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