l-Arginine Decreases Inflammation and Modulates the Nuclear Factor-κB/Matrix Metalloproteinase Cascade in Mdx Muscle Fibers
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.071009
ISSN1525-2191
AutoresKarim Hnia, Jérôme Gayraud, Gérald Hugon, Michèle Ramonatxo, Sabine de la Porte, Stéfan Matecki, Dominique Mornet,
Tópico(s)Sirtuins and Resveratrol in Medicine
ResumoDuchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of l-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether l-arginine inhibits inflammatory signaling cascades. We demonstrate that l-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-κB levels and activity. NF-κB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-κB signaling in both mdx and DMD muscle. Moreover, l-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-κB. We show that the inhibitory effect of l-arginine on the NF-κB/MMP cascade reduces β-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which l-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-κB-related signaling cascades could be potential therapeutic targets for DMD management. Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of l-arginine, the substrate of nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. However, little is known about signaling events that occur in dystrophic muscle with l-arginine treatment. Considering the implication of inflammation in dystrophic processes, we asked whether l-arginine inhibits inflammatory signaling cascades. We demonstrate that l-arginine decreases inflammation and enhances muscle regeneration in the mdx mouse model. Classic stimulatory signals, such as proinflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α, are significantly decreased in mdx mouse muscle, resulting in lower nuclear factor (NF)-κB levels and activity. NF-κB serves as a pivotal transcription factor with multiple levels of regulation; previous studies have shown perturbation of NF-κB signaling in both mdx and DMD muscle. Moreover, l-arginine decreases the activity of metalloproteinase (MMP)-2 and MMP-9, which are transcriptionally activated by NF-κB. We show that the inhibitory effect of l-arginine on the NF-κB/MMP cascade reduces β-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Collectively, our results clarify the molecular events by which l-arginine promotes muscle membrane integrity in dystrophic muscle and suggest that NF-κB-related signaling cascades could be potential therapeutic targets for DMD management. Duchenne muscular dystrophy (DMD) is the most common muscle wasting disease and it leads to early disability and death in young boys.1Deconinck N Dan B Pathophysiology of Duchenne muscular dystrophy: current hypotheses.Pediatr Neurol. 2007; 36: 1-7Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar The absence of dystrophin is a key factor in developing this disease.2Koenig M Hoffman PE Bertelson JC Monaco PA Feener C Kunkel ML Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.Cell. 1987; 50: 509-517Abstract Full Text PDF PubMed Scopus (2051) Google Scholar This protein is the central component of the dystrophin-glycoprotein complex that links the actin cytoskeleton to the extracellular matrix, thus maintaining muscle fiber membrane integrity.3Ibraghimov-Beskrovnaya O Ervasti MJ Leveille JC Slaughter AC Sernett WS Campbell PK Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1208) Google Scholar Although the primary genetic defect is known, the dystrophic process (eg, necrosis, exhaustible regeneration, and secondary fibrosis) has not been definitively identified. The mdx mouse, a genetically homologous DMD model, is frequently used to study the disease pathogenesis, despite relevant clinical and pathological differences.4Bulfield G Siller GW Wight AP Moore JK X chromosome-linked muscular dystrophy (mdx) in the mouse.Proc Natl Acad Sci USA. 1984; 81: 1189-1192Crossref PubMed Scopus (1437) Google Scholar, 5Sicinski P Geng Y Ryder-Cook SA Barnard AE Darlison GM Barnard JP The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.Science. 1989; 244: 1578-1580Crossref PubMed Scopus (1046) Google Scholar Compared to human disease, the murine model shows slower disease progression with repetitive degeneration regeneration cycles occurring between 2 and 12 weeks of age, particularly in the diaphragm that closely reflects the human pathology.6Stedman HH Sweeney LH Shrager BJ Maguire CH Panettieri AR Petrof B Narusawa M Leferovich MJ Sladky TJ Kelly MA The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.Nature. 1991; 352: 536-539Crossref PubMed Scopus (759) Google Scholar On the other hand, utrophin (a fetal homolog of dystrophin) is overexpressed in mdx muscles whereas its level seems lower in DMD. These observations suggest that genetically or pharmacologically utrophin induction could be an interesting therapeutic strategy to compensate for dystrophin deficiency. Several lines of evidence suggest the involvement of oxidative stress in the dystrophic process7Tidball GJ Wehling-Henricks M The role of free radicals in the pathophysiology of muscular dystrophy.J Appl Physiol. 2007; 102: 1677-1686Crossref PubMed Scopus (178) Google Scholar and underline the fact that dystrophic muscle cells are more susceptible to reactive oxygen intermediates. Oxidants are traditionally considered to exert their effects via a direct toxic action on target cells, but recent studies have suggested their contributory role in gene induction.8Yang XY Muqit MM Latchman SD Induction of parkin expression in the presence of oxidative stress.Eur J Neurosci. 2006; 24: 1366-1372Crossref PubMed Scopus (26) Google Scholar, 9Ito Y Oumi S Nagasawa T Nishizawa N Oxidative stress induces phosphoenolpyruvate carboxykinase expression in H4IIE cells.Biosci Biotechnol Biochem. 2006; 70: 2191-2198Crossref PubMed Scopus (27) Google Scholar Nuclear factor (NF)-κB is a pleiotropic transcription factor activated by low reactive oxygen intermediate levels and inhibited by antioxidants.10Jamaluddin M Wang S Boldogh I Tian B Brasier RA TNF-alpha-induced NF-kappaB/RelA Ser(276) phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway.Cell Signal. 2007; 19: 1419-1433Crossref PubMed Scopus (165) Google Scholar This factor regulates the expression of a plethora of genes involved in the inflammatory, immune, and acute stress response. In fact, NF-κB, after proteosomal degradation of the inhibitory protein I-κ-B (IκB-α), translocates to the nucleus and binds target DNA elements in the promoter of genes expressing cytokine, chemokine, cell adhesion molecules, immunoreceptors, and inflammatory enzymes such as nitric oxide synthase (nNOS), matrix metalloproteinase (MMPs), and phospholipase A2.11Lianxu C Hongti J Changlong Y NF-kappaBp65-specific siRNA inhibits expression of genes of COX-2. NOS-2 and MMP-9 in rat IL-1beta-induced and TNF-alpha-induced chondrocytes.Osteoarthritis Cartilage. 2006; 14: 367-376Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Several recent observations have suggested a possible role of NF-κB in the muscle-wasting process.12Kramer FH Goodyear JL Exercise, MAPK, and NF-{kappa}B signaling in skeletal muscle.J Appl Physiol. 2007; 103: 388-395Crossref PubMed Scopus (286) Google Scholar NF-κB activity and level have been demonstrated to be increased in muscle of either DMD patients or mdx mice.13Messina S Altavilla D Aguennouz M Seminara P Minutoli L Monici CM Bitto A Mazzeo A Marini H Squadrito F Vita G Lipid peroxidation inhibition blunts nuclear factor-kappaB activation, reduces skeletal muscle degeneration, and enhances muscle function in mdx mice.Am J Pathol. 2006; 168: 918-926Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Kumar A Lnu S Malya R Barron D Moore J Corry BD Boriek MA Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma.FASEB J. 2003; 17: 1800-1811Crossref PubMed Scopus (85) Google Scholar, 15Kumar A Boriek MA Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy.FASEB J. 2003; 17: 386-396Crossref PubMed Scopus (234) Google Scholar Monici and colleagues16Monici CM Aguennouz M Mazzeo A Messina C Vita G Activation of nuclear factor-kappaB in inflammatory myopathies and Duchenne muscular dystrophy.Neurology. 2003; 60: 993-997Crossref PubMed Scopus (151) Google Scholar observed increased immunoreactivity for NF-κB in the cytoplasm of all regenerating fibers and 20 to 40% of necrotic fibers in DMD as well as in inflammatory myopathies. NF-κB is activated in response to several secreted inflammatory molecules such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), whose levels have been found to be elevated in dystrophin-deficient muscle and other types of muscular dystrophies.17Stuerenburg HJ Jung R Schoser GB Age effects on interleukin-6 and interleukin-1beta responses to endurance exercise in patients with neuromuscular diseases.Arch Gerontol Geriatr. 1999; 29: 21-27Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar On the other hand, NF-κB also regulates myogenesis, and systemic administration of the NF-κB inhibitor curcumin stimulates muscle regeneration after traumatic injury, suggesting a beneficial effect of NF-κB blockade on muscle repair.18Durham JW Arbogast S Gerken E Li PY Reid BM Progressive nuclear factor-kappaB activation resistant to inhibition by contraction and curcumin in mdx mice.Muscle Nerve. 2006; 34: 298-303Crossref PubMed Scopus (43) Google Scholar Moreover, reduced necrosis was observed in mdx muscle when TNF-α action was blocked with etanercept.19Hodgetts S Radley H Davies M Grounds DM Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with Etanercept in mdx mice.Neuromuscul Disord. 2006; 16: 591-602Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar l-arginine, the substrate of nNOS, was proposed as a powerful pharmacological tool by reducing the necrotic zone in mdx lower limbs.20Archer DJ Vargas CC Anderson EJ Persistent and improved functional gain in mdx dystrophic mice after treatment with L-arginine and deflazacort.FASEB J. 2006; 20: 738-740PubMed Google Scholar Nevertheless, the mechanistic effect of the l-arginine on dystrophic muscle and its impact on necrotic associated pathways has not yet been investigated. Here we assessed whether l-arginine treatment of 5-week-old mice could affect the NF-κB pathway and investigated the impact of this regulation on the protein stability of mdx sarcolemma. Our study showed that l-arginine treatment inhibited IL-1β, IL-6, and TNF-α secretion by macrophages and other inflammatory infiltrating cells in mdx diaphragm muscle, leading to down-regulation of the NF-κB/MMP cascade. This effect decreased 43-kDa β-dystroglycan cleavages into a ∼30-kDa form and significantly improved dystrophic patterns. Full-length β-dystroglycan anchors and stabilizes utrophin within a glycoprotein-complex located at the plasmatic membrane, thus relocating nNOS to the subsarcolemmal compartment. Five-week-old male dystrophin-deficient (mdx) mice were purchased from Jackson Laboratory, Bar Harbor, ME (S/C57 BL/10 SC MAJAX SN 1801), and bred in our animal facilities. Mice were housed in plastic cages in a temperature-controlled environment with a 12-hour light/dark cycle and free access to food and water. The investigation complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animals were euthanized by rapid cervical dislocation and experiments were carefully designed to minimize the number of animals and their suffering. Animals (mdx control, n = 7; and mdx treated, n = 7) were treated for 2 weeks with intraperitoneal injections. The control group was injected with (SS) physiological saline solution (mdx-SS). The treated group was injected with l-arginine, (Sigma, St. Louis, MO), 20 μl vol/g at a cc of 200 mg/kg (mdx-l-Arg). After beheading, the diaphragm was dissected into two parts. One part was immediately frozen in liquid nitrogen-cooled isopentane and stored at −80°C and the other part was analyzed immediately to determine the enzymatic activities. Rabbit polyclonal antibodies that detect the macrophage α/CCL3 protein, IL-1β, IL-6, and TNF-α were purchased from Abcam, Cambridge, UK. Polyclonal antibodies against NF-κB, IκB, and BcL2 protein family (BAX and BcL2) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Polyclonal antibodies against β-dystroglycan (LG5) and utrophin (K7) were produced and determined to be specific after testing in competition with their corresponding peptides, as previously described.21Chazalette D Hnia K Rivier F Hugon G Mornet D Alpha7B integrin changes in mdx mouse muscles after L-arginine administration.FEBS Lett. 2005; 579: 1079-1084Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar The polyclonal anti-actin antibody was from Sigma and the nNOS commercial antibody was from BD Transduction Laboratories (Lexington, KY). Pan- and phospho-monoclonal antibodies which, respectively, recognize the nonphosphorylated and phosphorylated forms of p38 MAPK, ERK1/2, and JNK1/2, were purchased from R&D Systems (Minneapolis, MN). All antibodies were tested according to the manufacturer's recommendations using positive protein controls. Ten-μm transverse cryostat sections of mdx and wild-type muscles were stained by hematoxylin and eosin (H&E). Morphometric analysis was performed on six cross-sections of each muscle. The histological parameters were evaluated and treated according to previously described suitable parameters.21Chazalette D Hnia K Rivier F Hugon G Mornet D Alpha7B integrin changes in mdx mouse muscles after L-arginine administration.FEBS Lett. 2005; 579: 1079-1084Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 22De Luca A Nico B Liantonio A Didonna PM Fraysse B Pierno S Burdi R Mangieri D Rolland FJ Camerino C Zallone A Confalonieri P Andreetta F Arnoldi E Courdier-Fruh I Magyar PJ Frigeri A Pisoni M Svelto M Camerino CD A multidisciplinary evaluation of the effectiveness of cyclosporine A in dystrophic mdx mice.Am J Pathol. 2005; 166: 477-489Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 23Briguet A Courdier-Fruh I Foster M Meier T Magyar PJ Histological parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse.Neuromuscul Disord. 2004; 14: 675-682Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar Stained sections were viewed under a Nikon optiphot-2 microscope and images were analyzed using Histolab program version 5-13-1 (license number 2497; Microvision Instruments, Evry, France). All diaphragm images were obtained under identical conditions and at the same magnification. The shape of each muscle fiber was accurately defined (on images ×400%) to obtain a schematic diagram of each H&E-stained diaphragm section, assigning black or white colors to fibers with peripheral or internalized nuclei, respectively. These schematic representations of each muscle section were later analyzed using Histolab software, as described above. On each schematic diagram, the percentage of nonmuscle area (colored in light gray) and black or white fibers were analyzed to obtain related surfaces, relative fiber percentages, and variance of Feret's diameters, respectively. Data were averaged per ∼1500 muscle fibers from each dissected diaphragm obtained after l-arginine treatment of mdx mice and statistically compared using the Mann-Whitney test. Displayed data (mean ± SD) were normalized to the staining intensity of normal diaphragm muscle fibers from untreated mdx mice and comparisons were considered significant when *P < 0.05. Ten-μm unfixed cryostat sections were incubated with the primary antibody used at the appropriate dilution for 1 hour at room temperature or O/N at 4°C, according to the manufacturer's recommendations (Santa Cruz Biotechnology, Santa Cruz, CA). After washing in phosphate-buffered saline solution, sections were incubated with a secondary antibody (Cy3-goat or fluorescein isothiocyanate-goat anti-rabbit IgG; Chemicon International, Temecula, CA). For negative controls, only the second antibody was applied. Muscles (0.1 g) were homogenized in 150 μl of 5% sodium dodecyl sulfate (SDS) buffer (50 mmol/L Tris/HCl, pH 8.0, 10 mmol/L ethylenediaminetetraacetic acid, 5% SDS) supplemented with 1% trypsin inhibitor, 1% saponin, and 15 μg/ml of leupeptin. After centrifugation (10 minutes at 13,000 × g), the protein concentration was estimated in the supernatant using the BCA protein assay kit (Pierce, Rockford, IL). Protein homogenates recovered from the supernatant obtained from each sample were denatured for 5 minutes at 95°C in reducing buffer (50 μl of SDS buffer containing 5% SDS, 0.01% bromophenol blue, 10% glycerol, and 5% β-mercaptoethanol). Protein extracts were submitted, in duplicate, to SDS-polyacrylamide gel electrophoresis (3 to 10% or 5 to 15%) with prestained standard proteins (Bio-Rad, Hercules, CA) to achieve a more accurate molecular weight determination. The resulting gel was transferred onto a 0.2-μm nitrocellulose membrane using the transfer buffer (25 mmol/L Tris-HCl, pH 8.3, 192 mmol/L glycine, and 20% methanol). The membranes were stained with Ponceau S (0.005% in 1% acetic acid) to confirm that equal amounts of protein had been loaded and were blocked with Tris-buffer, 0.1% Tween 20 (TBST) containing 3% bovine serum albumin (w/v) for 1 hour at room temperature. All membranes were incubated with primary antibodies followed by several washings in Tris-buffer and 0.1% Tween 20 (TBST). The membranes were incubated with peroxidase-conjugated antibodies (Chemicon International) for 1 hour and washed several times with the washing buffer, as described above, and finally revealed by the enhanced chemiluminescence system according to the manufacturer's protocol (Amersham, Little Chalfont, Buckinghamshire, UK). The protein signals were quantified by scanning densitometry using the National Institutes of Health (Bethesda, MD) program package. The results from each experimental group were expressed as integrated intensities relative to the control samples. Equal loading of proteins was assessed on stripped blots by immunodetection using the β-actin antibody. Zymography was performed as described by Heussen and Dowdle.24Heussen C Dowdle BE Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.Anal Biochem. 1980; 102: 196-202Crossref PubMed Scopus (1878) Google Scholar Zymogram gel (5 to 15% polyacrylamide) was impregnated with gelatin (1 mg/ml). After electrophoresis, the gel was washed in 2.5% Triton X-100 solution at room temperature and incubated for 24 hours in a substrate buffer (50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L CaCl2, 0.02% NaN3) at 37°C. MMPs are secreted in a latent form and require cleavage of a peptide from their NH2 terminus for activation. However, exposure of proenzymes of tissue extracts to SDS during the gel separation procedure leads to activation without proteolytic cleavage. The gel was stained in Coomassie Blue R250 for 1 hour and destained in water overnight. Gelatin-degrading enzymes were visualized as clear bands, indicating proteolysis of the substrate protein. The gel was treated in black/white and the MMP bands were quantified using the National Institutes of Health image analysis program. Total RNA was isolated from muscle with an SV total RNA isolation kit (Promega Corporation, Madison, WI) according to the manufacturer's instructions. We used 0.1 μg of total RNA to generate first strand cDNA sequences with random hexanucleotide primers and the M-MLV reverse transcriptase (Invitrogen Corporation, Carlsbad, CA). Twelve μl of the RT reaction was subsequently used for each PCR reaction using the Taq polymerase (Qbiogene SA, Illkrich, France). Three PCR reactions were performed to amplify the mouse Up395 isoform using the primers (mUp-ex17/20-F and mUp-ex17/20-F) described previously.25Hnia K Hugon G Masmoudi A Mercier J Rivier F Mornet D Effect of beta-dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane.Neuroscience. 2006; 141: 607-620Crossref PubMed Scopus (16) Google Scholar, 26Wilson J Putt W Jimenez C Edwards YH Up71 and up140, two novel transcripts of utrophin that are homologues of short forms of dystrophin.Hum Mol Genet. 1999; 8: 1271-1278Crossref PubMed Scopus (37) Google Scholar The mouse β-dystroglycan (β-DG) gene fragment was amplified using the following primers: Fdag1, 5′-GGAGGCTGTTCCCACCGTGGT-3′ and Rdag1, 5′-CTCTGCATTCTGTTCAACAGATCG-3′. The obtained PCR products were 383 bp (mUp395) and 474 bp (dag1), respectively. The PCR conditions included 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and a final extension of 72°C for 8 minutes. Experiments were performed in duplicate using the housekeeping gene GAPDH to normalize the expression level of Up395 and β-DG mRNA, as described previously.25Hnia K Hugon G Masmoudi A Mercier J Rivier F Mornet D Effect of beta-dystroglycan processing on utrophin/Dp116 anchorage in normal and mdx mouse Schwann cell membrane.Neuroscience. 2006; 141: 607-620Crossref PubMed Scopus (16) Google Scholar, 27Hnia K Hugon G Rivier F Masmoudi A Mercier J Mornet D Modulation of p38 mitogen-activated protein kinase cascade and metalloproteinase activity in diaphragm muscle in response to free radical scavenger administration in dystrophin-deficient Mdx mice.Am J Pathol. 2007; 170: 633-643Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Negative controls were performed, with total RNA replaced by either RNAase-free water or reaction mixtures without RT (for DNA contaminations). PCR products were visualized on 1.5% agarose gel. One hundred-bp molecular mass markers (Promega) were used to estimate the molecular mass of the PCR products. The results are expressed as mean ± SD. Statistical analysis was performed using the unpaired Student's test and multiple statistical comparisons between groups were performed by one-way analysis of variance followed by Bonferroni's t-test posthoc correction to obtain a better evaluation of the intra- and intergroup variabilities and avoid false-positives using the Statview program (version 5.0; SAS Institute Inc., Cary, NC). Statistical significance was set at *P < 0.01. In accordance with previous studies,28Chaubourt E Voisin V Fossier P Baux G Israel M Porte DLS The NO way to increase muscular utrophin expression?.C R Acad Sci III. 2000; 323: 735-740Crossref PubMed Scopus (16) Google Scholar, 29Voisin V Sebrie C Matecki S Yu H Gillet B Ramonatxo M Israel M Porte DLS L-arginine improves dystrophic phenotype in mdx mice.Neurobiol Dis. 2005; 20: 123-130Crossref PubMed Scopus (84) Google Scholar the morphological aspect of l-arginine-treated mdx diaphragm is significantly improved. The analyzed muscles share also a significant decrease in the number of infiltrated cells (macrophages and lymphocytes). Analysis of the H&E-stained section (Figure 1A) shows a decrease in the percentage (∼20%) of nonmuscle area in treated mdx diaphragm sections (Figure 1A, histogram 1, panel 1). However, there was an overall increase in the number of fibers with internalized nuclei (more than 20%), with a concomitant increase in the variance coefficient of the Feret's diameter (Figure 1, Figure 2, Figure 3). To determine the specific impact of the treatment on histomorphological features of muscle fibers, we measured the variance coefficient for the Feret's diameter and the average diameter in two different fiber populations: fibers with internal nuclei, ie, regenerating fibers (desmin-positive fibers, not shown) and fibers with peripheral nuclei (drawing boards; Figure 1, Figure 2, respectively). We noted in the first population (regenerating fibers) that both the Feret's diameter variance coefficient and the average diameter were significantly increased after treatment. However, these two parameters were not affected in the second population (fibers with peripheral nuclei). This indicated, in accordance with the results presented above, that the treatment enhances the regenerative process in mdx muscle while also protecting new muscle fibers from degeneration or/and necrosis.Figure 2A: TNF-a, IL-1β, and IL-6 staining of mdx diaphragm sections and co-localization with macrophages (inset). The enlarged views (macro) correspond to specific fields with high fluorescent staining. Comparison between untreated mdx diaphragms (left) and l-arginine-treated samples (right) are relatively similar when stained with TNF-α (first lane), IL-1β (middle lane), and IL-6 (last lane). B: Western blot detection of TNF-α, IL-1β, and IL-6, in mdx diaphragm samples from mdx-SS (control) and mdx-l-Arg groups. β-Actin revelation is shown at the bottom of the blots to indicate equivalent protein concentrations between all samples. The results are presented as a histogram (bottom) and allow comparison of treated (black) and untreated (white) mdx diaphragms. Data are presented as mean ± SD. Statistical significance was *P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Western blot detections of NF-κB and IκB-α from treated (mdx-l-Arg) and control (mdx-SS) mdx diaphragm. Immunoblot detection of β-actin on stripped nitrocellulose membranes was recorded to show the equivalent protein quantity between samples. Blots were analyzed and the results are presented as a histogram (bottom) and allow comparison of treated (black) and untreated (white) muscle extracts. Data are presented as mean ± SD. Statistical significance was *P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was suggested that proinflammatory cytokines secreted by macrophages and other immune cells are involved in the necrotic phase in dystrophic muscle. It is also demonstrated that muscle regeneration occurs in response to injury and inflammation and that modulation of this inflammatory process would predictably decrease, rather than increase, the regenerative capacity of skeletal muscle. We thus focused on the impact of l-arginine on these proinflammatory cytokines. Co-staining of macrophage markers with TNF-α, IL-1β, and IL-6, showed a decrease in the levels of these inflammatory cytokines in mdx-l-Arg diaphragms (Figure 2A, and corresponding inset) with a concomitant reduction in the number of macrophages in necrotic zones. This result was confirmed by Western blot analyses, showing a decreased total level of TNF-α (∼62%), IL-1β (∼56%), and IL-6 (∼54%), as shown in Figure 2B (blots and associated histograms). We next sought to determine the mechanism by which l-arginine mediates its action on dystrophic myofibers. Several lines of evidence have suggested that NF-κB-mediated inflammation has an important role in mdx and DMD muscles. Our results showed that l-arginine treatment has an inhibitory effect on the total NF-κB level in mdx diaphragm and concomitant inhibition of IκB-α degradation (Figure 3). In line with previous reports,30Kabouridis SP Hasan M Newson J Gilroy WD Lawrence T Inhibition of NF-kappa B activity by a membrane-transducing mutant of I kappa B alpha.J Immunol. 2002; 169: 2587-2593Crossref PubMed Scopus (48) Google Scholar we suggest that the decreased level of inflammatory cytokines (TNF-α, IL-1β, and IL-6) after l-arginine treatment could directly affect the expression level and the activity of NF-κB in dystrophic muscle myofibers. Moreover, one of the mechanisms by which NF-κB signaling appears to contribute to dystrophy is by promoting chronic inflammation. Because of the wide range of NF-κB target genes, including cytokines and chemokines, we speculate that NF-κB-mediated transcription might serve as an amplification signal for a persistent immune response in dystrophic muscle, as supported by the noted reduction in the inflammatory response in dystrophic muscle after l-arginine administration. Besides immune cells, injured or necrotic muscle fibers could also act as a source of cytokines and chemokines involved in immune cell chemotaxis. Having gained insight into the r
Referência(s)