Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Rutin and rutin-conjugated gold nanoparticles ameliorate collagen-induced arthritis in rats through inhibition of NF-κB and iNOS activation Anum Gul a , Bimal Kunwar b , Maryam Mazhar a , Shaheen Faizi b , Dania Ahmed b , Muhammad Raza Shah b , Shabana U. Simjee a,b, ⁎ a Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan b H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan ARTICLE INFO Keywords: Rheumatoid arthritis autoimmune arthritic score animal model ABSTRACT Numerous studies have suggested that nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) are important mediators of inflammatory response in human and animal models of arthritis. Besides, oxidative stress markers, nitric oxide (NO) and peroxide (PO) are also major contributors in the pathogenesis of rheumatoid arthritis (RA). Over expression of these inflammatory mediators leads to the extracellular matrix degradation, and excessive cartilage and bone resorption, ultimately leading to the irreversible damage to joints. The aim of the present study was to investigate the anti-arthritic mechanism of bioflavonoids, rutin and rutin-conjugated gold nanoparticles (R-AuNPs) by determining their role in the modulation of NF-κB and iNOS expression in collagen-induced arthritis (CIA) model of rats. Arthritis was induced by the subcutaneous administration of bovine type II collagen. Treatment was started with rutin, indomethacin + rutin (I + R) and R-AuNPs on the day of CIA induction. The severity of arthritis was determined by measuring the arthritic score on alternate days until mean arthritic score of 4 was observed. The NO and PO levels were also analyzed in serum samples. NF-κB and iNOS expression levels were determined in spleen tissue samples by real time RT-PCR and im- munohistochemistry. Marked reduction in the arthritic score as well as in the NO and PO levels was observed in the treated groups. A significant downregulation in the NF-κB and iNOS expression levels was also observed in the treatment groups compared to the arthritic control group. Collectively, the findings suggest potential clinical role of rutin and R-AuNPs in the treatment of rheumatoid arthritis. 1. Introduction Rheumatoid arthritis (RA) is an autoimmune disorder characterized by synovial joint inflammation, hyperplasia, irreversible cartilage and bone damage, autoantibody production and systemic complications such as pulmonary, cardiovascular and skeletal disorders [1]. Recent advancements in the treatment modalities of RA, especially the use of biological agents have shown remarkable improvement in the symp- toms and outcomes associated with the progression of RA [2]. Despite these advancements, RA patients suffer from serious side effects and infections associated with the long term use of these medications [3]. Therefore, there is an unmet need to identify new therapeutic agents having less side effects and high efficacy. Reactive oxygen and nitrogen species (ROS and RNS) are involved in normal cellular metabolism, but over production of these oxidative stress markers is responsible for tissue injury, ultimately leading to various diseases such as cancer, diabetes, cardiovascular pathologies and rheumatoid arthritis. High levels of free radicals such as nitric oxide (NO), peroxide (PO) and superoxide anion at the inflammatory site are responsible for damage to cartilage, DNA, proteins, and mem- brane lipids [4–6]. NF-κB is a central regulator of inflammatory response in innate and adaptive immune systems. Dysregulated NF-κB activation has been as- sociated with the pathogenesis of many inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease (IBD), rheumatoid ar- thritis, systemic lupus erythematosus and type I diabetes [7,8]. NF-κB mediated iNOS regulation is an important phenomenon of the in- flammatory response [9]. In many diseases including RA, NF-κB is highly activated at the inflammatory site and leads to the induction of iNOS, Cox-2, MMPs, chemokines and pro-inflammatory cytokines [10]. Therefore, it is important to understand the mechanism behind the activation and pro-inflammatory role of NF-κB to develop more tar- geted therapeutic strategies for inflammatory disorders. Natural products have been used since long in medicine for the https://doi.org/10.1016/j.intimp.2018.04.017 Received 14 November 2017; Received in revised form 7 April 2018; Accepted 10 April 2018 ⁎ Corresponding author. E-mail address: shabana.simjee@iccs.edu (S.U. Simjee). International Immunopharmacology 59 (2018) 310–317 1567-5769/ © 2018 Elsevier B.V. All rights reserved. T