Abstract
The currently available treatment options for muscle injuries are suboptimal and often delay muscle recovery. In this study, the effects of curcumin on inflammation and skeletal muscle regeneration after contusion-induced injury in mice were investigated. The mice were randomly assigned to 4 groups, namely normal control (NC), with induced injury (mass-drop injury, MDI) and without treatment (MDI [M]), with induced injury and diclofenac (DCF) treatment (MDI + DCF [M + D]), and with induced injury and curcumin treatment (MDI + curcumin [M + C]). Contusion-induced injury was inflicted on the left gastrocnemius muscle, and DCF or curcumin was orally administered after injury once per day for 7 days. The M group exhibited significantly higher lipid peroxidation, myeloperoxidase (MPO), and desmin than the NC group. The M + D and M + C groups have lower lipid peroxidation and neutrophils (decrease in MPO protein) and higher muscle satellite cell regeneration (increase in desmin protein) than the M group. Additionally, for the contusion-induced muscle injury, curcumin could affect the specific proteins of inflammation, neutrophils, and differentiation of satellite cells, including Ikk-α/ß, MPO, and myogenin. In conclusion, curcumin potentially accelerates muscle recovery; therefore, it may be a potential candidate for further research as an effective treatment to enhance muscle repair.
Damage to skeletal muscle is accompanied with capillary rupture, infiltrative bleeding, inflammation, oxidative stress, and fibrosis. Inflammation plays a key role in regulating the repair process. Therefore, inflammation is the most suitable target for developing effective treatments for injured muscles. Modulating inflammation is one therapy for improving muscle regeneration. 1,2 During the inflammatory response, neutrophils and macrophages are attracted to the injury site for phagocytosis by chemotactic signals. 3 -5 Neutrophils, macrophages, and satellite cells release free radicals, which cause oxidative stress. Oxidative stress directly damages the tissue surrounding the injury site, which is unaffected by the primary injury, thus causing secondary muscle damage. 3,6 In addition, both neutrophils and macrophages stimulate T cells to release the cytokines interleukin (IL)-1, IL-6, and IL-8, and recruit muscle satellite cells to regenerate muscle. 3,7,8 Therefore, inflammation is a critical factor for postinjury muscle cell repair.
Curcumin, extracted from the root of the curcuma plant, is a natural polyphenolic compound. It has been widely used over the past 60 years to treat numerous diseases. 9 Curcumin is used in the traditional Chinese and Indian systems of medicine for its anti-inflammatory, anticarcinogenic, and antioxidant properties. 10 -12 Curcumin exhibits anti-inflammatory effects by downregulating NF-κB expression and suppressing COX-2 production, both of which play a vital role in the inflammatory cascade. 13,14 The anti-inflammatory effects of curcumin are used to treat several diseases, including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and diabetes. 15 Davis et al 14 showed that curcumin supplementation reduces the levels of IL-6 and tumor necrosis factor (TNF)-α in mouse muscle following downhill running-induced muscle damage. Kawanishi et al 16 reported that oral curcumin suppresses hydrogen peroxide and oxidative stress in mouse skeletal muscles after downhill running. Curcumin may therefore be a treatment option for muscle injury in clinical settings after contusion-induced injury. However, to the best of our knowledge, no prior study has investigated curcumin treatment for contusion-induced muscle injury in mice. Thus, this study evaluated the potential therapeutic effects of curcumin treatment on contusion-induced muscle injury in mice.
The effects of curcumin supplementation on the final body weight and diet intake after contusion-induced muscle injury are presented in Table 1. The final body weights of the NC (normal control), M (MDI, mass-drop injury), M + D (MDI + DCF, mass-drop injury with diclofenac), and M + C (MDI + curcumin, mass-drop injury with curcumin) groups were 38.3 ± 0.5, 38.6 ± 1.1, 38.0 ± 1.0, and 38.6 ± 0.6, respectively. The final body weight values did not differ significantly among the 4 groups. Therefore, the contusion-induced gastrocnemius injury of the mice did not affect their growth. In addition, food intake in the M (6.35 ± 0.08 g), M + D (7.08 ± 0.03 g), and M + C (7.05 ± 0.06 g) groups was significantly lower than that in the NC group (7.31 ± 0.04 g) by 13.1% (P < 0.05), 3.1% (P < 0.05), and 3.6% (P < 0.05), respectively. However, food intake in the M + D and M + C groups was significantly higher (11.5% and 11.0%, respectively) than that of the M group (P < 0.05). Therefore, contusion-induced gastrocnemius damage may cause pain and affect feeding.
Effects of Curcumin Treatment on Body Weight, Food Intake, and Water Intake After Contusion-Induced Muscle Injury in Mice.
b.w., body weight; NC, normal control, mice treated with RO water and without injury; M, mass-drop injury (MDI), mice treated using reverse osmosed water after MDI; M + D, MDI + DCF, mice treated using DCF (diclofenac, a nonsteroidal anti-inflammatory drug) after MDI; M + C, MDI + curcumin, mice treated using curcumin after MDI.
Data are the mean ± standard error of the mean (n = 8 mice/group). Values in the same row with different superscript letters (a, b, c) differed significantly, P < 0.05, in one-way analysis of variance.
One-way analysis of variance (ANOVA) indicated no significant differences in liver, kidney, heart, and lung masses of the M, M + D, and M + C groups compared with those of the NC group (Table 2). In the NC group, the muscle mass was 0.18 ± 0.01 g. In mice with contusion-induced muscle injury, muscle mass decreased to 0.16 ± 0.01 g. Therefore, the muscle mass of the M group decreased by 11.1% compared with that of the NC group (P < 0.05). However, the M + C group exhibited an 18.8% increase in muscle mass; hence, the muscle mass was significantly higher in the M + C group than in the M group.
Effect of Curcumin Treatment on Tissue Weights After Contusion-Induced Muscle Injury in Mice.
NC, normal control, mice treated with reverse osmosed (RO) water and without injury; M, mass-drop injury (MDI), mice treated with RO water after MDI; M + D, MDI + DCF, mice treated with DCF (diclofenac, a nonsteroidal anti-inflammatory drug) after MDI; M + C, MDI + curcumin, mice treated with curcumin after MDI.
Data are the mean ± standard error of the mean (n = 8 mice/group). Values in the same row with different superscript letters (a, b) differed significantly, P < 0.05, in one-way analysis of variance. Muscle mass includes both the gastrocnemius and soleus muscles at the back of the lower legs.
In the NC group, the footprint area was 52.6% ± 4.6%. In the mice with contusion-induced muscle damage, the footprint area was reduced to 45.5% ± 4.2%. Therefore, after contusion caused by muscle injury, contusion slightly affected the mice’s walking, but there was no significant difference. However, the M + D (48.7% ± 4.1%) and M + C (53.9% ± 2.7%) groups slightly improved the functional recovery of the gait compared to the M group (Figure 1).
Effect of curcumin treatment on footprint after contusion-induced muscle injury in mice. The mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water and without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), consisting of mice treated using DCF after MDI; and (4) MDI + curcumin (M + C), consisting of mice treated using curcumin after MDI. Data are the mean ± standard error of the mean (n = 8 mice/group). The same letter (a) indicates no significant difference between the 4 groups in one-way analysis of variance.
In the NC group, the serum uric acid (UA) level was 0.79 ± 0.15 mg/dL. In the M group, the serum UA level rose to 1.81 ± 0.09 mg/dL. Therefore, the serum UA level in the M group increased by 129.1% compared with that in the NC group (P < 0.05). As shown in Figure 2, the serum UA levels in the M + D (1.45 ± 0.10 mg/dL) and M + C (0.84 ± 0.17 mg/dL) group were significantly lower than that in the M group (1.81 ± 0.09 mg/dL) (P < 0.05). Therefore, compared with the M group, treatment with DCF and curcumin significantly reduced serum UA levels by 19.9% (P < 0.05) and 53.6% (P < 0.05), respectively.
Effect of curcumin treatment on serum blood uric acid (UA) levels after contusion-induced muscle injury in mice. The mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water and without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), consisting of mice treated using DCF after MDI; and (4) MDI + curcumin (M + C), consisting of mice treated using curcumin after MDI. Data are the mean ± standard error of the mean (n = 8 mice/group). Letters (a, b, c) indicate a significant difference at P < 0.05 in one-way analysis of variance.
Contusion-induced injury can cause physical damage; subsequently, physical damage results in sarcomeric damage and muscle cell necrosis. 17 During or after muscle damage, the muscle cells release creatine kinase (CK) into the blood. Thus, CK level is considered an accurate indicator of structural damage to muscle cells in clinical settings. As shown in Figure 3, the serum CK level in the M group (1239 ± 199 U/L) was 140.6% higher than that in the NC group (515 ± 48 U/L) (P < 0.05). However, the serum CK level in the M + D group (969 ± 80 U/L) was 21.8% lower than that in the M group, and the serum CK level in the M + C group (788 ± 137 U/L) was 36.4% lower than that in the M group (P = 0.0175). Therefore, the M + C group had significantly lower serum CK levels than did those in the M group, which indicated that curcumin can reduce muscle damage.
Effect of curcumin treatment on serum creatine kinase (CK) levels after contusion-induced muscle injury in mice. Mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water and without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), consisting of mice treated using DCF after MDI; (4) MDI + curcumin (M + C), consisting of mice treated using curcumin after MDI. Data are represented as mean ± standard error of the mean (n = 8 mice/group). Letters (a, b, c) indicate a significant difference at P < 0.05 in one-way analysis of variance.
As shown in Figure 4(a), the level of MDA, the end product of lipid peroxidation, was significantly increased after contusion-induced muscle injury compared to the vehicle control group (P < 0.05). However, treatment with curcumin slightly reduced the MDA level in the muscle compared with those in the MDI group. These results suggest that oxidative stress induced by contusion was slightly blocked by the supplementation of curcumin. The protein expression levels of Ikk-α/ß, myeloperoxidase (MPO), CD206, and myogenin are shown in Figure 4(b). The protein expression levels of Ikk-α/ß, MPO, CD206, and myogenin were markedly increased in the M group compared with those in the NC group. However, treatment with curcumin decreased the protein levels of Ikk-α/ß, MPO, CD206, and myogenin compared with that in the M group. Thus, for the contusion-induced muscle injury, curcumin could affect the specific markers of neutrophils, M2a macrophages, inflammation, and differentiation of satellite cells, including MPO, CD206, Ikk-α/ß, and myogenin. Therefore, curcumin improved contusion-induced muscle injury through inhibiting the production of neutrophils, M2a macrophages, and inflammation in contusion-induced muscle injury.
Effect of curcumin treatment on (a) TBARS and (b) protein expressions of muscle tissues after contusion-induced muscle injury in mice. Mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water and without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), consisting of mice treated using DCF after MDI; (4) MDI + curcumin (M + C), consisting of mice treated using curcumin after MDIs. Data are represented as mean ± standard error of the mean (n = 8 mice/group). Letters (a, b) indicate a significant difference at P < 0.05 in one-way analysis of variance.
As shown in Figure 5(a), the M, M + D, and M + C groups did not differ from the NC group in histological liver characteristics. Figure 5(b) shows the therapeutic effects of curcumin supplementation against contusion-induced muscle injury through H&E staining. Histological evaluation of contusion-induced injured muscle tissues revealed that the drop-mass method markedly disrupted and damaged the muscle fibers, thus causing accumulation of red blood cells in the interstitial spaces. However, DCF and curcumin could mitigate disruption of the muscle tissues in contusion-induced muscle injury. In addition, the M + C group exhibited the most optimal recovery effect, and the therapeutic effect of curcumin was superior to that of DCF (the positive control).
Effect of curcumin treatment on pathological histology of (a) the liver and (b) muscles, and immunohistochemistry staining of (c) desmin protein and (d) MPO protein in muscle tissues on contusion-induced muscle injury in mice. The mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), mice treated using DCF after MDI; (4) MDI + curcumin (M + C), mice treated using curcumin after MDI. Black arrow shows muscle fibers.
Desmin, a protein, is an indicator for the regeneration of muscle satellite cells. As shown in Figure 5(c), desmin expression in the M group increased compared with the NC group. However, the M + C group had a higher desmin level than did the M group; therefore, curcumin could regenerate muscle satellite cells during muscle recovery after contusion-induced muscle injury. In addition, desmin expression levels after curcumin treatment were higher than those after DCF treatment. Our data indicated that curcumin resulted in more effective muscle repair after contusion-induced injury than did the nonsteroidal anti-inflammatory drug DCF. Desmin expression also increased during the 7 days following contusion-induced injury in the M group. This result corresponds to the findings of George et al 18 , who reported that desmin expression steadily increased after injury, and desmin expression peaked 7 days after injury. In addition, they reported that Prosopis glandulosa significantly increased desmin expression, thereby accelerating muscle recovery. Therefore, in this study, curcumin increased desmin expression after contusion-induced muscle injury, which indicated that curcumin could promote muscle recovery.
In acutely injured muscle mice, the number of neutrophils begins to increase, 19 and their function mostly involves phagocytic activity to remove debris but also product MPO that induces muscle membrane damage and increases macrophage proinflammatory activity. 20 -22 As shown in Figure 5(d), MPO was assessed using IHC. MPO protein expression of the MDI-alone group showed an increase compared to the vehicle control. However, animals treated with curcumin reduced MPO protein expression compared to the MDI-alone group. In addition, MPO protein expression with curcumin was less than that with DCF. Furthermore, data indicate that curcumin resulted in more effective muscle repair after a contusion than the NSAID, DCF. These results indicate that mice with a contusion-induced muscle injury treated with curcumin exhibited decreased MPO protein expression compared to the MDI-alone group; so curcumin could decrease neutrophils in the repair of muscle after contusion-induced muscle injury.
Curcumin, a natural dietary polyphenol extracted from Curcuma longa, possesses numerous pharmacological properties, including antioxidant, 23 anti-inflammatory, 24 -28 immunomodulatory, 29 anticancer, 30 antipruritic, 31 antidepressant, 32,33 and antiarthritic 34 effects. In addition, curcumin has been demonstrated to have beneficial effects on injured skeletal muscle. Davis et al 14 reported that curcumin diminished IL-6, TNF-α, and CK activity and caused increased spontaneous activity in skeletal muscles after downhill running. Kawanishi et al 16 reported that curcumin administration effectively suppressed downhill running-induced hydrogen peroxide production and NADPH-oxidase expression in skeletal muscles. In addition, curcumin effectively reduced delayed onset muscle soreness (DOMS) in humans by blocking the NF-кB signaling pathway. 35,36 Nicol et al 37 demonstrated that curcumin can ameliorate DOMS symptoms and heal muscle injury in humans. Ono et al 38 demonstrated that curcumin alleviates streptozotocin-induced skeletal muscle atrophy in mice with type 1 diabetes mellitus by inhibiting protein ubiquitination. Thaloor et al 13 demonstrated that the intraperitoneal injection of curcumin regenerates muscle fibers in mouse masseter muscles with freeze-induced injury.
Soft tissue injury is the commonest type of injury in sport, and it constitutes up to 35% to 55% of all injuries in sports. 39 Soft tissue injury can result in severe pain, swelling, and bruising, which ultimately lead to impaired muscle function. 40 The pathophysiology of muscle injury is accompanied with several complex stages, including degeneration, inflammation, regeneration, and the formation of fibrotic scar tissue. 3,41 -43 In cases of severe skeletal muscle damage capillary rupture, infiltrative bleeding, inflammation, oxidative stress, and fibrosis may occur simultaneously. Inflammatory cytokines mediate the cellular environment that largely controls the progress of repair processes. Thus, inflammation plays a vital role in muscle recovery. Therefore, inflammation may be the most suitable target for developing effective treatments for faster repair. Curcumin can block NF-κB activation in several inflammatory stimuli. 44 Therefore, curcumin supplementation has potential for use to treat contusion-induced muscle injury.
Plant derivatives with antioxidant properties effectively inhibit neutrophil infiltration into the injury site by reducing neutrophil extravasation from the blood, 45 reducing neutrophil migration, 45 inhibiting immune cell activation, 46 and inhibiting neutrophil chemotaxis. 47 Neutrophil attraction, adhesion, and migration can be affected by reactive oxygen species (ROS) generation. 48 Therefore, an antioxidant can scavenge ROS, which alleviates excessive ROS production and reduces attraction, adhesion, and migration of circulating neutrophils. 49,50 Curcumin is a widely known antioxidant; therefore, curcumin supplementation may mitigate the damage caused by contusion-induced muscle injury by reducing ROS production and neutrophil attraction, adhesion, and migration.
Curcumin was proven to be effective in injured skeletal muscle, but the effects of curcumin on contusions remain unclear. We investigated the effects of curcumin on contusion-induced muscle injury in a mouse model. As shown in Table 2, curcumin significantly increased the muscle mass after contusion-induced muscle injury. In our study, although curcumin administration increased the muscle mass compared with the M group, it did not significantly increase the body weight (Tables 1,2). Moreover, the mice’s walking was slightly affected after contusion-induced muscle injury, however, treatment with curcumin slightly improved the functional recovery of the gait (Figure 1). Figure 5(b) also shows that the morphological and pathological characteristics of muscle (visualized through H&E staining) in the M + C group were superior to the M + D group, which constituted the positive control group. Thaloor et al 13 reported that systemic treatment with curcumin accelerates the recovery of normal muscle architecture after traumatic injury.
Contusion leads to oxidative stress in the muscle as evidenced by increasing lipid peroxidation, as shown in Figure 4(a). However, treatment with curcumin significantly decreased the lipid peroxidation against contusion-induced muscle injury of the muscle in mice. Thus, curcumin reduced muscle injury through reducing oxidative damage. In the present study, we also found that Ikk-α/ß, MPO, and CD206 proteins in the muscle were significantly increased by contusion (Figure 4(b)). However, treatment with curcumin decreased Ikk-α/ß, MPO, and CD206 proteins and thus inhibited the production of neutrophils, M2a macrophages, and inflammation in contusion-induced muscle injury.
As shown in Figure 5(c), curcumin significantly increased desmin expression compared with the M group. These results reveal that curcumin can promote desmin expression after contusion-induced muscle injury in mice by facilitating muscle satellite cell regeneration. However, desmin expression after treatment with DCF was significantly lower than that after treatment with curcumin. Davis 14 reported that curcumin can promote muscle regeneration and improve behaviors associated with DOMS in mice. Thaloor et al 13 pointed out that mouse masseter muscles that were subjected to freeze injury and were treated with curcumin possessed large, centrally nucleated regenerating fibers at the damage site, whereas the control mice did not have regenerating fibers. Moreover, Figure 5(d) shows curcumin and DCF significantly decreased MPO protein expression after contusion-induced muscle injury. The results show that curcumin can reduce the production of MPO, thereby reducing muscle membrane damage and macrophage inflammatory activity.
In the present study, the serum CK activities were attenuated through curcumin administration after contusion-induced muscle injury (Figure 3). The increase in CK activity in the blood indicates plasma membrane disruption in the muscle fibers, and CK activity is a common marker of muscle fiber damage. 51 Raastad et al 52 reported that as morphological disruption of myofibrils increased, muscle function decreased. As shown in Figure 3, the increase in serum CK activity in the M + C group was less than that in the M group, which suggests that myofibril damage was attenuated through curcumin administration. McFarlin et al 53 reported that curcumin supplementation resulted in significantly smaller increases in CK, TNF-α, and IL-8 levels following an eccentric exercise. Drobnic et al 54 found that curcumin administration significantly reduced pain in the right and left anterior thighs and the levels of the markers of muscle damage and inflammation. Delecroix et al 55 reported that curcumin supplementation can offset some of the physiological markers of muscle soreness after elite rugby players had undergone intense workouts.
In conclusion, curcumin supplementation significantly increased desmin expression and inhibited MPO expression, indicating that curcumin can promote muscle satellite cell regeneration and inhibit the production of neutrophils after contusion-induced injury. The contusion also increases Ikk-α/β, MPO, and CD206 proteins and increases muscle TBARS. Therefore, contusions severely induce muscle damage. The present study showed, for the first time, that curcumin has the potential to ameliorate muscle damage as evidenced by the significant reduction in lipid peroxidation and the decreases of neutrophils, M2a macrophages, and inflammation induced by contusion. Thus, curcumin has great potential for muscle repair after contusion-induced injury.
Experimental
Animals and Treatments
Eight-week-old male ICR mice (n = 32, weighing 30-40 g) were purchased from BioLASCO (Yilan, Taiwan); the mice were given 1 week to acclimatize to their new environment and diet. The mice were maintained on a regular cycle (12 hours light-dark) at room temperature (24°C ± 2°C) and 60% to 70% humidity; they were fed a chow diet (No. 5001; PMI Nutrition International, Brentwood, MO) and were provided with distilled water ad libitum. All the mice received humane care in accordance with the Guidelines for the Institutional Animal Care and Use Committee of National Taiwan Sports University (IACUC-10504-M).
As shown in Figure 6, the mice were randomly divided into the following 4 groups (8 mice per group): (1) normal control (NC), consisting of mice without injury and treated with reverse osmosed (RO) water; (2) mass-drop injury (MDI [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (Sigma Chemical Co., St Louis, MO) (M + D), consisting of mice treated using DCF (a nonsteroidal anti-inflammatory drug) after MDI; and (4) MDI + curcumin (Sigma Chemical Co.) (M + C), consisting of mice treated using curcumin after MDI. DCF and curcumin were dissolved in RO water and orally administered at doses of 10 mg/kg body weight (b. w.) and 5 mg/kg b. w., respectively, after injury once a day for 7 days. All mice were sacrificed on postinjury day 7, and the liver, kidneys, heart, lungs, and muscles of each mouse were collected and weighed.
Schedule of curcumin treatment for contusion-induced muscle injury in mice. The mice were randomly assigned to 4 groups (8 mice/group): (1) normal control (NC), consisting of mice treated using reverse osmosed (RO) water and without injury; (2) mass-drop injury (MDI, [M]), consisting of mice treated using RO water after MDI; (3) MDI + diclofenac (DCF) (M + D), consisting of mice treated using DCF after MDI; and (4) MDI + curcumin (M + C), consisting of mice treated using curcumin after MDI.
Induction of Experimental Contusion-Induced Muscle Injury and Sample Collection
The mice were anesthetized through inhalation of 4% to 5% isoflurane. Contusion muscle injury was induced in the mice as described in a previous study (with a slight modification) by dropping a 50 g weight from a height of 60 cm onto the medial surface of the left gastrocnemius muscle. 56 This MDI is a medium-intensity injury and does not cause bone injury or gait abnormality.
Walking Functional Assessment
The hind feet of mice were dipped into red ink, and the mice were allowed to walk across a plastic tunnel so that the footprints could be recorded on paper placed at the bottom of the tunnel. The area of footprints between the second to fourth footprints was measured on the experimental side (left) using ImageJ software (National Institutes of Health, Bethesda, MD).
Blood Biochemical Assessments
Following the experiments, the mice were sacrificed through CO2 inhalation after an 8-hour fast. Blood samples were collected through cardiac puncture, and the serum was collected through centrifugation at 1500 × g and 4°C for 10 minutes. The serum levels of UA and CK were measured using an autoanalyzer (Hitachi 7060; Hitachi High Technologies Co., Tokyo, Japan).
Measurement of TBARS
The content of lipid peroxidation products in the muscle was measured using commercial kits for TBARS. The absorbance at 535 nm was recorded, and the amounts of lipid peroxidation products were expressed as malondialdehyde (MDA) equivalents, that is, μM of MDA per μg protein.
Analysis of the Proteins in the Muscle
The protein levels of Ikk-α/ß, MPO, CD206, and myogenin in the muscle were determined using western blot analysis according to our previous method. 57
Pathological Histology and Immunohistochemistry of Muscle Tissues
The livers and muscles of the mice were removed and fixed in 10% formalin and neutral buffered saline dehydrated for 24 hours before the tissues were processed for histopathological analysis as described in a previous study. 17 The tissues were embedded in paraffin and cut into 4-μm-thick sections, stained with hematoxylin and eosin (H&E), and examined using a light microscope equipped with a charge coupled device camera (BX-51, Olympus, Tokyo, Japan) to determine the morphological and pathological characteristics of the tissues.
The formalin-fixed, paraffin-embedded tissue samples were cut into 5-μm-thick sections. The sections were deparaffinized using two changes of xylene for 10 minutes, rehydrated using an alcohol-to-water gradient, treated with boiling water for 15 minutes, and then incubated in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The sections were treated with the rabbit polyclonal desmin antibody or the rabbit polyclonal MPO antibody, and incubated overnight at 4°C. For antigen retrieval, the sections were immunostained using the VECTASTAIN ABC kit (Vector Laboratories, Inc., Burlingame, CA) in accordance with the manufacturer’s specifications. The sections were processed using diaminobenzidine for staining development and then counterstained using H&E.
Statistical Analysis
All results are expressed as means ± standard error of the mean (n = 8). The significance of the difference obtained through one-way ANOVA was determined using Duncan’s post hoc test. The differences were considered significant at P < 0.05.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by an institutional grant to Dr Tsai (grant No. TTCRD105-03 from Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation), and partly supported by the Ministry of Science and Technology of Taiwan (grant No. MOST 105-2314-B-303-004 to Dr Tsai).
