Molecular and Cellular Biochemistry

, Volume 394, Issue 1–2, pp 67–75 | Cite as

N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats

  • Kahkashan Perveen
  • Farina Hanif
  • Huma Jawed
  • Siddiqua Jamall
  • Shabana Usman Simjee


Toll-like receptors (TLRs) are key recognition structures of immune system and recently emerged as potential contributors to the inflammation observed in human and rodent models of arthritis. Present study aims to investigate the effect of N-(2-hydroxy phenyl)-acetamide (NA-2) on modulation of TLRs in the development of adjuvant-induced arthritis. Arthritis was induced by intradermal administration of heat-killed Mycobacterium tuberculosis H37Ra. The treatment of NA-2 (5 mg/kg) and indomethacin (5 mg/kg) was started in their respective group on the day of arthritis induction. Body weights, paw volume measurements, and nociception sensation (Plantar test) were done on alternate days to monitor the progression of the disease until arthritis score of four was observed in arthritic control group. Along with the clinical signs, histopathology of knee joints was also performed. The splenocytes cultures were prepared from each group; TLR-2 and TLR-4 mRNAs were analyzed in 48-h cultured splenocytes using RT-PCR; and the supernatants were used to determine IL-1β and TNF-α by ELISA. A significant reversal of deficit seen in body weights of the arthritic control group was observed in NA-2-treated animals with a parallel decrease in paw edema and transmission of nociception. Remission of the clinical signs and nociception was associated with improved histology. Compared with arthritic control, NA-2 treatment significantly decreased the level of IL-1β (p < 0.003) and TNF-α (p < 0.001) in the supernatants of cultured splenocytes. Likewise, NA-2 also reduced the expression of TLRs mRNA. Our findings suggest that NA-2 affects AIA in a pleiotropic manner, suppressing TLRs-mediated joint inflammation and related symptoms.


Toll-like receptor Tumor necrosis factor Interleukin-1 Inflammation N-(2-hydroxy phenyl) acetamide Rheumatoid arthritis 


Conflicts of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


  1. 1.
    Firestein GS, Zvaifler NJ (1991) Immunopathogenesis of rheumatoid arthritis (review). Immunol Ser 54:141–161PubMedGoogle Scholar
  2. 2.
    Firestein GS (2003) Evolving concepts of rheumatoid arthritis. Nature 423:356–361PubMedCrossRefGoogle Scholar
  3. 3.
    Arend WP (2001) The innate immune system in rheumatoid arthritis. Arthritis Rheum 44:2224–2234PubMedCrossRefGoogle Scholar
  4. 4.
    Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA, Takahashi K, Holers VM, Walport M, Gerard C, Ezekowitz A, Carroll MC, Brenner M, Weissled RV, Erbeek JS, Duchatelle V, Degott C, Benoist C, Mathis D (2002) Arthritis critically dependent on innate immune system players. Immunity 16:157–168PubMedCrossRefGoogle Scholar
  5. 5.
    Hopkins PA, Sriskandan S (2005) Mammalian Toll-like receptors: to immunity and beyond. Clin Exp Immunol 140:395–407PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bobacz K, Sunk IG, Hofstaetter JG, Amoyo L, Toma CD, Akira S, Weichhart TM, Saemann M, Smolen JS (2007) Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of Toll-like receptor 4 and antagonized by bone morphogenetic protein 7. Arthritis Rheum 56:1880–1893PubMedCrossRefGoogle Scholar
  7. 7.
    Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, Chow JC, Strauss JF (2001) The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 276:10229–10233PubMedCrossRefGoogle Scholar
  8. 8.
    Johnson GB, Brunn GJ, Kodaira Y, Platt JL (2002) Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol 168:5233–5239PubMedCrossRefGoogle Scholar
  9. 9.
    Trinchieri G, Sher A (2007) Cooperation of Toll-like receptor signals in innate immune defence. Nature Rev Immunol 7:179–190CrossRefGoogle Scholar
  10. 10.
    Asea A, Rehlim M, Kabingum E, Bochm JA, Barem O, Auronm PE, Stevensonm MA, Calderwood SK (2002) Novel signal transduction pathway utilized by extracellular HSP70: role of Toll-like receptor and TLR-4. J Biol Chem 277:15028–15034PubMedCrossRefGoogle Scholar
  11. 11.
    Schaefer L, Babelova A, Kiss E, Hausser HJ, Baliova M, Krzyzankova M, Marsche G, Young MF, Mihalik D, Götte M, Malle E, Schaefer RM, Gröne HJ (1991) The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest 115:2223–2233CrossRefGoogle Scholar
  12. 12.
    Krutzik SR, Modlin RL (2004) The role of Toll-like receptors in combating mycobacteria. Semin Immunol 16:35–41PubMedCrossRefGoogle Scholar
  13. 13.
    Coulombe F, Divangahi M, Veyrier F, de Léséleuc L, Gleason JL, Yang Y, Kelliher MA, Pandey AK, Sassetti CM, Reed MB, Behr MA (2009) Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide. J Exp Med 206(13):1709–1716PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC (2002) Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4. J Exp Med 195:99–111PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124:783–801PubMedCrossRefGoogle Scholar
  16. 16.
    Iwahashi M, Yamamura M, Aita T, Okamoto A, Ueno A, Ogawa N, Akashi S, Miyake K, Godowski PJ, Makino H (2004) Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis. Arthritis Rheum 50:1457–1467PubMedCrossRefGoogle Scholar
  17. 17.
    Zhu W, Meng L, Jiang C, He X, Hou W, Xu P, Du H, Holmdahl R, Lu S (2011) Arthritis is associated with T-cell induced upregulation of Toll like receptor 3 on synovial fibroblasts. Arthritis Res Ther 13(3):R103PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Seibl R, Birchl T, Loeliger S, Hossle JP, Gay RE, Saurenmann T, Michel BA, Seger RA, Gay S, Lauener RP (2003) Expression and regulation of Toll-like receptor 2 in rheumatoid arthritis synovium. Am J Pathol 162:1221–1227PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Radstake TR, Roelofs MF, Jenniskens YM, Oppers-Walgreen B, van Riel PL, Barrera P, Joosten LA, van den Berg WB (2004) Expression of Toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin- 18 via interferon γ. Arthritis Rheum 50:3856–3865PubMedCrossRefGoogle Scholar
  20. 20.
    Huang Q, Ma Y, Adebayo A, Pope RM (2007) Increased macrophage activation mediated through Toll-like receptors in rheumatoid arthritis. Arthritis Rheum 56:2192–2201PubMedCrossRefGoogle Scholar
  21. 21.
    O’Neill LA (2008) When signaling pathways collide: positive and negative regulation of Toll-like receptor signal transduction. Immunity 29:12–20PubMedCrossRefGoogle Scholar
  22. 22.
    Ospelt C, Brentano F, Rengel Y, Stanczyk J, Kolling C, Tak PP, Gay RE, Gay S, Kyburz D (2008) Over expression of Toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis. Arthritis Rheum 58:3684–3692PubMedCrossRefGoogle Scholar
  23. 23.
    Jawed H, Shah SU, Jamall S, Simjee SU (2010) N-(2-hydroxy phenyl) acetamide inhibits Inflammation-related cytokines and ROS in adjuvant-induced arthritic (AIA) rats. Int Immunopharmacol 10:900–905PubMedCrossRefGoogle Scholar
  24. 24.
    Boissier MC, Semerano L, Challal S, Saidenberg-Kermanac’h N, Falgarone G (2012) Rheumatoid arthritis: from autoimmunity to synovitis and joint destruction. J Autoimmun 39(3):222–228PubMedCrossRefGoogle Scholar
  25. 25.
    Lee DG, Woo JW, Kwok SK, Cho ML, Park SH (2013) MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis. Exp Mol Med 45:e20. doi: 10.1038/emm.2013.39 PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Severn PS, Fraser SG (2006) Bilateral cataracts and glaucoma induced by long-term use of oral prednisolone bought over the internet. Lancet 368:618PubMedCrossRefGoogle Scholar
  27. 27.
    Biskupiak JE, Brixner DI, Howard KB, Oderda GM (2006) Gastrointestinal complications of over-the-counter nonsteroidal antiinflammatory drugs. J Pain Palliat Care Pharmacother. 20:7–14PubMedCrossRefGoogle Scholar
  28. 28.
    Hinz B, Brune K (2004) Pain and osteoarthritis: new drugs and mechanisms. Curr Opin Rheumatol 16:628–633PubMedCrossRefGoogle Scholar
  29. 29.
    Meyer-Kirchrath J, Schrör K (2000) Cyclooxygenase-2 inhibition and side-effects of non-steroidal anti-inflammatory drugs in the gastrointestinal tract. Curr Med Chem 7:1121–1129PubMedCrossRefGoogle Scholar
  30. 30.
    Lester RS, Knowles SR, Shear NH (1998) The risks of systemic corticosteroid use. Dermatol Clin 16:277–287PubMedCrossRefGoogle Scholar
  31. 31.
    Santana-Sabagun E, Weisman MH (2001) Nonsteroidal anti-inflammatory drugs. In: Ruddy S, Harris JED, Sledge CB, Budd RC, Sergent JS (eds) W.B. Kelly’s textbook of rheumatology, vol 1. Saunders Company, Philadelphia, pp 799–822Google Scholar
  32. 32.
    Newman NM, Ling RSM (1985) Acetabular bone destruction related to non-steroidal anti-inflammatory drugs. Lancet 2:11–14PubMedCrossRefGoogle Scholar
  33. 33.
    Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff MH, Keystone EC et al (2000) A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. N Engl J Med 343:1586–1593PubMedCrossRefGoogle Scholar
  34. 34.
    Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pavelka K, van Vollenhoven R et al (2006) The PREMIER study: a multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis Rheum 54:26–37PubMedCrossRefGoogle Scholar
  35. 35.
    Caporali R, Caprioli M, Bobbio-Pallavicini F, Bugatti S, Montecucco C (2009) Long term treatment of rheumatoid arthritis with rituximab. Autoimmun Rev 8:591–594PubMedCrossRefGoogle Scholar
  36. 36.
    Castillo J, Milani C, Mendez-Allwood D (2009) Ofatumumab, a second-generation anti-CD20 monoclonal antibody, for the treatment of lymphoproliferative and autoimmune disorders. Expert Opin Investig Drugs 18:491–500PubMedCrossRefGoogle Scholar
  37. 37.
    Ruslan M, Paula P, Charles A, Janeway Jr (1997) Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397CrossRefGoogle Scholar
  38. 38.
    Takeda K, Kaisho T, Akira S (2003) Toll-like receptors. Annu Rev Immunol 21:335–376PubMedCrossRefGoogle Scholar
  39. 39.
    O’Neill LA (2008) Primer: Toll-like receptor signaling pathways - what do rheumatologists need to know? Nature Clin Prac Rheumatol 4:319–327CrossRefGoogle Scholar
  40. 40.
    Liesu M, Wenhua Z, Congshan J, Xiaojing H, Weikun H, Fang Z, Rikard H, Shemin L (2010) Toll-like receptor 3 upregulation In macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats. Arth Res Therapy 12.doi:  10.1186/ar3034
  41. 41.
    Van Lent PL, van de Loo FA, Holthuysen AE, van den Bersselaar LA, Vermeer H (1995) Major role for interleukin-1 but not tumor necrosis factor in early cartilage damage in immune complex arthritis in mice. J Rheumatol 22:2250–2258PubMedGoogle Scholar
  42. 42.
    Johnson GB, Brunn GJ, Kodaira Y, Platt JL (2002) Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol 168:5233–5239PubMedCrossRefGoogle Scholar
  43. 43.
    Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA, Takahashi K, Holers VM, Walport M, Gerard C, Ezekowit A, Carroll MC, Brenner M, Weissleder R, Verbeek JS, Duchatelle V, Degott C, Benoist C, Mathis D (2002) Arthritis critically dependent on innate immune system players. Immunity 16:157–168PubMedCrossRefGoogle Scholar
  44. 44.
    Joosten LAB, Helsen MMA, van de Loo FAJ, van den Berg WB (1996) Anti cytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNF-α anti-IL-1α/ß, and IL-1ra. Arthritis Rheum 39:797–809PubMedCrossRefGoogle Scholar
  45. 45.
    Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11:373–384PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Kahkashan Perveen
    • 1
  • Farina Hanif
    • 1
  • Huma Jawed
    • 2
  • Siddiqua Jamall
    • 3
  • Shabana Usman Simjee
    • 1
    • 2
  1. 1.Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological SciencesUniversity of KarachiKarachiPakistan
  2. 2.H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological SciencesUniversity of KarachiKarachiPakistan
  3. 3.Department of BiochemistryUniversity of KarachiKarachiPakistan

Personalised recommendations