Attenuation of Gouty Arthritis by Emodinol in Monosodium Urate Crystal-Treated Mice

• Introduction
• Materials and Methods
• Plant materials
• Reagents and drugs
• Extraction, isolation, and purification of emodinol
• Animals
• Crystal-induced ankle arthritis model and behavioral testing
• Mouse serum cytokines profiling
• Histological analyses
• Statistical analyses
• Supporting information
• Results
• Discussion
• Acknowledgements
• References

Abstract

A series of studies have recently demonstrated that the release of interleukin 1β induced by monosodium urate crystals is central to the experimental gouty arthritis. Elaeagnus pungens has been traditionally used for the treatment of gouty arthritis in China for more than thousands years. However, there is still little known about the active ingredients and mechanisms of E. pungens against gouty arthritis. Emodinol, as a major triterpene compound in E. pungens, has been seldom reported to have an effect on gouty arthritis. Therefore, the potential beneficial effects and mechanisms of emodinol on gouty arthritis were investigated in this study. Results showed that it significantly ameliorated the hyperalgesia, inflammation, and levels of multiple proinflammatory cytokines in monosodium urate crystals-treated mice. These findings elucidate that emodinol exhibits a prominent effect on improving symptoms of acute gouty arthritis induced by monosodium urate crystals through inhibiting the generation of proinflammatory cytokines.

Introduction

Acute attacks of gout are often triggered by specific events such as tumor, surgery, excess alcohol intake, or others [1]. Gouty arthritis is characterized as an inflammatory arthritis by periods of intense inflammatory response with lower grade systemic inflammation in the period between acute attacks [2]. It is caused by the deposits of needle-like crystals of MSU in the joints, resulting in inflammation with severe pain in the affected joint. This disease is the most common form of inflammatory arthritis in men older than 40 years and affects 1–2 % of adults in developed countries [3].

Nowadays, a number of anti-gout agents including nonsteroidal anti-inflammatory drugs such as indomethacin and naproxen are frequently used as the first-line therapies for acute gout. However, the associated adverse effects, including gastrointestinal and renal toxicity, as well as gastrointestinal bleeding, always limit their clinical uses.

Recently, there are reports of many herbal drugs and their active ingredients being able to protect against MSU crystals-induced gouty arthritis [4], [5], [6], [7]. The species EP, a constituent of a traditional Chinese medicine named Niunaizi, belongs to the Elaeagnaceae family. It has been traditionally used in the treatment of various diseases in China for more than thousand years. Its beneficial effects can be found in antiasthmatic, anti-inflammatory action and improvement of gouty arthritis [8]. Triterpenes are reported as the main components in EP. Although EP is clinically applied to treat gouty arthritis, there is still little known about the active ingredients and mechanisms of EP against gouty arthritis. To understand whether triterpenes contribute to the anti-gouty arthritis activity of EP, we purified and identified a major triterpene compound from EP, namely emodinol ([Fig. 1 A]). Emodinol has been reported to have beta-glucuronidase inhibitory activity in vitro [9]. Our previous studies also showed that emodinol was able to inhibit the production of proinflammatory cytokines in MSU crystal-treated RAW264.7 cells (unpublished data). Therefore, this study was carried out to further investigate the potential effect and mechanism of emodinol against gouty arthritis in MSU crystal-treated mice.

Materials and Methods

Plant materials

The raw materials of the fresh rhizome of EP were collected in July 2009 in Fujian Province, China. The plant materials were identified by Prof. Dingrong Wan, College of Pharmaceutical Sciences, South-Central University for Nationalities, China. The voucher specimen (No: 20090718) has been deposited at the herbarium situated at the College of Pharmaceutical Sciences. All the materials were dried at room temperature to constant weight. The coarse powder of the rhizome was stored in a well-closed vessel for use.

Reagents and drugs

Colchicine tablets (each tablet contains colchicine in a dosage of 1 mg) were purchased from Simcere drugstore in Nanjing, China. Homogenous suspensions of emodinol and colchicine were made with 0.5 % carboxy methyl cellulose in phosphate buffered saline. Fresh suspensions were prepared before each experiment. All other reagents used were standard laboratory reagents of analytical grade and were purchased locally.

MSU crystals were prepared by crystallization of the supersaturated solution of uric acid (Aldrich Chemical Company, Inc.) with minor modifications according to the previous research [10]. Briefly, 250 mg uric acid were dissolved in 45 mL of boiling distilled water containing 300 µL of 5 M NaOH. The solution was passed through an 0.2-µm filter, and 1 mL of 5 M NaCl was added to the hot solution. After that, the solution was cooled gradually and then stored at 26 °C to allow crystallization. Seven days later, the resulting MSU crystals (needle-shaped, 5 to 20 µm in length) were washed with ethanol and acetone over a Buchner flask and were allowed to air-dry under sterile conditions. MSU crystals were confirmed as endotoxin-free by a commercial test kit of limulus amebocyte lysate assay (< 0.01 EU/10 mg).

Extraction, isolation, and purification of emodinol

The dried powder of EP (10 kg) was extracted with ethanol (60 %, 2 × 75 L, 2 h each time). After being filtered and centrifuged, the filtrates were concentrated under reduced pressure to obtain 720 g ethanol extract (7.2 %, w/w). Then the ethanol extract was suspended in hot water and was extracted successively with petroleum ether, chloroform, ethyl acetate, and n-butanol. Each subfraction was then concentrated under reduced pressure to obtain a petroleum ether fraction (15 g, 2.1 %, w/w), chloroform fraction (72 g, 10 %,w/w), ethyl acetate fraction (114 g, 15.8 %,w/w), n-butanol fraction (320 g, 44.4 %, w/w), and a residual ethanol mother solution fraction (199 g, 27.7 %, w/w). The n-butanol fraction (100 g) was subjected to vacuum liquid chromatography on silica gel (200 mesh, 6 × 50 cm) and eluted with a chloroform-methanol gradient solvent system 50 : 1 (1 L), 40 : 1 (2 L), 30 : 1 (2 L), 20 : 1 (4 L), and 10 : 1 (2 L). The flow rate was 10 mL/min. 250 mL eluant at a time was collected and monitored by TLC. Finally, 10.0 g emodinol ([Fig. 1 A]) was obtained in the fraction of chloroform–methanol (20 : 1). The spectral and physicochemical data (Supporting Information) of the isolated compound agreed with those of the literature [9]. The purity of emodinol (> 98 %) was determined by HPLC.

Animals

Male BALB/c mice (SPF) weighing 18–22 g were purchased from the Experimental Animal Center, Institute of Health and Epidemic Prevention (Wuhan, China). They were maintained in a room controlled at 23 ± 2 °C with a relative humidity of 50–55 % and a 12-h light/dark cycle. Fresh tap water was provided ad libitum. After a 7-day acclimatization period, weight-matched animals were randomized into six experimental groups (n = 15 for each group). All the mice were fed with standard food and received humane care in accordance with the animal care provision. Animal experimentation and the corresponding protocol (NO: 2012–0036; Date: 10/05/2012) were approved by the Animal Ethics Committee of South-Central University for Nationalities (Wuhan, China). All the procedures were in strict accordance with the PR China legislation on the use and care of laboratory animals.

Crystal-induced ankle arthritis model and behavioral testing

MSU crystals (0.2 mg) suspended in 20 µL endotoxin-free PBS or PBS control were injected into the tibiotarsal joint (ankle) of mice anesthetized with 2.5 % isoflurane. Group I mice injected with PBS served as the control group. In group II, inflammation was induced by injection of 20 µL MSU crystal suspension. Mice in group I and group II were gavage fed (using a feeding tube) with PBS. Group III comprised of MSU crystal-treated mice which were gavage fed (using a feeding tube) with colchicine (1 mg/kg body weight). Groups IV, V, and VI comprised MSU crystal-treated mice which were gavage fed (using a feeding tube) with emodinol (20, 40, and 80 mg/kg body weight, respectively). Emodinol and colchicine were given orally 1 h before the MSU crystal suspension injection (single dose) and then once daily for 3 days.

Before behavioral testing, mice were acclimatized to the testing room for at least 1 h. All behavioral measures were taken by experimenters blind to treatment. Baseline readings were obtained for each measure before injections.

To study thermal hyperalgesia after ankle injections, the Hargreavesʼ test was performed as previously described [11]. Briefly, a radiant heat source was applied to the heel, and the paw withdrawal latency was recorded. For each mouse, three readings were obtained, and the median latencies were used. The results were presented as the percentage change from the baseline readings.

To test willingness to bear weight on injected ankles, the weight bearing test was performed using an incapacitance meter (NatureGene Corp.). Each hind paw was placed on a transducer pad, and the weight distribution, in grams, was recorded over 5 s. The results were presented as percentage weight distributed to the affected limb.

The diameters of the ankle joints were measured with calipers while mice were anesthetized with 2.5 % isoflurane. The results are presented as percentage change from baseline diameter.

Mouse serum cytokines profiling

Six mice per group were killed one day after ankle injection, and blood was collected by cardiac puncture. Whole blood samples were collected by cardiac puncture from anesthetized mice and were allowed to clot for approximately 1 h at room temperature and then centrifuged at 2500 × g for 10 min to obtain the serum. The levels of TNF-α, IL-1β, and IL-6 were measured using ELISA kits (R & D CO.), according to manufacturerʼs instructions.

Histological analyses

For histological analyses, six mice per group were killed one day after ankle injection. The ankle region was isolated and decalcified in 100 g/L ethylene dinitrilotetraacetic acid, embedded in paraffin and sectioned at 5 µm, followed by staining with hematoxylin and eosin. Histopathologic severity was scored using three different parameters as previously described [12]: synovial inflammation, synovial hyperplasia, and cartilage surface erosion. All parameters were scored on a scale of 0–3 (0, normal; 3, severe) by two independent observers in a blinded manner.

Statistical analyses

All values were expressed as the mean ± SEM and were analyzed using ANOVA and post hoc tests to probe significant effects (Bonferroni tests for one- or two-way ANOVAs; Tukey HSD tests for three-way ANOVAs).

Supporting information

Spectral and physicochemical data of emodinol and histological analyses of ankle joints are available as Supporting Information.

Results

Mice exhibited thermal hyperalgesia for 3 days after an intra-articular ankle injection of MSU crystal suspension, peaking one day after injection. The thermal hyperalgesia responses of mice were significantly reduced in emodinol groups compared with the control group. No hyperalgesia responses were detected in mice with an ankle injection of PBS ([Fig. 1 B]).

Ankle injections of MSU crystal suspension, but not PBS, induced a significant shift in weight bearing away from the affected limb in the control group, peaking one day after injection. In contrast, mice in emodinol groups showed significantly less weight redistribution ([Fig. 1 C]).

Moreover, after injections of MSU crystal suspension, mice developed ankle swelling that peaked after the first day. Mice in both the emodinol and colchicine groups showed significantly less swelling ([Fig. 1 D]).

Histological assessments of joint sections from mice ankles were also conducted. The results demonstrated that emodinol and colchicine could improve synovial hyperplasia, reduce the infiltration of inflammatory cells in the synovium and diminish erosive damage in the cartilage ([Fig. 2]; Fig. 1S, Supporting information). These joint histological results were consistent with the findings that EP and colchicine were capable of reducing clinical symptoms of gout.

To identify the alterations of proinflammatory cytokines after ankle injection of MSU crystal suspension, as well as to study the role of IL-1β in the disease process, we determined the levels of proinflammatory cytokines in the serum samples collected 24 h after ankle injection. The results confirmed that levels of IL-1β, IL-6, and TNF-α were upregulated in the serum of MSU crystal-treated mice. Significant attenuations of MSU crystal-induced increases in the levels of proinflammatory cytokines were observed in mice after the treatments with emodinol and colchicine ([Fig. 3]).

Discussion

Gouty arthritis is usually characterized by intense pain, swelling, and reddening around the joints and connective tissues. Recent studies indicate that MSU crystals are among the most potent proinflammatory stimuli and an innate immune inflammatory response to the crystalsʼ surface is intimately involved in the pathology of gouty arthritis [13]. In our present study, emodinol treatments reversed MSU crystal-induced elevation in ankle swelling, and the onset of amelioration was seen at hour 8 after MSU crystal suspension injection and remained in subsequent hours. In additional, histological evaluations indicated that emodinol treatments led to the reduction of joint inflammation and the amelioration of cartilage destruction and bone erosion, which contributed to a protective effect against gouty arthritis.

Several lines of evidence claim that MSU crystals stimulate the synthesis and release of IL-1β [14]. Overproduction of IL-1β upon the activation of inflammasome by MSU crystals provides significant insight into the mechanisms controlling inflammation in gouty arthritis. Previous studies have demonstrated that the release of IL-1β by the mediation of NLRP3 inflammasome activation induced by MSU crystals was central to the experimental gouty inflammation [15], [16]. Torres et al. [17] showed convincingly that IL-1 was a major trigger of joint inflammation in a new animal model of gouty arthritis involving injection of MSU crystals directly into the mouse ankle joint. In addition, central roles of TNF-α and IL-6 in the experimental model of acute gouty arthritis in vivo have also been documented. MSU crystals stimulate the secretion of proinflammatory cytokines including TNF-α, IL-1β, and IL-6, by synovial cells, monocytes-macrophages, and neutrophils, which results in an acute inflammation [18]. This concept has been strengthened by the findings of an open study on the effects of anakinra in gout [19]. Therefore, the treatment of inflammation is the ideal therapeutic approach against gouty arthritis. Our data indicated that emodinol significantly reduced the levels of proinflammatory cytokines including IL-1β, TNF-α, and IL-6 in MSU crystal-treated mice, which contributed to the improvement of inflammatory responses induced by MSU crystals. Colchicine is used as first-line therapy for acute inflammation in gouty arthritis; it also reduces the levels of proinflammatory cytokines induced by MSU crystals in mice. Similar to colchicine, emodinol may serve as an effective agent against gouty arthritis mediated by inhibition of proinflammatory cytokines secretion. However, the effect of emodinol on the pathogenesis of gouty arthritis in an animal model should be further investigated.

In conclusion, the present study has shown that emodinol could ameliorate the gouty arthritis induced by MSU crystals. The mode of action of emodinol seems to be owing to the inhibition of the synthesis and release of proinflammatory cytokines. The present results provide an assessment on the potent protective effect of emodinol against gouty arthritis for the first time. These insights are relevant to further develop the therapeutic potential of emodinol for the treatment of gouty arthritis.

Acknowledgements

The project was supported by the Chinese National Project of “Twelfth Five-Year” Plan for Science & Technology Support (2012BAI27B06) and the Special Fund for Basic Scientific Research of Central Colleges, South-Central University for Nationalities (No: CZQ11035).

Conflict of Interest

The authors declare that there are no conflicts of interest.

^* These authors contributed equally to this work.

• References

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Correspondence

• References

• 1 Sabina EP, Chandal S, Rasool MK. Inhibition of monosodium urate crystal-induced inflammation by withaferin A. J Pharm Pharm Sci 2008; 11: 46-55
  PubMedSearch in Google Scholar
• 2 Pascual E. Persistence of monosodium urate crystals and low-grade inflammation in the synovial fluid of patients with untreated gout. Arthritis Rheum 1991; 34: 141-145
  CrossrefPubMedSearch in Google Scholar
• 3 Krishnan E, Lienesch D, Kwoh CK. Gout in ambulatory care settings in the United States. J Rheumatol 2008; 35: 498-501
  PubMedSearch in Google Scholar
• 4 Huang J, Zhu M, Tao Y, Wang S, Chen J, Sun W, Li S. Therapeutic properties of quercetin on monosodium urate crystal-induced inflammation in rat. J Pharm Pharmacol 2012; 64: 1119-1127
  CrossrefPubMedSearch in Google Scholar
• 5 Silva CR, Fröhlich JK, Oliveira SM, Cabreira TN, Rossato MF, Trevisan G, Froeder AL, Bochi GV, Moresco RN, Athayde ML, Ferreira J. The antinociceptive and anti-inflammatory effects of the crude extract of Jatropha isabellei in a rat gout model. J Ethnopharmacol 2013; 145: 205-213
  CrossrefPubMedSearch in Google Scholar
• 6 Jiang Y, You XY, Fu KL, Yin WL. Effects of extract from Mangifera indica leaf on monosodium urate crystal-induced gouty arthritis in rats. Evid Based Complement Alternat Med 2012;
  PubMedSearch in Google Scholar
• 7 Jung SM, Schumacher HR, Kim H, Kim M, Lee SH, Pessler F. Reduction of urate crystal-induced inflammation by root extracts from traditional oriental medicinal plants: elevation of prostaglandin D2 levels. Arthritis Res Ther 2007; 9: R64
  CrossrefPubMedSearch in Google Scholar
• 8 State Administration of Traditional Chinese Medicine of Peopleʼs Republic of China (Ed.). Zhong-hua-ben-cao. Shanghai: Shanghai Science and Technology Publisher; 1999: 125-126
  Search in Google Scholar
• 9 Riaz N, Anis I, Aziz-ur-Rehman, Malik A, Ahmed Z, Muhammad P, Shujaat S. Atta-ur-Rahman. Emodinol, beta-glucuronidase inhibiting triterpene from Paeonia emodi . Nat Prod Res 2003; 17: 247-251
  CrossrefPubMedSearch in Google Scholar
• 10 Akahoshi T, Namai R, Murakami Y, Watanabe M, Matsui T, Nishimura A, Kitasato H, Kameya T, Kondo H. Rapid induction of peroxisome proliferator-activated receptor gamma expression in human monocytes by monosodium urate monohydrate crystals. Arthritis Rheum 2003; 48: 231-239
  CrossrefPubMedSearch in Google Scholar
• 11 Sluka KA, Milton MA, Willis WD, Westlund KN. Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation. Br J Pharmacol 1997; 120: 1263-1273
  CrossrefPubMedSearch in Google Scholar
• 12 Lu Y, Xiao J, Wu ZW, Wang ZM, Hu J, Fu HZ, Chen YY, Qian RQ. Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance. Phytomedicine 2012; 19: 882-889
  CrossrefPubMedSearch in Google Scholar
• 13 Popa-Nita O, Naccache PH. Crystal-induced neutrophil activation. Immunol Cell Biol 2010; 88: 32-40
  CrossrefPubMedSearch in Google Scholar
• 14 Roberge CJ, Grassi J, De Médicis R, Frobert Y, Lussier A, Naccache PH, Poubelle PE. Crystal-neutrophil interactions lead to interleukin-1 synthesis. Agents Actions 1991; 34: 38-41
  CrossrefPubMedSearch in Google Scholar
• 15 Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J. NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 2004; 20: 319-325
  CrossrefPubMedSearch in Google Scholar
• 16 Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006; 440: 237-241
  CrossrefPubMedSearch in Google Scholar
• 17 Torres R, Macdonald L, Croll SD, Reinhardt J, Dore A, Stevens S, Hylton DM, Rudge JS, Liu-Bryan R, Terkeltaub RA, Yancopoulos GD, Murphy AJ. Hyperalgesia, synovitis, and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis. Ann Rheum Dis 2009; 68: 1602-1608
  CrossrefPubMedSearch in Google Scholar
• 18 Di Giovine FS, Malawista SE, Nuki G, Duff GW. Interleukin 1 (IL-1) as a mediator of crystal arthritis. Stimulation of T cell and synovial fibroblast mitogenesis by urate crystal-induced IL-1. J Immunol 1987; 138: 3213-3218
  PubMedSearch in Google Scholar
• 19 So A, Desmedt T, Revaz S, Tschopp J. A pilot study of IL-1 inhibition by anakinra in acute gout. Arthritis Res Ther 2007; 9: R28
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material