Gold nanoparticles and/or N-acetylcysteine mediate carrageenan-induced inflammation and oxidative stress in a concentration-dependent manner Marcos M. S. Paula,1 Fabricia Petronilho,2 Francieli Vuolo,3 Gabriela K. Ferreira,1 ,1 Leandro De Costa,1 Giulia P. Santos,4 Pauline S. Effting,4 Felipe Dal-Pizzol,3 Alexandre G. Dal-Bo 1 4 4 Tiago E. Frizon, Paulo C. L. Silveira, Ricardo A. Pinho 1
rio De Sıntese De Complexos Multifuncionais, Programa De Po s-Graduac¸a ~o Em Cie ^ncia E Engenharia De Materiais, Laborato ma, Santa Catarina, Brazil Universidade Do Extremo Sul Catarinense, Criciu 2 rio De Fisiopatologia Clinica E Experimental, Programa De Po s-Graduac¸a ~o Em Cie ^ncias Da Sau de, Universidade Do Laborato ~ o, Santa Catarina, Brazil Sul De Santa Catarina, Tubara 3 rio De Fisiopatologia Experimental, Programa De Po s-Graduac¸a ~ o Em Cie ^ncias Da Sau de, Universidade Do Extremo Laborato ma, Santa Catarina, Brazil Sul Catarinense, Criciu 4 Laboratory of Physiology and Biochemistry of Exercise, Post-Graduate Program in Health Sciences, Health Sciences Unit, ma, Santa Catarina, Brazil Universidade Do Extremo Sul Catarinense, Criciu Received 3 February 2015; revised 19 March 2015; accepted 24 March 2015 Published online 28 April 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35469 Abstract: We report the effect of gold nanoparticles (AuNP) in an acute inflammation model induced by carrageenan (CG) and compared this effect with those induced by the antioxidant N-acetylcysteine (NAC) alone and by the synergistic effect of NAC and AuNP together. Male Wistar rats received saline or saline containing CG administered into the pleural cavity, and some rats also received NAC (20 mg/kg) subcutaneously and/or AuNP administered into the pleural cavity immediately after surgery. Four hours later, the rats were sacrificed and pleural exudates obtained for evaluation of cytokine levels and myeloperoxidase activities. Oxidative stress parameters were also evaluated in the lungs. The results demonstrated that the inflammatory process caused by the administration of CG into the pleural cavity resulted in
a substantial increase in the levels of tumor necrosis factor-a, interleukin-1b, and myeloperoxidase and a reduction in interleukin-10 levels. These levels seem to be reversed after different treatments in animals. Antioxidant enzymes exhibited positive responses after treatment of NAC 1 AuNP, and all treatments were effective at reducing lipid peroxidation and oxidation of thiol groups induced by CG. These findings suggest that small compounds, such as NAC plus AuNP, may be useful in the treatment of conditions associated with local C 2015 Wiley Periodicals, Inc. J Biomed Mater Res inflammation. V Part A: 103A: 3323–3330, 2015.
Key Words: carrageenan, gold nanoparticles, inflammation, oxidative stress, pleurisy
How to cite this article: Paula MMS, Petronilho F, Vuolo F, Ferreira GK, De Costa L, Santos GP, Effting PS, Dal-Pizzol F, Dal-Bo AG, Frizon TE, Silveira PCL, Pinho RA. 2015. Gold nanoparticles and/or N-acetylcysteine mediate carrageenan-induced inflammation and oxidative stress in a concentration-dependent manner. J Biomed Mater Res Part A 2015:103A:3323–3330.
INTRODUCTION
Carrageenan (CG) is a high-molecular-weight, sulfated polysaccharide used to assess the contribution of mediators involved in vascular changes associated with acute inflammation (paw edema and pleurisy).1 Injection of CG into the pleural space results in pleurisy, characterized by an immediate neutrophil movement out of the circulation into the inflamed tissue to function in the breakdown and remodeling of injured tissue.2 This local event induced by CG is linked to the production of neutrophil-derived reactive oxygen species (ROS), such as hydrogen peroxide, superoxide,
and hydroxyl radicals, and to the release of other neutrophil-derived mediators.3 Thus, neutrophil recruitment and activation result in parenchymal lung damage and subsequent lung dysfunction by cell death mediated by oxidative damage to DNA and proteins and lipid peroxidation in the cell membranes.4 Furthermore, in the lungs, several inflammatory stimuli have been shown to activate the transcription of nuclear factor (NF)-jB, implicating this pathway as a focal point for induction of lung inflammation.5 In normal conditions, NFjB, which is mainly composed of the subunits p50 and p65,
Correspondence to: M. M. S. Paula; e-mail: [email protected] Contract grant sponsor: Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) ~o de Aperfeic¸oamento de Pessoal de Nıvel Superior (CAPES) Contract grant sponsor: Coordenac¸a Contract grant sponsor: Universidade do Extremo Sul Catarinense (UNESC)
C 2015 WILEY PERIODICALS, INC. V
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is found in the cytoplasm of cells in its inactive form bound to its inhibitor IjB. After an inflammatory stimulus, IjB kinases phosphorylate IjB, which is degraded, and release NF-jB, which translocates to the nucleus to induce the transcription of genes involved in the early onset of the inflammatory response, such as proinflammatory cytokines.6 Over the past few decades, inorganic nanoparticles, with structures that exhibit significantly novel and distinct physical, chemical, and biological properties, have elicited much interest because of their potential for biological and pharmaceutical applications.7 Gold nanoparticles (AuNP), also called nanogold, have been actively investigated in a wide variety of biomedical applications because of their biocompatibility and easy conjugation to biomolecules.8,9 Gold compounds have received great attention as anti-inflammatory agents because they can inhibit expression of NF-jB and subsequent inflammatory reactions.10,11 According to Tsai et al.,12 nanogold decreases the levels of proinflammatory cytokines and macrophage infiltration in a model of arthritis. BarathManiKanth et al.13 also demonstrated that AuNP are antioxidative agents that inhibit the formation of ROS and scavenge free radicals to improve antioxidant defense enzymes. Conversely, antiinflammatory and antioxidant effects can be enhanced when the nanoparticles are associated with antioxidant compounds. Our group has demonstrated the therapeutic potential of AuNP isolated or linked to N-acetylcysteine (NAC) in experimental models of muscle damage.14–16 However, it remains uncertain whether the isolated use of AuNP or the use of AuNP together with compound thiols such as NAC is more effective in acute inflammatory models. Within this context, this study was designed to investigate the effects of AuNP alone and together with NAC on lung injury associated with CG-induced pleurisy. MATERIALS AND METHODS
AuNP preparation and characterization AuNP (20 nm in diameter) was synthesized as described by Turkevich et al.,17 with minor modifications. Tetrachloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (St. Louis, MO), and sodium citrate (Na3C6H5O7.2H2O), a reducing agent and stabilizer, was acquired from Nuclear (Diadema, SP, Brazil). AuNP size was controlled using distinct sodium citrate concentrations. Initially, 100 mL of 0.50 mM tetrachloroauric acid was transferred to a round-bottom flask and heated to 90 C with mechanical stirring at 700 rpm. Sodium citrate solution was then added, and the system was maintained at 90 C for another 20 min with stirring. Sodium citrate solution of 63.5 mM was used to obtain AuNP. The pH of 5.8 was adjusted to physiologic pH with a buffer solution. Then, the AuNP solutions were centrifuged (13,000 rpm for 15 min), washed twice with ultrapure water, and dispersed in a saline solution. The electronic spectra of the AuNP and NAC interactions were obtained by using a Shimadzu instrument model UV-1800 (Shimadzu Corp., Kyoto, Japan). The vibrational spectra of AuNP and AuNP plus NAC in KBr pellets were obtained in a Fourier transform infrared Shimadzu IRAffinity-1S. X-ray diffraction measurements were performed to determine the mean diameter for AuNP, which was calculated using Scherer’s
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equation on the signal at 2h 5 38 (major relative intensity).18 These values were confirmed using transmission electron microscopy analysis of images obtained using a JEOL Titan 80–300 kV. Atomic force microscopy (AFM) was used to investigate the morphological aspects of AuNP. The AuNP was placed on mica surface and evaporated under environmental conditions. The AFM measurements were performed using Shimadzu SPM-9700 equipment with dynamic mode scanning. The Au concentration was measured by atomic absorption spectroscopy (Varian model AA 240Z; Varian Medical Systems, Palo Alto, CA). The values obtained for both original solutions were 10, 25, and 50 mg L21. The zeta potentials of the solutions were also measured (Zeta Potential Analyser, Zeta PALS Brookhaven Instruments, Holtsville, NY) at 25 C, and a value of 232 mV was obtained for AuNP. Animals Adult male Wistar rats (weighing 250–350 g) obtained from the Central Animal House of Universidade do Extremo Sul Catarinense were used for induction of pleurisy. The rats were caged in groups of five, with free access to food and water, and maintained on a 12-h light–dark cycle (lights on 7:00 a.m.) at a temperature of 22 C 6 1 C. All experimental procedures involving animals were performed in accordance with the National Institutes of Health (Bethesda, MD) Guide for the Care and Use of Laboratory Animals, with the approval of Ethics Committee from Universidade do Extremo Sul Catarinense. Induction of pleurisy Pleurisy was induced by CG as previously described.19 The rats were anesthetized with ketamine hydrochloride and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 mL) or saline containing 2% k-CG (0.2 mL) was injected into the pleural cavity. The animals received saline or NAC (20 mg/kg)20 subcutaneously or AuNP 10, 25, or 50 mg/kg into the pleural cavity, and the skin incision was closed with a suture. Four hours after the induction of pleurisy, the animals were sacrificed, and approximately 1 mL of pleural exudates from each animal was obtained for cytokine level determination. Lung was also separated to evaluate myeloperoxidase (MPO) activity and oxidative damage parameters. Experimental groups The animals were divided randomly into nine groups (n 5 5 animals per group): group 1 (saline 1 saline); group 2 (CG 2% 1 saline); group 3 (CG 1 NAC); group 4 (CG 1 AuNP 10 mg/kg); group 5 (CG 1 AuNP 25 mg/kg); group 6 (CG 1 AuNP 50 mg/kg); group 7 (CG 1 AuNP 10 mg/ kg 1 NAC); group 8 (CG 1 AuNP 25 mg/kg 1 NAC); and group 9 (CG 1 AuNP 50 mg/kg 1 NAC). Inflammatory parameters The concentrations of the cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-10 were determined in pleural exudates by a standard sandwich enzyme-linked
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immunosorbent assay, using commercially available kits (R&D Systems, Minneapolis, MN). The MPO activity in lung was measured in homogenized tissue (50 mg/mL) in 0.5% hexadecyltrimethylammonium bromide centrifuged at 15,000g for 40 min. The suspension was then sonicated three times for 30 s. An aliquot of supernatant was mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM H2O2. MPO activity was measured spectrophotometrically as the change in absorbance at 650 nm at 37 C.21 Antioxidant system Superoxide dismutase (SOD) activity was measured by adrenalin oxidation inhibition according to Bannister and Calabrese.21 Muscle samples were homogenized in glycine buffer. Volumes of 5, 10, and 15 mL were retrieved, to which 5 mL catalase (0.0024 mg/mL distilled water), 175–185 mL glycine buffer (0.75 g in 200 mL distilled water at 32 C, pH 10.2), and 5 mL adrenaline (60 mM in distilled water plus 15 mL/mL fuming HCl) were added. Readings were conducted for 180 s at 10-s intervals and measured in plate readers at 480 nm. Values were expressed as SOD units per milligram of protein. Glutathione peroxidase (GPX) activity was determined according to Flohe and Gunzler.22 A 10 mL volume was aliquoted from the sample (tissue homogenized in specific buffer), and 10 mL of tert-butylhydroperoxide (tBuOOH) was added to a mixture formed by a 10-mL reaction medium (40 mL NADPH 100 mM, 3.1 mg glutathione (GSH), 4.5 mL GR 0.4 U/mL, and completed with phosphate buffer 50 mM plus EDTA 1 mM plus Azida 1 mM, pH 7.4). The method consisted of dismutating tBuOOH by GSH oxidation and oxidized glutathione (GSSG) formation catalyzed by GPX, which resulted in the oxidation of NADPH measured in a plate reader at 340 nm. Values were expressed as mM of NADPH per minute per milligram of protein. Catalase activity was determined based on the hydrogen peroxide (H2O2) decomposition rate by the enzyme present in the sample using an H2O2 10 mM solution in potassium phosphate buffer, pH 7.0.24 The maximum H2O2 decomposition rate was measured in a spectrophotometer at 240 nm, and values were expressed as catalase units per milligram of protein. Oxidative damage parameters Lipid peroxidation processes in lung tissues were determined from the formation of reactive substances during an acid-heating reaction of thiobarbituric acid.25 Briefly, samples were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, samples were heated in a boiling water bath for 30 min. Malondialdehyde equivalents were determined by absorbance at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. Results were expressed as malondialdehyde equivalents (nmol/mg protein). Sulfhydryl groups in lung tissue Total thiol content in lungs was determined using the 5,5dithiobis(2-nitrobenzoic acid) (2-nitrobenzoic acid) (DTNB) method. The conditions of DTNB measurement were as
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described in Ref. [21, with some modifications. Briefly, 30 mL of a sample was mixed with 1 mL of phosphatebuffered saline/1 mM EDTA (pH 7.5). The reaction was initialized by the addition of 30 lL of 10 mM DTNB stock solution in phosphate-buffered saline. Control samples, which did not include DTNB or protein, were run simultaneously. After 30 min of incubation at room temperature, absorbance at 412 nm was measured, and the amounts of TNB formed (equivalent to the amounts of sulfhydryl (SH) groups) were calculated. Protein determination All biochemical measures were normalized to total protein content, with bovine albumin as the standard.26 Statistical analysis Results are expressed as means 6 standard errors of mean, and p < 0.05 was considered significant. Differences between groups were determined by one-way analysis of variance followed by a Tukey’s test. All statistical analyses were performed with SPSS 20.0 for Windows (SPSS, Chicago, IL). All data are presented as means and standard errors of the mean. Differences among experimental groups were determined by two-way analysis of variance followed by the Tukey’s post hoc test. In all comparisons, statistical significance was set at p < 0.05. RESULTS
AuNP characterization and NAC interactions The AuNP used in this study had an average diameter of 20 nm, as shown in Figure 1(A), as obtained by transmission electron microscopy. The measured interplanar spacing was 0.2212 nm, typical of gold. These results were consistent with the X-ray diffraction pattern of the observed AuNP sample and AFM [Fig. 1(B)]. Figure 1(C) shows the dependence between the electronic spectra and zeta potential of AuNP after successive additions of NAC. The electronic spectrum of pure AuNP displayed a surface plasmon resonance (SPR) band with wavelength of maximum absorption at 520 nm and zeta potential of 235 mV. The successive addition of NAC in the AuNP solution caused a strong bathochromic shift in the SPR band close to 90 nm. Similarly, there was a substantial change in the zeta potential from 235 mV to 21.0 mV. The interaction between NAC and AuNP was further reinforced by analysis of Fourier transform infrared spectroscopy. Figure 1(D) shows the overlay of vibrational spectra of AuNP and AuNP plus NAC. As seen, most of the bands related to NAC were preserved after interaction with AuNP. However, the vibrational modes at 2548 cm21 (tSH), 1718 cm21 (tC@OAc), 1586–1536 cm21 (most likely COOA), and 1304–1277 cm21 (tCAOAc) were attenuated. Resonance Raman spectroscopy studies are being performed to provide more information regarding these interactions. Inflammatory parameters Initially, we verified through the analysis of proinflammatory mediators that increased IL-1b and TNF-a levels were
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FIGURE 1. A: Representative image of a transmission electron microscopy analysis of AuNP (300 kV) of 20 nm. The inset corresponds to the histogram depicting the particle size distribution. B: Micrography of AuNP obtained by atomic force microscopy. C: Plot of kmax and zeta potential with the addition of different concentrations of NAC. D: An overlay of the vibrational spectra of AuNP and AuNP plus NAC in KBr pellets.
observed after exposure to CG [Fig. 2(A,B), respectively]. The IL-1b levels decreased in all treatments, whereas the TNF-a levels were reduced at all doses of AuNP but remained as high as the values obtained in untreated group when treated with 25 mg/kg NAC. IL-10 [Fig. 2(C)], a cytokine with anti-inflammatory profile, was reduced by CG, and only AuNP 10 mg/kg and 50 mg/kg plus NAC seemed to reverse this change. Following the assessment of inflammatory mediators, we evaluated MPO activity, indicative of neutrophil infiltration in the lung tissue, and observed an increase of MPO in the group that received CG and a significant decrease in all treatments together with the proinflammatory cytokine response [Fig. 2(D)].
Oxidative stress parameters Figure 3(A–C) shows the profiles of SOD, CAT, and GPX, respectively. The SOD activity increased only when the animals were treated with NAC and AuNP at doses of 10 mg/ kg or NAC plus AuNP at doses of 25 and 50 mg/kg relative to the control group [Fig. 3(A)]. However, the catalase activity remained unaltered compared with the control group
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and decreased when the animals received NAC or AuNP at a dose of 50 mg/kg relative to the CG group [Fig. 3(B)]. In addition, the GPX activity increased only when NAC was administered together with AuNP at doses of 25 and 50 mg/kg [Fig. 3(C)]. The administration of CG increased levels of oxidative damage in lipids [Fig. 4(A)] and the oxidation of thiol [Fig. 4(B)]. All treatments were effective at reducing lipid peroxidation, and the oxidation of thiol groups induced by CG was reversed at a dose of 50 mg/kg of NAC plus AuNP.
DISCUSSION
In this study, we report on the effects of AuNP alone and together with a well-known NAC antioxidant compound in a well-established murine model for acute inflammation induced by CG. Gold is a transition metal that can use its d orbital to bond with several atoms, particularly pyridinic nitrogen, amine groups, carboxylic acids, and sulfur in thiol groups, which are often present in biomolecules.9 The high fraction of atoms in a AuNP surface and the strong interaction with these groups allow the formation of clusters with
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FIGURE 2. IL-1b, TNF-a, IL-10, and MPO (A, B, C, and D, respectively) levels in pleural exudates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
molecules of biological interest. In this sense, the increased zeta potential and bathochromic shift observed in the SPR band resulted from the interaction between ASH and ACOOH groups present in the NAC with the d orbital of gold atoms. This result can be supported by reducing the
vibrational mode of the band 2548 cm21 corresponding to group ASH. The reduction in the band intensities at 1586 and 1536 cm21 are also attributed to these ACOOH groups.27 In addition, the redox potential of NAC could potentially be displaced, making it more reductive,
FIGURE 3. SOD, CAT, and GPX antioxidant enzymes activity levels (A, B, and C, respectively) in lung tissue homogenates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
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FIGURE 4. Lipid peroxidation and SH protein integrity (A and B, respectively) levels in lung tissue homogenates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
explaining the enhancement of antioxidant effects in the NAC-AuNP association observed in this study. The schema shown in Figure 5 illustrates the interaction between the ASH and ACOOH groups of AuNP with NAC. The high fraction of atoms in an AuNP surface and the strong interaction with carboxylic and thiol groups allows the formation of clusters with molecules of biological interest that can be exploited for the development of biosensors, drug delivery systems, and analytical methods. Recently, a new method was reported for the visual detection of D-amino acids by employing 4-mercaptobenzoic acid-modified AuNP. The interaction with AuNP was so intense that the reaction could be monitored by UV-vis spectrophotometry or even the naked eye.28 A colorimetric analytical method for detecting tetracycline antibiotics in fluidic samples based on the formation of AuNP from the reduction of gold salt was reported
FIGURE 5. Interaction of the molecules of NAC with AuNP.
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earlier29 based on the appearance of a SPR band at 526 nm. This method was applied in the colorimetric detection of the antibiotic isoniazid.30 AuNP modified with aminoglycosidic antibiotics for use as a drug-delivery system was developed by Rastogi et al.31 The interaction between the functional groups of the amino-glycoside and AuNP was identified by Fourier transform infrared spectroscopy. Inflammation is commonly characterized by activation of resident macrophages and epithelial cells for recruitment and activation of cell types such as neutrophils, eosinophils, monocytes, and lymphocytes.32 Thus, cellular immune responses seem to be the predominant mechanisms involved in the pathogenesis of most inflammatory pleural effusions. Acute inflammation occurs in a relatively short time and is a necessary protection tool that removes foreign bodies and damaged tissues to prevent further damage. If uncontrolled, inflammation results in serious problems, including autoimmune, infectious, neurological, cardiovascular, and metastatic diseases. In this sense, CGinduced pleurisy is a well-established experimental model of inflammation characterized by the accumulation of fluid (edema) with many PMNs.33 During acute inflammation, serum leukocytes migrate to areas of tissue injury.34 CG-induced pleurisy has been extensively used to investigate the mechanisms involved in acute inflammation and to assess the effectiveness of anti-inflammatory drugs.35–37 Our group has shown similar results of AuNP in tendinitis and muscle damage models.38–40 In addition, the intrapleural injection of CG induced MPO activity because of inflammatory reaction, but treatment with NAC, AuNP, or NAC plus AuNP maintained MPO activity close to control levels. The decrease of MPO activity may also corroborate the inhibitory effect on neutrophils, because this proinflammatory enzyme can reflect the activation of neutrophils at the site of injury in a mouse model of pleurisy induced by CG. Accordingly, our research group has used pharmacological tools to demonstrate the anti-inflammatory properties of AuNP.39,40 In response to the presence of inflammatory mediators and the activation of phagocytic cells, elevated levels of superoxide anion are observed in different tissues.15,40 The
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radicals generated by these cells determine the secondary formation of other oxygen radicals, which react with biological targets to promote endothelial cell damage and recruit neutrophils at sites of inflammation, lipid peroxidation, and DNA single-strand damage.41 Endogenous antioxidant enzymes play an important role in the oxidative balance to reduce the possibility of oxidative damage. The GPX catalyze the reduction of hydrogen peroxide using cellular GSH as the reducing reagent. Therefore, the availability of GSH is generally the limiting factor for GPX activity. In this sense, NAC is a cell-permeable compound that acts as a source of cysteine for the synthesis of intracellular GSH.42 Therefore, administration of NAC replenishes GSH levels43 and explains the observed increase in GPX activity. The imbalance between oxidants and antioxidants results in possible damage to biomolecules, and the biochemical mechanism of action of AuNP remains unclear. The results demonstrated that treatment with AuNP and/or NAC was effective in decreasing lipid peroxidation. In addition, the administration of AuNP at a dose of 50 mg/kg associated with NAC resulted in a more evident effect of increased integrity of SH groups. The effects observed against oxidative damage from AuNP are possibly related to different mechanisms of antioxidant defense, because isolated effects were not observed with AuNP alone on SOD, catalase, and GPX activities. The ability of AuNP to inhibit the lipid from peroxidation to prevent ROS generation potentially restores the oxidative imbalances induced by CG that may be directly related to decreased secretion of the proinflammatory cytokines TNF-a and IL1-b13 observed in this study. In addition, NAC may also provide SH-groups and scavenge ROS itself.14 In conclusion, in this work, we observed that acute administration of AuNP alone or together with NAC exhibited pronounced anti-inflammatory actions, as characterized by the inhibition of IL-1b and TNF-a and an increase in levels of IL-10 in the pleural exudate of an acute model of inflammation caused by intrapleural administration of CG. Moreover, AuNP elicited important actions against oxidative damage in biomolecules, including the addition of free SH groups associated with the decreased profile of antioxidant enzymes. Taken together, these findings suggest that the administration of AuNP could effectively interfere with the inflammatory process in pleurisy induced by CG and that small compounds, such as NAC plus AuNP, may be useful in treating conditions associated with local inflammation. Thus, the dose-dependent results demonstrate that AuNP could efficiently enhance the activity of the NAC antioxidant, which represents a potential strategy for antioxidant design with novel perspectives on inflammatory disease.
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ACKNOWLEDGMENTS
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25. Riddles PW, Blakeley RL, Zerner B. Reassessment of Ellman’s reagent. Methods Enzymol 1983;91:49–60. 26. Lowry OH, Rosebrough AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–275. 27. Boeckx B, Ramaekers R, Maes G. A theoretical and matrixisolation FT-IR investigation of the conformational landscape of N-acetylcysteine. J Mol Spectrosc 2010;261:73–81. 28. Yuan LF, He YJ, Zhao H, Zhou Y, Gu P. Colorimetric detection of d-amino acids based on anti-aggregation of gold nanoparticles. Chinese Chem Lett 2014;25:995–1000. 29. Shen L, Chen J, Li N, He P, Li Z. Rapid colorimetric sensing of tetracycline antibiotics with in situ growth of gold nanoparticles. Anal Chim Acta 2014;839:83–90. 30. Zargar B, Hatamie A. Localized surface plasmon resonance of gold nanoparticles as colorimetric probes for determination of isoniazid in pharmacological formulation. Spectrochim Acta Part A: Mol Biomol Spectrosc 2013;106:185–189. 31. Rastogi L, Jyothi AK, Arunachalam J. Highly stable, protein capped gold nanoparticles as effective drug delivery vehicles for amino-glycosidic antibiotics. Mater Sci Eng C 2012;32:1571–1577. 32. Lunardelli A, Leite CE, Pires MGS, Oliveira JR. Extract of the bristles of Dirphia sp. increases nitric oxide in a rat pleurisy model. Inflamm Res 2006;55:129–135. 33. Saleh TSF, Calixto JB, Medeiros YS. Effects of antiinflammatory drugs upon nitrate and myeloperoxidase levels in the mouse pleurisy induced by carrageenan. Peptides 1999;20:949–956. 34. Paul EL, Lunardelli A, Caberlon E, Oliveira CB, Santos RCV, Biolchi V, Bastos CMA, Moreira KB, Nunes FB, Gosmann G, Oliveira JR. Anti-inflammatory and immunomodulatory effects of Baccharis trimera aqueous extract on induced pleurisy in rats and lymphoproliferation in vitro. Inflammation 2009;32:419–425.
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35. Vinegar R, Truax JF, Selph JL, Voelker FA. Pathway of onset, development, and decay of carrageenan pleurisy in the rat. Fed Proc 1982;41:2588–2595. 36. Costa R, Fernandes ES, Menezes-de-Lima O, Campos MM, Calixto JB. Effect of novel selective non-peptide kinin b1 receptor antagonists on mouse pleurisy induced by carrageenan. Peptides 2006;27:2967–2975. 37. Froede TS, Buss ZS, dos Reis GO, Medeiros YS. Evidence of antiinflammatory effects of pioglitazone in the murine pleurisy model induced by carrageenan. Int Immunopharmacol 2009;9:1394–1400. 38. Dohnert MB, Ferreira GK, Silveira PC, Zanoni ET, Dohnert LH, de Souza CT, Paula MMS. Inflammatory cytokines content in Achilles tendinopathy after phonophoresis treatment combined with gold nanoparticles and diclophenac diethylammonium in rats. Inflammation 2014;1:1–6. ^ncio M, Possato JC, Zeferino RC, Dohnert LH, 39. Dohnert MB, Vena Zugno AI, Souza D, Paula CT, Luciano MMTF. Gold nanoparticles and diclofenac diethylammonium administered by iontophoresis reduce inflammatory cytokines expression in Achilles tendinitis. Int J Nanomed 2012;7:1651–1657. 40. MacNee W. Oxidative stress and lung inflammation in airways disease. Eur J Pharmacol 2001;429:195–207. 41. Salvemini D, Riley DP, Lennon PJ, Wang ZQ, Currie MG, Macarthur H, Misko TP. Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage. Br J Pharmacol 1999;127:685–692. 42. Pinho RA, Silveira PC, Silva LA, Luiz Streck E, Dal-Pizzol F, Moreira JCF. N-Acetylcysteine and deferoxamine reduce pulmonary oxidative stress and inflammation in rats after coal dust exposure. Environ Res 2005;99:355–360. 43. Atkuri KR, Mantovani JJ, Herzenberg LA, Herzenberg LAN. Acetylcysteine—A safe antidote for cysteine/glutathione deficiency. Curr Opin Pharmacol 2007;7:355–359.
AUNP AND N-ACETYLCYSTEINE MEDIATE INFLAMMATION AND OXIDATIVE STRESS
rio De Sıntese De Complexos Multifuncionais, Programa De Po s-Graduac¸a ~o Em Cie ^ncia E Engenharia De Materiais, Laborato ma, Santa Catarina, Brazil Universidade Do Extremo Sul Catarinense, Criciu 2 rio De Fisiopatologia Clinica E Experimental, Programa De Po s-Graduac¸a ~o Em Cie ^ncias Da Sau de, Universidade Do Laborato ~ o, Santa Catarina, Brazil Sul De Santa Catarina, Tubara 3 rio De Fisiopatologia Experimental, Programa De Po s-Graduac¸a ~ o Em Cie ^ncias Da Sau de, Universidade Do Extremo Laborato ma, Santa Catarina, Brazil Sul Catarinense, Criciu 4 Laboratory of Physiology and Biochemistry of Exercise, Post-Graduate Program in Health Sciences, Health Sciences Unit, ma, Santa Catarina, Brazil Universidade Do Extremo Sul Catarinense, Criciu Received 3 February 2015; revised 19 March 2015; accepted 24 March 2015 Published online 28 April 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35469 Abstract: We report the effect of gold nanoparticles (AuNP) in an acute inflammation model induced by carrageenan (CG) and compared this effect with those induced by the antioxidant N-acetylcysteine (NAC) alone and by the synergistic effect of NAC and AuNP together. Male Wistar rats received saline or saline containing CG administered into the pleural cavity, and some rats also received NAC (20 mg/kg) subcutaneously and/or AuNP administered into the pleural cavity immediately after surgery. Four hours later, the rats were sacrificed and pleural exudates obtained for evaluation of cytokine levels and myeloperoxidase activities. Oxidative stress parameters were also evaluated in the lungs. The results demonstrated that the inflammatory process caused by the administration of CG into the pleural cavity resulted in
a substantial increase in the levels of tumor necrosis factor-a, interleukin-1b, and myeloperoxidase and a reduction in interleukin-10 levels. These levels seem to be reversed after different treatments in animals. Antioxidant enzymes exhibited positive responses after treatment of NAC 1 AuNP, and all treatments were effective at reducing lipid peroxidation and oxidation of thiol groups induced by CG. These findings suggest that small compounds, such as NAC plus AuNP, may be useful in the treatment of conditions associated with local C 2015 Wiley Periodicals, Inc. J Biomed Mater Res inflammation. V Part A: 103A: 3323–3330, 2015.
Key Words: carrageenan, gold nanoparticles, inflammation, oxidative stress, pleurisy
How to cite this article: Paula MMS, Petronilho F, Vuolo F, Ferreira GK, De Costa L, Santos GP, Effting PS, Dal-Pizzol F, Dal-Bo AG, Frizon TE, Silveira PCL, Pinho RA. 2015. Gold nanoparticles and/or N-acetylcysteine mediate carrageenan-induced inflammation and oxidative stress in a concentration-dependent manner. J Biomed Mater Res Part A 2015:103A:3323–3330.
INTRODUCTION
Carrageenan (CG) is a high-molecular-weight, sulfated polysaccharide used to assess the contribution of mediators involved in vascular changes associated with acute inflammation (paw edema and pleurisy).1 Injection of CG into the pleural space results in pleurisy, characterized by an immediate neutrophil movement out of the circulation into the inflamed tissue to function in the breakdown and remodeling of injured tissue.2 This local event induced by CG is linked to the production of neutrophil-derived reactive oxygen species (ROS), such as hydrogen peroxide, superoxide,
and hydroxyl radicals, and to the release of other neutrophil-derived mediators.3 Thus, neutrophil recruitment and activation result in parenchymal lung damage and subsequent lung dysfunction by cell death mediated by oxidative damage to DNA and proteins and lipid peroxidation in the cell membranes.4 Furthermore, in the lungs, several inflammatory stimuli have been shown to activate the transcription of nuclear factor (NF)-jB, implicating this pathway as a focal point for induction of lung inflammation.5 In normal conditions, NFjB, which is mainly composed of the subunits p50 and p65,
Correspondence to: M. M. S. Paula; e-mail: [email protected] Contract grant sponsor: Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) ~o de Aperfeic¸oamento de Pessoal de Nıvel Superior (CAPES) Contract grant sponsor: Coordenac¸a Contract grant sponsor: Universidade do Extremo Sul Catarinense (UNESC)
C 2015 WILEY PERIODICALS, INC. V
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is found in the cytoplasm of cells in its inactive form bound to its inhibitor IjB. After an inflammatory stimulus, IjB kinases phosphorylate IjB, which is degraded, and release NF-jB, which translocates to the nucleus to induce the transcription of genes involved in the early onset of the inflammatory response, such as proinflammatory cytokines.6 Over the past few decades, inorganic nanoparticles, with structures that exhibit significantly novel and distinct physical, chemical, and biological properties, have elicited much interest because of their potential for biological and pharmaceutical applications.7 Gold nanoparticles (AuNP), also called nanogold, have been actively investigated in a wide variety of biomedical applications because of their biocompatibility and easy conjugation to biomolecules.8,9 Gold compounds have received great attention as anti-inflammatory agents because they can inhibit expression of NF-jB and subsequent inflammatory reactions.10,11 According to Tsai et al.,12 nanogold decreases the levels of proinflammatory cytokines and macrophage infiltration in a model of arthritis. BarathManiKanth et al.13 also demonstrated that AuNP are antioxidative agents that inhibit the formation of ROS and scavenge free radicals to improve antioxidant defense enzymes. Conversely, antiinflammatory and antioxidant effects can be enhanced when the nanoparticles are associated with antioxidant compounds. Our group has demonstrated the therapeutic potential of AuNP isolated or linked to N-acetylcysteine (NAC) in experimental models of muscle damage.14–16 However, it remains uncertain whether the isolated use of AuNP or the use of AuNP together with compound thiols such as NAC is more effective in acute inflammatory models. Within this context, this study was designed to investigate the effects of AuNP alone and together with NAC on lung injury associated with CG-induced pleurisy. MATERIALS AND METHODS
AuNP preparation and characterization AuNP (20 nm in diameter) was synthesized as described by Turkevich et al.,17 with minor modifications. Tetrachloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (St. Louis, MO), and sodium citrate (Na3C6H5O7.2H2O), a reducing agent and stabilizer, was acquired from Nuclear (Diadema, SP, Brazil). AuNP size was controlled using distinct sodium citrate concentrations. Initially, 100 mL of 0.50 mM tetrachloroauric acid was transferred to a round-bottom flask and heated to 90 C with mechanical stirring at 700 rpm. Sodium citrate solution was then added, and the system was maintained at 90 C for another 20 min with stirring. Sodium citrate solution of 63.5 mM was used to obtain AuNP. The pH of 5.8 was adjusted to physiologic pH with a buffer solution. Then, the AuNP solutions were centrifuged (13,000 rpm for 15 min), washed twice with ultrapure water, and dispersed in a saline solution. The electronic spectra of the AuNP and NAC interactions were obtained by using a Shimadzu instrument model UV-1800 (Shimadzu Corp., Kyoto, Japan). The vibrational spectra of AuNP and AuNP plus NAC in KBr pellets were obtained in a Fourier transform infrared Shimadzu IRAffinity-1S. X-ray diffraction measurements were performed to determine the mean diameter for AuNP, which was calculated using Scherer’s
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equation on the signal at 2h 5 38 (major relative intensity).18 These values were confirmed using transmission electron microscopy analysis of images obtained using a JEOL Titan 80–300 kV. Atomic force microscopy (AFM) was used to investigate the morphological aspects of AuNP. The AuNP was placed on mica surface and evaporated under environmental conditions. The AFM measurements were performed using Shimadzu SPM-9700 equipment with dynamic mode scanning. The Au concentration was measured by atomic absorption spectroscopy (Varian model AA 240Z; Varian Medical Systems, Palo Alto, CA). The values obtained for both original solutions were 10, 25, and 50 mg L21. The zeta potentials of the solutions were also measured (Zeta Potential Analyser, Zeta PALS Brookhaven Instruments, Holtsville, NY) at 25 C, and a value of 232 mV was obtained for AuNP. Animals Adult male Wistar rats (weighing 250–350 g) obtained from the Central Animal House of Universidade do Extremo Sul Catarinense were used for induction of pleurisy. The rats were caged in groups of five, with free access to food and water, and maintained on a 12-h light–dark cycle (lights on 7:00 a.m.) at a temperature of 22 C 6 1 C. All experimental procedures involving animals were performed in accordance with the National Institutes of Health (Bethesda, MD) Guide for the Care and Use of Laboratory Animals, with the approval of Ethics Committee from Universidade do Extremo Sul Catarinense. Induction of pleurisy Pleurisy was induced by CG as previously described.19 The rats were anesthetized with ketamine hydrochloride and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 mL) or saline containing 2% k-CG (0.2 mL) was injected into the pleural cavity. The animals received saline or NAC (20 mg/kg)20 subcutaneously or AuNP 10, 25, or 50 mg/kg into the pleural cavity, and the skin incision was closed with a suture. Four hours after the induction of pleurisy, the animals were sacrificed, and approximately 1 mL of pleural exudates from each animal was obtained for cytokine level determination. Lung was also separated to evaluate myeloperoxidase (MPO) activity and oxidative damage parameters. Experimental groups The animals were divided randomly into nine groups (n 5 5 animals per group): group 1 (saline 1 saline); group 2 (CG 2% 1 saline); group 3 (CG 1 NAC); group 4 (CG 1 AuNP 10 mg/kg); group 5 (CG 1 AuNP 25 mg/kg); group 6 (CG 1 AuNP 50 mg/kg); group 7 (CG 1 AuNP 10 mg/ kg 1 NAC); group 8 (CG 1 AuNP 25 mg/kg 1 NAC); and group 9 (CG 1 AuNP 50 mg/kg 1 NAC). Inflammatory parameters The concentrations of the cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and IL-10 were determined in pleural exudates by a standard sandwich enzyme-linked
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immunosorbent assay, using commercially available kits (R&D Systems, Minneapolis, MN). The MPO activity in lung was measured in homogenized tissue (50 mg/mL) in 0.5% hexadecyltrimethylammonium bromide centrifuged at 15,000g for 40 min. The suspension was then sonicated three times for 30 s. An aliquot of supernatant was mixed with a solution of 1.6 mM tetramethylbenzidine and 1 mM H2O2. MPO activity was measured spectrophotometrically as the change in absorbance at 650 nm at 37 C.21 Antioxidant system Superoxide dismutase (SOD) activity was measured by adrenalin oxidation inhibition according to Bannister and Calabrese.21 Muscle samples were homogenized in glycine buffer. Volumes of 5, 10, and 15 mL were retrieved, to which 5 mL catalase (0.0024 mg/mL distilled water), 175–185 mL glycine buffer (0.75 g in 200 mL distilled water at 32 C, pH 10.2), and 5 mL adrenaline (60 mM in distilled water plus 15 mL/mL fuming HCl) were added. Readings were conducted for 180 s at 10-s intervals and measured in plate readers at 480 nm. Values were expressed as SOD units per milligram of protein. Glutathione peroxidase (GPX) activity was determined according to Flohe and Gunzler.22 A 10 mL volume was aliquoted from the sample (tissue homogenized in specific buffer), and 10 mL of tert-butylhydroperoxide (tBuOOH) was added to a mixture formed by a 10-mL reaction medium (40 mL NADPH 100 mM, 3.1 mg glutathione (GSH), 4.5 mL GR 0.4 U/mL, and completed with phosphate buffer 50 mM plus EDTA 1 mM plus Azida 1 mM, pH 7.4). The method consisted of dismutating tBuOOH by GSH oxidation and oxidized glutathione (GSSG) formation catalyzed by GPX, which resulted in the oxidation of NADPH measured in a plate reader at 340 nm. Values were expressed as mM of NADPH per minute per milligram of protein. Catalase activity was determined based on the hydrogen peroxide (H2O2) decomposition rate by the enzyme present in the sample using an H2O2 10 mM solution in potassium phosphate buffer, pH 7.0.24 The maximum H2O2 decomposition rate was measured in a spectrophotometer at 240 nm, and values were expressed as catalase units per milligram of protein. Oxidative damage parameters Lipid peroxidation processes in lung tissues were determined from the formation of reactive substances during an acid-heating reaction of thiobarbituric acid.25 Briefly, samples were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, samples were heated in a boiling water bath for 30 min. Malondialdehyde equivalents were determined by absorbance at 532 nm using 1,1,3,3-tetramethoxypropane as an external standard. Results were expressed as malondialdehyde equivalents (nmol/mg protein). Sulfhydryl groups in lung tissue Total thiol content in lungs was determined using the 5,5dithiobis(2-nitrobenzoic acid) (2-nitrobenzoic acid) (DTNB) method. The conditions of DTNB measurement were as
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described in Ref. [21, with some modifications. Briefly, 30 mL of a sample was mixed with 1 mL of phosphatebuffered saline/1 mM EDTA (pH 7.5). The reaction was initialized by the addition of 30 lL of 10 mM DTNB stock solution in phosphate-buffered saline. Control samples, which did not include DTNB or protein, were run simultaneously. After 30 min of incubation at room temperature, absorbance at 412 nm was measured, and the amounts of TNB formed (equivalent to the amounts of sulfhydryl (SH) groups) were calculated. Protein determination All biochemical measures were normalized to total protein content, with bovine albumin as the standard.26 Statistical analysis Results are expressed as means 6 standard errors of mean, and p < 0.05 was considered significant. Differences between groups were determined by one-way analysis of variance followed by a Tukey’s test. All statistical analyses were performed with SPSS 20.0 for Windows (SPSS, Chicago, IL). All data are presented as means and standard errors of the mean. Differences among experimental groups were determined by two-way analysis of variance followed by the Tukey’s post hoc test. In all comparisons, statistical significance was set at p < 0.05. RESULTS
AuNP characterization and NAC interactions The AuNP used in this study had an average diameter of 20 nm, as shown in Figure 1(A), as obtained by transmission electron microscopy. The measured interplanar spacing was 0.2212 nm, typical of gold. These results were consistent with the X-ray diffraction pattern of the observed AuNP sample and AFM [Fig. 1(B)]. Figure 1(C) shows the dependence between the electronic spectra and zeta potential of AuNP after successive additions of NAC. The electronic spectrum of pure AuNP displayed a surface plasmon resonance (SPR) band with wavelength of maximum absorption at 520 nm and zeta potential of 235 mV. The successive addition of NAC in the AuNP solution caused a strong bathochromic shift in the SPR band close to 90 nm. Similarly, there was a substantial change in the zeta potential from 235 mV to 21.0 mV. The interaction between NAC and AuNP was further reinforced by analysis of Fourier transform infrared spectroscopy. Figure 1(D) shows the overlay of vibrational spectra of AuNP and AuNP plus NAC. As seen, most of the bands related to NAC were preserved after interaction with AuNP. However, the vibrational modes at 2548 cm21 (tSH), 1718 cm21 (tC@OAc), 1586–1536 cm21 (most likely COOA), and 1304–1277 cm21 (tCAOAc) were attenuated. Resonance Raman spectroscopy studies are being performed to provide more information regarding these interactions. Inflammatory parameters Initially, we verified through the analysis of proinflammatory mediators that increased IL-1b and TNF-a levels were
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FIGURE 1. A: Representative image of a transmission electron microscopy analysis of AuNP (300 kV) of 20 nm. The inset corresponds to the histogram depicting the particle size distribution. B: Micrography of AuNP obtained by atomic force microscopy. C: Plot of kmax and zeta potential with the addition of different concentrations of NAC. D: An overlay of the vibrational spectra of AuNP and AuNP plus NAC in KBr pellets.
observed after exposure to CG [Fig. 2(A,B), respectively]. The IL-1b levels decreased in all treatments, whereas the TNF-a levels were reduced at all doses of AuNP but remained as high as the values obtained in untreated group when treated with 25 mg/kg NAC. IL-10 [Fig. 2(C)], a cytokine with anti-inflammatory profile, was reduced by CG, and only AuNP 10 mg/kg and 50 mg/kg plus NAC seemed to reverse this change. Following the assessment of inflammatory mediators, we evaluated MPO activity, indicative of neutrophil infiltration in the lung tissue, and observed an increase of MPO in the group that received CG and a significant decrease in all treatments together with the proinflammatory cytokine response [Fig. 2(D)].
Oxidative stress parameters Figure 3(A–C) shows the profiles of SOD, CAT, and GPX, respectively. The SOD activity increased only when the animals were treated with NAC and AuNP at doses of 10 mg/ kg or NAC plus AuNP at doses of 25 and 50 mg/kg relative to the control group [Fig. 3(A)]. However, the catalase activity remained unaltered compared with the control group
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and decreased when the animals received NAC or AuNP at a dose of 50 mg/kg relative to the CG group [Fig. 3(B)]. In addition, the GPX activity increased only when NAC was administered together with AuNP at doses of 25 and 50 mg/kg [Fig. 3(C)]. The administration of CG increased levels of oxidative damage in lipids [Fig. 4(A)] and the oxidation of thiol [Fig. 4(B)]. All treatments were effective at reducing lipid peroxidation, and the oxidation of thiol groups induced by CG was reversed at a dose of 50 mg/kg of NAC plus AuNP.
DISCUSSION
In this study, we report on the effects of AuNP alone and together with a well-known NAC antioxidant compound in a well-established murine model for acute inflammation induced by CG. Gold is a transition metal that can use its d orbital to bond with several atoms, particularly pyridinic nitrogen, amine groups, carboxylic acids, and sulfur in thiol groups, which are often present in biomolecules.9 The high fraction of atoms in a AuNP surface and the strong interaction with these groups allow the formation of clusters with
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FIGURE 2. IL-1b, TNF-a, IL-10, and MPO (A, B, C, and D, respectively) levels in pleural exudates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
molecules of biological interest. In this sense, the increased zeta potential and bathochromic shift observed in the SPR band resulted from the interaction between ASH and ACOOH groups present in the NAC with the d orbital of gold atoms. This result can be supported by reducing the
vibrational mode of the band 2548 cm21 corresponding to group ASH. The reduction in the band intensities at 1586 and 1536 cm21 are also attributed to these ACOOH groups.27 In addition, the redox potential of NAC could potentially be displaced, making it more reductive,
FIGURE 3. SOD, CAT, and GPX antioxidant enzymes activity levels (A, B, and C, respectively) in lung tissue homogenates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
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FIGURE 4. Lipid peroxidation and SH protein integrity (A and B, respectively) levels in lung tissue homogenates of rats subjected to CG administration and treated with NAC or AuNP 10, 25, and 50 mg/kg alone and with NAC. Results are expressed as means 6 SD, n 5 8 per group. *p < 0.05 compared with the sal 1 sal group. #p < 0.05 compared with the CG 1 sal group.
explaining the enhancement of antioxidant effects in the NAC-AuNP association observed in this study. The schema shown in Figure 5 illustrates the interaction between the ASH and ACOOH groups of AuNP with NAC. The high fraction of atoms in an AuNP surface and the strong interaction with carboxylic and thiol groups allows the formation of clusters with molecules of biological interest that can be exploited for the development of biosensors, drug delivery systems, and analytical methods. Recently, a new method was reported for the visual detection of D-amino acids by employing 4-mercaptobenzoic acid-modified AuNP. The interaction with AuNP was so intense that the reaction could be monitored by UV-vis spectrophotometry or even the naked eye.28 A colorimetric analytical method for detecting tetracycline antibiotics in fluidic samples based on the formation of AuNP from the reduction of gold salt was reported
FIGURE 5. Interaction of the molecules of NAC with AuNP.
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earlier29 based on the appearance of a SPR band at 526 nm. This method was applied in the colorimetric detection of the antibiotic isoniazid.30 AuNP modified with aminoglycosidic antibiotics for use as a drug-delivery system was developed by Rastogi et al.31 The interaction between the functional groups of the amino-glycoside and AuNP was identified by Fourier transform infrared spectroscopy. Inflammation is commonly characterized by activation of resident macrophages and epithelial cells for recruitment and activation of cell types such as neutrophils, eosinophils, monocytes, and lymphocytes.32 Thus, cellular immune responses seem to be the predominant mechanisms involved in the pathogenesis of most inflammatory pleural effusions. Acute inflammation occurs in a relatively short time and is a necessary protection tool that removes foreign bodies and damaged tissues to prevent further damage. If uncontrolled, inflammation results in serious problems, including autoimmune, infectious, neurological, cardiovascular, and metastatic diseases. In this sense, CGinduced pleurisy is a well-established experimental model of inflammation characterized by the accumulation of fluid (edema) with many PMNs.33 During acute inflammation, serum leukocytes migrate to areas of tissue injury.34 CG-induced pleurisy has been extensively used to investigate the mechanisms involved in acute inflammation and to assess the effectiveness of anti-inflammatory drugs.35–37 Our group has shown similar results of AuNP in tendinitis and muscle damage models.38–40 In addition, the intrapleural injection of CG induced MPO activity because of inflammatory reaction, but treatment with NAC, AuNP, or NAC plus AuNP maintained MPO activity close to control levels. The decrease of MPO activity may also corroborate the inhibitory effect on neutrophils, because this proinflammatory enzyme can reflect the activation of neutrophils at the site of injury in a mouse model of pleurisy induced by CG. Accordingly, our research group has used pharmacological tools to demonstrate the anti-inflammatory properties of AuNP.39,40 In response to the presence of inflammatory mediators and the activation of phagocytic cells, elevated levels of superoxide anion are observed in different tissues.15,40 The
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radicals generated by these cells determine the secondary formation of other oxygen radicals, which react with biological targets to promote endothelial cell damage and recruit neutrophils at sites of inflammation, lipid peroxidation, and DNA single-strand damage.41 Endogenous antioxidant enzymes play an important role in the oxidative balance to reduce the possibility of oxidative damage. The GPX catalyze the reduction of hydrogen peroxide using cellular GSH as the reducing reagent. Therefore, the availability of GSH is generally the limiting factor for GPX activity. In this sense, NAC is a cell-permeable compound that acts as a source of cysteine for the synthesis of intracellular GSH.42 Therefore, administration of NAC replenishes GSH levels43 and explains the observed increase in GPX activity. The imbalance between oxidants and antioxidants results in possible damage to biomolecules, and the biochemical mechanism of action of AuNP remains unclear. The results demonstrated that treatment with AuNP and/or NAC was effective in decreasing lipid peroxidation. In addition, the administration of AuNP at a dose of 50 mg/kg associated with NAC resulted in a more evident effect of increased integrity of SH groups. The effects observed against oxidative damage from AuNP are possibly related to different mechanisms of antioxidant defense, because isolated effects were not observed with AuNP alone on SOD, catalase, and GPX activities. The ability of AuNP to inhibit the lipid from peroxidation to prevent ROS generation potentially restores the oxidative imbalances induced by CG that may be directly related to decreased secretion of the proinflammatory cytokines TNF-a and IL1-b13 observed in this study. In addition, NAC may also provide SH-groups and scavenge ROS itself.14 In conclusion, in this work, we observed that acute administration of AuNP alone or together with NAC exhibited pronounced anti-inflammatory actions, as characterized by the inhibition of IL-1b and TNF-a and an increase in levels of IL-10 in the pleural exudate of an acute model of inflammation caused by intrapleural administration of CG. Moreover, AuNP elicited important actions against oxidative damage in biomolecules, including the addition of free SH groups associated with the decreased profile of antioxidant enzymes. Taken together, these findings suggest that the administration of AuNP could effectively interfere with the inflammatory process in pleurisy induced by CG and that small compounds, such as NAC plus AuNP, may be useful in treating conditions associated with local inflammation. Thus, the dose-dependent results demonstrate that AuNP could efficiently enhance the activity of the NAC antioxidant, which represents a potential strategy for antioxidant design with novel perspectives on inflammatory disease.
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ACKNOWLEDGMENTS
The authors thank CNPq and UNESC for their fellowship support. REFERENCES 1. Lentsch AB, Ward PA. Regulation of experimental lung inflammation. Respir Physiol 2001;28:17–21. 2. Nantel F, Denis D, Gordon R, Northey A, Cirino M, Metters KM, Chan CC. Distribution and regulation of cyclooxygenase-2 in
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AUNP AND N-ACETYLCYSTEINE MEDIATE INFLAMMATION AND OXIDATIVE STRESS
