Andrographolide attenuates skeletal muscle dystrophy in mdx mice and increases efficiency of cell therapy by reducing fibrosis
© Cabrera et al.; licensee BioMed Central Ltd. 2014
Received: 22 October 2013
Accepted: 26 February 2014
Published: 21 March 2014
Duchenne muscular dystrophy (DMD) is characterized by the absence of the cytoskeletal protein dystrophin, muscle wasting, increased transforming growth factor type beta (TGF-β) signaling, and fibrosis. At the present time, the only clinically validated treatments for DMD are glucocorticoids. These drugs prolong muscle strength and ambulation of patients for a short term only and have severe adverse effects. Andrographolide, a bicyclic diterpenoid lactone, has traditionally been used for the treatment of colds, fever, laryngitis, and other infections with no or minimal side effects. We determined whether andrographolide treatment of mdx mice, an animal model for DMD, affects muscle damage, physiology, fibrosis, and efficiency of cell therapy.
mdx mice were treated with andrographolide for three months and skeletal muscle histology, creatine kinase activity, and permeability of muscle fibers were evaluated. Fibrosis and TGF-β signaling were evaluated by indirect immunofluorescence and Western blot analyses. Muscle strength was determined in isolated skeletal muscles and by a running test. Efficiency of cell therapy was determined by grafting isolated skeletal muscle satellite cells onto the tibialis anterior of mdx mice.
mdx mice treated with andrographolide exhibited less severe muscular dystrophy than untreated dystrophic mice. They performed better in an exercise endurance test and had improved muscle strength in isolated muscles, reduced skeletal muscle impairment, diminished fibrosis and a significant reduction in TGF-β signaling. Moreover, andrographolide treatment of mdx mice improved grafting efficiency upon intramuscular injection of dystrophin-positive satellite cells.
These results suggest that andrographolide could be used to improve quality of life in individuals with DMD.
KeywordsAndrographolide mdx DMD Fibrosis Skeletal muscle Cell therapy
Muscular dystrophies are a group of genetic muscular diseases. The most severe is Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3,500 births for which there is no effective therapy . DMD is caused by the absence of dystrophin, a cytoskeletal protein that anchors the muscle fiber to the extracellular matrix (ECM). The absence of this protein increases susceptibility to muscle fiber rupture caused by the continuous cycles of contraction and relaxation [2, 3]. Thus, children with this condition gradually and progressively lose muscle strength, typically requiring the use of a wheel chair from the age of ten and dying in the late second or early third decade of life as a result of cardiorespiratory arrest. One of the causes of muscle damage and loss of function is the development of fibrosis, which is characterized by excessive accumulation of extracellular matrix (ECM) that replaces muscle tissue with connective tissue, dramatically affecting the environment of the fibers and normal muscle physiology [4–9].
Pathologic features of DMD include myofiber atrophy, fatty degeneration, necrosis and fibrosis, but only fibrosis has been shown through clinical studies to correlate with poor motor outcome, evaluated by muscle strength and age at loss of ambulation . This finding supports the notion that fibrosis directly contributes to progressive muscle dysfunction and the lethal phenotype of DMD . Therefore, the development of new drugs and therapies with anti-fibrotic activity is crucial.
Andrographolide, a bicyclic diterpenoid lactone, is the major constituent of Andrographis paniculata, a plant indigenous to Southeast Asian countries that has been used as an official herbal medicine in China for many years . Traditionally, it is used for the treatment of colds, fever, laryngitis, and other infections, with no or minimal side effects. It has been reported to be particularly efficient at regulating immune responses [12, 13] and possesses anti-inflammatory properties by reducing the generation of reactive oxygen species in human neutrophils . Andrographolide not only regulates inflammation, but is also effective against the fibrotic pathology observed in chronic liver and kidney diseases [14–16]. Mechanistically, andrographolide forms a covalent adduct with NF-κB, thus blocking the binding of NF-κB to nuclear proteins . NF-κB is an important transcription factor in the progression of skeletal muscular dystrophic diseases [18–20].
Because dystrophic disorders such as DMD have genetic origins there is a great effort to restore gene expression through gene and/or cell therapies. Nevertheless, these therapies represent a major challenge because muscle is the most abundant tissue in the body. Moreover, fibrosis strongly reduces the efficacy of these approaches [6, 9, 21], therefore, even if current trials are successful, they are unlikely to elicit a significant benefit when extended to people with more advanced stages of the disease and enhanced fibrosis [21, 22]. Therefore, understanding the cellular and molecular mechanisms underlying muscle fibrogenesis associated with muscular dystrophies is critical to the development of an effective anti-fibrotic therapy for this type of disease.
In this study, we investigated the effects of andrographolide on the onset of dystrophy in mdx mice, an animal model used to study DMD. We demonstrated that andrographolide treatment of dystrophic mice prevented damage and fibrosis progression as reflected by reduced collagen and fibronectin deposition through a mechanism that involves a decrease in the expression of transforming growth factor type beta (TGF-β) and its downstream mediator connective tissue growth factor (CTGF/CCN2). The reduction of fibrosis was associated with enhanced muscle strength and improved exercise performance. Finally, we determined that the use of andrographolide increased the efficiency of cell therapy through fibrosis reduction.
Animals and experimental exercise
We used 12-week-old control or mdx male mice of the C57BL/10 ScSn strain. The animals were kept at room temperature with a 24-hour night-day cycle and were fed with pellets and water ad libitum. Experimental exercise involved running the mice on a treadmill three times per week for 30 minutes each session at 12 m/minute over three or four months [6, 23–25]. Two experimental groups were designed: animals in the first group were injected intraperitoneally (ip) with andrographolide (1.0 mg/kg/day) and animals in the second group were treated with vehicle alone. At the end of the experiment the muscles were dissected and removed under anesthesia (isofluorane gas) and then the animals were sacrificed. Tissues were used for electrophysiological measurement or rapidly frozen and stored at −80°C until processing. All protocols were conducted in strict accordance with guidelines and with the formal approval of the Animal Ethics Committee of the Pontificia Universidad Católica de Chile.
Skeletal muscle histology
Evans blue dye (EBD) uptake
Animals were sacrificed 24 hours after injection with EBD (1% in PBS). The tibialis anterior (TA) muscles were snap-frozen in isopentane, sectioned in 7-μm cryosections, and fixed in 4% paraformaldehyde . Muscle cross-sections were visualized under a Nikon Diaphot Eclipse-600 (Nikon, Melville, NY, USA) inverted microscope equipped for epifluorescence. The percentage of EBD-positive fibers was manually counted in a blinded manner .
Serum creatine kinase measurement
Mice were anesthetized and blood was obtained from the periorbital vascular plexus directly into 70 μl microhematocrit tubes (Fisher Scientific, Loughborough, UK). Serum was obtained by allowing the blood to clot at room temperature for 30 minutes followed by centrifugation at 1,700 × g for ten minutes. Serum creatine kinase (CK) was measured using an enzymatic system (Valtek, Santiago, Chile) according to the manufacturer’s instructions .
Muscles were homogenized in ten volumes of Tris-EDTA buffer with 1 mM PMSF as described previously . Briefly, protein concentration in aliquots of muscle extract was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) using BSA as a standard. Aliquots (50 to 100 μg) were subjected to SDS gel electrophoresis in 8% or 10% polyacrylamide gels, electrophoretically transferred onto PVDF membranes (Schleicher & Schuell, Keene, NH, USA), and probed with specific antibodies against fibronectin (Sigma, St. Louis, MO, USA), collagen III (Rockland, Gilbertsville, PA USA) and GAPDH (Millipore, Billerica, MA, USA), tubulin (Sigma, St. Louis, MO, USA) as described previously . All immunoreactions were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). Densitometric analysis and quantification were performed using ImageJ software (NIH, Bethesda, MD, USA) .
For immunofluorescence, 7-μm cryosections were fixed in 4% paraformaldehyde, blocked for one hour in 10% goat serum in PBS and incubated for one hour at room temperature with specific antibodies against fibronectin (Sigma, St. Louis, MO, USA), collagen I (Chemicon, Temecula, CA, USA), F4/80 (Abcam, Cambridge, MA, USA), p-Smad-2 (Abcam, Cambridge, MA, USA), and dystrophin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). FITC-conjugated goat anti-rabbit IgG and rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For monoclonal anti-mouse antibodies, all incubations were performed with mouse IgG-blocking solution from the MOM kit (Vector Lab, Burlingame, CA, USA) diluted in 0.01% Triton X-100/PBS. For nuclear staining, sections were incubated with 1 μg/ml Hoechst 33258 in PBS for ten minutes. After rinsing, the coverslips were mounted using Fluoromount (Dako, Carpinteria, CA, USA) and observed under a Nikon Diaphot inverted microscope equipped for epifluorescence .
NF-κB detection in vivo by Southwestern blotting
Synthetic sense DNA 5′-AGTTGAGGGGACTTTCCCAGGC-3′, which contains a consensus sequence for NF-κB, was used as the probe. After annealing with complementary DNA (80°C for two minutes), the probe was labeled with digoxigenin (DIG) oligonucleotide 3′-end labeling (Boehringer Mannheim, Mannheim, Germany). Paraffin-embedded muscle sections were dewaxed, rehydrated, and fixed with 0.2% paraformaldehyde for 30 minutes at 28°C. Sections were subsequently digested with 433 U/mg pepsin A (Sigma, St. Louis, MO, USA), washed twice with buffer 1 (10 mmol/L HEPES, 40 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L EDTA, 0.25% BSA, pH 7.4), treated with 0.1 mg/mL DNase I and washed with buffer 2 (10 mmol/L HEPES, 40 mmol/L NaCl, 1 mmol/L DTT, 10 mmol/L EDTA, 0.25% BSA, pH 7.4) to stop the reaction. Labeled probe (100 pmol/L) diluted in buffer 1 containing 0.5 mg/mL poly dl-dC (Pharmacia, New York, NY, USA) was applied overnight at 37°C. After washing, sections were incubated for one hour in blocking solution (0.01× SSC, 0.01% SDS, 0.03% Tween 20, 0.1 mol/L maleic acid, 0.15 mol/L NaCl, pH 7.5) and then overnight at 4°C with rabbit anti-digoxigenin antibody (1:250 in blocking solution; Invitrogen, Carlsbad, CA, USA). The samples were washed and incubated with a secondary Alexa fluor 568 anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA). Nuclear staining with Hoechst 33258 was performed as described above. We used the following negative controls: (a) absence of probes, (b) DIG-labeled mutant NF-κB probe (sense: 5′-AGTTGAGGCTCCTTTCCCAGGC-3′), at the same concentration as labeled probe, (c) competition assays with 100-fold excess of unlabeled NF-κB probe, followed by incubation with the respective labeled probe .
RNA isolation and quantitative real-time PCR analysis
During tissue collection, one TA muscle from each animal was rapidly frozen in liquid nitrogen and used for RNA isolation. Total RNA was isolated with TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Total RNA (500 ng) from each sample was reverse transcribed to cDNA using Super Script Reverse Transcriptase II (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR (qPCR) reactions were performed using SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA). Levels of TGF-β and CTGF were determined as described before . The real-time amplification of genes was measured with the iCycler thermocycler system and iQ5 optical system software (Bio-Rad, Hercules, CA, USA) .
The isometric force of isolated muscles was measured as described previously [6, 25, 26]. Briefly, optimum muscle length (Lo) and stimulation voltage were determined from micromanipulation of muscle length to produce maximum isometric twitch force. Maximum isometric tetanic force (Po) was determined from the plateau of the frequency-force relationship after successive stimulations at 1 to 200 Hz for 450 ms, with two-minute rests between stimuli. After determination of isometric contractile properties, muscles were subjected to a three repeated tetanic stimulation protocol. Muscles at Lo were maximally stimulated for 450 ms once every five seconds. After functional testing, muscles were removed from the bath, trimmed of their tendons and any adhering non-muscle tissue, blotted once on filter paper and weighed [33–35]. Muscle mass and Lo were used to calculate specific net force (force normalized per total muscle fiber cross-sectional area (CSA), mN/mm2) [6, 26].
Mice were subjected to a running test for 15 minutes at 15 m/minute on a treadmill. The number of times that mice were retarded to the first one third of the moving platform (detentions) was counted [6, 25].
Single myofiber isolation and satellite cell grafting
Single myofibers were isolated essentially as described previously [6, 36]. Briefly, extensor digitorum longus (EDL) and soleus muscles from six-week-old C57-BL10 mice were dissected and digested in 0.2% (w/v) collagenase type 1 (Sigma, St. Louis, MO, USA) in DMEM (Gibco, Grand Island, NY, USA) with 4 mM L-glutamine (Sigma, St. Louis, MO, USA) and 1% penicillin and streptomycin solution (Sigma, St. Louis, MO, USA) for 90 minutes in a 37°C water bath. Satellite cells were separated from the myofibers by physical trituration using the method of Collins et al. . The isolated intact fibers were suspended in 10 ml of complete medium and triturated with a 19 G needle mounted on a 1 ml syringe. The suspension was sequentially passed through a 70-μm and 40-μm cell sieve (BD Biosciences, San Jose, CA, USA) to remove debris. The satellite cell suspension was centrifuged for 15 minutes at 450 × g. The pellet was resuspended in physiologic serum (0.9% NaCl). An aliquot was stained with 1 μg/ml Hoechst and 1 μg/ml cholera toxin subunit B conjugated to Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for five minutes, washed with PBS and incubated with trypan blue. Cells were counted using a hemocytometer. Double-stained cells that exclude trypan blue were counted as viable cells and the concentration of cells was adjusted to 25 cells/μl. To confirm the purity of the isolated satellite cells, an aliquot was seeded onto Matrigel (1 mg/ml) (Sigma, St. Louis, MO, USA) and cultured overnight in complete medium for 18 hours before immunocytochemistry for myogenic markers. For grafting, 500 satellite cells were grafted into both TA muscles of seven-month-old mdx mice in a C57-BL10 background under anesthesia using an 8-mm 30 G needle under microscopic observation.The number of dystrophin positive fibers was determined as described previously .
Determination of the grafted-satellite cell survival
The determination of the survival of the grafted satellite cells was performed as previously described . Briefly, 500 freshly isolated satellite cells from a C57-EGFP mice, as described in the previous section, were grafted into both TA muscles of seven-month-old mdx mice in a C57-BL10 background under anesthesia using an 8-mm 30 G needle under microscopic observation. The muscle genomic DNA was extracted with a DNA purification kit (QiAamp DNA from Quiagen). Then the purified DNA were subjected to amplification by real time PCR using TaqMan PCR Universal Master Mix, in an Illumina Eco real time PCR (Illumina, USA), Primer and Taqman probe for EGFP and b-actin (endogenous control) were from Applied Biosystems, (USA).
The statistical significance of differences between the means of the experimental groups was evaluated using one-way analysis of variance (ANOVA) with a post hoc Bonferroni multiple-comparison test or two tailed t-tests (Prism 3.0, GraphPad, San Diego, CA, USA). A difference was considered statistically significant at P < 0.05.
Andrographolide treatment improves histology and reduces muscle damage in dystrophic skeletal muscle
Cumulative muscle damage in exercised mdx mice treated or not with andrographolide
mdx + vehicle
7.63 ± 0.85
42.14 ± 4.03
49.77 ± 4.61
mdx + andrographolide
4.14 ± 0.51b
30.23 ± 2.86a
34.37 ± 2.97a
Fibrosis in dystrophic skeletal muscle is reduced by andrographolide treatment
Andrographolide reduces the activity of NF-κB in vivo
Andrographolide reduces the level of the pro-fibrotic factor TGF-β and the downstream Smad-dependent signaling pathway in mdx skeletal muscles
Andrographolide treatment improves skeletal muscle strength and exercise performance in mdx mice
Reduction of fibrosis by andrographolide improves the efficiency of in vivo cell therapy
In this paper, we show that andrographolide treatment reduced skeletal muscle damage and fibrosis in mdx mice. We observed that this reduction was associated with an increase in muscle functionality. Moreover, we showed that the improved skeletal muscle phenotype of dystrophic mice favored the incorporation of dystrophin-positive muscle cells after intramuscular injection of satellite cells derived from WT skeletal muscle. To the best of our knowledge, this is the first report of the use of andrographolide on a model of muscular dystrophy.
Several anti-fibrotics have been tested to decrease fibrosis associated to dystrophic skeletal muscle . Among them neutralizing antibodies against all three forms of TGF-β importantly reduced hydroxyproline levels and plasma creatine kinase, improved respiratory function and grip strength . Halofunginone has been tested in mdx mice, reducing collagen content and improving respiratory and heart function. It has been suggested that it inhibits p-Smad-3 in response to TGF-β1 [45–47]. The use of inhibitors and antagonists of the renin-angiotensin system have been shown to decrease fibrosis and improve skeletal muscle function . Infusion of angiotensin 1-7, which signals through the Mas receptor, has been shown to importantly decrease fibrosis, TGF-β mediated signaling and increase skeletal muscle strength . It is difficult to compare which of these drugs, including andrographolide, have a better effect on dystrophic skeletal muscle, since some of them may also have other undesired side effects. Furthermore, the same readouts are not always determined in each case. Nevertheless a comparative study, under the same experimental conditions would be very valuable.
Corticosteroids are currently the most effective treatment for DMD [50–52]. They act by blocking transcription factors such as NF-κB and AP-1 to down-regulate a vast group of pro-inflammatory genes. However, the use of corticosteroids is associated with unwanted side effects and a significant proportion of patients are steroid-resistant , therefore there is an urgent need to develop novel anti-inflammatory drugs to replace or complement current therapy. In this study, we have preliminary data that indicate that parallel treatment with prednisone or andrographolide in mdx three-month-old animals for three months produce the same histological improvement together with a decrease in the amount of collagen (Additional file 4: Figure S4).
Andrographolide has been used in acute and chronic diseases such as the common cold and rheumatoid arthritis with no observable side effects (for a review, see reference ). Therefore, it is reasonable to consider the use of alternative treatments such as andrographolide to improve skeletal muscle physiology in patients suffering from skeletal muscular dystrophies such as DMD. Although our findings suggest that andrographolide might be used to improve quality of life in individuals with DMD, the effectiveness of andrographolide in other skeletal muscle dystrophies requires further investigation. Extrapolation of studies in small animals, such as mice, to clinical treatment of humans can be very difficult. The use of andrographolide as shown in this paper is promising because andrographolide has been used alone or as a botanical extract for the treatment of colds, fever, laryngitis and other infections with no or minimal side effects. Andrographolide not only regulates inflammation, but also suppresses the fibrotic pathology observed in chronic liver and kidney diseases [14–16]. Other advantages include the low cost of the drug, or a botanical extract highly enriched in andrographolide, and the fact that it can be given orally in capsules, thus providing hope for future therapeutic options of an oral formulation with no undesired side effects for patients with muscular disorders.
Fibrosis is one of the main features of dystrophic muscle. A high increase in ECM deposition is found in biopsies from DMD patients and in different animal models of the disease [4, 39, 54, 55]. Although enormous efforts have been made to restore dystrophin expression in DMD patients through different approaches, such as gene and cell therapies, it has been proposed that fibrosis is an important barrier to the success of these approaches [9, 21, 56–58]. Therefore, targeting fibrosis is an important strategy to generate an environment that facilitates cell migration and regeneration in the dystrophic muscle. This is supported by a report that local injection of fibroblasts secreting metalloproteinases reduced collagen deposition, thereby allowing efficient subsequent therapy with intra-arterial injection of WT mesoangioblasts in dystrophic muscles , and the observation that models with reduced activity of a pro-fibrotic growth factor showed an increase in the number of grafted dystrophin-positive skeletal muscle fibers in mdx muscle .
Different experimental approaches have been attempted to decrease fibrosis in dystrophic muscle and other myopathies [59, 60]. One of the main pro-fibrotic cytokines is TGF-β, therefore blocking TGF-β promotes histological recovery of muscle tissue and also significantly decreases the levels of ECM proteins, thus promoting and increasing muscular functionality [41, 60, 61]. In the same way, our results show that inhibition of fibrosis is correlated with a decrease in TGF-β. Furthermore, our results showed that andrographolide treatment inhibited the TGF-β canonical signaling pathway, as evidenced by a reduced number of nuclei positive for the key TGF-β intracellular mediators p-Smad-2 and p-Smad-3. The decrease seems to affect, in higher proportion, interstitial cells than regenerating fibers. These observations suggest a novel mechanism of the action for andrographolide.
Andrographolide is an anti-inflammatory molecule that acts through specific inhibition of NF-κB. NF-κB predominantly functions as a heterodimer of p65 and p50, in which p65 contains the transactivation domain and p50 is involved in the recognition of NF-κB DNA element responses. It has been reported that andrographolide inhibits NF-κB by covalent binding with a cysteine residue in the p50 subunit, thus inhibiting the binding of NF-κB to DNA . Contrary to our report, mdx mice heterozygous for the p50 subunit do not present any significant histological changes; however, mdx mice heterozygous for the p65 subunit present a mild dystrophic phenotype . A probable explanation is that p50 bound to andrographolide sequesters the p65 subunits thus preventing transactivation activity of this protein on target genes. However, this hypothesis needs to be confirmed.
Moreover, we showed that the inhibition of fibrosis by andrographolide correlates with a decrease in TGF-β and CTGF expression. Both growth factors can directly induce fibrosis in skeletal muscle and specific reduction of either of them improves the dystrophic phenotype in mdx mice [6, 40]. It is known that TGF-β is a strong inducer of CTGF activity [7, 42, 62]. Furthermore, it has been shown that CTGF strongly synergizes with TGF-β to induce fibrosis . CTGF expression requires NF-κB activity because the CTGF promoter contains a functional and specific NF-κB response element . Therefore, is plausible that andrographolide down-regulates CTGF expression through inhibition of NF-κB because andrographolide decreased NF-κB activity in vivo.
In conclusion, our preclinical evaluation of andrographolide in a mouse model of DMD showed promising improvements in dystrophic skeletal muscles by preventing damage and fibrosis progression. The reduction of fibrosis was associated with enhanced muscle strength and increased efficiency of cell therapy.
one-way analysis of variance
bovine serum albumin
connective tissue growth factor
Duchenne Muscular Dystrophy
Dulbecco’s modified Eagle’s medium
Evans blue dye
extensor digitorum longus
nuclear factor kappa-light-chain-enhancer of activated B cells
quantitative real-time polymerase chain reaction
transforming growth factor type beta
This study was supported by research grants from CARE PFB12/2007. FONDEF: D07I1051, VIU 110070. FONDECYT: 1110426, 11110010, 1120380, 3140396, 3130593. CONICYT: Graduate student scholarship (DC), AT-24091098, 79090027. Fundación Chilena para Biología Celular Proyecto MF-100. Association-Française Contre Les Myopathies AFM 16670. UNAB-DI-281-13/R.
- Kapsa R, Kornberg AJ, Byrne E: Novel therapies for Duchenne muscular dystrophy. Lancet Neurol 2003, 2: 299-310. 10.1016/S1474-4422(03)00382-XView ArticlePubMedGoogle Scholar
- Blau HM, Webster C, Pavlath GK: Defective myoblasts identified in Duchenne muscular dystrophy. Proc Natl Acad Sci USA 1983, 80: 4856-4860. 10.1073/pnas.80.15.4856PubMed CentralView ArticlePubMedGoogle Scholar
- Allen DG, Whitehead NP: Duchenne muscular dystrophy - what causes the increased membrane permeability in skeletal muscle? Int J Biochem Cell Biol 2011, 43: 290-294. 10.1016/j.biocel.2010.11.005View ArticlePubMedGoogle Scholar
- Alvarez K, Fadic R, Brandan E: Augmented synthesis and differential localization of heparan sulfate proteoglycans in Duchenne muscular dystrophy. J Cell Biochem 2002, 85: 703-713. 10.1002/jcb.10184View ArticlePubMedGoogle Scholar
- Wynn TA: Cellular and molecular mechanisms of fibrosis. J Pathol 2008, 214: 199-210. 10.1002/path.2277PubMed CentralView ArticlePubMedGoogle Scholar
- Morales MG, Gutierrez J, Cabello-Verrugio C, Cabrera D, Lipson KE, Goldschmeding R, Brandan E: Reducing CTGF/CCN2 slows mdx muscle dystrophy and improves cell-therapy. Hum Mol Genet 2013, 22: 4938-4951. 10.1093/hmg/ddt352View ArticlePubMedGoogle Scholar
- Brandan E, Gutierrez J: Role of proteoglycans in the regulation of the skeletal muscle fibrotic response. FEBS J 2013, 280: 4109-4117. 10.1111/febs.12278View ArticlePubMedGoogle Scholar
- Varga J, Brenner DA, Phan SH: Fibrosis Research: Methods and Protocols. Totowa, NJ: Humana Press; 2005:392.View ArticleGoogle Scholar
- Zhou L, Lu H: Targeting fibrosis in Duchenne muscular dystrophy. J Neuropathol Exp Neurol 2010, 69: 771-776. 10.1097/NEN.0b013e3181e9a34bPubMed CentralView ArticlePubMedGoogle Scholar
- Desguerre I, Mayer M, Leturcq F, Barbet JP, Gherardi RK, Christov C: Endomysial fibrosis in Duchenne muscular dystrophy: a marker of poor outcome associated with macrophage alternative activation. J Neuropathol Exp Neurol 2009, 68: 762-773. 10.1097/NEN.0b013e3181aa31c2View ArticlePubMedGoogle Scholar
- Shen YC, Chen CF, Chiou WF: Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect. Br J Pharmacol 2002, 135: 399-406. 10.1038/sj.bjp.0704493PubMed CentralView ArticlePubMedGoogle Scholar
- Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R: Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata . J Exp Ther Oncol 2003, 3: 147-158. 10.1046/j.1359-4117.2003.01090.xView ArticlePubMedGoogle Scholar
- Calabrese C, Berman SH, Babish JG, Ma X, Shinto L, Dorr M, Wells K, Wenner CA, Standish LJ: A phase I trial of andrographolide in HIV positive patients and normal volunteers. Phytother Res 2000, 14: 333-338. 10.1002/1099-1573(200008)14:5<333::AID-PTR584>3.0.CO;2-DView ArticlePubMedGoogle Scholar
- Lee TY, Lee KC, Chang HH: Modulation of the cannabinoid receptors by andrographolide attenuates hepatic apoptosis following bile duct ligation in rats with fibrosis. Apoptosis 2010, 15: 904-914. 10.1007/s10495-010-0502-zView ArticlePubMedGoogle Scholar
- Lee MJ, Rao YK, Chen K, Lee YC, Chung YS, Tzeng YM: Andrographolide and 14-deoxy-11,12-didehydroandrographolide from Andrographis paniculata attenuate high glucose-induced fibrosis and apoptosis in murine renal mesangial cell lines. J Ethnopharmacol 2010, 132: 497-505. 10.1016/j.jep.2010.07.057View ArticlePubMedGoogle Scholar
- Ye JF, Zhu H, Zhou ZF, Xiong RB, Wang XW, Su LX, Luo BD: Protective mechanism of andrographolide against carbon tetrachloride-induced acute liver injury in mice. Biol Pharm Bull 2011, 34: 1666-1670. 10.1248/bpb.34.1666View ArticlePubMedGoogle Scholar
- Xia YF, Ye BQ, Li YD, Wang JG, He XJ, Lin X, Yao X, Ma D, Slungaard A, Hebbel RP, Key NS, Geng JG: Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50. J Immunol 2004, 173: 4207-4217. 10.4049/jimmunol.173.6.4207View ArticlePubMedGoogle Scholar
- Acharyya S, Villalta SA, Bakkar N, Bupha-Intr T, Janssen PM, Carathers M, Li ZW, Beg AA, Ghosh S, Sahenk Z, Weinstein M, Gardner KL, Rafael-Fortney JA, Karin M, Tidball JG, Baldwin AS, Guttridge DC: Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J Clin Invest 2007, 117: 889-901. 10.1172/JCI30556PubMed CentralView ArticlePubMedGoogle Scholar
- Peterson JM, Guttridge DC: Skeletal muscle diseases, inflammation, and NF-kappaB signaling: insights and opportunities for therapeutic intervention. Int Rev Immunol 2008, 27: 375-387. 10.1080/08830180802302389View ArticlePubMedGoogle Scholar
- Peterson JM, Kline W, Canan BD, Ricca DJ, Kaspar B, Delfín DA, DiRienzo K, Clemens PR, Robbins PD, Baldwin AS, Flood P, Kaumaya P, Freitas M, Kornegay JN, Mendell JR, Rafael-Fortney JA, Guttridge DC, Janssen PM: Peptide-based inhibition of NF-kappaB rescues diaphragm muscle contractile dysfunction in a murine model of Duchenne muscular dystrophy. Mol Med 2011, 17: 508-515.PubMed CentralView ArticlePubMedGoogle Scholar
- Gargioli C, Coletta M, De Grandis F, Cannata SM, Cossu G: PlGF-MMP-9-expressing cells restore microcirculation and efficacy of cell therapy in aged dystrophic muscle. Nat Med 2008, 14: 973-978. 10.1038/nm.1852View ArticlePubMedGoogle Scholar
- Rowland LP: Pathogenesis of muscular dystrophies. Arch Neurol 1976, 33: 315-321. 10.1001/archneur.1976.00500050001001View ArticlePubMedGoogle Scholar
- De Luca A, Pierno S, Liantonio A, Cetrone M, Camerino C, Fraysse B, Mirabella M, Servidei S, Ruegg UT, Conte Camerino D: Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of taurine and insulin-like growth factor-1. J Pharmacol Exp Ther 2003, 304: 453-463. 10.1124/jpet.102.041343View ArticlePubMedGoogle Scholar
- De Luca A, Nico B, Liantonio A, Didonna MP, Fraysse B, Pierno S, Burdi R, Mangieri D, Rolland JF, Camerino C, Zallone A, Confalonieri P, Andreetta F, Arnoldi E, Courdier-Fruh I, Magyar JP, Frigeri A, Pisoni M, Svelto M, Conte Camerino D: A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice. Am J Pathol 2005, 166: 477-489. 10.1016/S0002-9440(10)62270-5PubMed CentralView ArticlePubMedGoogle Scholar
- Cabello-Verrugio C, Morales MG, Cabrera D, Vio CP, Brandan E: Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles. J Cell Mol Med 2012, 16: 752-764. 10.1111/j.1582-4934.2011.01354.xPubMed CentralView ArticlePubMedGoogle Scholar
- Morales MG, Cabello-Verrugio C, Santander C, Cabrera D, Goldschmeding R, Brandan E: CTGF/CCN-2 over-expression can directly induce features of skeletal muscle dystrophy. J Pathol 2011, 225: 490-501. 10.1002/path.2952View ArticlePubMedGoogle Scholar
- Grounds MD, Torrisi J: Anti-TNFalpha (Remicade) therapy protects dystrophic skeletal muscle from necrosis. FASEB J 2004, 18: 676-682. 10.1096/fj.03-1024comView ArticlePubMedGoogle Scholar
- Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A: Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis 2008, 31: 1-19. 10.1016/j.nbd.2008.03.008PubMed CentralView ArticlePubMedGoogle Scholar
- Straub V, Rafael JA, Chamberlain JS, Campbell KP: Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J Cell Biol 1997, 139: 375-385. 10.1083/jcb.139.2.375PubMed CentralView ArticlePubMedGoogle Scholar
- Hernandez-Presa MA, Gomez-Guerrero C, Egido J: In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry. Kidney Int 1999, 55: 209-214. 10.1046/j.1523-1755.1999.00226.xView ArticlePubMedGoogle Scholar
- Morales MG, Cabrera D, Cespedes C, Vio CP, Vazquez Y, Brandan E, Cabello-Verrugio C: Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2). Cell Tissue Res 2013, 353: 173-187. 10.1007/s00441-013-1642-6View ArticlePubMedGoogle Scholar
- Villalta SA, Rinaldi C, Deng B, Liu G, Fedor B, Tidball JG: Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype. Hum Mol Genet 2011, 20: 790-805. 10.1093/hmg/ddq523PubMed CentralView ArticlePubMedGoogle Scholar
- Gregorevic P, Plant DR, Leeding KS, Bach LA, Lynch GS: Improved contractile function of the mdx dystrophic mouse diaphragm muscle after insulin-like growth factor-I administration. Am J Pathol 2002, 161: 2263-2272. 10.1016/S0002-9440(10)64502-6PubMed CentralView ArticlePubMedGoogle Scholar
- Barton ER, Morris L, Kawana M, Bish LT, Toursel T: Systemic administration of L-arginine benefits mdx skeletal muscle function. Muscle Nerve 2005, 32: 751-760. 10.1002/mus.20425View ArticlePubMedGoogle Scholar
- Bogdanovich S, McNally EM, Khurana TS: Myostatin blockade improves function but not histopathology in a murine model of limb-girdle muscular dystrophy 2C. Muscle Nerve 2008, 37: 308-316. 10.1002/mus.20920View ArticlePubMedGoogle Scholar
- Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE: Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 2005, 122: 289-301. 10.1016/j.cell.2005.05.010View ArticlePubMedGoogle Scholar
- Gillis JM: Multivariate evaluation of the functional recovery obtained by the overexpression of utrophin in skeletal muscles of the mdx mouse. Neuromuscul Disord 2002,12(Suppl 1):S90-S94.View ArticlePubMedGoogle Scholar
- Radley HG, Davies MJ, Grounds MD: Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment. Neuromuscul Disord 2008, 18: 227-238. 10.1016/j.nmd.2007.11.002View ArticlePubMedGoogle Scholar
- Caceres S, Cuellar C, Casar JC, Garrido J, Schaefer L, Kresse H, Brandan E: Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle. Eur J Cell Biol 2000, 79: 173-181. 10.1078/S0171-9335(04)70020-5View ArticlePubMedGoogle Scholar
- Andreetta F, Bernasconi P, Baggi F, Ferro P, Oliva L, Arnoldi E, Cornelio F, Mantegazza R, Confalonieri P: Immunomodulation of TGF-beta 1 in mdx mouse inhibits connective tissue proliferation in diaphragm but increases inflammatory response: implications for antifibrotic therapy. J Neuroimmunol 2006, 175: 77-86. 10.1016/j.jneuroim.2006.03.005View ArticlePubMedGoogle Scholar
- Cohn RD, van Erp C, Habashi JP, Soleimani AA, Klein EC, Lisi MT, Gamradt M, ap Rhys CM, Holm TM, Loeys BL, Ramirez F, Judge DP, Ward CW, Dietz HC: Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states. Nat Med 2007, 13: 204-210. 10.1038/nm1536PubMed CentralView ArticlePubMedGoogle Scholar
- Vial C, Zuniga LM, Cabello-Verrugio C, Canon P, Fadic R, Brandan E: Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/CCN2), which induces their dedifferentiation. J Cell Physiol 2008, 215: 410-421. 10.1002/jcp.21324View ArticlePubMedGoogle Scholar
- De Luca A: Pre-clinical drug tests in the mdx mouse as a model of dystrophinopathies: an overview. Acta Myol 2012, 31: 40-47.PubMed CentralPubMedGoogle Scholar
- Nelson CA, Hunter RB, Quigley LA, Girgenrath S, Weber WD, McCullough JA, Dinardo CJ, Keefe KA, Ceci L, Clayton NP, McVie-Wylie A, Cheng SH, Leonard JP, Wentworth BM: Inhibiting TGF-beta activity improves respiratory function in mdx mice. Am J Pathol 2011, 178: 2611-2621. 10.1016/j.ajpath.2011.02.024PubMed CentralView ArticlePubMedGoogle Scholar
- Huebner KD, Jassal DS, Halevy O, Pines M, Anderson JE: Functional resolution of fibrosis in mdx mouse dystrophic heart and skeletal muscle by halofuginone. Am J Physiol Heart Circ Physiol 2008, 294: H1550-H1561. 10.1152/ajpheart.01253.2007View ArticlePubMedGoogle Scholar
- Turgeman T, Hagai Y, Huebner K, Jassal DS, Anderson JE, Genin O, Nagler A, Halevy O, Pines M: Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone. Neuromuscul Disord 2008, 18: 857-868. 10.1016/j.nmd.2008.06.386View ArticlePubMedGoogle Scholar
- Pines M, Halevy O: Halofuginone and muscular dystrophy. Histol Histopathol 2011, 26: 135-146.PubMedGoogle Scholar
- MacDonald EM, Cohn RD: TGFbeta signaling: its role in fibrosis formation and myopathies. Curr Opin Rheumatol 2012, 24: 628-634. 10.1097/BOR.0b013e328358df34View ArticlePubMedGoogle Scholar
- Acuna MJ, Pessina P, Olguin H, Cabrera D, Vio CP, Bader M, Munoz-Canoves P, Santos RA, Cabello-Verrugio C, Brandan E: Restoration of muscle strength in dystrophic muscle by angiotensin-1-7 through inhibition of TGF-beta signalling. Hum Mol Genet 2014, 23: 1237-1249. 10.1093/hmg/ddt514View ArticlePubMedGoogle Scholar
- Wehling-Henricks M, Lee JJ, Tidball JG: Prednisolone decreases cellular adhesion molecules required for inflammatory cell infiltration in dystrophin-deficient skeletal muscle. Neuromuscul Disord 2004, 14: 483-490. 10.1016/j.nmd.2004.04.008View ArticlePubMedGoogle Scholar
- Hoffman EP, Reeves E, Damsker J, Nagaraju K, McCall JM, Connor EM, Bushby K: Novel approaches to corticosteroid treatment in Duchenne muscular dystrophy. Phys Med Rehabil Clin N Am 2012, 23: 821-828. 10.1016/j.pmr.2012.08.003PubMed CentralView ArticlePubMedGoogle Scholar
- Wong BL, Christopher C: Corticosteroids in Duchenne muscular dystrophy: a reappraisal. J Child Neurol 2002, 17: 183-190. 10.1177/088307380201700306View ArticlePubMedGoogle Scholar
- Lim JC, Chan TK, Ng DS, Sagineedu SR, Stanslas J, Wong WS: Andrographolide and its analogues: versatile bioactive molecules for combating inflammation and cancer. Clin Exp Pharmacol Physiol 2012, 39: 300-310. 10.1111/j.1440-1681.2011.05633.xView ArticlePubMedGoogle Scholar
- Cabello-Verrugio C, Acuna MJ, Morales MG, Becerra A, Simon F, Brandan E: Fibrotic response induced by angiotensin-II requires NAD(P)H oxidase-induced reactive oxygen species (ROS) in skeletal muscle cells. Biochem Biophys Res Commun 2011, 410: 665-670. 10.1016/j.bbrc.2011.06.051View ArticlePubMedGoogle Scholar
- Mezzano V, Cabrera D, Vial C, Brandan E: Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-beta and CTGF/CCN2. J Cell Commun Signal 2007, 1: 205-217. 10.1007/s12079-008-0018-2PubMed CentralView ArticlePubMedGoogle Scholar
- Li G, Xie Q, Shi Y, Li D, Zhang M, Jiang S, Zhou H, Lu H, Jin Y: Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats. J Gene Med 2006, 8: 889-900. 10.1002/jgm.894View ArticlePubMedGoogle Scholar
- Guha M, Xu ZG, Tung D, Lanting L, Natarajan R: Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes. FASEB J 2007, 21: 3355-3368. 10.1096/fj.06-6713comView ArticlePubMedGoogle Scholar
- Klingler W, Jurkat-Rott K, Lehmann-Horn F, Schleip R: The role of fibrosis in Duchenne muscular dystrophy. Acta Myol 2012, 31: 184-195.PubMed CentralPubMedGoogle Scholar
- Leask A, Abraham DJ: TGF-beta signaling and the fibrotic response. FASEB J 2004, 18: 816-827. 10.1096/fj.03-1273revView ArticlePubMedGoogle Scholar
- Ng CM, Cheng A, Myers LA, Martinez-Murillo F, Jie C, Bedja D, Gabrielson KL, Hausladen JM, Mecham RP, Judge DP, Dietz HC: TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. J Clin Invest 2004, 114: 1586-1592. 10.1172/JCI200422715PubMed CentralView ArticlePubMedGoogle Scholar
- Fukushima K, Badlani N, Usas A, Riano F, Fu F, Huard J: The use of an antifibrosis agent to improve muscle recovery after laceration. Am J Sports Med 2001, 29: 394-402.PubMedGoogle Scholar
- de Winter P, Leoni P, Abraham D: Connective tissue growth factor: structure-function relationships of a mosaic, multifunctional protein. Growth Factors 2008, 26: 80-91. 10.1080/08977190802025602View ArticlePubMedGoogle Scholar
- Wang Q, Usinger W, Nichols B, Gray J, Xu L, Seeley TW, Brenner M, Guo G, Zhang W, Oliver N, Lin A, Yeowell D: Cooperative interaction of CTGF and TGF-beta in animal models of fibrotic disease. Fibrogenesis Tissue Repair 2011, 4: 4. 10.1186/1755-1536-4-4PubMed CentralView ArticlePubMedGoogle Scholar
- Chaqour B, Yang R, Sha Q: Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation. J Biol Chem 2006, 281: 20608-20622. 10.1074/jbc.M600214200View ArticlePubMedGoogle Scholar
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