Cytokine 65 (2014) 231–235 Contents lists available at ScienceDirect Cytokine journal homepage: www.journals.elsevier.com/cytokine Attenuation of adjuvant-induced arthritis in rats by phonophoresis with an aqueous gel of the Amazonian plant Elaeoluma nuda (Sapotaceae) Lilian Regiani Merini a,e,f, Silvânia da Conceição Furtado b,e, Marcelo Miguel Brito de Oliveira c, Ana Lúcia Basílio Carneiro d, Antonio Luiz Boechat a,e,⇑, José Fernando Marques Barcellos a,b a Postgraduate Program in Basic and Applied Immunology, Institute of Biological Sciences, Federal University of Amazonas, Brazil Morphology Department, Institute of Biological Sciences, Federal University of Amazonas, Brazil c Undergraduate Science Project, Morphology Department, Institute of Biological Sciences, Federal University of Amazonas, Brazil d Morphology Department, Center for Health Sciences, Federal University of Paraíba, Brazil e Amazonian Group for the Study of Inflammation and Autoimmunity (GAIA), Brazil f Médio Solimões Institute of Health and Biotechnology, Coari Campus, Federal University of Amazonas, Brazil b a r t i c l e i n f o Article history: Received 15 February 2013 Received in revised form 21 October 2013 Accepted 28 October 2013 Available online 23 November 2013 Keywords: Elaeoluma nuda Phonophoresis Adjuvant-induced arthritis a b s t r a c t Background: Various species of the genus Pouteria (Elaeoluma) are used by the native population of Brazil because of, among other factors, their anti-inflammatory properties. The anti-inflammatory properties of the extract of the Amazonian plant Elaeoluma nuda were recently identified in prospective pharmacological studies. Objectives: The objective of this study was to assess the anti-inflammatory effect of phonophoresis with aqueous gel extract of E. nuda in rat adjuvant-induced arthritis. Methodology: Arthritis was induced in Lewis rats with an adjuvant. Phonophoresis with E. nuda gel was then administered daily and the results compared with those obtained with phonophoresis of diclofenac diethylammonium gel and ultrasound therapy without phonophoresis. Arthritis in the different groups was evaluated by plethysmometry. Proinflammatory cytokines TNF-a and IL-1a were quantified by cytometric bead array (CBA). Results: The effect of phonophoresis of aqueous gel with E. nuda extract on arthritis in rats’ paws (a 33% reduction compared with the controls) was the same as that produced by phonophoresis with diclofenac diethylammonium. Ultrasound therapy without phonophoresis produced no significant effect on the 21st day of therapy. There was a significant reduction in TNF-a and IL-1a levels in the group treated with phonophoresis with E. nuda gel (p = 0.0042; p = 0.0003, respectively). Conclusion: Our results demonstrate the anti-inflammatory effect of phonophoresis with E. nuda gel on cytokines TNF-a, IL-1a and adjuvant-induced arthritis. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Many South American plant species, particularly those from the Amazon, have been studied and have proven to have good prospects for application in human health. In Brazil, various species from genus Pouteria are used because of their disinfectant, laxative, vermifugal, aphrodisiac and anti-dysenteric properties [1]. Genus Pouteria belongs to the family Sapotaceae and includes approxi⇑ Corresponding author. Address: Disciplina de Imunologia, Programa de PósGraduação em Imunologia Básica e Aplicada, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General Rodrigo Otávio Jordão Ramos, 3000 Coroado, Mini Campus, Setor Sul, Manaus CEP 69077-000, Brazil. Tel.: +55 92 3305 4276. E-mail address: antonioluiz.boechat@gmail.com (A.L. Boechat). 1043-4666/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2013.10.007 mately 80 species. Elaeoluma nuda (Baehni) Aubrév, whose known basionym is Pouteria nuda, is a Sapotaceae that was identified in the Adolpho Ducke Forestry Reservation, in Manaus, AM. Species from genus Pouteria have also been investigated for antiretroviral potential due to the HIV cell entry inhibition on classical viral assays and also due the triterpenoids in the extracts that inhibits the virus replication [2]. The ethanolic and methanolic extracts are active against Trypanossoma cruzi (in vitro) and Plasmodium bergei reducing the induced infection in rats [1]. In vitro assays have shown that many Pouteria species has antibacterial activity for Staphylococcus aureus, Pseudmonas aeruginosa and Mycobacterium smigamatis [3]. Antifungal against Candida albicans was also detected [3]. Anti-inflammatory effects on carrageenan on rat air pouch have been described [4]. 232 L.R. Merini et al. / Cytokine 65 (2014) 231–235 Phonophoresis is the application of topical drugs to the external layer of the skin with the aid of pulsed ultrasound [5,6]. The use of low-frequency ultrasound in phonophoresis increases the concentration of the drug in the tissue being treated by increasing the permeability of the skin to molecules with low or high molecular weights [6]. This property appears to be especially significant when low-frequency ultrasound is used [7]. Low-frequency phonophoresis has also been successfully used with plant extracts in animal models of inflammation [8–10]. Adjuvant-induced arthritis in Lewis rats is an inflammation model used for investigating the immunopathogenesis of autoimmunity and assessing new therapies [11–13]. In light of the ethnopharmacological importance of the genus Pouteria in Brazil and the pharmacological potential of E. nuda in in vitro studies, this study sought to investigate the anti-inflammatory effect of phonophoresis with E. nuda gel on arthritis induced by Freund’s adjuvant. isoflurane. From the 15th day of the experiment, when signs of arthritis became evident, treatment with phonophoresis was started. This was administered daily to both rear paws by a qualified physiotherapist. On the 21st day of the experiment the animals were sacrificed by inhalation of isoflurane, and blood samples was collected by heart puncture [15]. The 2% E. nuda gel was administered transdermally to the plantar region of the rear paws with therapeutic ultrasound equipment (Sonacel Expert 1 MHz with a reduced head, BiosetÒ, Brazil) using slow circular movements and the following parameters: application time – 1 min; frequency – 1.0 MHz; intensity – 0.5 W/cm2; ERA - 0.8 cm2; pulse mode; one daily session in the afternoon; total number of sessions per animal – 10. The diclofenac diethylammonium gel (Cataflan EmulgelÒ, Novartis) was also applied in ten sessions using the same protocol. While the animal was being treated it was gently restrained by a trained researcher so that it was subjected to as little stress as possible. 2. Methodology 2.4. Assessment of edema in the paw 2.1. Aqueous extract of E. nuda The aqueous extract used in this study was obtained from the Amazonian plant E. nuda, which was collected in the Adolpho Ducke Forestry Reservation (Amazonas State, Brazil). The exsiccata is stored at the National Amazonian Institute for Research (INPA) under reference no. 179316. The extract was obtained by a method described elsewhere [3,14]. Briefly, the aqueous extract was obtained using the dried ground material by infusion followed by filtration and total evaporation of the filtrate. 2.2. Preparation of the gel The gel was prepared at INPA and had the following composition: 87.65% of demineralized water, 0.15% Nipagin, 4.00% propylene glycol, triethanolamine, 5% deionized water and 2% aqueous extract of lyophilized E. nuda, which were mixed until completely dispersed. Next, 1.2% carbopol was pulverized, added slowly and left to hydrate for 30 min. The substances were then mixed and heated to no more than 60 °C. Triethanolamine and 5% demineralized water were added and homogenized to a final pH of 7.0. 2.3. Animals Thirty 8-week-old Lewis rats weighing between 250 and 350 g, donated and maintained by the biotherium at the Federal University of Amazonas (UFAM), were used in the experiment. The animals had ad libitum access to water and food and were kept in a light-controlled (12 h light/dark cycle) and temperature-controlled (22 °C) environment. Thirty animals were divided into five groups and two animals were kept in each cage. All the experiments were carried out with the approval of the Institutional Committee for Ethics in Animal Experiments under reference no. 001/2010 (CEEA) UFAM. The Anti-inflammatory Effect of E. nuda phonophoresis on Arthritis Induced with Freund’s complete adjuvant. For this experiment the animals were divided into five groups: Group 1 – negative control (no arthritis and no treatment); Group 2 – positive control (arthritis without treatment); Group 3 – ultrasound with an aqueous placebo gel (UST); Group 4 – phonophoresis with diclofenac diethylammonium (DDP); and Group 5 – phonophoresis with 2% E. nuda gel (ENP). Arthritis was induced on the first day of the experiment by ID injection of 0.1 mL of Freund’s complete adjuvant (DifcoÒ, USA) at the base of the tail [12] after brief inhalatory anesthesia with Paw swelling was assessed by a trained observer who had not been involved in the planning of the experiment (blind assessment). Paw volumes (mL) were measured on days 0, 7, 14 and 21 of the experiment with a digital plethysmometer (InsightÒ, Brazil). 2.5. Quantification of cytokines TNF-a and IL-1a Serum concentrations of cytokines TNF-a and IL-1a were measured by cytometric bead array using CBA Flex SetsÒ for TNF-a and IL-1a (BD Biosciences) in accordance with the manufacturer’s instructions. A FACSCaliburÒ flow cytometer (BD Biosciences) was used to read the samples. The concentrations and mean fluorescence intensities (MFIs) of each cytokine were calculated using FCAP Array™ version 1.0.1 software. 2.6. High cytokine producer signatures High-cytokine producer signatures were identified by taking the median MFI value obtained in the flow cytometry for each cytokine for the whole population, as described previously [15,16]. These values were used as cut-off points, and each animal in each group was then classified as a high or low cytokine producer. 2.7. Statistical analysis Results were expressed as means and standard deviations, and a 95% confidence interval was used. One-way ANOVA was used to compare means, and Holm–Sidak’s test with correction for multiple comparisons was used for post hoc analysis. For the last test, adjusted p-values were reported for each comparison. The linear trend test was applied to investigate whether there was a significant trend line among the mean cytokine concentrations [16]. A significance level of p = 0.05 was used. In the analysis to identify high cytokine producers, the results were considered statistically Table 1 Volume of paws means on the 21st day of the experiment as measured by plethysmometry. Group Average ± SD (mL) 95% CI No treatment UST DDP ENP No arthritis 2.14 ± 0.390 1.80 ± 0.350 1.35 ± 0.058 1.35 ± 0.060 1.03 ± 0.095 1.51–2.75 1.25–2.31 1.25–1.44 1.26–1.45 0.87–1.18 233 L.R. Merini et al. / Cytokine 65 (2014) 231–235 Table 2 Quantification of cytokines TNF-a and IL-1a on the 21st day of the experiment. Group No treatment UST DDP ENP No arthritis TNF-a (qg/mL) IL-1a (qg/mL) Average ± SD 95% CI Average ± SD 95% CI 12.13 ± 1.67 11.65 ± 1.05 10.13 ± 2.22 7.40 ± 2.76 7.03 ± 2.19 10.38–13.88 10.54–12.75 7.80–12.46 4.51–10.30 4.76–9.300 18.45 ± 2.13 15.44 ± 1.73 14.82 ± 2.18 13.18 ± 1.60 12.38 ± 1.14 16.21–20.69 13.28–17.60 12.53–17.10 11.50–14.86 11.18–13.58 significant when the frequency of high producers shifted the group to a different quartile (25% interval) in relation to the reference value [17,18]. In the last case, v2 test for trend was also applied. Post-hoc power calculations was performed using GPower Software (http://www.psycho.uni-duesseldorf.de/abteilungen/aap/ gpower3). lower mean paw volume (1.35 ± 0.06 mL) than the control group (a 33% reduction compared with the controls). This finding was statistically significant (p = 0.001, Table 2). However, when compared with the group treated with DDP, the mean difference was equal (0.79 mL), showing that the effect observed on the last day of the experiment was the same for both treatments (Fig. 1, Panels A and B). A post hoc power calculation shows 98% of statistical power. 3. Results 3.3. Quantification of TNF-a and IL-1a 3.1. Yield of the aqueous extract The total mass of the ground dried leaves was 52.51 g while the mass of the lyophilized extract was 8.33 g, corresponding to a yield of 15.86%. 3.2. Effect of phonophoresis with E. nuda gel on arthritis induced by Freund’s complete adjuvant Table 1 shows the mean paw volumes on the 21st day of the experiment. It can be seen that the group treated with ENP had a Table 2 shows the results for the quantification of cytokines TNF-a and IL-1a. It can be seen that the groups of animals treated with ENP had lower TNF-a (p = 0.0042) and IL-1a (p = 0.0003) levels than the positive control group (ANOVA analysis showed in Table 3). It should be stressed that the mean difference of cytokine quantification for both cytokines is higher for ENP than other groups. Post-test analysis with the linear trend test revealed a statistically significant trend toward lower TNF-a and IL-1a values compared with the positive control and DDP group (Fig. 1, Panels C and D). This was more pronounced in the group of animals trea- A B C D Fig. 1. The results are expressed as mean paw volume (mL) or mean cytokine levels (qg/mL) and standard deviation (SD). Panel A: Kinetics of arthritis for the different experimental groups. Positive control represents the untreated arthritis animals and negative control represents no arthritis and not treated animals. Panel B: ANOVA analysis of paw volumes means on the 21st day of the experiment as measured by plethysmometry. Panel C and D: Quantification of cytokines TNF-a and IL-1a. Post-test analysis with the linear trend test revealed a statistically significant trend toward lower TNF-a and IL-1a values compared with the positive control (p = 0.0125 and p = 0.0086, respectively). This was more pronounced in the group of animals treated with ENP. 234 L.R. Merini et al. / Cytokine 65 (2014) 231–235 Table 3 ANOVA analysis with multiple comparisons between the groups. Comparisons a b c Mean difference 3.4. Analysis of high cytokine producer signatures Adjusted p-values ANOVA analysis for volume paws (mL)a No treatment vs. UST 0.340 No treatment vs. DDP 0.790 No treatment vs. ENP 0.790 No treatment vs. no arthritis 1.115 0.0643 0.0010 0.0010 <0.0001 ANOVA analysis for TNF-a (qg/mL)b No treatment vs. UST 0.488 No treatment vs. DDP 2.000 No treatment vs. ENP 4.728 No treatment vs. no arthritis 5.107 0.9936 0.4595 0.0042 0.0019 ANOVA analysis for IL-1a (qg/mL)c No treatment vs. UST No treatment vs. DDP No treatment vs. ENP No treatment vs. no arthritis 0.0742 0.0147 0.0003 <0.0001 3.010 3.633 5.276 6.067 p < 0.0001. p = 0.0003. p < 0.0001. Differences in baseline serum cytokine levels between individuals are important and must be considered. Categorical analysis (using the experiment median value for categorization) of cytokine production (high or low) by the animals therefore represents a good strategy for analyzing the effects of the different treatments as it minimizes the impact of individual values and makes the data more homogeneous [19]. This strategy allows the number of high TNF-a and IL-1a producers in each group to be determined and a direct comparison to be made between the groups. The cytokine production signature (% of high cytokine producers) in the group treated with ENP was similar to that of the negative control, and there were fewer high producers in the groups in which arthritis had been induced (v2 test for trend, p < 0.001). The group treated with UST had a high-producer signature similar to that of the positive control group (Fig. 2, Panels A and B). 4. Discussion ted with ENP (p = 0.004 and 0.002, respectively). A post hoc power calculation shows 98% and 96% of statistical power for IL-1a and TNF-a, respectively. This study is the first to show the anti-inflammatory action of the species E. nuda in vivo. However, prior to this study, phytochemical evaluation of genus Pouteria had revealed the presence of biochemical compounds of considerable pharmacological Individual Samples s N N o N o tr ea tm en t EN P D U ea tm en tr o N iti 0 th r 0 P 5 ar 5 o 10 EN 10 D P 15 D 15 ar th rit is 20 D P 20 ST 25 t 25 ST B U A % High Producers C D 100 *p<0.0001 80 60 40 20 o is P ar th rit o P D D ST U EN N tr ea tm ar th rit en t is P P D D ST U EN N o N N o tr ea tm en t 0 Fig. 2. Panel A and B: Individual values and discrimination of high cytokine producers in each group. Panel C and D: Percentages of high cytokine producers in each group. v2 test for trend. L.R. Merini et al. / Cytokine 65 (2014) 231–235 interest such as terpenoids, flavonoids and saponins [19]. Preliminary phytochemical screening revealed that E. nuda extracts contain phenols, flavonoids, tannins, saponins, alkaloids and anthrones (Carneiro, A. L. B., data not published) [14]. In the present study, the effectiveness of treatment with ENP was comparable to that of treatment with phonophoresis of diclofenac gel, a well-known anti-inflammatory agent. These results show that, like other species of Elaeoluma, E. nuda also exhibits anti-inflammatory activity when tested using an inflammation model. A retrospective review study of the animal models used to evaluate approved Rheumatoid Arthritis (RA) therapies found that the tree most commonly used models were adjuvant-induced arthritis in rats, collagen-induced arthritis in rats and mice [13]. The attenuation of adjuvant-induced arthritis in rats produced by ENP indicates the potential value of this technique in the treatment of arthritis. The use of low-frequency ultrasound in phonophoresis increases the concentration of the drug in the tissue being treated by increasing the permeability of the skin to molecules with low or high molecular weights [5,6]. Thus, phonophoresis could have potentiated the anti-inflammatory actions of aqueous gel of ENP. Recently, some authors have been isolated the pouterin, a cytotoxic lectin-like protein from Pouteria torta, that is capable to cause a time-dependent decrease of the expression of p65 nuclear kappa B (NFjB) subunit [20] and may play a role in pro-inflammatory cytokine decreasing. However, to date no bioactive compound in the E. nuda extract has been identified as responsible for this effect. TNF-a and IL-1a are pleiotropic cytokines that play a key role in the immunopathogenesis of adjuvant-induced and rheumatoid arthritis [21,22]. The main source of proinflammatory cytokines TNF-a and IL-1a are dendritic cells, activated macrophages. When secreted, these cytokines promote endothelial activation, cellular activation and the induction of acute-phase proteins [23]. ENP was able to reduce serum levels of these cytokines as well as the number of animals that were high producers of these cytokines and had a significant attenuation effect on edema in rats’ paws. Inhibition of TNF-a and IL-1a production can promote various important effects, such as reduced expression of MHC molecules, reduced prostaglandin production, reduced expression of adhesion molecules and chemokines [24]. Many therapies designed to inhibit these cytokines are current in clinical trials, or even, in clinical using for RA [13]. These findings suggest that the anti-inflammatory effect of ENP occurs mainly because of its as yet uncharacterized action on TNF-a- and IL-1a-producing cells. 5. Conclusion The components of E. nuda play an anti-inflammatory role when used in topical treatment. Since the results of this study show that phonophoresis of 2% E. nuda gel has an attenuation effect on adjuvant-induced arthritis in Lewis rats and reduce the proinflammatory cytokine levels. UST was not effective in producing an attenuation effect on arthritis induced by Freund’s complete adjuvant. Acknowledgment Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for the financial support. 235 References [1] Silva C, Simeoni L, Silveira D. Genus Pouteria: chemistry and biological activity. Rev Bras de Farmacognosia 2009;19:501–9. [2] Bedoya LM, Alvarez A, Bermejo M, Gonzalez N, Beltran M, Sanchez-Palomino S, et al. Guatemalan plants extracts as virucides against HIV-1 infection. Phytomed: Int J Phytotherapy and Phytopharm 2008;15:520–4. [3] Carneiro ALB, Teixeira MFS, Oliveira VMA, Fernandes OCC, Cauper GSB, Pohlit AM. Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity. 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