Curcumin Alleviates Dystrophic Muscle Pathology in mdx Mice

Mol. Cells, Vol. 25, No. 4, pp. 531-537

Molecules and Cells ©KSMCB 2008

Curcumin Alleviates Dystrophic Muscle Pathology in mdx Mice Ying Pan1,2, Chen Chen1, Yue Shen2, Chun-Hua Zhu3, Gang Wang4, Xiao-Chun Wang2, Hua-Qun Chen3,*, and Min-Sheng Zhu1,* 1

Model Animal Research Center, Medical School, Nanjing University, Nanjing 210061, People’s Republic of China; Huadong Research Institute for Medical Biotechnics, Nanjing 210002, People’s Republic of China; 3 Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing 210046, People’s Republic of China; 4 Nanjing Children’s Hospital, Nanjing 210005, People’s Republic of China. 2

(Received October 29, 2007; Accepted December 12, 2007)

Abnormal activation of nuclear factor kappa B (NF-κB) probably plays an important role in the pathogenesis of Duchenne’s muscular dystrophy (DMD). In this report, we evaluated the efficacy of curcumin, a potent NF-κB inhibitor, in mdx mice, a mouse model of DMD. We found that it improved sarcolemmic integrity and enhanced muscle strength after intraperitoneal (i.p.) injection. Histological analysis revealed that the structural defects of myofibrils were reduced, and biochemical analysis showed that creatine kinase (CK) activity was decreased. We also found that levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) in the mdx mice were decreased by curcumin administration. EMSA analysis showed that NF-κB activity was also inhibited. We thus conclude that curcumin is effective in the therapy of muscular dystrophy in mdx mice, and that the mechanism may involve inhibition of NF-κB activity. Since curcumin is a nontoxic compound derived from plants, we propose that it may be useful for DMD therapy. Keywords: Creatine Kinase Activity; Curcumin; Interleukin-1beta; i.p. Injection; mdx Mice; Muscular Dystrophy; NF-κB; Tumor Necrosis Factor alpha.

der caused by mutations of the gene encoding dystrophin (Hoffman et al., 1987; 1988; Koenig et al., 1987; Monaco et al., 1986). In DMD muscle, absence of dystrophin leads to loss of all the dystrophin-associated proteins and the dystrophin-glycoprotein complex (DGC) in muscle sarcolemma. This results in intrinsic mechanical defects that are accelerated by toxic substances produced by active contraction of muscle (Blake, et al., 1996; Matsumura et al., 1994; Sadoulet-Puccio et al., 1996; Tinsley et al., 1994). One hypothesis for the progressive pathology of DMD is that dystrophin deficiency renders membranes susceptible to injury resulting from mechanical contraction, oxidative free radicals, nitric oxide and other toxic molecules (Disatnik et al., 2000; Pasternak et al., 1995; Rando et al., 1998). This hypothesis implies that the introduction of DGC-associated components, such as neuronal nitric oxide synthase (nNOS), integrin and Nacetylgalactosamine (GalNAc) transferase, should partially prevent sarcolemmic impairment and alleviate muscular dystrophy in mdx mice. However, to our knowledge and according to a recent review (Nowak et al., 2004), no clinical trials have yet applied this strategy successfully. Thus, an alternative strategy for developing drugs to inhibit DMD progression is desirable. The dystrophin complex has emerged as a scaffold responsible for membrane docking of signaling proteins,

Introduction Duchenne’s Muscular Dystrophy (DMD), characterized by progressive muscle weakness, is a fatal genetic disor* To whom correspondences should be addressed. Tel: 86-25-58641529; Fax: 86-25-58641500 E-mail: [email protected] (MSZ)/ [email protected] (HQC)

Abbreviations: CK, creatine kinase; DGC, dystrophin-glycoprotein complex; DMD, Duchenne’s muscular dystrophy; DTT, dithiothreitol; EBD, evans blue dye; EDL, extensor digitorum longus; EMSAs, electrophoretic mobility shift assays; GalNAc, N-acetylgalactosamine; i.p., intraperitoneal; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor kappa B; nNOS, neuronal nitric oxide synthase; NRCMM, national resource center for mutant mice; PMSF, phenylmethyl-sulfonyl fluoride; TNF-α, tumor necrosis factor alpha.

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Curcumin Attenuates DMD Pathology

and loss of dystrophin may disturb the signaling network in dystrophic muscle (Lapidos et al., 2004; Oak et al., 2003; Spence et al., 2004). Recent data show that NF-κB is activated in dystrophic muscles and that mechanical stresses may intensify its activation (Kumar et al., 2003). Several reports suggest that NF-κB plays an important role in the process of muscle wasting in DMD (Acharyya et al., 2007; Cai et al., 2004; Hunter et al., 2004). NF-κB transcription factors are homo- and heterodimers consisting of proteins of the Rel/NF-κB family. In mammals, the NF-κB family includes five proteins: p50/105, p52/100, p65, RelB and c-Rel. NF-κB regulates the expression of a plethora of genes, especially those involved in inflammatory and acute stress responses (Ghosh et al., 1998). In skeletal muscle, NF-κB activation can cause severe muscle wasting (Cai et al., 2004), and disruption of the nfkb1 gene inhibits skeletal muscle atrophy (Hunter et al., 2004). NF-κB activity is elevated in unloaded dystrophic skeletal muscle in a Ca2+ channel-independent manner, and, in response to NF-κB activation there is increased expression of several inflammatory genes such as TNF-α, IL-1β and iNOS (Kumar et al., 2003), which is believed to be harmful to dystrophic muscle. In this report, we evaluated the effect of curcumin, an NF-κB inhibitor (Das et al., 2002; Pan et al., 2000; Shishodia et al., 2003), on the mdx mouse model. We found that it alleviated muscular dystrophy and inhibited NF-κB activation.

Materials and Methods Reagents and animals Reagents: Curcumin, Evans blue dye (EBD) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Anti-NF-κB p50, anti-iNOS antibodies and secondary antibodies were from Santa Cruz. Animal: mdx and wild-type C57BL/10 mice (SPF grade) were obtained from the Jackson Laboratory and maintained in the National Resource Center for Mutant Mice (NRCMM) (PR China). Animal protocols were approved by the NRCMM. Animal treatment: 18-day-old mdx mice were treated for 10 d with i.p. injection of different dosages of curcumin (0.1 mg/kg body weight, 0.5 mg/kg body weight or 1 mg/kg body weight, respectively) in 150–200 μl vehicle (1 ml DMSO in 10 ml PBS/kg body weight) or vehicle, every other day. At the end of the experiments, the mice were subjected to muscle strength measurement and fiber integrity assessment, and tissues and blood samples were collected for various tests. EBD staining of muscle fibers (Bonuccelli et al., 2003; Straub et al., 1997) To detect damaged muscle fibers, EBD (stock solution: 10 mg/ml in PBS) was injected (1 ml/kg body weight in PBS) into the right peritoneal cavity without anesthesia, and the mice were killed 8 h post injection. The muscles under the thigh joints of the hind legs were dissected, rinsed in PBS, fixed in

10% formaldehyde and evaluated macroscopically. Sections (6 μm) of extensor digitorum longus (EDL) muscle were examined under a fluorescent microscope with a rhodamine filter (Leica) as described (Bonuccelli et al., 2003; Grounds et al., 2004). The presence of an autofluorescent red signal after EBD staining indicates damaged myofibrils. Evaluation of muscle strength Mice were weighed and tested for grip strength by the method of Meyer et al. (Belzung et al., 2001; Meyer et al., 1979). The grip strength apparatus was constructed in Nanjing University. Basically, it consists of a 4 × 3 cm rectangle brass net threaded smoothly on a standard weight loading on a balancer and an adjustable trough. The animal is placed into the trough with forepaws inside the triangular grasping net. Using one hand, the operator grasps the base of the tail and pulls it steadily (~2 cm/s) away from the net until the grip is broken. Typically, five successive readings are taken for each animal, with an intertrial interval of 10 s. The grip strength is normalized by body weight and the results are expressed as mean values of grip strength. The traction test was performed as described (Murugesan et al., 2001; Villar et al., 1992) with minor modifications. Briefly, a mouse is hung by its front paws on a metallic wire string (diameter 1.5 mm) placed horizontally 20 cm over the floor. The longest time for which the mouse is able to cling onto the horizontal string and the drop times with 3 min are recorded. Histologic studies EDL muscles were dissected, fixed with 10% formaldehyde and embedded in paraffin. Sections (6 μm) were stained with haematoxylin/eosin. Necrotic fibers, central nucleated fibers (indicative of regenerated fibers) and regenerating fibers were determined by morphometric techniques as described by St-Pierre et al. (2004). The histological and morphological assessments were performed double-blind. Measurement of creatine kinase activity Blood samples were collected in dry tubes prior to killing the animals and the sera were stored at –20°C. Serum creatine kinase activity was measured with a spectrophotometric analysis kit supplied by the Jiancheng Biotechnology Institute (PR China). Electrophoretic mobility shift assays (EMSAs) To determine NF-κB activity in muscle tissue, EMSAs were performed according to Kumar et al. (2003) with minor modifications. Briefly, EDL muscles were immediately frozen in dry ice and suspended in 18 μl low salt lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethyl-sulfonyl fluoride (PMSF), 2.0 mg/mL aprotinin) per mg of wet tissue, followed by grinding mechanically and swelling on ice for 5 min. Two freeze-thaw cycles were performed. The lysates were centrifuged for 10 min at 14,000 rpm and the supernatants were saved as cytoplasmic extracts at –70°C. The pellets were resuspended in 1 μl of ice cold high-salt nuclear extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM EDTA, 1 mM EGTA,

Ying Pan et al. 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin) per mg of original muscle weight. The mixtures were incubated on ice for 45 min followed by centrifugation for 5 min at 14,000 rpm. The supernatants were stored at –70°C as nuclear extracts. Protein concentrations were quantified by the BCA Protein Assay kit (Bio-Rad). 4 μg of nuclear protein was incubated with 1.7 fmol of the NF-κB consensus oligonucleotide 5′-AGTTGAGGGGACTTTCCCAGG-3′ labeled with [γ−32P] ATP, and the mixture was resolved on a 5% non-denaturing polyacrylamide gel. The running buffer contained 50 mM Tris, 200 mM glycine (pH8.5), and 1 mM EDTA. The dried gel was exposed to Kodak X-ray film at –70°C and the visualized bands were recorded. To determine the DNA binding specificity of NF-κB, antip50 antibody and NF-κB cold probe were used. Western blot assay EDL muscle extracts were prepared as described previously (Bonuccelli et al., 2003). They were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore). Western blot analysis was performed using polyclonal antibodies to iNOS, followed by HRP-labeled goat anti-rabbit secondary antibody. Signals were visualized by ECL chemiluminescence as described previously (Zhu et al., 2004). Measurement of TNF-α and IL-1β in sera ELISA kits (JingMed Company, PR China) were used to quantify TNF-α and IL-1β levels in serum following exactly the manufacturer’s protocol. Statistics analyses Data are presented as means ± SEM. All results were derived from 5–6 animals per group. Groups were compared using the two-tailed Student’s t-test. Statistical significance was set at p < 0.05.

Results Curcumin alleviates the pathological severity of dystrophic muscle The accumulation of EBD stain within fibers (blue fibers) is indicative of the loss of sarcolemmal integrity (Straub et al., 1997). In C57BL/10 mice, the muscles of the thigh joint of the hind legs showed almost no EBD staining. However, in mdx mice, blue EBD staining was apparent. After treatment with curcumin, the area of blue fibers clearly decreased. In the group receiving 1 mg curcumin/kg body weight, the mdx mice exhibited much less staining, almost as little as the control mice (Fig. 1A). However, with doses of curcumin ranging from 5 mg/kg to 10 mg/kg body weight, the amount of blue fiber staining did not change further (our unpublished data). We therefore used dosage ranging from 0.1 mg/kg to 1 mg/kg body weight in subsequent experiments. Next we examined cross-sections of EDL muscle under a fluorescent microscope. Consistent with the observation under bright field, the red autofluorescence of EBD was strong in sections of mdx mice treated with vehicle (Fig. 1B), indicative of damaged muscle fibers. In the muscle

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A B

Fig. 1. Curcumin alleviates muscular dystrophy symptoms in mdx mice as evaluated by EBD staining. A. EBD stained muscles from under the thigh joint of hind legs (blue color). B. EBD stained EDL sections visualized with a fluorescent microscope (red color).

fibers of curcumin-treated mdx mice, the areas of red autofluorescence were reduced, suggesting that muscle impairment was greatly ameliorated by curcumin. Curcumin inhibits the morphological changes of dystrophic muscle To assess the histological changes in muscle, sections of the EDL muscles were stained with hematoxylin/eosin. The mdx mice treated with vehicle showed the typical abnormal dystrophic muscles, including fiber hypertrophy, small regeneration fibers, necrosis, and centrally located nuclei. After administration of curcumin, the extent of necrosis and variability of myofiber size were reduced. As shown in Fig. 2, at a dose of 1 mg/kg body weight, the proportion of centrally nucleated fibers decreased, and the number of fibers of uniform diameter increased. These results indicate that curcumin can protect mdx muscle, probably by inhibiting spontaneous necrosis and promoting muscle regeneration. Improvement of muscular function in curcumin treated mdx mice There was no significant difference of body weight between the animals receiving or not receiving curcumin treatment (data not shown). Muscle strength was determined by Meyer’s method. The grip strength of the control C57BL/10 mice normalized to body weight was much greater than that of the mdx mice, and administration of curcumin increased the grip strength of the mdx mice in a dose-dependent manner. At a curcumin dose of 1 mg/kg of body weight, the grip strength of the mdx mice was almost the same as control (p < 0.01) (Fig. 3A). Furthermore, the time they could hold onto a string was longer (p < 0.01) and the drop times with 3 min from the string was reduced (p < 0.01) (Figs. 3B and 3C). Taken together, these results show that curcumin can improve the muscle contractility of mdx mice. CK activity is decreased in mdx mice administered

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B Fig. 2. Histological assessment of H&E stained EDL fibers of mdx mice after administration of curcumin. In vehicle-treated mdx mice, EDL fibers showed myopathic changes including necrosis and central nucleated fibers, as well as variable fiber diameters compared with control C57BL/10 mice (upper panel). Curcumin greatly reduced the pathology of muscle in the mdx mice (lower panel).

with curcumin CK is a serological marker of skeletal muscle injury. CK activity was very low in normal C57BL/10 mice, and increased significantly in mdx mice (Fig. 4). When mdx mice were treated with curcumin, activity declined in a dose-dependent manner. At a dose of 1 mg/kg body weight, the effect of curcumin was substantial (from 120 ± 14 unit/ml to 73 ± 27 units/ml) (p < 0.01). NF-κB activation in mdx muscle is suppressed by curcumin To assess NF-κB activity in the dystrophic muscle of mdx mice, we performed EMSAs. Consistent with a previous report (Kumar et al., 2003), we found that NFκB DNA binding activity was increased (Fig. 5A). This increase was reduced in a dose-dependent manner in the muscle of curcumin-treated mice (Fig. 5B). iNOS expression in mdx muscle is reduced by curcumin administration iNOS has the ability to catalyse the production of NO, an important free radical playing with complex effects on many physiological and pathological processes (Salvemini et al., 1998). The abnormal production of NO may be harmful to muscles. As a target of NFκB, iNOS protein may be expressed when NF-κB is activated (Ghosh et al., 1998). We detected weak iNOS expression in mdx mice, and none in control mice (Fig. 6A), consistent with another report (Louboutin et al., 2001). When mdx mice were treated with curcumin, the iNOS level in the mdx muscle declined. Serum levels of TNF-α and IL-1β are reduced by curcumin in mdx mice TNF-α is up regulated in DMD patients (Lundberg et al., 1995) and may cause dystrophic muscle by enhancing inflammatory responses or by direct

C

Fig. 3. Muscle function is improved by curcumin. A. Grip strength was enhanced in curcumin-treated mdx mice. B. The longest time holding onto a string increased in the curcumintreated mdx mice. C. The drop times in 3 min on string decreased in the curcumin-treated mdx mice. * p < 0.05, ** p < 0.01 vs. mdx mice treated with vehicle.

cytotoxicity. TNF-α and IL-1β are NF-κB-regulated proinflammatory factors that in turn may augment NF-κB activation. We measured serum levels of TNF-α and IL1β, and found that both were high in mdx mice and were reduced by curcumin treatment.

Discussion Currently, the disease interventions available to DMD patients include the administration of a subclass of glucocorticoids or PTC124 (Hamed, 2006). However, a number of severe side effects limit their clinical usefulness. Thus,

Ying Pan et al.

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A

B

Fig. 4. CK activity is decreased in curcumin-administered mdx mice. Blood samples of the mice were collected and serum CK activity was measured. * p < 0.05, ** p < 0.01 vs. vehicletreated mdx mice.

A

B

Fig. 5. NF-κB DNA binding activity in mdx muscle is inhibited by curcumin. A. DNA binding activity of NF-κB increases in mdx muscle. B. DNA-binding activity of NF-κB in mdx muscle is reduced by curcumin administration. To demonstrate the DNA binding specificity of NF-κB, anti-NF-κB p50 antibody and cold probe were added to the reaction buffer.

the development of a non-toxic drug for halting DMD progression is crucial. In this report, we showed that curcumin, a natural substance, alleviated muscular dystrophy and improved muscular contraction function in mdx mice. Although our results were obtained from an animal model, they suggest that curcumin is a promising candidate for DMD therapy. Data from clinical trials demonstrate no obvious toxicity of long-term oral administration of curcumin (Aggarwal et al., 2003). We have tested the toxicity of curcumin administrated to mice by i.p. injection over two months and found no obvious toxic effect at low (2 mg/kg body weight), medium (6 mg/kg body weight) and high (18 mg/kg body weight) doses (data not shown). NF-κB may have toxic effects by inducing the expression of target genes (such as TNF-α, IL-1β and iNOS). In this report, abnormally high levels of TNF-α, IL-1β and

C

Fig. 6. High levels of expression of iNOS, TNF-α and IL-1β are suppressed by curcumin administration. A. Curcumin supressed iNOS expression in the EDL of mdx mice. Coomassie blue staining of beta-actin showed that equal amounts of protein were loaded. B, C. Curcumin decreased serum levels of TNF-α (B) and IL-1β (C). The serum samples were diluted with assay buffer, and TNF-α and IL-1β were measured with ELISA kits. * p < 0.05 vs. vehicle-treated mdx mice.

iNOS were detected in mdx mice, and these were reduced by administration of curcumin. It is well documented that TNF-α is a crucial factor in muscle wasting (Kumar et al., 2003) and iNOS may cause cytotoxicity by producing nitric oxide and its derivatives (Griscavage et al., 1995). In addition, TNF-α, IL-1β and free radicals may activate NF-κB in a feedback manner, and elevated NF-κB activity may initiate the expression of several NF-κB regulated inflammatory genes whose products may also augment NF-κB activity. These positive feedback cycles may well drive the progression of muscular dystrophic pathology, and the therapeutic efficacy of curcumin demonstrated here may be due to the blockade of such feedback cycles. Like many other chemical compounds, curcumin has a variety of biological activities, such as inhibition of the

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sarcoplasmic reticulum Ca++-ATPase, suppression of cyclooxygenase-2 expression and inhibition of c-Jun Nterminal kinase activity (Mahmmoud et al., 2005). It is usually used as an anti-inflammatory and anti-carcinogenic agent or a free radical scavenger (Ammon et al., 1991; Araujo et al., 2001). Our data showed a close correlation between phenotypic attenuation and NF-κB inactivation after curcumin intervention, which strongly suggests that NF-κB is a significant therapeutic target for DMD. This conclusion is also supported by the recent report that dietary administration of curcumin does not reduce the elevated NF-κB activity in mdx mice and that, consistent with this, no phenotypic attenuation was observed (Durham et al., 2006). Since orally administrated curcumin has lower bioavailability than i.p. injected curcumin (Yang et al., 2006), our results suggest that the route of administration would be crucial for curcumin treatment of muscular dystrophy.

Acknowledgments This work was supported by National Natural Science Foundation of China (30570911). We thank Drs. J. Chen, C.F. Li, and Y.L. He for valuable comments on the manuscripts, and Dr. Z. Chang for help with mouse handling.

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