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The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats Shu-Kun Jiang, Miao Zhang, Zhi-Ling Tian, Meng Wang, Rui Zhao, Lin-Lin Wang, Shan-Shan Li, Min Liu, Jiao-Yong Li, Meng-Zhou Zhang, Da-Wei Guan n Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, Liaoning Province, PR China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 4 January 2015 Received in revised form 10 April 2015 Accepted 15 April 2015
Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and proinflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors. & 2015 Published by Elsevier B.V.
Keywords: Monoacylglycerol lipase Inflammation Fibrosis Skeletal muscle injury Wound repair Chemical compounds studied in this article: JZL184 (PubChem CID: 25021165) AM281 (PubChem CID: 4302962) AM630 (PubChem CID: 4302963)
1. Introduction Muscle injuries are one of the most common injuries, which are usually caused by contusion, strain or laceration (Jarvinen et al., 2007). After injury, muscle repair process can be temporally divided into three overlapping phases, the acute inflammatory and degenerative phase, the repair phase, and the remodeling phase. Inflammatory cells play an important role in the event. They phagocytize necrotic tissue and activate myogenic cells to differentiate and fuse into new myofibers (Baoge et al., 2012). However, the early inflammatory response after injury may be excessive and cause edema, resulting in anoxia and further cell death (Paoloni et al., 2009). Monoacylglycerol lipase (MAGL) is a serine hydrolase that preferentially hydrolyzes monoacylglycerols to glycerol and fatty acid. In the endogenous cannabinoid system, MAGL is mainly responsible for hydrolyzing 2-arachidonoyl glycerol (2-AG), an endogenous ligand
n Correspondence to: Department of Forensic Pathology, China Medical University School of Forensic Medicine, No.77, Puhe Road, Shenyang North New Area, Shenyang 110122, Liaoning Province, PR China. Tel.: þ 86 24 23256666x5483; fax: þ86 24 23267698. E-mail addresses: [email protected], [email protected] (D.-W. Guan).
for cannabinoid CB1 and CB2 receptors (Labar et al., 2010; Mulvihill and Nomura, 2013). MAGL releases arachidonic acid for the synthesis of pro-inflammatory eicosanoids by hydrolyzing 2-AG in certain tissues such as the brain, liver, and lung (Nomura et al., 2011), which suggests that MAGL is closely involved in inflammatory response. Long et al. (2009a, 2009b) described a potent and selective inhibitor for MAGL termed (4-nitrophenyl) 4-[bis(1,3-benzodioxol-5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) in mice, which decreases 2-AG hydrolysis both in the central nervous system and peripheral tissues. Accumulated evidence demonstrates the protective and therapeutic effect of JZL184 on diseases having an inflammatory component. JZL184 treatment reduces inflammatory nociception through the activation of both cannabinoid CB1 and CB2 receptors in mice and rats (Ghosh et al., 2013; Guindon et al., 2011). In addition, JZL184 mitigates the inflammatory response in trinitrobenzene sulfonic acid-induced colitis and lipopolysaccharideinduced acute lung injury by a cannabinoid CB1 and CB2 receptordependent manner (Alhouayek et al., 2011; Costola-de-Souza et al., 2013). Since muscle wound healing is a typical inflammation-evoked event, we hypothesized that MAGL inhibition might alleviate inflammatory response of injured skeletal muscles, which may subsequently affect the skeletal muscle wound healing. In this study, we verified the hypothesis using JZL184 in a rat muscle contusion model.
http://dx.doi.org/10.1016/j.ejphar.2015.04.018 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Using selective cannabinoid CB1/CB2 receptor antagonists, we also attempted to determine whether the effects observed after MAGL inhibition by JZL184 treatment were associated with activation of the cannabinoid receptors.
2. Materials and methods 2.1. Animal model of skeletal muscle contusion All animal protocols were conformed to the “Principles of Laboratory Animal Care” (National Institutes of Health Publication no. 85-23, revised 1985) that sought to minimize both the number of animals used in a procedure and any suffering that they might experience, and were performed according to the Guidelines for the Care and Use of Laboratory Animals of China Medical University. A reproducible muscle contusion model in rats was described previously (Yu et al., 2010). Briefly, adult Sprague–Dawley male rats weighing 280–300 g were anesthetized by intraperitoneal injection with 2% sodium pentobarbital (30 mg/kg). The right hindlimb was positioned on a board in a prone position by extending the knee and dorsiflexing the ankle to 901, and a single impact at velocity of 3 m/s was delivered to the gastrocnemius and soleus of the right posterior limb. The size of impact interface of the counterpoise (weighing 500 g) was 1.127 cm2. After injury, each rat was housed individually and kept under a 12 h light–dark cycle. Rats were fed with commercial rat chow and water ad libitum. Rats were killed by intraperitoneal injection of an overdose of sodium pentobarbital. Gastrocnemius was taken and equally divided into two blocks. One block was used for morphological evaluation, and another was used for molecular biological assays. No bone fracture was detected at dissection. 2.2. Experimental protocol and grouping Experiment 1: 60 rats were randomly divided into 2 groups (30 rats/group) according to treatments with JZL184 (Cayman Chemical, MI, USA) or vehicle alone. JZL184 (10 mg/kg, 2 ml/kg) was injected intraperitoneally immediately after contusion once a day for 5 days. Rats injected with equal volume of vehicle were used as control. JZL184 was dissolved in a vehicle containing saline:DMSO:Tween-80 in an 18:1:1 ratio. JZL184 dose and treatment were based on previous studies (Kerr et al., 2013; Long et al., 2009a, 2009b; Sciolino et al., 2011). Rats were killed at 1, 3, 5, 7, 9 and 14 days after contusion (5 rats at each time point in vehicle- or JZL184-treated group). Experiment 2: 30 rats were divided into two groups that received JZL184 (10 mg/kg, 2 ml/kg) or vehicle. Within each group, the rats were further divided into 3 groups that received one of the following treatments 30 min before JZL184 or vehicle (5 rats in each group): 1(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N00 -morpholin-4ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist, Tocris Bioscience, Ellisville, MO, USA, 3 mg/kg, 2 ml/kg), [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist, Tocris Bioscience, Ellisville, MO, USA, 3 mg/kg, 2 ml/kg) or vehicle. AM281 and AM630 were dissolved in the same vehicle as JZL184. AM281 and AM630 were chosen and administered based on previous studies (Arevalo-Martin et al., 2012; Costola-deSouza et al., 2013; Sciolino et al., 2011; Yang et al., 2014). The rats were killed at 3 days after injury. 2.3. Immunohistochemical staining and morphometric analysis The skeletal muscle specimens were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). Fixed muscle specimens were embedded in paraffin. 5 μm-thick sections
were prepared. The primary antibodies against rabbit antimyeloperoxidase (MPO) polyclonal antibody (dilution 1:500; ab65871, Abcam, Cambridge, UK), mouse anti-CD68 monoclonal antibody (dilution 1:100; ab31630, Abcam, Cambridge, UK) or mouse anti-alpha smooth muscle actin (α-SMA) monoclonal antibody (dilution 1:200; ab7817, Abcam, Cambridge, UK) were used. Histostain-Plus kit (Zymed Laboratories, South San Francisco, CA, USA) was used according to the manufacturer's instructions. As immunohistochemical controls, some sections were incubated with normal rabbit/mouse IgG or phosphate-buffered saline (pH 7.4) in place of the primary antibodies. Nuclei were counterstained with hematoxylin. In addition, hematoxylin-eosin (H&E) staining and Masson's trichrome staining were conventionally conducted. Sections containing the largest contusion area were evaluated. MPO, CD68 or α-SMA positive cells were counted under the 400fold magnification in the contusion zones. Five fields in the contused zones were randomly chosen for the calculations or evaluations in each section. The nucleus number of regenerating myofibers and collagen deposition areas were analyzed under the 200-fold magnification in the contusion zones. All measurements and data analysis were performed independently by two pathologists in a blind manner. Morphometrical analysis was performed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, USA). 2.4. Protein extraction and immunoblotting assay The skeletal muscle samples were ground into powder with liquid nitrogen using a grinder and homogenized with a sonicator in RIPA buffer (sc-24948, Santa Cruz Biotechnology, CA, USA) containing protease inhibitors at 4 1C. After being prepared via standard procedures, protein samples (30 mg) were separated on 12% sodium dodecyl sulfate polyacrylamide electrophoresis gel and were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then they were used to perform immunoblotting. Rabbit anti-IL-1β polyclonal antibody (dilution 1:1000; ab1832p, Chemicon, ON, Canada), rabbit anti-IL-6 polyclonal antibody (dilution 1:500; ab6672, Abcam, Cambridge, UK) and rabbit anti-TNF-α polyclonal antibody (dilution 1:1000; ab1837p, Chemicon, ON, Canada) were applied as primary antibodies, and rabbit anti-GAPDH polyclonal antibody (dilution 1:1000; ab37168, Abcam, Cambridge, UK) was used for relative protein quantification. The horseradish peroxidase conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology, CA, USA) was diluted to 1:2000 and applied. The blotting was visualized with western blotting luminol reagent (sc-2048, SantaCruz Biotechnology, CA, USA) and by the Electrophoresis Gel Imaging Analysis System (MF-ChemiBIS 3.2, DNR Bio-Imaging Systems, ISR). Subsequently, densitometric analyses of the bands were semi-quantitatively conducted using Scion Image software (Scion Corporation, MD, USA). 2.5. Cytokine and chemokine analysis A Quantibody Rat Inflammation Array 1 (QAR-INF-1-4, RayBiotech, Inc, Norcross, GA) was used to simultaneously detect the expression of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, monocyte chemoattractant protein 1 (MCP-1), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the contused muscles. The assays were performed according to the manufacturer's instructions. Each cytokine was arrayed in quadruplicate, together with positive and negative controls. Fluorescent signals were visualized with a laser scanner (Axon GenePix; Molecular Devices, Sunnyvale, CA) set. Data were extracted with RayBio Q Analyzer software (RayBiotech, Inc, Norcross, GA). After subtracting background signals and normalization to positive controls, comparison of signal intensities for antigen-specific antibody spots between groups was utilized to determine relative differences in expression levels of each sample.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 1. H&E staining of muscle sections harvested 1 day (A and B) and 3 days (C and D) post-injury. Hemorrhage, edema, and necrosis are observed in contused skeletal muscles in both vehicle- and JZL184-treated groups. Many polymorphonuclear cells appear at contused zones at 1 day post-injury. Numerous round-shaped mononuclear cells and a few spindle-shaped fibroblastic cells are present in the injured tissue at 3 days post-injury (original magnification, 200). Scale bar: 100 μm.
2.6. Total RNA extraction and quantitative real-time PCR (qPCR)
3. Results
Total RNA was isolated from the skeletal muscle specimens with RNAiso Plus (9108, Takara Biotechnology, Shiga, Japan) according to the manufacturer's instructions. OD values of each RNA sample were measured by an ultraviolet spectrophotometer. A260/A280 ranged from 1.8 to 2.0. Then, the RNA (250 ng) was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (RR037A, Takara Biotechnology, Shiga, Japan). The resulting cDNA was used for quantitative real-time PCR with the sequence-specific primer pairs for Col1a1, Col3a1 and Gapdh (Takara Biotechnology, Shiga, Japan). PCRgenerated fragments for Col1a1, Col3a1 and Gapdh primer sets were 98, 108 and 100 bp, respectively. The PCR primers were as follows: Col1a1 (sense 50 -ACTGGTACATCAGCCCAAAC-30 and antisense 50 GGAACCTTCGCTTCCATACTC-30 ); Col3a1 (sense 50 -CAGGCCAATGGCAATGTAAAG-30 and antisense 50 -GCCATCCTCTAGAACTGTGTAAG-30 ); Gapdh (sense 50 -CATCTCCCTCACAATTCCATCC-30 and antisense 50 GAGGGTGCAGCGAACTTTAT-30 ). qPCR amplification was performed by an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) using a SYBRs PrimeScript™ RT-PCR Kit (RR081A, Takara Biotechnology, Shiga, Japan) followed by the parameters: 2 μL cDNA was loaded to each qPCR reaction. A denaturing step was at 95 1C for 30 s followed by 15 s at 95 1C, 34 s at 60 1C, 15 s at 95 1C, 60 s at 60 1C, and 15 s at 95 1C for 40 cycles. To exclude any potential contamination, negative controls were also performed with dH2O instead of cDNA during each run. No amplification product was detected. The qPCR procedure was repeated at least three times for each sample.
3.1. Effects of JZL184 on inflammatory response
2.7. Statistical analysis Data were expressed as mean 7standard deviation (S.D.). Statistical significance was determined by two-way analysis of variance using GraphPad PRISM 6.0 software (GraphPad Software, Inc., La Jolla, CA). P o0.05 was considered significantly different.
Hemorrhage, edema, necrosis and inflammatory cell infiltration were observed in both JZL184- and vehicle-treated groups at 1 and 3 days post-injury in H&E stained sections (Fig. 1A–D). Evaluation of the neutrophil or macrophage infiltration was performed by immunostaining of MPO or CD68. A large number of MPO-positive cells (neutrophils) appeared at wound zones at 1 day post-injury and numerous CD68positive cells (macrophages) accumulated in the wounds at 3 days postinjury. Morphometrically, neutrophils decreased significantly in number after JZL184 treatment at 1 day post-injury (Po0.05; Fig. 2A, B, and E), and macrophages decreased in number at 1, 3 and 5 days post-injury in the JZL184-treated group (Po0.05; Fig. 2C, D, and F). In both JZL184- and vehicle-treated groups, MCP-1 and TNF-α expressions decreased, IL-1β expression increased from 1 to 5 days post-injury, and IL-6 expression peaked at 3 days post-injury. JZL184 treatment decreased TNF-α expression at 1 and 3 days post-injury, respectively (Po0.05; Fig. 3A), as well as MCP-1 expression at 1 day post-injury (Po0.05; Fig. 3B). Reduced IL-1β and IL-6 expressions were found in JZL184-treated group at 3 days post-injury (Po0.05; Fig. 3C and D). No significant differences were observed between the two groups for IL-1α, IL-2, IL-4, IL-10, IL-13 or IFN-γ expressions. 3.2. Effects of JZL184 on muscle regeneration In sections stained with H&E, a large number of centronucleated regenerating myofibers concomitant with fibroblastic cells were observed in JZL184- and vehicle-treated groups at 5 days post-injury (Fig. 4A and B). The centronucleated regenerating myofibers gradually increased with extension of posttraumatic interval (Fig. 4C–F). The nucleus number of regenerating myofibers in JZL184-treated group was significantly fewer than in vehicle-treated group at 9 (Po0.05) and 14 (Po0.01) days post-injury (Fig. 4G).
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 2. Immunohistochemical analysis of MPO positive (A, B) or CD68 positive (C, D) cells in contused muscles treated with vehicle or JZL184. MPO and CD68 positive cells are indicated as neutrophils and macrophages, respectively (original magnification, 200). Scale bar: 100 μm. The number of neutrophils and macrophages is shown in E and F, respectively. Data are presented as mean 7S.D. *Po 0.05 vs. vehicle.
3.3. Effects of JZL184 on fibrosis H&E and Masson staining were used to observe fibrotic tissue formation after injury. Fibrotic tissue filled the contused sites at 14 days post-injury, and muscles contained more fibrotic tissue in JZL184-treated group than in vehicle-treated group (P o0.05; Fig. 5A, B, and E). Immunohistochemical staining of α-SMA was used to detect activated fibroblasts (myofibroblasts). Myofibroblasts emerged at 5 days post-injury in JZL184- and vehicletreated groups. In relation to vehicle-treated group, more myofibroblasts were observed in contusion zones in JZL184-treated group at 9 and 14 days post-injury (P o0.05; Fig. 5C, D, and F). The findings were consistent with the results of qPCR analysis. mRNA expressions of Col1a1 and Col3a1 were significantly increased in contused muscles following JZL184 treatment at 3 days (P o0.05) and 5 days (Po 0.05 and P o0.001, respectively) post-injury (Fig. 5G and H). 3.4. Effects of AM281 and AM630 pre-treatment before JZL184 on contused muscles In experiment 2, administration of JZL184 had the same antiinflammatory effect as in experiment 1 and effectively reduced macrophage infiltration (P o0.05; Fig. 6A and B), as well as IL-1β
(P o0.05; Fig. 7B), IL-6 (P o0.05; Fig. 7C) and TNF-α (P o0.05; Fig. 7D) expressions at 3 days post-injury. AM630 pre-treatment effectively abolished the effect of JZL184 on macrophage infiltration (Fig. 6C). AM281 pre-treatment completely abrogated the JZL184-induced decrease of IL-1β expression (P o0.01; Fig. 7B), and AM630 pre-treatment blocked JZL184-induced decrease of IL1β (P o0.05; Fig. 7B), IL-6 (P o0.05; Fig. 7C) and TNF-α (P o0.05; Fig. 7D) expressions. When used alone, AM281, but not AM630, attenuated the expressions of IL-1β (Po0.05; Fig. 7B), IL-6 (P o0.01; Fig. 7C) and TNF-α (Po 0.05; Fig. 7D).
4. Discussion The present study investigated the effects of JZL184 on contused skeletal muscles. We found that JZL184 attenuated neutrophil and macrophage infiltration, and decreased TNF-α, IL-1β, IL-6, and MCP1 expressions, which demonstrates the anti-inflammatory effect of JZL184 on contused muscles. In addition, impeded muscle regeneration and promoted fibrosis were observed in JZL184-treated group, which suggests that JZL184 treatment interferes with the repair of injured skeletal muscles. Besides, cannabinoid CB1 and CB2 receptors might have a relevant role in anti-inflammatory effect induced by JZL184.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 3. The relative expressions of cytokines (TNF-α, IL-1β and IL-6) and chemokine (MCP-1) in contused muscles treated with vehicle or JZL184. Reduced TNF-α, MCP-1, IL-1β and IL-6 expressions are shown in A, B, C and D, respectively. Data are presented as mean 7 S.D. *P o0.05 vs. vehicle.
Damage to skeletal muscle due to blunt trauma presents pathologically a typical inflammatory process with neutrophil and macrophage infiltration. Following the onset of neutrophils invasion, macrophages begin to invade into injured tissue. Neutrophils promote the cytolytic capacity of macrophages to kill muscle cells (Nguyen and Tidball, 2003). CD68 is a specific marker for the M1 macrophage and a receptor for oxidized low-density lipoproteins (LDLs). When oxidized LDLs bind to CD68, the phagocytosis and the pro-inflammatory cytokines production of M1 macrophages are promoted (Tidball and Villalta, 2010). We found fewer neutrophils and CD68-positive macrophages after treatment with JZL184 in the acute inflammatory and degenerative phase, indicating the antiinflammatory effect of JZL184. To further explore anti-inflammatory effect of JZL184, we analyzed the expressions of pro-inflammatory cytokines/chemokines in contused muscles. Reduced TNF-α, IL-1β, IL-6, and MCP-1 expressions were observed after JZL184 treatment. These molecules are essential for both inflammation establishment and phagocyte activation. TNF-α plays important roles in activation inflammatory cells which are associated with promoting muscle damage and secretion of the other pro-inflammatory cytokines in injured muscles (Karalaki et al., 2009; Tidball and Villalta, 2010). IL6 is considered a cytokine that strongly activates the immune system and enhances inflammatory response (Wojdasiewicz et al., 2014). IL-1β and TNF-α are able to increase IL-6 expression and release in cultured skeletal muscle cells (Langen et al., 2001; Luo et al., 2003; Prelovsek et al., 2006). Furthermore, MCP-1 attracts monocytes to the inflammatory site (Luster, 1998), causes neutrophil recruitment in lung following Escherichia coli infection in mice (Balamayooran et al., 2011). MCP-1 blockage leads to a dramatic decrease of macrophage infiltration and TNF-α and IL-6 production (Baeck et al., 2012; Lloyd et al., 1997). In conclusion, JZL184 is able to attenuate inflammatory response in injured skeletal muscles by decreasing neutrophil and macrophage infiltration, and the proinflammatory cytokine/chemokine production. The alleviated neutrophil and macrophage infiltration might be attributed to the reduced MCP-1 expression.
Inflammatory response is essential for muscle regeneration. It has been reported that reduction of phagocytic leukocytes slows the removal of cellular debris and muscle regeneration (Arnold et al., 2007; Teixeira et al., 2003). Observations from acute muscle injury in TNF-α receptor knockout mice suggest that TNF-α may promote muscle regeneration (Chen et al., 2005; Warren et al., 2002). IL-1β increases the mitogenic activity of myogenic cells (Otis et al., 2014). IL-6 knockout mice show slowed muscle growth in a compensatory muscle hypertrophy model (Otis et al., 2014). Muscle precursor cells and macrophages express the chemokine CCR2 receptor (MCP-1 receptor). Disruption of signaling through MCP-1 receptor leads to slower muscle regeneration (Otis et al., 2014; Tidball and Villalta, 2010). Since inflammatory response is essential for muscle regeneration, and alleviated inflammatory response with impeded muscle regeneration was observed by JZL184 treatment, it is suggested that the anti-inflammatory effect of JZL184 on contused muscles interferes with the healing process, which leads to decreased muscle regeneration. Fibrosis is an inevitable result of wound repair in many organs and tissues. The fibrotic tissue in the injured area hampers the recovery of normal tissue architecture and function. There is an antagonism between muscle regeneration and fibrosis. When the regeneration process goes awry, the fibrosis may be promoted (Moyer and Wagner, 2011). In our study, increased myofibroblasts and deposition of collagen were detected with retarded muscle regeneration in the repair phase after JZL184 delivery, which was in line with up-regulated levels of Col I and Col ІІІ mRNA. We presume that impeded muscle regeneration induced by JZL184 aggravates the fibrogenesis. After muscle injury, a series of inflammation-related symptoms, such as pain and vasodilation are produced by the release of inflammatory metabolites. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often prescribed to relive pain in sports medicine (Gharaibeh et al., 2012; Paoloni et al., 2009; Urso, 2013). NS-398, a member of NSAIDs, reduces the neutrophil and macrophage infiltration in the injured muscles, but slows the proliferation
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 4. H&E staining of muscle sections harvested 5 days (A, B), 7 days (C, D) and 14 days post-injury (E, F; original magnification, 200). Scale bar: 100 μm. The nucleus number of regenerating myofibers is shown in G. Data are presented as mean 7 S.D. *P o0.05, **Po 0.01 vs. vehicle.
and maturation of differentiated myogenic precursor cells, which delays the regenerative myogenesis process (Shen et al., 2005). Though JZL184 is able to alleviate inflammation in muscle injury, similar to NS-398, it may interfere with muscle regeneration, suggesting that JZL184 may not be a potential option for primary care of skeletal muscle injuries. 2-AG is a signaling lipid which binds to cannabinoid CB1 and CB2 receptors (Sugiura et al., 2006). Inhibiting MAGL with JZL184 increases the level of endocannabinoid 2-AG (Long et al., 2009a). We administrated the cannabinoid CB1 and CB2 receptors selective antagonists before JZL184 treatment to analyze the involvement of
cannabinoid CB1 and CB2 receptors in the anti-inflammatory effect induced by JZL184. AM281 pre-treatment blocked the effect of JZL184 on IL-1β expression. AM630 pre-treatment reversed the effects of JZL184 on macrophage infiltration, and TNF-α, IL-1β and IL-6 expressions. The results indicated the participation of cannabinoid CB1 and CB2 receptors in the anti-inflammatory effect of JZL184. However, other mechanisms may be involved in cannabinoid actions besides cannabinoid CB1 and CB2 receptors activation, such as adenosine receptor activation (Ribeiro et al., 2012). JZL184 partially inhibits fatty acid amide hydrolase which mainly hydrolyzes anandamide, another endocannabinoid (Long et al., 2009a).
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 5. Detection of fibrosis by Masson trichrome staining, immunohistochemical procedure and qPCR. (A and B) The deposition of collagen in vehicle and JZL184 groups at 14 days post-injury, respectively. Fibrotic tissue is stained blue (original magnification, 200). (E) The percentage of fibrosis areas at 9 and 14 days post-injury. (C and D) Myofibroblasts infiltration in vehicle and JZL184 groups at 14 days post-injury, respectively (original magnification, 200). (F) The number of myofibroblasts. (G and H) The dynamic changes of Col1a1 and Col3a1 mRNA expressions, respectively. Data are presented as mean7 S.D. *Po 0.05, ***Po 0.001 vs. vehicle. Scale bar: 100 μm.
Furthermore, the release of arachidonic acid from hydrolyzed 2-AG and/or anandamide leads to eicosanoid (leukotriene) formation in neutrophils (Chouinard et al., 2011). Though these mechanisms may explain the anti-inflammatory effect of JZL184, the results similar to ours were observed in colitis and acute lung injury with co-administration of cannabinoid CB1 or CB2 receptors antagonists with JZL184 (Alhouayek et al., 2011; Costola-de-Souza et al., 2013). Our observations suggest that simultaneous activation
of both cannabinoid receptors is needed to maintain the antiinflammatory effect of JZL184. AM281 effectively blocked the effect on IL-1β expression induced by JZL184. Interestingly, decreased expressions of IL-1β, IL-6 and TNF-α were observed when AM281 was administered alone. The mechanisms underlying the anti-inflammatory effect of AM281 are not fully elucidated. It has been reported that AM281 significantly improved intestinal microcirculation by reducing leukocyte adhesion
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 6. Immunohistochemical analysis of CD68 positive cells in contused muscles at 3 days post-injury. Cannabinoid CB2 receptor antagonism AM630 prevents the effect of JZL184 on macrophage infiltration (original magnification, 200). Scale bar: 100 μm. The number of macrophages is shown in G. Data are presented as mean 7 S.D. #Po 0.05 vs. vehicle, **Po 0.01 vs. JZL184.
and increasing functional capillary density in acute endotoxemia in rats (Kianian et al., 2013). The anti-inflammatory effect of AM281 alone may be attributed to the endocannabinoids signaling through cannabinoid CB2 receptor in the presence of inactivated cannabinoid CB1 receptor (Schafer et al., 2008).
In conclusion, the present findings demonstrate that JZL184 significantly inhibits inflammatory response and muscle regeneration, promotes fibrosis in contused muscles. cannabinoid CB1 and CB2 receptors appear to be the likely mechanism involved in the anti-inflammatory effect induced by JZL184.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 7. Analysis of IL-1β, IL-6, TNF-α and GAPDH protein from skeletal muscle specimens by western blotting. (A) The bands of L-1β, IL-6, TNF-α and GAPDH in vehicle, JZL184, JZL184þ AM630, JZL184þ AM281, AM630 and AM281 groups, respectively. The relative quantity of IL-1β (B), IL-6 (C) and TNF-α (D) to GAPDH is shown. Data are presented as mean 7 S.D. #Po 0.05, ##Po 0.01 vs. vehicle, *Po 0.05, **P o0.01 vs. JZL184.
Acknowledgments The study was financially supported in part by grants from research fund for the Doctoral Program funded by Ministry of Education of China (20122104110025) and from projects funded by National Natural Science Foundation of China (81273342) and Shenyang Scientific and Technological Plan (F12-277-1-03).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats Shu-Kun Jiang, Miao Zhang, Zhi-Ling Tian, Meng Wang, Rui Zhao, Lin-Lin Wang, Shan-Shan Li, Min Liu, Jiao-Yong Li, Meng-Zhou Zhang, Da-Wei Guan n Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, Liaoning Province, PR China
ar t ic l e i nf o
a b s t r a c t
Article history: Received 4 January 2015 Received in revised form 10 April 2015 Accepted 15 April 2015
Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and proinflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors. & 2015 Published by Elsevier B.V.
Keywords: Monoacylglycerol lipase Inflammation Fibrosis Skeletal muscle injury Wound repair Chemical compounds studied in this article: JZL184 (PubChem CID: 25021165) AM281 (PubChem CID: 4302962) AM630 (PubChem CID: 4302963)
1. Introduction Muscle injuries are one of the most common injuries, which are usually caused by contusion, strain or laceration (Jarvinen et al., 2007). After injury, muscle repair process can be temporally divided into three overlapping phases, the acute inflammatory and degenerative phase, the repair phase, and the remodeling phase. Inflammatory cells play an important role in the event. They phagocytize necrotic tissue and activate myogenic cells to differentiate and fuse into new myofibers (Baoge et al., 2012). However, the early inflammatory response after injury may be excessive and cause edema, resulting in anoxia and further cell death (Paoloni et al., 2009). Monoacylglycerol lipase (MAGL) is a serine hydrolase that preferentially hydrolyzes monoacylglycerols to glycerol and fatty acid. In the endogenous cannabinoid system, MAGL is mainly responsible for hydrolyzing 2-arachidonoyl glycerol (2-AG), an endogenous ligand
n Correspondence to: Department of Forensic Pathology, China Medical University School of Forensic Medicine, No.77, Puhe Road, Shenyang North New Area, Shenyang 110122, Liaoning Province, PR China. Tel.: þ 86 24 23256666x5483; fax: þ86 24 23267698. E-mail addresses: [email protected], [email protected] (D.-W. Guan).
for cannabinoid CB1 and CB2 receptors (Labar et al., 2010; Mulvihill and Nomura, 2013). MAGL releases arachidonic acid for the synthesis of pro-inflammatory eicosanoids by hydrolyzing 2-AG in certain tissues such as the brain, liver, and lung (Nomura et al., 2011), which suggests that MAGL is closely involved in inflammatory response. Long et al. (2009a, 2009b) described a potent and selective inhibitor for MAGL termed (4-nitrophenyl) 4-[bis(1,3-benzodioxol-5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) in mice, which decreases 2-AG hydrolysis both in the central nervous system and peripheral tissues. Accumulated evidence demonstrates the protective and therapeutic effect of JZL184 on diseases having an inflammatory component. JZL184 treatment reduces inflammatory nociception through the activation of both cannabinoid CB1 and CB2 receptors in mice and rats (Ghosh et al., 2013; Guindon et al., 2011). In addition, JZL184 mitigates the inflammatory response in trinitrobenzene sulfonic acid-induced colitis and lipopolysaccharideinduced acute lung injury by a cannabinoid CB1 and CB2 receptordependent manner (Alhouayek et al., 2011; Costola-de-Souza et al., 2013). Since muscle wound healing is a typical inflammation-evoked event, we hypothesized that MAGL inhibition might alleviate inflammatory response of injured skeletal muscles, which may subsequently affect the skeletal muscle wound healing. In this study, we verified the hypothesis using JZL184 in a rat muscle contusion model.
http://dx.doi.org/10.1016/j.ejphar.2015.04.018 0014-2999/& 2015 Published by Elsevier B.V.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Using selective cannabinoid CB1/CB2 receptor antagonists, we also attempted to determine whether the effects observed after MAGL inhibition by JZL184 treatment were associated with activation of the cannabinoid receptors.
2. Materials and methods 2.1. Animal model of skeletal muscle contusion All animal protocols were conformed to the “Principles of Laboratory Animal Care” (National Institutes of Health Publication no. 85-23, revised 1985) that sought to minimize both the number of animals used in a procedure and any suffering that they might experience, and were performed according to the Guidelines for the Care and Use of Laboratory Animals of China Medical University. A reproducible muscle contusion model in rats was described previously (Yu et al., 2010). Briefly, adult Sprague–Dawley male rats weighing 280–300 g were anesthetized by intraperitoneal injection with 2% sodium pentobarbital (30 mg/kg). The right hindlimb was positioned on a board in a prone position by extending the knee and dorsiflexing the ankle to 901, and a single impact at velocity of 3 m/s was delivered to the gastrocnemius and soleus of the right posterior limb. The size of impact interface of the counterpoise (weighing 500 g) was 1.127 cm2. After injury, each rat was housed individually and kept under a 12 h light–dark cycle. Rats were fed with commercial rat chow and water ad libitum. Rats were killed by intraperitoneal injection of an overdose of sodium pentobarbital. Gastrocnemius was taken and equally divided into two blocks. One block was used for morphological evaluation, and another was used for molecular biological assays. No bone fracture was detected at dissection. 2.2. Experimental protocol and grouping Experiment 1: 60 rats were randomly divided into 2 groups (30 rats/group) according to treatments with JZL184 (Cayman Chemical, MI, USA) or vehicle alone. JZL184 (10 mg/kg, 2 ml/kg) was injected intraperitoneally immediately after contusion once a day for 5 days. Rats injected with equal volume of vehicle were used as control. JZL184 was dissolved in a vehicle containing saline:DMSO:Tween-80 in an 18:1:1 ratio. JZL184 dose and treatment were based on previous studies (Kerr et al., 2013; Long et al., 2009a, 2009b; Sciolino et al., 2011). Rats were killed at 1, 3, 5, 7, 9 and 14 days after contusion (5 rats at each time point in vehicle- or JZL184-treated group). Experiment 2: 30 rats were divided into two groups that received JZL184 (10 mg/kg, 2 ml/kg) or vehicle. Within each group, the rats were further divided into 3 groups that received one of the following treatments 30 min before JZL184 or vehicle (5 rats in each group): 1(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N00 -morpholin-4ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist, Tocris Bioscience, Ellisville, MO, USA, 3 mg/kg, 2 ml/kg), [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist, Tocris Bioscience, Ellisville, MO, USA, 3 mg/kg, 2 ml/kg) or vehicle. AM281 and AM630 were dissolved in the same vehicle as JZL184. AM281 and AM630 were chosen and administered based on previous studies (Arevalo-Martin et al., 2012; Costola-deSouza et al., 2013; Sciolino et al., 2011; Yang et al., 2014). The rats were killed at 3 days after injury. 2.3. Immunohistochemical staining and morphometric analysis The skeletal muscle specimens were immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). Fixed muscle specimens were embedded in paraffin. 5 μm-thick sections
were prepared. The primary antibodies against rabbit antimyeloperoxidase (MPO) polyclonal antibody (dilution 1:500; ab65871, Abcam, Cambridge, UK), mouse anti-CD68 monoclonal antibody (dilution 1:100; ab31630, Abcam, Cambridge, UK) or mouse anti-alpha smooth muscle actin (α-SMA) monoclonal antibody (dilution 1:200; ab7817, Abcam, Cambridge, UK) were used. Histostain-Plus kit (Zymed Laboratories, South San Francisco, CA, USA) was used according to the manufacturer's instructions. As immunohistochemical controls, some sections were incubated with normal rabbit/mouse IgG or phosphate-buffered saline (pH 7.4) in place of the primary antibodies. Nuclei were counterstained with hematoxylin. In addition, hematoxylin-eosin (H&E) staining and Masson's trichrome staining were conventionally conducted. Sections containing the largest contusion area were evaluated. MPO, CD68 or α-SMA positive cells were counted under the 400fold magnification in the contusion zones. Five fields in the contused zones were randomly chosen for the calculations or evaluations in each section. The nucleus number of regenerating myofibers and collagen deposition areas were analyzed under the 200-fold magnification in the contusion zones. All measurements and data analysis were performed independently by two pathologists in a blind manner. Morphometrical analysis was performed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, USA). 2.4. Protein extraction and immunoblotting assay The skeletal muscle samples were ground into powder with liquid nitrogen using a grinder and homogenized with a sonicator in RIPA buffer (sc-24948, Santa Cruz Biotechnology, CA, USA) containing protease inhibitors at 4 1C. After being prepared via standard procedures, protein samples (30 mg) were separated on 12% sodium dodecyl sulfate polyacrylamide electrophoresis gel and were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then they were used to perform immunoblotting. Rabbit anti-IL-1β polyclonal antibody (dilution 1:1000; ab1832p, Chemicon, ON, Canada), rabbit anti-IL-6 polyclonal antibody (dilution 1:500; ab6672, Abcam, Cambridge, UK) and rabbit anti-TNF-α polyclonal antibody (dilution 1:1000; ab1837p, Chemicon, ON, Canada) were applied as primary antibodies, and rabbit anti-GAPDH polyclonal antibody (dilution 1:1000; ab37168, Abcam, Cambridge, UK) was used for relative protein quantification. The horseradish peroxidase conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology, CA, USA) was diluted to 1:2000 and applied. The blotting was visualized with western blotting luminol reagent (sc-2048, SantaCruz Biotechnology, CA, USA) and by the Electrophoresis Gel Imaging Analysis System (MF-ChemiBIS 3.2, DNR Bio-Imaging Systems, ISR). Subsequently, densitometric analyses of the bands were semi-quantitatively conducted using Scion Image software (Scion Corporation, MD, USA). 2.5. Cytokine and chemokine analysis A Quantibody Rat Inflammation Array 1 (QAR-INF-1-4, RayBiotech, Inc, Norcross, GA) was used to simultaneously detect the expression of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, monocyte chemoattractant protein 1 (MCP-1), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the contused muscles. The assays were performed according to the manufacturer's instructions. Each cytokine was arrayed in quadruplicate, together with positive and negative controls. Fluorescent signals were visualized with a laser scanner (Axon GenePix; Molecular Devices, Sunnyvale, CA) set. Data were extracted with RayBio Q Analyzer software (RayBiotech, Inc, Norcross, GA). After subtracting background signals and normalization to positive controls, comparison of signal intensities for antigen-specific antibody spots between groups was utilized to determine relative differences in expression levels of each sample.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 1. H&E staining of muscle sections harvested 1 day (A and B) and 3 days (C and D) post-injury. Hemorrhage, edema, and necrosis are observed in contused skeletal muscles in both vehicle- and JZL184-treated groups. Many polymorphonuclear cells appear at contused zones at 1 day post-injury. Numerous round-shaped mononuclear cells and a few spindle-shaped fibroblastic cells are present in the injured tissue at 3 days post-injury (original magnification, 200). Scale bar: 100 μm.
2.6. Total RNA extraction and quantitative real-time PCR (qPCR)
3. Results
Total RNA was isolated from the skeletal muscle specimens with RNAiso Plus (9108, Takara Biotechnology, Shiga, Japan) according to the manufacturer's instructions. OD values of each RNA sample were measured by an ultraviolet spectrophotometer. A260/A280 ranged from 1.8 to 2.0. Then, the RNA (250 ng) was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (RR037A, Takara Biotechnology, Shiga, Japan). The resulting cDNA was used for quantitative real-time PCR with the sequence-specific primer pairs for Col1a1, Col3a1 and Gapdh (Takara Biotechnology, Shiga, Japan). PCRgenerated fragments for Col1a1, Col3a1 and Gapdh primer sets were 98, 108 and 100 bp, respectively. The PCR primers were as follows: Col1a1 (sense 50 -ACTGGTACATCAGCCCAAAC-30 and antisense 50 GGAACCTTCGCTTCCATACTC-30 ); Col3a1 (sense 50 -CAGGCCAATGGCAATGTAAAG-30 and antisense 50 -GCCATCCTCTAGAACTGTGTAAG-30 ); Gapdh (sense 50 -CATCTCCCTCACAATTCCATCC-30 and antisense 50 GAGGGTGCAGCGAACTTTAT-30 ). qPCR amplification was performed by an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) using a SYBRs PrimeScript™ RT-PCR Kit (RR081A, Takara Biotechnology, Shiga, Japan) followed by the parameters: 2 μL cDNA was loaded to each qPCR reaction. A denaturing step was at 95 1C for 30 s followed by 15 s at 95 1C, 34 s at 60 1C, 15 s at 95 1C, 60 s at 60 1C, and 15 s at 95 1C for 40 cycles. To exclude any potential contamination, negative controls were also performed with dH2O instead of cDNA during each run. No amplification product was detected. The qPCR procedure was repeated at least three times for each sample.
3.1. Effects of JZL184 on inflammatory response
2.7. Statistical analysis Data were expressed as mean 7standard deviation (S.D.). Statistical significance was determined by two-way analysis of variance using GraphPad PRISM 6.0 software (GraphPad Software, Inc., La Jolla, CA). P o0.05 was considered significantly different.
Hemorrhage, edema, necrosis and inflammatory cell infiltration were observed in both JZL184- and vehicle-treated groups at 1 and 3 days post-injury in H&E stained sections (Fig. 1A–D). Evaluation of the neutrophil or macrophage infiltration was performed by immunostaining of MPO or CD68. A large number of MPO-positive cells (neutrophils) appeared at wound zones at 1 day post-injury and numerous CD68positive cells (macrophages) accumulated in the wounds at 3 days postinjury. Morphometrically, neutrophils decreased significantly in number after JZL184 treatment at 1 day post-injury (Po0.05; Fig. 2A, B, and E), and macrophages decreased in number at 1, 3 and 5 days post-injury in the JZL184-treated group (Po0.05; Fig. 2C, D, and F). In both JZL184- and vehicle-treated groups, MCP-1 and TNF-α expressions decreased, IL-1β expression increased from 1 to 5 days post-injury, and IL-6 expression peaked at 3 days post-injury. JZL184 treatment decreased TNF-α expression at 1 and 3 days post-injury, respectively (Po0.05; Fig. 3A), as well as MCP-1 expression at 1 day post-injury (Po0.05; Fig. 3B). Reduced IL-1β and IL-6 expressions were found in JZL184-treated group at 3 days post-injury (Po0.05; Fig. 3C and D). No significant differences were observed between the two groups for IL-1α, IL-2, IL-4, IL-10, IL-13 or IFN-γ expressions. 3.2. Effects of JZL184 on muscle regeneration In sections stained with H&E, a large number of centronucleated regenerating myofibers concomitant with fibroblastic cells were observed in JZL184- and vehicle-treated groups at 5 days post-injury (Fig. 4A and B). The centronucleated regenerating myofibers gradually increased with extension of posttraumatic interval (Fig. 4C–F). The nucleus number of regenerating myofibers in JZL184-treated group was significantly fewer than in vehicle-treated group at 9 (Po0.05) and 14 (Po0.01) days post-injury (Fig. 4G).
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 2. Immunohistochemical analysis of MPO positive (A, B) or CD68 positive (C, D) cells in contused muscles treated with vehicle or JZL184. MPO and CD68 positive cells are indicated as neutrophils and macrophages, respectively (original magnification, 200). Scale bar: 100 μm. The number of neutrophils and macrophages is shown in E and F, respectively. Data are presented as mean 7S.D. *Po 0.05 vs. vehicle.
3.3. Effects of JZL184 on fibrosis H&E and Masson staining were used to observe fibrotic tissue formation after injury. Fibrotic tissue filled the contused sites at 14 days post-injury, and muscles contained more fibrotic tissue in JZL184-treated group than in vehicle-treated group (P o0.05; Fig. 5A, B, and E). Immunohistochemical staining of α-SMA was used to detect activated fibroblasts (myofibroblasts). Myofibroblasts emerged at 5 days post-injury in JZL184- and vehicletreated groups. In relation to vehicle-treated group, more myofibroblasts were observed in contusion zones in JZL184-treated group at 9 and 14 days post-injury (P o0.05; Fig. 5C, D, and F). The findings were consistent with the results of qPCR analysis. mRNA expressions of Col1a1 and Col3a1 were significantly increased in contused muscles following JZL184 treatment at 3 days (P o0.05) and 5 days (Po 0.05 and P o0.001, respectively) post-injury (Fig. 5G and H). 3.4. Effects of AM281 and AM630 pre-treatment before JZL184 on contused muscles In experiment 2, administration of JZL184 had the same antiinflammatory effect as in experiment 1 and effectively reduced macrophage infiltration (P o0.05; Fig. 6A and B), as well as IL-1β
(P o0.05; Fig. 7B), IL-6 (P o0.05; Fig. 7C) and TNF-α (P o0.05; Fig. 7D) expressions at 3 days post-injury. AM630 pre-treatment effectively abolished the effect of JZL184 on macrophage infiltration (Fig. 6C). AM281 pre-treatment completely abrogated the JZL184-induced decrease of IL-1β expression (P o0.01; Fig. 7B), and AM630 pre-treatment blocked JZL184-induced decrease of IL1β (P o0.05; Fig. 7B), IL-6 (P o0.05; Fig. 7C) and TNF-α (P o0.05; Fig. 7D) expressions. When used alone, AM281, but not AM630, attenuated the expressions of IL-1β (Po0.05; Fig. 7B), IL-6 (P o0.01; Fig. 7C) and TNF-α (Po 0.05; Fig. 7D).
4. Discussion The present study investigated the effects of JZL184 on contused skeletal muscles. We found that JZL184 attenuated neutrophil and macrophage infiltration, and decreased TNF-α, IL-1β, IL-6, and MCP1 expressions, which demonstrates the anti-inflammatory effect of JZL184 on contused muscles. In addition, impeded muscle regeneration and promoted fibrosis were observed in JZL184-treated group, which suggests that JZL184 treatment interferes with the repair of injured skeletal muscles. Besides, cannabinoid CB1 and CB2 receptors might have a relevant role in anti-inflammatory effect induced by JZL184.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 3. The relative expressions of cytokines (TNF-α, IL-1β and IL-6) and chemokine (MCP-1) in contused muscles treated with vehicle or JZL184. Reduced TNF-α, MCP-1, IL-1β and IL-6 expressions are shown in A, B, C and D, respectively. Data are presented as mean 7 S.D. *P o0.05 vs. vehicle.
Damage to skeletal muscle due to blunt trauma presents pathologically a typical inflammatory process with neutrophil and macrophage infiltration. Following the onset of neutrophils invasion, macrophages begin to invade into injured tissue. Neutrophils promote the cytolytic capacity of macrophages to kill muscle cells (Nguyen and Tidball, 2003). CD68 is a specific marker for the M1 macrophage and a receptor for oxidized low-density lipoproteins (LDLs). When oxidized LDLs bind to CD68, the phagocytosis and the pro-inflammatory cytokines production of M1 macrophages are promoted (Tidball and Villalta, 2010). We found fewer neutrophils and CD68-positive macrophages after treatment with JZL184 in the acute inflammatory and degenerative phase, indicating the antiinflammatory effect of JZL184. To further explore anti-inflammatory effect of JZL184, we analyzed the expressions of pro-inflammatory cytokines/chemokines in contused muscles. Reduced TNF-α, IL-1β, IL-6, and MCP-1 expressions were observed after JZL184 treatment. These molecules are essential for both inflammation establishment and phagocyte activation. TNF-α plays important roles in activation inflammatory cells which are associated with promoting muscle damage and secretion of the other pro-inflammatory cytokines in injured muscles (Karalaki et al., 2009; Tidball and Villalta, 2010). IL6 is considered a cytokine that strongly activates the immune system and enhances inflammatory response (Wojdasiewicz et al., 2014). IL-1β and TNF-α are able to increase IL-6 expression and release in cultured skeletal muscle cells (Langen et al., 2001; Luo et al., 2003; Prelovsek et al., 2006). Furthermore, MCP-1 attracts monocytes to the inflammatory site (Luster, 1998), causes neutrophil recruitment in lung following Escherichia coli infection in mice (Balamayooran et al., 2011). MCP-1 blockage leads to a dramatic decrease of macrophage infiltration and TNF-α and IL-6 production (Baeck et al., 2012; Lloyd et al., 1997). In conclusion, JZL184 is able to attenuate inflammatory response in injured skeletal muscles by decreasing neutrophil and macrophage infiltration, and the proinflammatory cytokine/chemokine production. The alleviated neutrophil and macrophage infiltration might be attributed to the reduced MCP-1 expression.
Inflammatory response is essential for muscle regeneration. It has been reported that reduction of phagocytic leukocytes slows the removal of cellular debris and muscle regeneration (Arnold et al., 2007; Teixeira et al., 2003). Observations from acute muscle injury in TNF-α receptor knockout mice suggest that TNF-α may promote muscle regeneration (Chen et al., 2005; Warren et al., 2002). IL-1β increases the mitogenic activity of myogenic cells (Otis et al., 2014). IL-6 knockout mice show slowed muscle growth in a compensatory muscle hypertrophy model (Otis et al., 2014). Muscle precursor cells and macrophages express the chemokine CCR2 receptor (MCP-1 receptor). Disruption of signaling through MCP-1 receptor leads to slower muscle regeneration (Otis et al., 2014; Tidball and Villalta, 2010). Since inflammatory response is essential for muscle regeneration, and alleviated inflammatory response with impeded muscle regeneration was observed by JZL184 treatment, it is suggested that the anti-inflammatory effect of JZL184 on contused muscles interferes with the healing process, which leads to decreased muscle regeneration. Fibrosis is an inevitable result of wound repair in many organs and tissues. The fibrotic tissue in the injured area hampers the recovery of normal tissue architecture and function. There is an antagonism between muscle regeneration and fibrosis. When the regeneration process goes awry, the fibrosis may be promoted (Moyer and Wagner, 2011). In our study, increased myofibroblasts and deposition of collagen were detected with retarded muscle regeneration in the repair phase after JZL184 delivery, which was in line with up-regulated levels of Col I and Col ІІІ mRNA. We presume that impeded muscle regeneration induced by JZL184 aggravates the fibrogenesis. After muscle injury, a series of inflammation-related symptoms, such as pain and vasodilation are produced by the release of inflammatory metabolites. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often prescribed to relive pain in sports medicine (Gharaibeh et al., 2012; Paoloni et al., 2009; Urso, 2013). NS-398, a member of NSAIDs, reduces the neutrophil and macrophage infiltration in the injured muscles, but slows the proliferation
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 4. H&E staining of muscle sections harvested 5 days (A, B), 7 days (C, D) and 14 days post-injury (E, F; original magnification, 200). Scale bar: 100 μm. The nucleus number of regenerating myofibers is shown in G. Data are presented as mean 7 S.D. *P o0.05, **Po 0.01 vs. vehicle.
and maturation of differentiated myogenic precursor cells, which delays the regenerative myogenesis process (Shen et al., 2005). Though JZL184 is able to alleviate inflammation in muscle injury, similar to NS-398, it may interfere with muscle regeneration, suggesting that JZL184 may not be a potential option for primary care of skeletal muscle injuries. 2-AG is a signaling lipid which binds to cannabinoid CB1 and CB2 receptors (Sugiura et al., 2006). Inhibiting MAGL with JZL184 increases the level of endocannabinoid 2-AG (Long et al., 2009a). We administrated the cannabinoid CB1 and CB2 receptors selective antagonists before JZL184 treatment to analyze the involvement of
cannabinoid CB1 and CB2 receptors in the anti-inflammatory effect induced by JZL184. AM281 pre-treatment blocked the effect of JZL184 on IL-1β expression. AM630 pre-treatment reversed the effects of JZL184 on macrophage infiltration, and TNF-α, IL-1β and IL-6 expressions. The results indicated the participation of cannabinoid CB1 and CB2 receptors in the anti-inflammatory effect of JZL184. However, other mechanisms may be involved in cannabinoid actions besides cannabinoid CB1 and CB2 receptors activation, such as adenosine receptor activation (Ribeiro et al., 2012). JZL184 partially inhibits fatty acid amide hydrolase which mainly hydrolyzes anandamide, another endocannabinoid (Long et al., 2009a).
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 5. Detection of fibrosis by Masson trichrome staining, immunohistochemical procedure and qPCR. (A and B) The deposition of collagen in vehicle and JZL184 groups at 14 days post-injury, respectively. Fibrotic tissue is stained blue (original magnification, 200). (E) The percentage of fibrosis areas at 9 and 14 days post-injury. (C and D) Myofibroblasts infiltration in vehicle and JZL184 groups at 14 days post-injury, respectively (original magnification, 200). (F) The number of myofibroblasts. (G and H) The dynamic changes of Col1a1 and Col3a1 mRNA expressions, respectively. Data are presented as mean7 S.D. *Po 0.05, ***Po 0.001 vs. vehicle. Scale bar: 100 μm.
Furthermore, the release of arachidonic acid from hydrolyzed 2-AG and/or anandamide leads to eicosanoid (leukotriene) formation in neutrophils (Chouinard et al., 2011). Though these mechanisms may explain the anti-inflammatory effect of JZL184, the results similar to ours were observed in colitis and acute lung injury with co-administration of cannabinoid CB1 or CB2 receptors antagonists with JZL184 (Alhouayek et al., 2011; Costola-de-Souza et al., 2013). Our observations suggest that simultaneous activation
of both cannabinoid receptors is needed to maintain the antiinflammatory effect of JZL184. AM281 effectively blocked the effect on IL-1β expression induced by JZL184. Interestingly, decreased expressions of IL-1β, IL-6 and TNF-α were observed when AM281 was administered alone. The mechanisms underlying the anti-inflammatory effect of AM281 are not fully elucidated. It has been reported that AM281 significantly improved intestinal microcirculation by reducing leukocyte adhesion
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 6. Immunohistochemical analysis of CD68 positive cells in contused muscles at 3 days post-injury. Cannabinoid CB2 receptor antagonism AM630 prevents the effect of JZL184 on macrophage infiltration (original magnification, 200). Scale bar: 100 μm. The number of macrophages is shown in G. Data are presented as mean 7 S.D. #Po 0.05 vs. vehicle, **Po 0.01 vs. JZL184.
and increasing functional capillary density in acute endotoxemia in rats (Kianian et al., 2013). The anti-inflammatory effect of AM281 alone may be attributed to the endocannabinoids signaling through cannabinoid CB2 receptor in the presence of inactivated cannabinoid CB1 receptor (Schafer et al., 2008).
In conclusion, the present findings demonstrate that JZL184 significantly inhibits inflammatory response and muscle regeneration, promotes fibrosis in contused muscles. cannabinoid CB1 and CB2 receptors appear to be the likely mechanism involved in the anti-inflammatory effect induced by JZL184.
Please cite this article as: Jiang, S.-K., et al., The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.04.018i
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Fig. 7. Analysis of IL-1β, IL-6, TNF-α and GAPDH protein from skeletal muscle specimens by western blotting. (A) The bands of L-1β, IL-6, TNF-α and GAPDH in vehicle, JZL184, JZL184þ AM630, JZL184þ AM281, AM630 and AM281 groups, respectively. The relative quantity of IL-1β (B), IL-6 (C) and TNF-α (D) to GAPDH is shown. Data are presented as mean 7 S.D. #Po 0.05, ##Po 0.01 vs. vehicle, *Po 0.05, **P o0.01 vs. JZL184.
Acknowledgments The study was financially supported in part by grants from research fund for the Doctoral Program funded by Ministry of Education of China (20122104110025) and from projects funded by National Natural Science Foundation of China (81273342) and Shenyang Scientific and Technological Plan (F12-277-1-03).
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