Ser552) in MDCK cells were repressed by NaOx, indicating inhibitory effects on Wnt/β-catenin signaling. An inhibition of glycogen synthase kinase-3β (GSK-3β) by SB216763 enhanced the abundance of β-catenin and p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in NaOx-treated MDCK cells. The results revealed a critical role of Wnt/β-catenin signaling in the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be a potential therapeutic target for the treatment of oxalate-linked renal stones.