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Clinical Nutrition 32 (2013) 472e475

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Clinical Nutrition

journal homepage: http: / /www.elsevier .com/locate/clnu

Short communication

N-Acetylcysteine treatment reduces TNF-a levels and myonecrosis in diaphragmmuscle of mdx mice

Rafael de Senzi Moraes Pinto, Renato Ferretti, Luis Henrique Rapucci Moraes, Humberto Santo Neto,Maria Julia Marques, Elaine Minatel*

Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo 13083-970, Brazil

a r t i c l e i n f o

Article history:Received 23 November 2011Accepted 1 June 2012

Keywords:N-AcetylcysteineTNF-aMdx miceDiaphragm muscle

* Corresponding author. Tel.: þ55 19 3521 6660; faE-mail address: minatel@unicamp.br (E. Minatel).

0261-5614/$ e see front matter � 2012 Elsevier Ltd ahttp://dx.doi.org/10.1016/j.clnu.2012.06.001

s u m m a r y

Background & aims: Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by theabsence of dystrophin. An established animal model of DMD is the mdx mouse, which is unable toexpress dystrophin. Inflammation, particularly the proinflammatory cytokine tumor necrosis factor alpha(TNF-a), strongly contributes to necrosis in the dystrophin-deficient fibers of the mdx mice and in DMD.In this study we investigated whether the antioxidant N-acetylcysteine (NAC) decreases TNF-a levels andprotects the diaphragm muscle of mdx mice against necrosis.Methods: Mdx mice (14 days old) received daily intraperitoneal injections of NAC for 14 days, followed byremoval of the diaphragm muscle. Control mdx mice were injected with saline.Results: NAC reduced TNF-a and 4-HNE-protein adducts levels, inflammation, creatine kinase levels, andmyonecrosis in diaphragm muscle.Conclusions: NAC may be used as a complementary treatment for dystrophinopathies. However, clinicaltrials are needed to determine the appropriate dose for patients with Duchenne muscular dystrophy.

� 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction

Duchenne muscular dystrophy (DMD) is a lethal X-linkedmuscle disease resulting from a defect in the subsarcolemmalprotein dystrophin. The disease affects 1 in every 3500male births.1

The mdx mouse is unable to express dystrophin and is widely usedas an animal model of DMD.2 In addition to the deregulation ofcalcium homeostasis, evidence indicates a crucial role of oxidativestress and inflammatory responses in the pathogenesis of thedisease.3,4

There is currently no effective therapy for DMD, but a widerange of anti-inflammatory drugs and nutritional interventionshave been tested in an attempt to reduce the severity of the disease.Previous studies have shown that N-acetylcysteine (NAC) canprotect limb and heart muscles of mdx mice against degenerationand inflammation.3,5 However, the effects of NAC on the mostaffected muscle in DMD, the diaphragm (DIA), were not investi-gated. Contrary to limb muscles, DIA muscles of mdx mice showprogressive myonecrosis and functional loss that closely resemblethe dystrophic phenotype seen in limb muscles of boys with DMD.6

In addition, the production of reactive oxygen species (ROS) seems

x: þ55 19 3521 6185.

nd European Society for Clinical N

to be higher in mdx DIA than in mdx limb muscles, a fact that maycontribute to the extensive fibrosis, weakness and fatigue of thismuscle.4

In the present study, we investigated whether NAC wouldprotect the DIA muscle of mdx mice against degeneration. Consid-ering the anti-inflammatory properties of NAC,3,7 we also deter-mined whether this agent would decrease the levels of tumornecrosis factor alpha (TNF-a), a key cytokine that stimulates theinflammatory cell response in limb and DIA muscles of mdxmice.8e10

2. Materials and methods

Mdx mice (14 days old) received daily intraperitoneal injectionsof NAC (SigmaeAldrich, Inc., St. Louis, MO) at a dose of 150 mg/kgbody weight diluted in 0.1 ml saline, or only saline for 14 days.Some C57BL/10 mice (control mice) were used for focused experi-ments. The animal experiments were performed in accordancewith the guidelines of the Brazilian College for Animal Experi-mentation (COBEA; process #1584-1).

For biochemical evaluation of muscle fiber degeneration, controlmice (n ¼ 10) and NAC-treated (n ¼ 10) and saline-treated (n ¼ 10)mdx mice were anesthetized with a mixture of ketamine hydro-chloride (130 mg/kg; Francotar, Virbac, Fort Worth, Texas) andxylazine hydrochloride (6.8 mg/kg; 2% Virbaxil, Virbac), and blood

utrition and Metabolism. All rights reserved.

Fig. 1. In (A) EBD-positive myofibers (arrow) indicate sarcolemmal leakage in saline-treated mdx diaphragm muscle fibers. In (B), quantification of EBD-positive fibers indiaphragm muscle of saline-treated mdx mice (mdxSAL) and N-acetylcysteine-treatedmdx mice (mdxNAC). Values are expressed as the percentage of the total number offibers in diaphragm muscle. In (C), a representative inflammation/regeneration area isindicated by the outline in NAC-treated mdx mice. In (D), quantification of theinflammatory area in diaphragm muscle of mdxSAL and mdxNAC. All values areexpressed as mean � SD (n ¼ 5, *P < 0.05 versus mdxSAL). Scale bar: 50 mm (A,C).

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samples were collected by cardiac puncture. After incubation atroom temperature for 1e2 h to allow for clotting, the samples weremicrocentrifuged at 936 g for 10 min and the supernatant (serum)was removed and used for analysis. The creatine kinase (CK) assaywas performed using a commercially available kit (CK CineticoCrystal, Bioclin, Quibasa, Minas Gerais, Brazil) and a Genesys 20spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA).Values are reported as international units (U/liter).

For morphological analysis and quantification of muscle fiberdamage, NAC-treated (n ¼ 5) and saline-treated (n ¼ 5) mdx micewere injected intraperitoneally with Evans blue dye (EBD). Twelvehours later, the mice were anesthetized as described above and theDIA muscle was dissected out, snap-frozen in n-hexane, cooled inliquid nitrogen, and stored at �80 �C. Cryostat cross-sections wereincubated in ice-cold acetone, washed with PBS, and mounted inDABCO (mounting medium for fluorescence microscopy; Sigma).EBD staining shows a bright red emission upon fluorescencemicroscopy. EBD-positive muscle fibers were counted with a handcounter in all sections and photographed under a Nikon fluores-cence microscope connected to a Hamamatsu video camera. Thenumber of EBD-positive muscle fibers is expressed as thepercentage of the total number of muscle fibers.

Other sections were stainedwith hematoxylin-eosin for analysisof the inflammatory cell infiltrate. The slides were examined undera Nikon Eclipse E400microscope connected to a personal computerand a video camera (Nikon Express Series). Nonoverlapping imagesof the entire muscle cross-section were taken and tiled togetherusing the ImagePro-Express software (Media Cybernetics, SilverSprings, MD). Inflammatory cells were identified based on nucleusmorphology and cell size characterized by basophilic nuclearstaining and scarce cytoplasm. Areas containing densely packedinflammatory cells were measured with the ImagePro-Expresssoftware and were calculated as the percentage of total musclearea in each section studied (4e5 sections per muscle). All countsand measurements were done by a blinded observer.

TNF-a and 4-hydroxynonenal (4-HNE)-protein adducts (abiomarker of lipid peroxidation11) were quantified by westernblotting in control mice (n ¼ 10) and in NAC-treated (n ¼ 10) andsaline-treated (n ¼ 10) mdx mice. Samples of the DIA muscle werelysed in lysis buffer containing freshly added protease and phos-phatase inhibitors. The samples were centrifuged at 12581 g for20 min and the soluble fraction was resuspended in 50 ml Laemmliloading buffer. Next, 30 mg total protein homogenate was loadedonto 12% SDS-polyacrylamide gels. Proteins were transferred fromthe gels to a nitrocellulose membrane using a submersion elec-trotransfer apparatus (Bio-Rad Laboratories, Hercules, CA).Membranes were blocked for 2 h at room temperature with 5%skim milk/TriseHCl buffer saline-Tween buffer (TBST; 10 mMTriseHCl, pH 8, 150 mM NaCl, and 0.05% Tween 20). Themembranes were incubated with the primary antibodies TNF-a (rabbit anti-mouse polyclonal, Millipore, California, USA) or 4-HNE (goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, Cal-ifornia, USA) overnight at 4 �C, washed in TBST, incubated with theperoxidase-conjugated secondary antibodies (mouse or rabbit IgGantibody, KPL, Gaithersburg, Maryland, USA) for 2 h at roomtemperature, and developed using the SuperSignal West PicoChemiluminescent Substrate kit (Pierce Biotechnology, Rockford,IL). To control for protein loading, blot transfer, and nonspecificchanges in protein levels, the blots were stripped and reprobed forglyceraldehyde-3-phosphate dehydrogenase (GAPDH; rabbit poly-clonal; Santa Cruz Biotechnology, Santa Cruz, California, USA). Bandintensities were quantified using the ImageJ 1.38X software(National Institutes of Health, Bethesda, MD).

All data are expressed as mean � standard deviation (SD).Statistical analysis for direct comparison between means of two

groups was performed by the Student t-test and ANOVA followedby Bonferroni test was used for multiple statistical comparisonsbetween groups. P � 0.05 was considered statistically significant.

3. Results

Body weight did not differ significantly between NAC-treated andsaline-treated mdx mice during the study period. Longitudinalcomparison showed an increase in body weight at the end of theexperiment inall animals. Thefinalmeanbodyweightwas15.0� 0.8gforNAC-treatedmdxmice and 14.6�1.5 g for saline-treatedmdxmice(P > 0.05, Student t-test). These results show that NAC treatment didnot interfere with the growth rate of youngmdxmice.

The levels of CK were significantly increased in saline-treatedmdx mice (1389.1 � 121.6 U/l) when compared to control mice(111.3 � 31.0 U/l). Administration of NAC markedly reduced thelevels of this enzyme (184.8 � 35.1 U/l; NAC mdx vs saline mdx,P < 0.05).

NAC caused a significant decrease of EBD staining, indicative ofsarcolemmal leakage and myonecrosis, in DIA muscle of mdx mice(Fig. 1A,B). This reduction was accompanied by a significantdecrease in the inflammatory area (Fig. 1C,D).

The levels of TNF-a were significantly higher in the dystrophicDIA muscle of saline-treated mdx mice compared to control mice(Fig. 2). NAC significantly reduced TNF-a levels in dystrophic DIAmuscle (Fig. 2).

Several bands of 4-HNE-protein adducts ranging from 26 to170 kDa were detected in the DIA muscle (Fig. 3). Proteins ofapproximately 170, 55 and 26 kDa exhibited elevated 4-HNE bindingin the dystrophic DIA muscle of saline-treated mdx compared tocontrol mice and NAC significantly reduced this increase (Fig. 3).

4. Discussion

NAC is a thiol-reducing agent that naturally occurs in vegetables.NAC acts as a cysteine donor and maintains the intracellular levels

Fig. 2. (A) Immunoblot analysis of TNF-a. (B) Graphs showing protein level in thecrude extracts of diaphragm muscles from C57BL/10 mice (Ctrl), saline-treated mdxmice (mdxSAL), and N-acetylcysteine-treated mdx mice (mdxNAC). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each group con-sisted of ten mice. Each band represents the pooled DIA extracts from five mice. All theexperiments were performed in triplicate, and the relative value of the band intensitywas quantified and normalized by the corresponding Ctrl. *P < 0.05 versus Ctrl;**P < 0.05 versus mdxSAL. Error bars, SD.

Fig. 3. (A) Immunoblot analysis showed several bands of 4-HNE-protein adducts,ranging from 26 to 170 kDa in the DIA muscle. (B) Graphs showing proteins levels inthe crude extracts of diaphragm muscles from C57BL/10 mice (Ctrl), saline-treated mdxmice (mdxSAL), and N-acetylcysteine-treated mdx mice (mdxNAC). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Each group con-sisted of ten mice. Each band represents the pooled DIA extracts from five mice. All theexperiments were performed in triplicate, and the relative value of the sum of thebands intensity between 170 and 26 kDa per lane was quantified and normalized bythe corresponding Ctrl. *P < 0.05 versus Ctrl; **P < 0.05 versus mdxSAL. Error bars, SD.

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of glutathione, which protects cells from free radicals. Severalstudies have demonstrated that NAC is able to protect againstoxidative stress and inflammation.7 In the present study, NACameliorated the DIA response to oxidative stress, as indicated bya decrease in the levels of 4-HNE-protein adducts. While furtherstudies are necessary to elucidate the proteins that were protectedby NAC from modification by 4HNE, one possibility could be thatthe membrane form of TNF (26 kDa),12 or calcium-binding proteins,such as the 55 kDa mitochondrial protein13 and calsequestrin-likeproteins (170 kDa)14 were affected.

NAChasbeenshowntoprotect limbandheartmusclesofmdxmiceagainstmyonecrosis.3,5 Thepresent studyadds further informationbyshowing that NAC is able to attenuatemyonecrosis and inflammationin the dystrophic DIA muscle. The elevated levels of TNF-a observedhere in mdx DIA muscle are in agreement with previous studies8e10

and are consistent with the role of this cytokine in the progressionof muscle dystrophy.8 In the present study, NAC treatment was foundto substantially reduceTNF-a levels in thedystrophicDIAmuscle. Thisfinding supports the view that the protective effects of NAC againstinflammation and myonecrosis inmdxmice are mediated, at least inpart, by a reduction of TNF-a levels in dystrophin-deficient skeletalmuscles. The inhibitory effects of NAC on TNF-a have also beendemonstrated under other experimental conditions.15

In conclusion, the present study demonstrated that NAC signifi-cantlyattenuated inflammation, lipidperoxidation andmyonecrosisin the DIA muscle of mdx mice, concomitant to a reduction in thelevels of TNF-a. NAC has been approved for use in humans and the

typical oral dose for clinical indications is 600e1800 mg/day.16 TheNAC dose used in the present study (150 mg/kg) is higher than therecommended dietary allowance in human patients. However, caremust be takenwhen correlating doses frommdxmice to dystrophicpatients, due to differences in the metabolic rate, duration of treat-ment and in the progression of dystrophy. Taken together, thepresent results suggest that NAC can be used as a complementarytreatment for dystrophinopathies. However, clinical trials areneeded to determine the appropriate dose for DMD patients.

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Conflict of interest

All the authors declare that they do not have any conflict ofinterest.

Statement of authorship

Rafael de Senzi Moraes Pinto conducted the study. Rafael deSenzi Moraes Pinto, Renato Ferretti and Luis Henrique RapucciMoraes analyzed the data and performed the statistical analysis.Elaine Minatel participated in the design of the study and coordi-nation. Elaine Minatel, Humberto Santo Neto and Maria Julia Mar-ques helped draft the manuscript. All authors read and approvedthe final manuscript.

Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP, grant 08/50731-3) and by Coor-denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-PROEX). H.S.N. and M.J.M. are recipients of fellowships from Con-selho Nacional de Pesquisas (CNPq; grants 302006/09-5 and301386/07-2). R.S.M.P. was the recipient of a CAPES fellowship, R.F.was recipients of a CNPq fellowship, and L.H.R.M. was the recipientof a FAPESP (grant 10/01087-4) fellowship. We thank Mrs. KerstinMarkendof for English revision of the manuscript.

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