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Ameliorating effect of TI-1-162, a hydroxyindenone derivative, against TNBS-induced rat colitis is mediated through suppression of RIP/ASK-1/MAPK signaling

https://doi.org/10.1016/j.ejphar.2018.03.027Get rights and content

Abstract

The pathogenesis of inflammatory bowel disease (IBD) is associated with production of immense pro-inflammatory cytokines including TNF-α. Once generated, TNF-α stimulates production of various pro-inflammatory cytokines and disrupts mucosal barrier by inducing inflamed mucosal epithelial cell death. In the present study, we investigated inhibitory effects of TI-1-162, a hydroxyindenone derivative, against TNF-α-induced and TNBS-induced colon inflammation. TI-1-162 showed inhibitory effect on the TNF-α-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells (IC50 = 0.83 ± 0.12 μM), which is an in vitro model representing the initial step of colitis. In addition, TI-1-162 suppressed TNF-α-stimulated caspase-3 activation and HT-29 cell apoptosis. These in vitro inhibitory activities of TI-1-162 correlated to recovery changes in in vivo colon tissues, such as downregulation of adhesion molecules (ICAM-1, VCAM-1) and chemokines (CCL11, CXCL1, CXCL2, CXCL3, CX3CL1) revealed by gene expression array and Western blot analyses. Such molecular recovery of colon epithelium from TNBS-treated rats corresponded to the recovery in body weight, colon weight/length, and myeloperoxidase level by TI-1-162 (10 and 30 mg/kg/day, orally). In relation to action mechanism, TI-1-162 did not disturb TNF-α binding to its receptor, but suppressed phosphorylation of RIP-1, ASK-1, JNK and p38, and nuclear translocation of NF-kB and AP-1, which corresponded to down regulation of inflammatory cytokines in TNF-α-treated cells (HT-29 and U937) and TNBS-treated rat colon tissues. Taken together, the results indicate that the protective effects of TI-1-162 against colon inflammation and epithelial cell death are associated with its inhibitory action in RIP/ASK-1/MAPK signaling pathway downstream to TNF receptor 1.

Introduction

Inflammatory Bowel Disease (IBD), encompassing Crohn's Disease (CD) and Ulcerative colitis (UC), describes chronic and progressive inflammation of the digestive tract (Yapali, 2007). Both CD and UC usually involve severe diarrhea, abdominal pain, fatigue and weight loss, which may lead to debilitating complications. The etiology of IBD has not been completely revealed, however, aberrant and prolonged immune response triggered by genetic and environmental factors are considered as the major etiology of the disease (Danese et al., 2004, Rocchi et al., 2012). IBD is often characterized by an infiltration of mucosal immune cells (macrophages, neutrophils and lymphocytes) into the intestinal tissue producing a wide range of pro-inflammatory cytokines, resulting in imbalance between pro- and anti-inflammatory cytokines (Neurath, 2014a, Rogler and Andus, 1998). The increased levels of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IFN-γ are mostly responsible for the pathogenesis of IBD (Schreiber et al., 1995, Zhang and Li, 2014).

In the inflamed mucosa of patients with IBD, TNF-α exerts various functions such as stimulating production of other pro-inflammatory cytokines and chemokines, disrupting barrier function, and promoting cell death of intestinal epithelial cells (Neurath, 2014a, Parameswaran and Patial, 2010). Binding of TNF-α to its receptor (TNFR) induces receptor trimerization and recruits downstream adaptor molecules, such as TNF receptor type 1-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), and receptor interacting protein-1 (RIP-1). In turn, RIP-1 recruits mitogen-activated protein kinase kinase and p38 to activate inhibitor of κB kinase (IKK) complex which phosphorylates IκB leading to IκB degradation and consequent nuclear translocation of NF-κB (Blüml et al., 2012, Parameswaran and Patial, 2010). Germinal center kinase (GCK) coupled to TRAF2 also activates mitogen-activated protein kinase/ERK kinase kinase 1, Jun N-terminal kinase (JNK), and p38 (Sethu and Melendez, 2011, Yuasa et al., 1998). Apoptosis-signaling kinase-1 (ASK-1) is also activated either by interaction with TRAF2 and TRADD or by reactive oxygen species generated after TNF receptor 1 ligation, which ultimately leads to activation of JNK and AP-1 (Geering et al., 2011, Geering and Simon, 2011, Papa et al., 2009, Parameswaran and Patial, 2010, Westwick et al., 1994). In addition, recruitment of TRADD and Fas associated protein with death domain (FADD) by trimerized TNF receptor 1 forms death-inducing signaling complex (DISC) to activate pro-caspase-8 leading to activation of apoptosis executioner, caspase-3 (Hsu et al., 1996, Sedger and McDermott, 2014).

Reflecting the critical role of TNF-α in the pathogenesis of IBD, biological drugs such as infliximab and adalimumab targeting and blocking TNF-α action have become a mainstay in the therapy of IBD over other orally available drugs such as aminosalicylate, glucocorticoids, and immunosuppressive drugs. However, the biological drugs have a high level of economic burden and low degree of administration compliance. In addition, the biological drugs exhibit deleterious side effects (Bernstein, 2015). In the current treatment regimens for IBD, it is required to develop orally available new drugs to have better efficacy including epithelium recovery and less side effect. Previously, we have investigated 2-benzylidene-2,3-dihydro-1H-inden-1-one and benzofuran-3(2H)-one derivatives for their structure-activity relationships (SAR) against TNF-α action (Kadayat et al., 2017). According to our previous study, TI-1–162 seems to be a promising lead compound for IBD drug development. However, no data exist that shed light on its efficacy and action mechanism. In the current study, we examined protective effects of TI-1-162 against inflammation and epithelial apoptosis in in vitro and in vivo IBD models, and its mode of action.

Section snippets

Materials

Unless otherwise stated, all the materials were purchased from Sigma-Aldrich (St. Louis, MO, U.S.). RPMI-1640, fetal bovine serum (FBS), penicillin/streptomycin and trizol reagent were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Trypsin/EDTA was purchased from Clonetics, Inc. (Walkersville, MD, USA). Antibodies directed against TNF-α, IL-1β, ICAM-1, VCAM-1, β-actin and Lamin B were purchased from Abcam (Cambridge, MA, USA) whereas antibodies of phospho-I-κB, I-κB and c-Fos

Inhibitory effects of TI-1-162 on monocyte-epithelial cell adhesion and TNF receptor down-stream signaling

TI-1-162 inhibited TNF-α-induced adhesion of U937 monocytic cells to HT-29 colonic epithelial cells in a concentration-dependent manner with IC50 of 0.83 ± 0.12 μM (Fig. 1B and Supplementary Fig. S1), which is about three orders of magnitude better than that of 5-aminosalicylic acid (5-ASA). To determine whether TI-1-162 directly inhibits TNF-α binding to TNF receptor, we performed TNF-α receptor binding assay using a human TNF-α biotinylated fluorokine flow cytometry kit. Similar to 5-ASA,

Discussion

Damaged colon epithelium losing its protective barrier function against invasion of microorganism produces pro-inflammatory cytokines such as TNF-α from activated macrophages as well as damaged epithelial cells themselves. The secreted pro-inflammatory cytokines induce excessive recruitment and accumulation of leukocytes in lesion sites of mucosa, which further aggravates inflammation and tissue damage. In the inflamed gut, an effective modulation of the immune response (suppression of

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2014R1A4A1071040), and by the grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C0542).

Conflict of interest

The authors declare that they have no conflict of interest.

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