Influence of adiponectin and inflammatory cytokines in fatty degenerative atrophic muscle

Tendon rupture and nerve injury cause fatty infiltration of the skeletal muscle, and the adipokines secreted from the infiltrated adipocytes are known to contribute to chronic inflammation. Therefore, in this study, we evaluated the effects of the adipokines on chronic inflammation using a rat sciatic nerve-crushed injury model. In vitro and in vivo experiments showed that the expression of adiponectin was decreased (0.3-fold) and the expression of Il6 (~ 3.8-fold) and Tnf (~ 6.2-fold) was increased in the nerve-crushed group compared to that in the control group. It was also observed that the administration of an adiponectin receptor agonist decreased the levels of Il6 (0.38-fold) and Tnf (0.28-fold) and improved cellular viability (~ 1.9-fold) in vitro. Additionally, in the fatty infiltrated skeletal muscle, low adiponectin levels were found to be associated with chronic inflammation. Therefore, the local administration of adiponectin receptor agonists would prevent chronic inflammation.


Results
Gastrocnemius muscle weight. The weight of the gastrocnemius muscle at 4 weeks post-operation was compared between the nerve-crushed model (refer to the Methods section, Fig. 1a, b), in which the sciatic nerve was crushed as described in a previous study 13 , and the control group, which had sham surgery wherein only a skin incision was made. The gastrocnemius muscle weight was significantly decreased on the affected side in the nerve-crushed group than on the unaffected side. The weight of the gastrocnemius muscle in the control group was not significantly different from those of the affected and unaffected sides. There was no significant difference in gastrocnemius muscle weight between the unaffected side of the nerve-crushed group and both sides of the control group (Fig. 1c). The ratio of the muscle weight on the affected side to that on the unaffected side in the nerve-crushed group was significantly lower than that in the control group, as shown in Fig. 1d.

Cell morphology evaluation. Morphological observation via microscopy and Hematoxylin-eosin (HE)
staining showed the formation of myotubes in the control group. Conversely, in the nerve-crushed group, the cells appeared small, with poorly formed myotubes (Fig. 2a). The number of myotubes formed per field of view (400 μm × 600 μm) was calculated as the average of four fields of view. The number of myotubes formed per field of view was 24.8 ± 2.3 in the control group and 6.6 ± 1.9 in the nerve-crushed group, and the number of myotubes formed was significantly lower in the nerve-crushed group (p = 0.0003).
Cell viability (cell proliferation assay). The water-soluble tetrazolium salt (WST) assay was used to measure the absorbance of reduced formazan at 450 nm. Thus, it was observed that the absorbance corresponding to the control group was significantly higher than that corresponding to the nerve-crushed group. Further, in the nerve-crushed group treated with AdipoRon, the absorbance showed a significant increase compared with the nerve-crushed group without AdipoRon treatment. However, the differences among all the AdipoRon doses were not significant (Fig. 2b). Fluorescent immunostaining. The cytoplasm of the adiponectin-positive cells was stained green, and the quantitative evaluation of the ratio of adiponectin-stained cells to blue-stained 4' ,6-diamidino-2-phenylindole (DAPI)-positive cells was determined (Fig. 3a). Thus, we observed that the ratio of green adiponectin-stained  Histological examination. HE staining showed that the diameter of muscle fibers corresponding to the rats in the nerve-crushed group was smaller than that corresponding to the rats in the control group. Furthermore, the mean cross-sectional area of each muscle fiber was significantly decreased in the nerve-crushed group, indicating that the muscles were atrophied four weeks after the nerve crush injury (Fig. 4a, b). Oil Red-O positive lipid droplets were observed in the muscle in both the control and nerve-crushed groups. The number of fat droplets quantitatively assessed in four non-overlapping areas by microscopy was significantly higher in the nerve-crushed group than in the control group (Fig. 5a, b). Furthermore, immunofluorescence staining showed that adiponectin was stained along the fascia, and the degree of staining corresponding to the nerve-crushed group was lower than that corresponding to the control group (Fig. 6a). Quantitative evaluation performed using the ratios of green adiponectin-and adiponectin receptor (AdipoR)-stained cells to blue DAPI-positive cells showed that the expression of adiponectin was significantly weaker in the nerve-crushed group than in the control group. Conversely, the two groups showed no significant differences with respect to the expression of AdipoR1 (Fig. 6b).   www.nature.com/scientificreports/ nerve-crushed group than in the control group, indicating the occurrence of an inflammatory reaction in the muscles after nerve injury. The expression of oxidative markers, NADPH oxidase1 (Nox1) and Nox4, was significantly increased in the nerve-crushed group. Furthermore, the expression levels of adipogenic markers, such as Pparg and Cebpa were significantly higher in the nerve-crushed group than in the control group, as shown in Fig. 7.
To evaluate the effect of AdipoRon administration in vitro, the nerve-crushed group received AdipoRon at concentrations of 0 μg/ml, 0.3 μg/ml, 1.0 μg/ml, and 2.0 μg/ml, respectively. Although the expression levels of myogenin and Myod, the markers of muscle atrophy, were not significantly different after the AdipoRon treatment, the expression levels of Atrogin1, Murf1, and Foxo1, the markers of muscle atrophy, were significantly decreased following AdipoRon treatment, the differences in group which were administered different concentrations were not significant. In terms of inflammatory cytokines, Il6 and Tnf were significantly downregulated after AdipoRon treatment. AdipoRon treatment did not bring about any significant changes in Cox1 expression. The expressions of Nox1 and Nox4, which are involved in oxidative stress, were significantly decreased by AdipoRon treatment. The groups that received different doses did not show significant differences (Fig. 8).

Discussion
In this study, we used a nerve-crushed rat model to verify the hypothesis that an imbalance in adipokine secretion owing to fatty degeneration associated with muscle atrophy triggers an inflammatory response. In the nerve-crushed group, the cross-sectional area of the muscle was decreased, fatty infiltration was observed by oil red staining, and the expression of adipogenic differentiation markers was increased. This was accompanied by decreased expression of Adipoq in the skeletal muscle and increased levels of inflammatory cytokines and Nox, which are involved in oxidative stress. However, the expression of adiponectin receptor, AdipoR1, was not significantly different between the control and nerve-crushed groups. It was also observed that in vitro agonist treatment significantly improved inflammatory response and increased cellular viability. To the best of our www.nature.com/scientificreports/ knowledge, there is no report on the effect of AdipoRon administration on muscle atrophy. In this in vitro study, AdipoRon administration showed anti-oxidant, anti-inflammatory, and anti-apoptotic effects.
Reportedly, muscle atrophy accompanied by the fatty degeneration of muscles is clinically observed after nerve injury or tendon rupture 4 . However, despite recent improvements in nerve and tendon repair techniques, chronic inflammation and muscle atrophy associated with fatty degeneration still limit postoperative function 6,14 . Muscle atrophy occurs when the breakdown of proteins in muscle cells exceeds their synthesis, resulting in a decrease in muscle mass 15 , and reportedly, inflammatory cytokines and oxidative stress enhance this muscle degradation process.
In the skeletal muscle, myokines secreted by the muscle and adipokines secreted by the white adipose tissue balance the metabolism 16 . Among these cytokines, IL6 and TNF are considered inflammatory cytokines, while www.nature.com/scientificreports/ adiponectin is considered an anti-inflammatory cytokine [17][18][19] . Although adiponectin was originally identified as a secretory protein in adipose tissue, it is now known to be expressed in the skeletal muscle 10 . Adiponectin in skeletal muscle has potent anti-inflammatory, antioxidant, and anti-apoptotic effects, as well as a significant inverse correlation with pro-inflammatory cytokines, like IL6 and TNF 20 . These adipokines are tightly regulated to maintain homeostasis in the skeletal muscle 16,21 ; however, when adipose tissue proliferates under nonphysiological conditions, such as is the case with obesity and degeneration, the adipokine secretion balance is disturbed 22 . Thus, the adipocytes that infiltrate skeletal muscle owing to the degeneration caused by trauma or tendon rupture (ectopic fat) possibly disrupt adipokine homeostasis, resulting in chronic pain 23 . Under such conditions, atrophied muscles with ectopic fat tissue secrete low amounts of adiponectin 23 , and the catabolism induced by TNF is particularly enhanced 24,25 . In addition, NOX-mediated activation of reactive oxygen species causes oxidative stress, which induces apoptosis via the FoxO1/MuRF-1/Atrogin-1 signaling pathway and contributes to muscle atrophy 26,27 . Adiponectin is thought to play an important role in antagonizing these inflammatory and oxidative stresses 20,28 . Two subtypes of adiponectin receptors (AdipoR1 and AdipoR2) have been identified via complementary DNA expression cloning 29 . Specifically, AdipoR1 is predominantly expressed in skeletal muscles, while AdipoR2, which functions as a major receptor for adiponectin in vivo, is predominantly expressed in the liver 28 . Additionally, AdipoR1 activates the activated protein kinase (AMPK) pathway, which promotes fatty acid burning and reduces inflammation 30,31 . Even though the anti-inflammatory and antioxidant effects of adiponectin via AdipoR1 have been evaluated in some previous studies 32 , reports on the expression of adiponectin and AdipoR1 in atrophied muscles are limited. In this study, the fluorescence immunostaining of muscle samples from the control group showed that adiponectin and its receptor, AdupoR1, were expressed along the fascia. Conversely, the nervecrushed group showed a significant decrease in adiponectin expression, even though the AdipoR1 expression was maintained. There was no significant difference in the expression of AdipoR1 between the control group and the nerve injury group. These results suggest that AdipoRon administration is useful for correcting the imbalance of adipokines after muscle atrophy. Furthermore, previous reports have demonstrated that it shows anti-inflammatory and anti-fibrotic actions in the liver as well as anti-inflammatory actions in the myocardium 33,34 . In this study, real-time PCR showed decreased Tnf and Il6 expression owing to AdipoRon treatment. However, this treatment had no effect on Cox1 expression. Therefore, the balance between TNF and IL6 as inflammatory cytokines and the secretion of adiponectin is important for the maintenance of skeletal muscle homeostasis. The expression of muscle atrophy markers and oxidative markers also showed a significant decrease following AdipoRon treatment. Furthermore, the anti-inflammatory and anti-apoptotic effects of adiponectin are expected to be sufficient to maintain the cytokine balance, even at low concentrations given at high concentrations (20 μM). AdipoRon administration to myocytes rather inhibits differentiation into myotubular cells, resulting in a decrease in muscle mass 35 . AdipoRon treatment decreased inflammatory, oxidative, and degenerative markers and increased cellular activity in the nerve-crushed group. These results suggest that AdipoRon has anti-inflammatory, antioxidant, and anti-apoptotic effects in vitro. The doses of AdipoRon used in this study were based on previous reports 36,37 . No cytotoxicity was observed within the range of doses administered. In addition, there was no significant difference in the effect of AdipoRon dose concentration. When examined in light of previous reports 35,36 , this may be due to saturation of AMPK pathway activation. Furthermore, atrophied muscles were used in this study, which may have lowered the response threshold to AdipoRon stimulation. The saturation of the AMPK pathway after AdipoRon administration was thought to lead to a concentration-independent response of anti-inflammatory and antioxidant effects. Therefore, administration of AdipoRon at a low dose concentration may be an effective treatment option for fatty degeneration-associated chronic inflammation and muscle atrophy.
This study had several limitations. The first is that the evaluation period was limited to a single time point (the subacute phase). This implies that the evaluation of the chronic phase needs to be considered in the future. Second, in this study, we evaluated adiponectin receptor agonists only in vitro. However, in vivo expression of adiponectin receptors was observed in the nerve-crushed group, suggesting that the administration of adiponectin may be effective. Therefore, in the future, it would be necessary to examine the dosage concentrations as well as other factors. Further research on the role of AdipoRon in chronic inflammation and muscle atrophy is also required. In vivo evaluation of the effect of AdipoRon on the activation of the AMPK pathway in injured muscle may lead to further confirmation of the therapeutic effect. Third, the present study did not measure differences in the isoforms of adiponectin in gastrocnemius muscle. Since adiponectin physiologically forms multimers 10 , further studies investigating isoforms are needed. Finally, adipokines from atrophied muscle and infiltrated fat cannot be strictly evaluated separately. The balance of adipkines in muscle is important, and correcting the imbalance is expected to prevent chronic pain and degeneration.
In this study, we observed muscle atrophy and fatty infiltration accompanied by a decreased expression of adiponectin which had anti-inflammatory and anti-apoptotic effects in the nerve-crushed group. Conversely, the secretion of inflammatory cytokines increased. This cytokine imbalance possibly resulted in chronic pain owing to the induction of apoptosis and inflammation in the muscle cells. Even though adiponectin expression was decreased in the nerve-crushed group, the expression of AdipoR1 was maintained. Furthermore, the in vitro administration of the adiponectin receptor agonist showed anti-inflammatory and anti-apoptotic effects, suggesting that the control of adipokines via the local administration of AdipoRon could lead to the prevention of fatty degeneration-associated chronic pain. www.nature.com/scientificreports/ experiments were conducted in accordance with the ARRIVE guidelines. Twenty-four SD rats (12-week-old) with a mean weight of 250 g were used in this study (CLEA Japan, Inc., Tokyo, Japan).

Materials and methods
Surgical procedure. All surgical interventions were performed under sterile conditions, with isoflurane, the intraperitoneal injection of pentobarbital sodium (50 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan), and the subcutaneous injection of lidocaine (2.5 mg/kg, Xylocaine®; AstraZeneca, London, UK) at the surgical site as anesthesia. The skin of the rats was incised at the right hind limbs, and the vastus lateralis and biceps femoris muscles were separated to expose the sciatic nerve. To establish the sciatic nerve injury model, the sciatic nerve was clamped using hemostatic forceps for one minute at a proximal distance of 5 mm from the bifurcation point of the peroneal and tibial nerves (Fig. 1a) 13 . Twelve rats were included in the nerve-crushed group. For the control rats, only a skin incision was made on the hind limbs. The skin incision was closed with a 4-0 nylon suture, and 4 weeks after the surgery, gastrocnemius muscle samples were harvested and analyzed (Fig. 1b).
Cell culture. After muscle weight measurement, the muscles were minced and treated with 0.4% collagenase for 2 h. After this collagenase treatment, the cells were washed with phosphate-buffered saline (PBS) and centrifuged at 1200 rpm for 5 min. Thereafter, the cells were plated in 100-mm diameter culture dishes and cultured in a monolayer mode using Dulbecco's modified eagle medium (DMEM, HyClone, Logan, UT, USA) mixed with 10% fetal bovine serum (FBS, Cansera, Rexdale, Ontario, Canada), 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were then evaluated after 2-3 passages and group comparisons were performed.

Protocol for in vitro AdipoRon administration. Myocytes corresponding to the nerve-crushed group
were treated with AdipoRon (AdipoGen Life Sciences, Liestal, Switzerland) 36 , a small molecule adiponectin receptor (AdipoR) agonist. Specifically, the AdipoRon was first dissolved in DMSO to obtain a 2 mM stock solution. Thereafter, it was administered at concentrations of 0.3, 1.0, and 2.0 μg/ml, as previously reported 36,37 , and the differences in the efficacy of this treatment among the treatment groups were determined. Specifically, cell viabilities at 24 h after treatment administration were compared using the WST assay, and the expression levels of Tnf, Il6, and Cox1 (inflammatory markers) and myogenin and Myod (muscle markers) and Murf1 and Atrogin1 (muscle atrophy markers) were evaluated via real-time PCR.
Evaluation method. Cell morphology evaluation. After three passages, 1.0 × 10 5 cells were seeded into 12-well plates, and the cell morphologies were evaluated via HE staining after 48 h.
Cell viability (cell proliferation assay). Cell viability was measured via a WST assay using a cell counting kit-8 (Dojindo, Kumamoto, Japan). A total of 5,000 cells were seeded into each well of a 96-well-plate and cultured in a DMEM medium for 48 h. Fluorescent immunostaining. The expression level of intracellular adiponectin in myocyte samples from the different groups was detected using an anti-adiponectin polyclonal antibody (NB100-65810F; Bios, Boston, MA, USA). The antibodies, which were used at a dilution ratio of 1:100, were incubated with myocytes (5 × 10 4 ) for 60 min in the dark at 37 °C. Thereafter, they were washed twice with PBS and nuclear staining was performed using DAPI solution for 10 min. The percentage of stained cells was then observed using a fluorescence microscope (BZ-8000 confocal microscope; Keyence, Osaka, Japan). For quantification, the number of DAPI-positive and Adiponectin-stained cells in five fields of view (0.75 mm × 1.0 mm) on each slide was counted, and the ratio of the mean values was calculated. The measurements were performed on randomly selected areas by two investigators who were blinded to each other.
Histological examination. The weight ratio of the gastrocnemius muscle at the affected side to that at the healthy side was measured. Specifically, gastrocnemius muscle samples were harvested and embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek USA, Inc., Torrance, CA), and stored at −80 °C for histochemical and immunohistochemical staining. Specifically, the gastrocnemius muscle samples in the OCT blocks were sectioned serially to have a thickness of 6 μm. Thereafter, they were was mounted on a silane-coated glass slide, and air-dried for 1 h before fixation with 4% paraformaldehyde at 4 °C for 5 min. The tissue sections were then stained with HE to observe the histological differences between the muscle samples from the control and nerve-crushed groups. Fat droplets were also stained with Oil Red-O solution (Mutoh Pure Chemical, Tokyo, Japan) to evaluate fat infiltration into the muscle. Gastrocnemius muscle was frozen using isopentane cooled with liquid nitrogen and stored at −80 °C until needed. As described in the previously published protocol 38 , the muscles were cryosectioned at a thickness of 10 μm and fixed in 4% paraformaldehyde. The sections were then stained with Oil Red-O and counterstained with hematoxylin.
For immunofluorescence staining, adiponectin antibody and AdipoR1 antibody were used at a dilution ratio of 1:100; the staining was performed at 25 ℃ room temperature for 1 h. To ensure nuclear staining, the DAPI solution was applied for 5 min. All sections were visualized using a fluorescence microscope (BZ-8000 confocal microscope; Keyence). Furthermore, the number of positively stained cells was counted in five randomly selected fields (250 × 250 mm).
Real-time PCR. Myocytes from both groups were seeded in 12-well culture plates at a density of 1.0 × 10 5 cells/ well and cultured in DMEM for 48 h. Thereafter, total RNA was extracted using an RNeasy Mini Kit (QIAGEN,

Adipoq
AAT CCT GCC CAG TCA TGA AG  CAT CTC CTG GGT CAC CCT TA   Il6  TCC TAC CCC AAC TTC CAA TGCTC  ACC CAG AGC GTA TCA TCC TTCAC   Tnf  AAA TGG GCT CCC TCT CAT CAG TTC  CCA ACT GAC TTT GAG CCA ACGAG   Cox1  TGC CAG TAT TAG CAG CAG GT  GAA TTG GGT CTC CAC CTC CA   Nox1  CTT CCT CAC TGG CTG GGA TA  TGA CAG CAT TTG CGC AGG CT   Nox4  AGT CAA ACA GAT GGG ATA  TGT CCC ATA TGA GTT GTT   Pparg  TGT GGA CCT CTC TGT GAT GG  CAT TGG GTC AGC TCT TGT