IL-10-deficient mice develop chronic intestinal inflammation,
which is in part caused by a loss of suppression of the mucosal immune response toward normal intestinal bacteria . Recent studies have reported that topical treatment with IL-10 is effective in preventing certain inflammatory diseases. Moreover, probiotics can exert a therapeutic effect mediated through an IL-10-dependent mechanism . It has been shown that oral administration of probiotics can prevent inflammation and mucosal ulcerations, which are associated with up-regulation of IL-10, which inhibits the increase of the CD4+ helper T cell population and down-regulates inflammatory cytokines . Probiotics can exert immunomodulatory activities by increasing IL-10 production, which can in turn help learn more prevent an excessive immune response. However, probiotic bacteria have multiple and diverse effects in the host, and not all probiotic strains act in this manner. The C. butyricum MIYAIRI II 588 stain has been used to prevent disturbances of microflora, treat diarrhea and enhance the humoral
immune response in the human intestine . However, the mechanisms by which C. butyricum treats and prevents diarrhea remain unclear. The aim of the present study was https://www.selleckchem.com/products/MLN-2238.html to assess whether C. butyricum achieves its beneficial effects via modulation of IL-10 production. Methods Bacterial strains and culture conditions C. butyricum MIYAIRI II very 588 used in this study was obtained from Miyarisan Pharmaceutical Co. Ltd, Tokyo, Japan. This strain is a butyric-acid producing, spore-forming and gram-positive rod bacterium . It was cultured in MRS broth at 28°C in an anaerobic environment.
Cell culture HT-29 human colonic epithelial cells were purchased from the cell bank of the type culture collection of the Chinese academy of sciences (Shanghai). Enterocyte-like HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin at 37°C in a humidified atmosphere of 5% CO2. SiRNA transient transfection One day before transfection, HT-29 cells (1 × 106 cells well−1) were allowed to attach and grow in 6-well culture plates (Corning, USA). When the selleckchem plated cells in medium without antibiotics were 30–50% confluent, IL-10-specific siRNA (small interfering RNA) synthesized by Ribobio (Guangzhou, China) was transfected into cells with Lipofectamine 2000 (Invitrogen). After 48 h, cells were treated with C. butyricum and assayed for transfection efficiency by real-time PCR. IL-10 neutralization IL-10 antibody-blocking was performed as described previously . To prevent the effects of IL-10, supernatants were treated with IL-10 antibody (5 μg ml−1; HuaAn, China). These treated cells were then cultured in 6-well plates at 1 × 106 cell well−1. After 48 h, the cells were stimulated with C. butyricum.