Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

https://doi.org/10.1016/j.taap.2015.06.017Get rights and content

Highlights

  • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA).

  • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS.

  • ASK1 may be used as a potential therapeutic target in RA.

  • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS.

  • Thymoquinone might serve as a potential small-molecule inhibitor in RA.

Abstract

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by cellular infiltration and proliferation of synovium, leading to progressive destruction of the joints (Ahmed et al., 2008, Smolen and Aletaha, 2009). Antigen-activated CD4 + T cells stimulate monocytes, macrophages, and synovial fibroblasts (FLS) to produce cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). These proinflammatory cytokines are master regulator of chronic inflammation and tissue destruction in RA (Choy and Panayi, 2001, Iwamoto et al., 2008, Jones et al., 2013). In response to these cytokines, FLS produce chemokines, matrix metalloproteinases (MMPs), and adhesion molecules that further promote inflammation, hyperplasia and cartilage destruction (Ahmed et al., 2006, Vaillancourt et al., 2011, Jones et al., 2013).

TNF-α is a potent cytokine that exerts diverse effects by stimulating a variety of cells (Abdel-Aziz et al., 2013). It is mainly produced by monocytes and macrophages, but also by B-cells, T-cells, and FLS (Zhu et al., 2014). TNF-α acts as a potent inducer of inflammatory responses through up-regulation of many genes, including cytokines, chemokines, and adhesion molecules (Liang et al., 2011, Tsou et al., 2012). TNF-α binds to cell surface receptors (TNFR1) to initiate multiple signal transduction pathways, including mitogen-activated protein (MAP) kinases and nuclear factor kappa B (NF-κB) pathways (Sabio and Davis, 2014). MAP kinase pathway includes central three-tiered core signaling proteins comprising of MAP kinase kinase kinase (MAP3K), MAP kinase kinase (MAP2K), and MAP kinase (MAPK) (Kyriakis and Avruch, 2012). C-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) are well characterized sub-groups of a large MAP kinase family. MAP3Ks, as the proteins upstream in the signaling cascade, sense the degree of stress-induced cell damage and determine cell fate by regulation of the downstream MAP kinase pathways (Cuevas et al., 2007). Apoptosis signal regulating kinase 1 (ASK1) is an important member of MAP3K family that activates both the JNK and p38 MAPK pathways in response to TNF-α stimulation (Nishitoh et al., 1998). ASK1 is activated by various types of stress, including oxidative stress, endoplasmic reticulum (ER) stress, calcium overload, and inflammatory cytokines such as TNF-α (Mnich et al., 2010, Philippe et al., 2013). However, the role of ASK1 in TNF-α signaling pathway to regulate IL-6 and IL-8 production, or the expression adhesion molecules in RA-FLS is still unknown.

Thymoquinone (TQ) is the major active compound derived from Nigella sativa (Woo et al., 2012). Recent animal studies support the potential of TQ for the treatment of a variety of inflammatory disorders like inflammatory bowel disease (IBD), RA, and osteoarthritis (OA) (Salem, 2005, Badr et al., 2011). We have previously shown that oral administration of TQ (5 mg/kg/day) significantly reduced the serum levels of IL-1β and TNF-α as well as a number of inflammatory mediators involved in RA pathogenesis (Umar et al., 2012). In this study, we evaluated the intracellular signaling mechanism by which TQ inhibits TNF-α-induced IL-6 and IL-8 production, and the expression of ICAM-1, VCAM-1, and cadherin-11 (Cad-11) in human RA-FLS.

Section snippets

Antibodies and reagents

Recombinant human TNF-α, goat polyclonal antibodies against human ICAM-1 and VCAM-1, IL-6 and IL-8 Duoset ELISA kits, and ASK1 inhibitor (TC ASK10) were purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibodies against phosphorylated ERK1/2, JNK/SAPK, and p38, cadherin-11, and anti-rabbit and anti-mouse horseradish peroxide-linked secondary antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Mouse anti p-ASK1 (Thr845) and TRAF-2 antibodies were purchased

Effect of TQ on RA-FLS viability

The results of an MTT-based viability assay showed that TQ (0.1–10 μM) had only modest effect on the cell viability of cultured RA-FLS (Fig. 1A; n = 4). We observed that TQ in combination with TNF-α also had slight change in the viability of RA-FLS in vitro (Fig. 1B; n = 4). Furthermore, we studied the effects of TQ on expression of Mcl-1, as its overexpression in RA-FLS is a major cause of their resistance to TNF-α-induced apoptosis (Ahmed et al., 2009). We found that the upregulating expression of

Discussion

Rheumatoid arthritis (RA) is an autoimmune disease that leads to inflammation and destruction of synovial joints (Jones et al., 2013). Curing RA is still out of our reach, despite the broad spectrum of anti-rheumatic drugs (Koenders and van den Berg, 2015). The inflammatory process is mediated through a complex cytokine network which is not yet completely understood. Current treatment strategies for RA include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying

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Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This study was supported by the NIH grant AR063104 (S.A.), the Arthritis Foundation Innovative Research Grant (S.A.), the start-up funds from Washington State University (S.A.), and the ASPET summer undergraduate research fellowship (SURF) award (O.H.). Authors also thank the National Disease Research Interchange (NDRI), Philadelphia and Co-operative Human Tissue Network (CHTN) for providing the synovial tissue for research.

References (37)

  • G. Sabio et al.

    TNF and MAP kinase signalling pathways

    Semin. Immunol.

    (2014)
  • M.L. Salem

    Immunomodulatory and therapeutic properties of the Nigella sativa L. seed

    Int. Immunopharmacol.

    (2005)
  • S. Umar et al.

    Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats

    Chem. Biol. Interact.

    (2012)
  • M. Valko et al.

    Free radicals, metals and antioxidants in oxidative stress-induced cancer

    Chem. Biol. Interact.

    (2006)
  • C.C. Woo et al.

    Thymoquinone: potential cure for inflammatory disorders and cancer

    Biochem. Pharmacol.

    (2012)
  • P. Adiseshaiah et al.

    A JNK-independent signaling pathway regulates TNF alpha-stimulated, c-Jun-driven FRA-1 protooncogene transcription in pulmonary epithelial cells

    J. Immunol.

    (2006)
  • S. Ahmed et al.

    Biological basis for the use of botanicals in osteoarthritis and rheumatoid arthritis: a review

    Evid. Based Complement. Alternat. Med.

    (2005)
  • S. Ahmed et al.

    Regulation of interleukin-1beta-induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts

    Arthritis Rheum.

    (2006)
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