Diosgenin inhibits macrophage-derived inflammatory mediators through downregulation of CK2, JNK, NF-κB and AP-1 activation

https://doi.org/10.1016/j.intimp.2010.06.004Get rights and content

Abstract

Diosgenin is a precursor of steroid hormones, which can be found in several plant species. Diosgenin has been shown to have a variety of biological activities including anti-inflammatory activity, but through a mechanism that is unclear. Especially, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of diosgenin on the production of inflammatory mediators in lipopolysaccharide (LPS)/interferon γ (IFN-γ)-activated murine macrophage. Macrophages pre-exposed to diosgenin (0.1–10 μΜ) were stimulated with LPS/IFN-γ. Pretreatment with diosgenin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In addition, diosgenin inhibits production of reactive oxygen species (ROS), interleukin-1 (IL-1), and IL-6, but not that of tumor necrosis factor-α (TNF-α). Inhibition of these inflammatory mediators appears to be at the transcriptional level, since diosgenin decreased LPS/IFN-γ-induced NF-κB and AP-1 activity. Diosgenin blocked CK2 activation and phosphorylation of c-Jun NH2-terminal kinase (JNK), but not that of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of inflammatory mediators in macrophages. Taken together the present data suggest that diosgenin reduces the production of inflammatory meditators by inhibiting LPS/IFN-γ-triggered CK2, JNK, NF-κB and AP-1 activation, thereby implicating a mechanism by which diosgenin may exert its immunosuppressive effects.

Introduction

There has been increasing interest in the discovery and development of novel pharmaceuticals from plants that have the same or better immunosuppressants accompanied by less side effects. Diosgenin (Fig. 1) is a steroidal saponin found in several plants including Solanum and Dioscorea species. Previous investigations have shown that diosgenin plays an important pharmacological role as an antidiabetic activity [1], antioxidant activity [2], the ability to lower plasma cholesterol levels [3], the antineoplastic and anti-inflammatory properties [4], [5]. However, the precise mechanism of action involved in the pharmacological activities induced by diosgensin remains unclear.

Recently, it has been shown that diosgenin modulates certain aspects of acquired immunity, including the enhancement of antigen-specific IgG2a and IFN-γ expression [6]. In addition, formosanin C, a diosgenin glycoside, showed immunomodulating effects on the proliferative response of mouse lymphocytes to concanavalin A [7]. However, the effect of diosgenin on macrophage function is less well understood.

Macrophages have been shown to be important components of the host defenses [8], [9]. Peritoneal macrophages can be stimulated by a variety of agents such as IFN-γ, lipopolysaccharides, or other microbial products [10], [11], [12] and some of these have also been shown to trigger the release of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, reactive oxygen species (ROS) and nitric oxide (NO) by the macrophages [13], [14], [15]. However, overexpression of inflammatory mediators such as cytokines and free radicals has been implicated in the pathogenesis of many disease processes. The control of macrophage overproduction of these mediators should greatly facilitate the treatment of many immunoinflammatory diseases such as septic shock, rheumatoid arthritis and autoimmune diabetes [16], [17]. In macrophages, inflammatory mediators are regulated primarily at the level of mRNA expression via the involvement of transcription factors such as NF-κB and AP-1. A lot of studies have shown that different signaling pathways participate in the activation of macrophages by various stimuli [18], [19], [20], [21]. Previous studies have demonstrated that the NF-κB, AP-1 and mitogen-activated protein kinase (MAPK) pathway mediate the activation of macrophages [22], [23], [24], [25]. In addition, MAPKs play an important role in the signal transduction pathways that regulate the response of macrophages to external stimuli including LPS/IFN-γ [18], [19], [20], [21], [22], [23], [24], [25]. However, the cellular signaling and molecular mechanisms responsible for pharmacological activity by diosgenin are not known in macrophages.

In the present study, we determine the effect of diosgenin on in vitro production of inflammatory mediators in order to gain a further insight into the mechanisms whereby this compound may mediate its immunosuppressive action in vivo.

Section snippets

Mice, chemicals and reagents

The C57BL/6 male mice (6–8 weeks old, 17–21 g) were obtained from Charles River Breeding Laboratories (Atsugi, Japan). Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Lipofetamine Plus, RPMI 1640 medium and fetal bovine serum were purchased from Life Technologies, Inc. (Carlsbad, CA). pGL3-NF-κB and the luciferase assay system were obtained from Promega (Madison, WI). pGL2-AP-1(PMA)-TA-luciferase and pCMV-β-gal were purchased from Clontech (Palo

Effect of diosgenin on NO production and iNOS expression in LPS/IFN-γ-stimulated macrophages

The effect of diosgenin pretreatment on LPS/IFN-γ-induced NO production in murine peritoneal macrophage or RAW264.7 cells was examined (Fig. 2). MTT assay showed that digosgenin was not cytotoxic to primary cultures of macrophages in concentrations from 0.1 to 40 μM, but concentrations > 20 μM were found to be cytotoxic to RAW264.7 cells (data not shown). We then selected three concentrations of diosgenin (0.1, 1 and 10 μM) for this in vitro study. Pretreatment of cells with diosgenin 2 h before

Discussion

The present study elucidates the ability and molecular mechanisms of diosgenin in inhibition of LPS/IFN-γ-induced inflammatory mediator production in macrophage. The inhibition by diosgenin of the LPS/IFN-γ-stimulated expressions of these molecules was not attributable to diosgenin cytotoxicity, as assessed by MTT assay and the expression of the housekeeping genes, GAPDH and ß-actin. To our knowledge, this is the first report showing the inhibitory effect of diosgenin on macrophage function.

It

Acknowledgement

This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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