Thymoquinone TQ is the major active compound derived from

Thymoquinone (TQ) is the major active 568 australia 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 (5mg/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.
Materials and methods
Results
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 anti-rheumatic drugs (DMARDS) and biologic response modifiers (Ahmed et al., 2005). Despite the availability of biological therapies to treat RA, rigors ongoing research for orally active small-molecule drugs are highly desirable to fulfill unmet medical need. p38 mitogen-activated protein kinases (MAPKs) in particular, have attracted considerable attention as potential therapeutic targets due to their ability to suppress the production of key inflammatory mediators (Thalhamer et al., 2008). The success of first p38 inhibitors to advance to phase IB clinical trials was hampered by hepatotoxicity and preclinical safety studies (Hammaker and Firestein, 2010). Apoptosis signal regulating kinase 1 (ASK1) is a member of MAP3K family that activates both the JNK and p38 MAPK pathways through a variety of mechanism, including oxidative stress, ER stress, calcium overload, and inflammatory cytokines such as tumor necrosis factor α (TNF-α) (Matsuzawa and Ichijo, 2008, Choi et al., 2011). Our finding suggests that ASK1 may serve as a promising therapeutic target in the regulation of pro-inflammatory cytokines release in autoimmune disorders especially in RA. TNF-α is a major pathologic mediator and a target of anti TNF-α therapies for RA (Choy and Panayi, 2001). In this study, we further identified TQ as a therapeutic molecule that regulates TNF-α signaling pathways to suppress the inflammatory cellular response. ASK1 activity is controlled by phosphorylation and interactions with other proteins (Yu et al., 2009). A previous study showed that ROS induces dephosphorylation of ASK1 at Ser967 as well as phosphorylation of Thr845 in the ASK1 activation loop, both of which are correlated with ASK1 activity (Valko et al., 2006). Here, we found that TNF-α enhanced phosphorylation of ASK1 at Thr845 and TQ was effective in inhibiting that phosphorylation, thereby decelerating downstream signaling pathways. TNF-α signaling also relies on TRAF-2 association with ASK1 in order to phosphorylate it in a TNF-α-dependent manner (Nishitoh et al., 1998). Our immunoprecipitation and immunofluorescence results also confirmed that TQ does not alter TRAF-2 and ASK1 interaction, but effectively inhibits ASK1 activation in RA-FLS. Using ASK1 inhibitor (TC-ASK10), we also validate a novel regulatory role of ASK1 in TNF-α-induced expression of adhesion molecules (ICAM-1/VCAM-1 and Cad-11). Our present study may have two-pronged relevance in the field of RA pathogenesis. First, we show that the therapeutic regulation of ASK1 is important in regulating adhesion molecules that are relevant in disease pathogenesis (Lee et al., 2007, Chang and Karin, 2001). Second, we also provide an evidence that TQ, a natural phytochemical, which inhibits ASK1 activation may have potential for its testing in pre-clinical models of RA and further development in this regard.