Quercetin reduced inflammation and increased antioxidant defense in rat adjuvant arthritis

https://doi.org/10.1016/j.abb.2015.08.008Get rights and content

Highlights

  • Administration of quercetin is investigated in a rat model of adjuvant arthritis.

  • Quercetin ameliorates several markers of inflammation and oxidative stress.

  • Quercetin inhibits inflammation mainly by suppressing NF-κB and ERK pathways.

Abstract

Novel therapies for rheumatoid arthritis also include the use of naturally occurring compounds possessing antioxidant properties. In the present work, the effects of oral administration of quercetin were investigated in a rat model of adjuvant arthritis. Arthritis was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experimental groups were treated with an oral daily dose of 150 mg/kg b.w. of quercetin for 28 days. Results indicated that quercetin was able to ameliorate all markers of inflammation and oxidative stress measured. Quercetin lowered levels of interleukin-1β, C-reactive protein, and monocyte chemotactic protein-1 and restored plasma antioxidant capacity. In addition, quercetin inhibited the enzymatic activity of pro-inflammatory 12/15-lipoxygenase in lung and liver and increased the expression of heme oxygenase-1 in joint and lung of arthritic rats. Finally, quercetin inhibited the 2-fold increase of NF-қB activity observed in lung, liver and joint after induction of arthritis.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approximately 1% of the whole world population. Patients with RA have a reduced life quality (joints and bones degeneration, muscle weakness, persistent pain) and require long-life therapy. A common effect of long-term therapy is the development of resistance to treatment and also an increased occurrence of adverse effects. Due to these reasons, a continuous need for new agents in the therapy of RA is envisaged. Primary and dominant processes in the etiopathogenesis of RA are immunological mechanisms, closely related to redox imbalance in the organism, which may potentiate chronic inflammatory processes [1]. Our studies [2], [3], [4] are in agreement with findings of other authors who referred to the important role of oxidative stress in the pathogenesis of RA [5], [6], [7].

In the last decade, the potential involvement of flavonoids with antioxidant properties in RA has been evaluated [8], [9], [10]. In this context, the limited side effects of quercetin (QUE) and its well-known pharmacological activities suggested a potential application as an adjuvant natural drug for the treatment of RA [10]. QUE (3,30,40,5,7-pentahydroxyflavone) is the major dietary flavonol found in fruits, vegetables and beverages, such as tea and red wine [11]. Several epidemiological and experimental studies support the antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative and pro-apoptotic effects of this molecule [12], [13], [14]. In Western populations, the estimated daily intake of total flavonols is in the range of 20–50 mg/day, of which about 15–20 mg correspond to QUE glycosides [15].

The existence of a functional link between the intake of QUE and other flavonoids and RA is supported by circumstantial evidence deriving from pre-clinical studies on primary cells and animal models, as well as clinical studies. Early in the 1997, it was reported that QUE suppressed the increase in the mRNA for interleukin 8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in cultured human synovial cells stimulated by tumor necrosis factor-alpha (TNF-α) in a dose dependent manner. TNF-α is present in synovial fluid and induces the expression of pro-inflammatory cytokines in synovial cells of patients with RA. The suppression was dose dependent and probably induced by the inhibition of TNF-α mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation [16]. A decade later, the anti-RA capacity of QUE was confirmed in synoviocytes isolated from rabbit where the molecule inhibited proliferation of 30–40% at very low micromolar concentrations (<10 μM). It must be considered that proliferation of synoviocytes in RA contributes to the establishment of the so-called “pannus formation”, a lesion accompanied by restriction of joint movement and the generation of pro-inflammatory cytokines [17]. In human rheumatoid synovial fibroblasts activated by interleukin 1 beta (IL-1β), QUE inhibited proliferation and induced apoptosis starting from 20 μM concentration. The mode of action was double: i. inhibition of both the expression of IL-1β-induced mRNA and protein of matrix metalloproteases MMP-1, MMP-3, and COX-2 and PGE2 production; ii. inhibition of extracellular signal-regulated kinases (ERK) signal pathways and NF-κB activation both mediated by IL-1β [18].

The anti-RA effect of QUE was confirmed in several animal models of experimentally induced arthritis. In a rat model of gouty arthritis, QUE treatment (100–400 mg/kg) ameliorated edema by decreasing histological signs of acute inflammation and attenuating several markers of inflammation [19]. QUE was more effective than hesperidin, but less than rutin (all tested at a dose of 80 mg/kg and administered intraperitoneally) in inhibiting acute and chronic inflammation in rats where experimental arthritis was induced following the method of adjuvant-carrageenan-induced inflammation [20]. Considering that rutin differs from QUE for the presence of rutinose in position 3, it is possible to hypothesize that the 2-fold higher efficacy of rutin than QUE in arthrogram scores can be attributed to pharmacokinetic factors [20]. In a subsequent work, the same group, comparing the effects of different flavonoids on different rats and mice models, confirmed that rutin was the only effective against chronic-like arthritis, principally in adjuvant arthritis (AA), but QUE resulted the most active in reducing the paw edema induced by carrageenan [21]. In AA induced in female Lewis rats by subcutaneous injection of inactivated Mycobacterium butyricum, oral administration of QUE (5 × 160 mg/kg) clearly decreased clinical signs of arthritis. Importantly, the dosage was selected to be comparable to that administered to patients affected by prostatitis who received QUE as dietary supplement 1 g/day [22]. When the molecule was given by intracutaneous injection in AA-induced rats at lower doses (5 × 60 mg/kg), the anti-arthritic effects were similar, while injection of relatively low doses (5 × 30 mg/kg) prior to AA induction significantly reduced arthritis signs, suggesting multiple approaches (different doses and modes of administrations) to exploit the clinical potentiality of QUE as an anti-arthritic agent. Finally, analysis of cumulated arthritic scores clearly indicated that high oral doses were most efficient in reducing arthritic signs, followed by lower intracutaneous therapeutic or preventive QUE doses [22].

More recently, the study of the effects of QUE in RA was extended to human subjects with contradictory results compared to pre-clinical studies. In a randomized controlled trial aimed to investigate the efficacy of antioxidant supplementation in RA patients, QUE was administered together with vitamin C (166 mg + 133 mg/capsule, respectively) for 4 weeks in 26 subjects. No changes in the levels of serum pro-inflammatory cytokines and C-reactive protein (CRP) in RA patients after supplementation were observed [23]. In a more recent work, 51 women affected by RA were supplemented with 500 mg/day of QUE for 8 weeks. As in the previous study, measurements of several markers of inflammation, such as plasmatic total antioxidant capacity, malondialdehyde, oxidized low density lipoprotein (Ox-LDL) and high sensitivity CRP did not show any significant difference between QUE and placebo groups [24]. On the opposite, in an ex vivo study, where neutrophils were isolated from RA patients versus healthy subjects and stimulated by in vitro prepared immune complex before treatment with 4 different flavonols (galangin, kaempferol, QUE, and myricetin), QUE was the most effective in reducing superoxide anion production with an IC50 of 1.71 μM [25]. It is worthwhile to note that the applied concentrations were in the physiological range of those measured in vivo after supplementation with QUE or other flavonols [15], [26]. Matsuno et al. [27] performed a study with osteoarthritic patients and RA patients, in which QUE was administered in form of glucosamine-chondrotin-QUE glucoside combination. The patients were treated for 3 months with oral doses of QUE glucoside (45 mg/day). Significant improvement in pain symptoms, daily activities (walking and climbing up and down stairs) and changes in the synovial fluid properties were observed in osteoarthritic patients. No beneficial effects were observed in RA subjects [27].

Therefore, in our study we re-investigated the effect of QUE orally administered in a dose of 150 mg/kg in AA with the aim to prove its anti-arthritic potential, as well as to study its mechanisms of action. We focused on the key two processes in arthritis: inflammation and oxidative stress. Both processes were evaluated in plasma and in selected tissues as joint, liver and lung homogenates.

Section snippets

Animals, experimental design and treatments

Male Lewis rats weighing 160–180 g were obtained from the Breeding Farm Dobra Voda (Slovakia). The rats had free access to standard pelleted diet and tap water. The experimental protocol was approved by the Ethics Committee of the Institute of the Experimental Pharmacology and Toxicology, by the Slovak State Veterinary and Food Administration in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, and by Slovak

Effect of QUE on clinical parameters and on selected inflammatory parameters in plasma

The ability of QUE to ameliorate the clinical parameters following induction of AA in rats was evaluated. As reported in Table 1, a significant decrease in body weight and an increase in arthritic score were observed at both days monitored in AA group. The administration of QUE in healthy and in arthritis animals did not change clinical parameters observed in this study. At day 28, the arthritic score almost doubled in the AA group compared to control and QUE treated groups and this increase

Discussion

The present manuscript highlights the capacity of QUE to improve the anti-inflammatory status and antioxidant defenses in AA rats. At systemic level, QUE increases the plasma antioxidant capacity (Fig. 1) at a dosage (150 mg/kg) corresponding to the approximate daily intake of QUE glycosides (15–20 mg/die) within a regular diet in the human population [15]. Considering the complex series of biotransformation reactions which QUE undergoes following its uptake and metabolism at intestinal and

Conclusion

This study demonstrated that QUE, orally administrated in a rat model of AA, ameliorated all markers of inflammation and oxidative stress measured. The molecule achieved this goal modulating key processes involved in cellular antioxidant defenses, including down-regulation of NF-κB pathway and inhibiting ERK phosphorylation. The ability of QUE to trigger multiple cellular pathways is in agreement with its functional pleiotropy and results in an improvement of inflammatory response and reduction

Conflict of interest

The authors of this paper declare no conflict of interest.

Acknowledgments

The study was supported by grants: VEGA 2/0045/11, VEGA 2/0044/15 and performed in the frame of two SAV-CNR bilateral projects coordinated by Dr. Katarina Bauerova (Slovakia) and Dr. Gian Luigi Russo (Italy) entitled: “In vitro and in vivo models of arthritic processes to study the mechanisms of inflammation and oxidative stress link-up: New perspectives for arthritis therapy” and “Phytochemicals in ameliorating rheumatoid arthritis therapy: from preclinical studies to clinical applications.”

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