Anti-inflammation response of astaxanthin on inhibiting nuclear factor kappa B activation

Abstract

minimize the ‘‘variations’’ during manufacturing. Glycosaminoglycans (GAGs) and proteoglycans are one of the major components of extracellular matrix including basement membrane in oral mucosa and havemany biological functions. Thus, they play an important role in homeostasis of the epithelium. Recently, CXylopyranoside derivatives is reported to increase GAGs and PGs biosynthesis and improve tissue morphogenesis in a skin organotypic culture model. Purpose: We hypothesized that one of the C-Xylopyranoside derivatives b-D-xylopyranoside-n-propane-2-one (XPP) stimulates the ability to produce basement membrane related molecules such as basement membrane components and integrins in oral mucosa keratinocytes and fibroblasts, resulting in improvement of epithelial structure. The present study aimed to assess the effects of XPP on the expression level of GAGs and basement membrane related molecules produced by oral keratinocytes and fibroblasts, and the differences of histological appearance in a reconstructed oral mucosa model. Methods: Primary oral keratinocytes and fibroblasts were isolated and serially cultured in EpiLife culture medium containing 0.06mMCa (growthmedium) and Dulbecco’s Modified Eagle Medium containing 10% serum (DMEM), respectively. First, we tested the cell viability of oral keratinocytes cultured in growth medium and EpiLife containing 1.2mMCa(hereafter differentiationmedium) as well as oral fibroblasts cultured in differentiation medium and DMEM supplemented with 0, 2 and 10mM XPP for up to 48 hours using a Cell-Counting Kit. Next, total amount of GAGs produced by oral keratinocytes and fibroblasts into the culture media was quantitated using Blyscan GAG assay. Concurrently, oral keratinocytes and fibroblasts were lysed and immunoblotting was performed to detect the expression levels of basement membrane related molecules. Using an organotypic culture system, three-dimensional oralmucosamodels (3DOMM) inwhich oral keratinocytes and fibroblasts were incorporated were fixed, embedded in paraffin, and then histologically and immunohistochemically examined. Results: The concentration of 2 and 10mM XPP did not affect cell viability of oral keratinocytes and fibroblasts grown in those culture media. Blyscan assay showed the amount of GAGs secreted from both cells into those culture media significantly increased when XPP was added, particularly from keratinocytes cultured in differentiation medium. The expression level of type zW collagen, Nidogen, Integrin a6 and b1 in oral keratinocytes increased when cultured in growth medium. While the histological examinations did not showed significant differences between the XPP-treated and untreated 3DOMMs, more intense expression of laminin and type zW collagen was seen in the XPP-treated one. Moreover, basal layer cells of the XPP treated 3DOMM strongly expressed integrin a6 and b1, ligands of laminin and type zW collagen, compared with untreated 3DOMM.