Icariin inhibits TNF-α/IFN-γ induced inflammatory response via inhibition of the substance P and p38-MAPK signaling pathway in human keratinocytes

Pro-inflammatory cytokines play a crucial role in the etiology of atopic dermatitis. We demonstrated that Herba Epimedii has anti-inflammatory potential in an atopic dermatitis mouse model; however, limited research has been conducted on the anti-inflammatory effects and mechanism of icariin, the major active ingredient in Herba Epimedii, in human keratinocytes. In this study, we evaluated the anti-inflammatory potential and mechanisms of icariin in the tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma)-induced inflammatory response in human keratinocytes (HaCaT cells) by observing these cells in the presence or absence of icariin. We measured IL-6, IL-8, IL-1 beta, MCP-1 and GRO-alpha production by ELISA; IL-6, IL-8, IL-1 beta, intercellular adhesion molecule-1 (ICAM-1) and tachykinin receptor 1 (TACR1) mRNA expression by real-time PCR; and P38-MAPK, P-ERK and P-JNK signaling expression by western blot in TNF-alpha/IFN-gamma-stimulated HaCaT cells before and after icariin treatment. The expression of INF-alpha-R1 and IFN-gamma-R1 during the stimulation of the cell models was also evaluated before and after icariin treatment. We investigated the effect of icariin on these pro-inflammatory cytokines and detected whether this effect occurred via the mitogen-activated protein kinase (MAPK) signal transduction pathways. We further specifically inhibited the activity of two kinases with 20 mu M SB203580 (a p38 kinase inhibitor) and 50 mu M PD98059 (an ERK1/2 kinase inhibitor) to determine the roles of the two signal pathways involved in the inflammatory response. We found that icariin inhibited TNF-alpha/IFN-gamma-induced IL-6, IL-8, IL-1 beta, and MCP-1 production in a dose-dependent manner; meanwhile, the icariin treatment inhibited the gene expression of IL-8, IL-1 beta, ICAM-1 and TACR1 in HaCaT cells in a time- and dose-dependent manner. Icariin treatment resulted in a reduced expression of p-P38 and p-ERK signal activation induced by TNF-alpha/IFN-gamma; however, only SB203580, the p38 alpha/beta inhibitor, inhibited the secretion of inflammatory cytokines induced by TNF-alpha/IFN-gamma in cultured HaCaT cells. The differential expression of TNF-alpha-R1 and IFN-gamma-R1 was also observed after the stimulation of TNF-alpha/IFN-gamma, which was significantly normalized after the icariin treatment. Collectively, we illustrated the anti-inflammatory property of icariin in human keratinocytes. These effects were mediated, at least partially, via the inhibition of substance P and the p38-MAPK signaling pathway, as well as by the regulation of the TNF-alpha-R1 and IFN-gamma-R1 signals. (C) 2015 Elsevier B.V. All rights reserved.