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Medicine & Science in Sports & Exercise:
doi: 10.1249/MSS.0b013e31828de99f
Basic Sciences

Catechins Suppress Muscle Inflammation and Hasten Performance Recovery after Exercise

HARAMIZU, SATOSHI; OTA, NORIYASU; HASE, TADASHI; MURASE, TAKATOSHI

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Author Information

Biological Science Laboratories, Kao Corporation, Tochigi, JAPAN

Address for Correspondence: Takatoshi Murase, Ph.D., Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan; E-mail: murase.takatoshi@kao.co.jp.

Submitted for publication July 2012.

Accepted for publication February 2013.

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Abstract

Introduction: Catechins, abundant in green tea, exhibit many biological actions for potential clinical applications. Our purpose was to explore the potential benefits of catechin ingestion on recovery of physical performance after downhill running.

Methods: Institute of Cancer Research mice were used to examine the effects of prior catechin ingestion (0.5% w/w in diet for 3 wk) on 1) wheel-running activity, 2) running endurance, 3) muscle force, and 4) muscle oxidative stress and inflammation after downhill running (16 m·min−1 for 5 min, 18 m·min−1 for 5 min, 20 m·min−1 for 10 min, and 22 m·min−1 for 130 min).

Results: Voluntary wheel-running activity and the contractile force of the isolated soleus muscle decreased (P < 0.05) after downhill running. Notably, catechin ingestion significantly alleviated the running-induced decrease in voluntary wheel-running activity by 35%; the catechin-treated mice maintained endurance running capacity (214 ± 9 vs 189 ± 10 min, P < 0.05). Furthermore, catechins alleviated (P < 0.05) the decrease in tetanic force evident in the soleus muscle after downhill running. Catechins suppressed the running-induced increases in plasma creatine phosphokinase levels by 52%; this was also true of the carbonylated protein content of the soleus muscle by 17% (P < 0.05), malondialdehyde levels by 32% in the gastrocnemius muscle, and myeloperoxidase activity of the gastrocnemius by 22% (P < 0.05). The levels of tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant protein-1 in the gastrocnemius muscle were significantly lower (P < 0.05) by 33%, 29%, and 35%, respectively, in treated mice; the expression levels of mRNAs encoding these fell in parallel.

Conclusion: Our results suggest that long-term intake of catechins, perhaps through their antioxidant properties, attenuates downhill running-induced muscle damage by suppressing muscle oxidative stress and inflammation, hastening recovery of physical performance in mice.

Understanding the postexercise impairment of muscle and developing a coping strategy are essential for both health maintenance and athletic performance. Unaccustomed or intensive exercise, particularly eccentric and lengthening exercises, temporarily decreases production of muscle contractile force (1,2,17). This decline in muscle force is caused by lack of energy, disordered homeostasis of ion channels, muscle damage that results in the release of soluble enzymes such as creatine kinase, and inflammation including leukocyte infiltration and the production of cytokines (17,18) and reactive oxygen species (ROS). The resulting impairment of muscle function contributes to the delay of recovery of physical performance after exercise.

ROS and inflammatory cytokines synthesized in working muscles, plasma, and indeed the brain are likely to affect both skeletal muscles and the CNS. These materials, acting together, decrease exercise performance as measured both by endurance capacity and muscle force production, and delay recovery after exercise (9,13,36). Extracellular ROS are generated principally via the actions of myeloperoxidase (MPO) and xanthine oxidase in neutrophils that infiltrate muscles after strenuous exercise (23,38). An excess of ROS in muscles causes oxidation of cellular components including lipids, proteins, and DNA, creating further damage in cells and tissues. ROS have been reported to impair the contractile properties of muscle via oxidative modification of myofibrillar proteins as reflected by an increase in the carbonylation levels of such proteins (39). Furthermore, N-acetylcysteine, an antioxidant, alleviates the muscle fatigue developing during prolonged exercise in humans partly by elevating the activity of the Na+, K+-pump, and curcumin enhances recovery after downhill running in mice (13,28). These findings suggest that alleviation of oxidative stress may attenuate muscle damage and promote recovery from diminished physical performance.

Green tea contains a group of polyphenols collectively termed catechins; the principal components are epigallocatechin gallate, epicatechin gallate, gallocatechin, and epigallocatechin. Catechins exhibit antioxidant activities (8) and have also been reported to possess several notable biological properties including antifungal activities (19), antiatherogenic effects (12), and a capacity to mitigate development of obesity (31). Moreover, both (unfractionated) green tea extract and epigallocatechin gallate protect muscles from the massive necrosis and contractile dysfunction normally evident in a mouse model of Duchenne muscular dystrophy (6,14). Thus, interest in the potential health benefits of catechins has grown (7).

We have investigated the biological functions of catechins for some years, and we earlier demonstrated that catechin intake combined with habitual exercise improved endurance capacity and prevented diet-induced obesity in mice; these benefits were achieved via enhancement of fat oxidation (29,30 and references therein). More recently, we found that catechin ingestion prevented the decrease in muscle force output normally observed in electrically stimulated soleus muscle isolated from both tail-suspended mice and senescence-accelerated mice (SAMP1), and reduced oxidative stress and alleviated inflammation (17,34). In comparison, a single oral administration of catechins did not alleviate inflammation, decrease muscle force, or ameliorate oxidative stress after downhill running in SAMP1 (17). Moreover, plasma levels of catechins increased after repeated administration in humans (16). These results suggest that repeated intakes seem to be required to garner beneficial effects of catechins. Such earlier work has suggested that the long-term intake of catechins intake hastens recovery from diminished physical performance due to downhill running through their antioxidative and anti-inflammatory properties.

In the present study, we explored the effect of catechins on recovery of physical performance after downhill running at the whole-body level. To this end, we examined the effect of catechin intake on whole-body physical performance (both voluntary and as assessed by forced running) and recovery after downhill running in Institute of Cancer Research (ICR) mice. Downhill running induces muscle injury, inflammation, and oxidative stress (13) and is used frequently as a model for exercise-induced muscle damage.

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METHODS

Materials

Catechins were prepared from green tea leaves and analyzed as previously described (31). The total catechins content was 81% (w/w). The composition of isolated catechins was epigallocatechin gallate (41% w/w), epigallocatechin (23%), epicatechin gallate (12%), epicatechin (9%), gallocatechin (7%), gallocatechin gallate (4%), and others (4%).

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Animals, Diets, and Training on Treadmill Running

Male 6-wk-old ICR mice were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan), and maintained under controlled conditions of temperature (23°C ± 2°C), humidity (55% ± 10%), and lighting (0700–1900). All animals were fed one of several defined diets (as described below) and had free access to drinking water for maintenance over 10 d before experimental testing. In this time, all mice were trained to run on a 10-lane motorized rodent treadmill (model MK-680; Muromachi Kikai Co., Ltd., Tokyo, Japan) set at an inclination of −14°. Training was conducted over 2 d; the distance covered by the animals on the treadmill was gradually increased during this interval. On day 1, mice ran on the treadmill at 6 m·min−1 for 2 min, 8 m·min−1 for 2 min, 10 m·min−1 for 3 min, and 12 m·min−1 for 3 min. On day 2, training was conducted at 12 m·min−1 for 2 min, 16 m·min−1 for 2 min, 20 m·min−1 for 3 min, and 22 m·min−1 for 3 min. We chose to keep the acclimatization program brief to minimize any adaptation to training that might have been caused by exercise. At the age of 7–8 wk, all mice were weighed, and those that were 3.8% heavier or lighter than the average were eliminated from the study. This was because we sought to minimize any inherent variation in body weight that could influence the load borne by weight-bearing muscles. In addition, we eliminated animals that started running but refused to run during downhill running training. Thereafter, the residual animals were divided into three groups (n = 8 per group), defined as follows: control diet + no downhill running (Non-Down) group; control diet + downhill running (Down) group; and catechin-containing diet + downhill running (Cat) group. The sample size of eight mice per group was determined based on our previous study (29) to provide adequate statistical power for this study. The mice in each group were of similar average body weight. Each animal was housed individually in either a regular plastic cage or such a cage equipped with a running wheel (model SW-15; MELQUEST, Toyama, Japan). Mice had access to unlimited access to either a control diet containing 10% (w/w) fat, 20% casein, 55.5% potato starch, 8.1% cellulose, 2.2% vitamins, 0.2% methionine, and 4% minerals or a catechin-containing diet that was the control diet supplemented with 0.5% (w/w) catechins for 3 wk before the downhill running tests. The Animal Care Committee of Kao Tochigi Institute approved the present study. Animal experiments in this study were conducted in accordance with the policy statement of the American College of Sports Medicine.

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Experimental Design

An overview of our experimental approach is shown in Figure 1.

Figure 1
Figure 1
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Voluntary wheel-running activity and treadmill running time after downhill running: experiment 1

Subsets of mice (Non-Down (n = 8), Down (n = 8), and Cat (n = 8)) were subjected to downhill running, and thereafter, we evaluated recovery of voluntary wheel-running activity and running endurance as described below. Wheel-running activity in all groups was monitored for 3 wk before downhill running was initiated. The baseline level of wheel-running activity was defined as the level of activity over each 24-h period of the last week only; this was because voluntary wheel-running activity increased during the first 10 d of the experiment but became constant thereafter. Immediately after the downhill running, the test was completed, each animal was returned to its cage, and the extent of voluntary wheel-running activity was measured over the following 24 h. Next, the ratio of wheel-running activity during this 24-h period compared with the baseline level was calculated for each mouse. The running capacities of animals in the Down and Cat groups 24 h after downhill exercise was completed were measured on a treadmill. The running task set was as follows: 20 m·min−1 for 5 min, 24 m·min−1 for 5 min, 28 m·min−1 for 5 min, 32 m·min−1 for 5 min, and 36 m·min−1 to the time when running ceased. The treadmill was set an inclination of 8°.

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Analysis of muscle contractile force: experiment 2

Subsets of mice (Non-Down (n = 16), Down (n = 16), and Cat (n = 16)) were used to explore contractile force and the extent of muscle damage after the downhill running. Half of the mice in the Down (n = 8) and Cat (n = 8) groups were anesthetized via the inhalation of sevoflurane (SEVOFRAN®; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) immediately after downhill running was completed, whereas half of all animals in the Non-Down group (n = 8) were anesthetized at rest. The other half of all animals in Non-Down (n = 8), Down (n = 8), and Cat (n = 8) were anesthetized 24 h after completion of the downhill running. Blood samples were collected from all mice. Next, the gastrocnemius, soleus, and plantaris muscles were dissected and weighed. Muscle samples were stored at −80°C until subsequent analyses.

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Downhill Running Protocol

Mice in the Down and Cat groups ran downhill on a treadmill following a protocol previously reported to induce muscle weakness in ICR animals (13) with a minor modification: 16 m·min−1 for 5 min, 18 m·min−1 for 5 min, 20 m·min−1 for 10 min, and 22 m·min−1 for 130 min. The treadmill was set at an inclination of –14°. Mice in the Non-Down group rested in their cages during the test interval.

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Biochemical Analysis

We determined erythrocytes and leukocytes numbers, hemoglobin concentrations, and hematocrit of heparinized blood samples with the aid of an automated hematocytometer (Celltac MEK-5258; Nihon Kohden, Tokyo, Japan). Plasma was obtained by centrifugation of whole blood at 2000g for 15 min. With assay kits (Nittobo Medical Co., Ltd., Tokyo, Japan), we measured the plasma concentrations of glucose with N-A Glu-UL, triglyceride with N-A L TG-H, nonesterified fatty acid (NEFA) with NEFA-HA, aspartate aminotransferase (AST) with N-A L GOT, alanine transaminase (ALT) with N-A L GPT, lactate dehydrogenase (LDH) with N-A L LDH, and ketone bodies with T-KB-H. CPKII-test kits (WAKO Pure Chemical Industries, Osaka, Japan) were used to assay plasma creatine phosphokinase (CPK) levels. Plasma malondialdehyde (MDA) concentrations were measured using OXI-TEK TBARS assay kits (Alexis, Lausen, Switzerland). All measurements were performed according to the instructions of the kit manufacturers.

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Contractile Properties Revealed by Electrical Stimulation

Muscle force measurements were performed as described previously (17). In brief, each isolated right soleus muscle was immersed in Krebs solution that was continuously bubbled with 95% O2/5% CO2 (v/v) at 37°C. The muscle was electrically stimulated by a stimulus isolation unit (SEN-3301, Nihon Kohden), and an optimal twitch length was set. Thereafter, tetanus responses were induced with a 0.2-ms pulse (140 Hz) for 330 ms once every 2 s; the responses were digitally recorded over the next 2 min by using a bridge amplifier and a data acquisition system (Quad-16I; World Precision Instruments, Inc., Sarasota, FL). Data were analyzed with the aid of a DATA-TRAX™ (World Precision Instruments, Inc.).

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Preparation of Muscle Homogenates

Frozen gastrocnemius and soleus muscles were placed into 10 mM Tris (pH 7.4) containing 250 mM sucrose and 2 mM ethylenediaminetetraacetic acid and then homogenized on ice by a Physcotron homogenizer (NS-310E; Microtech, Chiba, Japan). Subcellular debris was removed by centrifugation at 10,000g for 10 min at 4°C, and the levels of various components in the resulting supernatants were assayed in subsequent assays. Supernatant protein concentrations were determined using BCA protein assay kits (Pierce, Rockford, IL).

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Measurement of Carbonylated Protein and MDA Levels

The levels of carbonylated protein in soleus homogenates were measured by using Protein Carbonyl assay kits (Cayman Chemical, Ann Arbor, MI). Each value was normalized to the protein concentration of the relevant muscle. MDA levels in gastrocnemius muscle homogenates were assayed using OXI-TEK TBARS assay kits (Alexis).

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MPO Assay

MPO levels in the gastrocnemius muscle samples were measured as previously described (37). In brief, muscle samples were homogenized in ice-cold 20 mM potassium phosphate buffer (pH 7.4) containing 1 mM potassium ethylenediaminetetraacetic acid. After centrifugation (3300g, 4°C), each supernatant was decanted and the pellet suspended in 50 mM potassium phosphate (pH 6.0) containing 0.5% (w/v) hexadecyltrimethyl ammonium bromide. The suspension was homogenized and next centrifuged at 3300g for 15 min. MPO activity was determined by measuring the H2O2-dependent oxidation of N,N,N′,N′-tetramethylbenzine. One unit of enzyme activity was defined as that causing a change in absorbance at 655 nm of 1.0 U·min−1 at 37°C.

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Measurement of Interleukin-1β, Tumor Necrosis Factor-α, and Monocyte Chemoattractant Protein-1 Levels

Interleukin (IL)-1β and tumor necrosis factor (TNF)-α levels were measured using mouse IL-1β ELISA kit and mouse TNF-α ELISA kits (Invitrogen Corporation, Carlsbad, CA), respectively. Monocyte chemoattractant protein (MCP)-1 levels were assayed using mouse MCP-1 ELISA kits (Bender MedSystems, Vienna, Austria). Each value obtained was normalized to muscle protein concentration.

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RNA Extraction and Reverse Transcription Polymerase Chain Reaction

Total RNA isolation, cDNA production, and real-time polymerase chain reaction were performed as previously described (17). All mRNA levels were normalized to that of a housekeeping gene encoding a ribosomal protein (large, P0 [RPLP0/36B4]). The mouse-specific primer sequences used were as follows: TNF-α forward, 5′-CCCACGTCGTAGCAAACCAC-3′; TNF-α reverse, 5′-AAGGTACAACCCATCGGCTG-3′; IL-1β forward, 5′-TAACCTGCTGGTGTGTGACGTT-3′; IL-1β reverse, 5′-AGGTGGAGAGCTTTCAGCTCAT-3′; MCP-1 forward, 5′-AGCAGCAGGTGTCCCAAAGA-3′; MCP-1 reverse, 5′-TCATTTGGTTCCGATCCAGG-3′; CXC chemokine ligand (CXCL)-1 forward, 5′-ACGCACGTGTTGACGCTTC-3′; CXCL-1 reverse, 25′-CTTTGAACGTCTCTGTCCCGA-3′; lymphocyte antigen 6 complex, locus G (Ly6G) forward, 5′-TCATATGCCATATCCGAGCCT-3′; Ly6G reverse, 5′-GATGAGACCAAGGACAGCGG-3′; CD68 antigen forward, 5′-CCATGTTTCTCTTGCAACCGT-3′; CD68 reverse, 5′-TTGATTGTCGTCTGCGGGT-3′; F4/80 forward, 5′-GGCATTTTCCAGATTGGCC-3′; and F4/80 reverse, 5′-CATCCCGTACCTGACGGTTG-3′.

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Statistical Analysis

All values are presented as means ± SEM. Statistical analysis was conducted using the StatView® software package (SAS Institute Inc., Cary, NC). The wheel-running activity data (Fig. 2) were analyzed to detect effects of downhill running and catechin intake by using one-way ANOVA and Fisher protected least-significant difference test. The difference in running time between the Down and Cat groups (Fig. 2) was evaluated with the aid of the unpaired t-test. The among-group data (Fig. 3A and Tables 1–3) were compared with detect effects of downhill running and catechin intake immediately and 24 h after exercise by using the Tukey–Kramer post hoc test. The possible existence of a time–group interaction in tetanic force data (Fig. 3B) was explored between the Down and Cat groups using repeated-measures ANOVA, and the among-group data were compared with detect effects of downhill running and catechin intake immediately and 24 h after exercise by using the Tukey–Kramer post hoc test. A P value of less than 0.05 was considered to be statistically significant.

Table 1
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Table 2
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Table 3
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Figure 2
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Figure 3
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RESULTS

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Catechin ingestion hastens the recovery of physical performance after downhill running

We evaluated chronic catechin intake because prolonged consumption seems to be required to achieve the beneficial effects of catechins (17). In experiment 1, downhill running induced a significant reduction in voluntary wheel-running activity during the 24-h interval after downhill running (Fig. 2A). Catechins significantly alleviated the downhill running-induced decrease in the wheel-running activity by 35%. Moreover, the running time at 24 h after downhill running in the Cat group was significantly longer than that in the Down group (Down: 189 ± 10 min; Cat: 214 ± 9 min, Fig. 2B).

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Catechin ingestion suppresses the increases in plasma CPK, AST, ALT, and MDA levels after downhill running

The plasma and blood analysis results are shown in Table 1. Enhanced plasma LDH and CPK levels serve as indirect markers of muscle injury, and these increased significantly both immediately and 24 h after downhill running. The LDH increase was notable immediately after downhill running, whereas the CPK activity had risen notably by 24 h later. Catechin ingestion significantly alleviated the downhill running-induced increase in CPK level by 52% 24 h after running. An enhanced plasma MDA level is also indicative of oxidative stress and was significantly increased by 24 h after downhill running. The plasma AST and ALT levels also rose significantly by downhill running. Catechin ingestion significantly alleviated the downhill running-induced increments in some of the abovementioned parameters. The plasma NEFA level of the Cat group immediately after downhill running was significantly higher than that of the Non-Down group, consistent with data of a previous report to the effect that catechin ingestion causes serum NEFA levels to increase during exercise (31). Plasma glucose and ketone body levels significantly fell and rose, respectively, immediately after downhill running, and the levels of both returned to those measured before the downhill running test at 24 h after exercise. Catechin ingestion did not affect the levels of either glucose or ketone bodies. The red blood cell count of the Cat group immediately after downhill running was significantly higher than that of the Non-Down group.

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Catechin ingestion alleviates the decline in muscle contractile force after downhill running

The twitch forces of the Down group both immediately and 24 h after downhill running were significantly lower than those of the Non-Down group (Fig. 3A). The twitch force of the Cat group 24 h after downhill running was significantly higher than that of the Down group and did not differ significantly from that of the Non-Down group. The tetanic forces of the Down group both immediately and 24 h after downhill running were significantly lower (P < 0.05) than those of the Non-Down group over the interval 0–120 s. The tetanic forces of the Cat group 24 h after downhill running were significantly higher than those of the Down group over the interval 40–90 s (Fig. 3B). No significant difference between the Down and Cat groups was evident immediately after downhill running (Fig. 3B). Neither body nor muscle weight differed between these groups (data not shown). Therefore, the observed differences in muscle forces are not attributable to variations in muscle mass.

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Catechin ingestion reduces accumulation of carbonylated proteins in the soleus muscle after downhill running

The carbonylated protein level in the Non-Down group, Down and Cat groups immediately after exercise, and Down and Cat groups 24 h after exercise was 0.89 ± 0.03, 1.17 ± 0.05, 1.09 ± 0.06, 1.36 ± 0.08, and 1.12 ± 0.06, respectively. Downhill running induced significant accumulation of carbonylated proteins in the soleus muscle; this was most evident 24 h after downhill running. This result suggests that muscle proteins were oxidatively modified by ROS. Catechin ingestion significantly reduced the levels of carbonylated protein evident at this point.

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Catechin ingestion attenuates oxidative stress and inflammation in muscle after downhill running

To clarify the effects of catechins on oxidative stress and inflammation induced by downhill running, we measured MDA, TNF-α, IL-1β, and MCP-1 levels in the gastrocnemius muscle. Downhill running significantly increased muscle MDA levels 24 h after running, and this increase was attenuated by catechin ingestion (32% lower in treated mice, Table 2). Also, the levels of TNF-α, IL-1β, and MCP-1 had increased significantly 24 h after downhill running in the Down group, but those levels were significantly lower by 33%, 29%, and 35%, respectively, in mice that had ingested catechins before downhill running. We next examined the levels of mRNAs encoding mediators of inflammation. The levels of mRNAs encoding TNF-α, IL-1β, MCP-1, and CXCL-1 in the Down group had increased significantly immediately after downhill running, 24 h later, or both compared with the Non-Down group (Table 2). Catechin ingestion significantly attenuated the downhill running-induced increases in the levels of these mRNAs 24 h after downhill running.

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Catechin ingestion suppresses the increases in neutrophil and macrophage markers in muscle after downhill running

Compared with the Non-Down group, the levels of mRNA encoding Ly6G, a neutrophil marker, increased significantly both immediately and 24 h after downhill running; the levels of mRNAs encoding macrophage markers CD68 and F4/80 also had risen significantly by 24 h after downhill running (Table 3). Catechin ingestion significantly attenuated the increases in mRNAs encoding Ly6G and CD68 observed at 24 h after downhill running. MPO activity in the gastrocnemius muscle increased significantly 24 h after downhill running, and this rise was significantly attenuated by catechin ingestion (22% lower in the treated mice).

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DISCUSSION

In the present study, we examined the effect of catechin ingestion on postexercise muscle properties and physical performance and found that such ingestion attenuated the downhill running-induced decrease in wheel-running activity, contributed to running endurance, and enhanced recovery from loss of muscle contractile force. At least in part, these effects were mediated via suppression of muscle oxidative stress and inflammation. Our findings thus suggest that the long-term catechin intake would effectively attenuate downhill running-induced muscle damage and hasten recovery from such loss in physical performance.

Voluntary wheel-running activity decreased significantly after downhill running (Fig. 2), concomitant with significant increases in the plasma levels of CPK, LDH, and AST. The activities of some serum components, including CPK, LDH, and AST, all of which are released from muscle and are considered to be indirect markers of muscle damage, increase during strenuous exercise (33). Both our results and those of Nozaki et al. suggest that the observed decrease in voluntary wheel-running activity is associated with muscle damage. However, we found that ingestion of catechins significantly alleviated the observed fall in wheel-running activity and the rise in plasma CPK level. Moreover, endurance was significantly higher in catechin-fed mice, in terms of running performance 24 h after downhill running. Our results thus suggest that catechin ingestion may effectively attenuate loss in physical performance induced by downhill running, partly by reducing the extent of muscle damage inflicted by such exercise.

Loss of contractile force in isolated muscles has been used as an indicator of muscle function (17). In the present study, the contractile force of the soleus muscle decreased significantly immediately after downhill running and had not recovered fully even 24 h later. However, catechin ingestion significantly alleviated such loss in contractile force production (Fig. 3). Our results thus suggest that catechin ingestion hastens recovery from decreased muscle performance after downhill running. In our previous study, we found that catechin ingestion over 3 wk improved neither endurance capacity nor muscle contractile force, although a combination of such ingestion with habitual exercise did in fact enhance endurance (29). These results suggest that the earlier recovery from exercise-induced loss in physical performance noted in animals that had experienced catechin intake was not attributable to any improvement in basal physical performance but rather to an alteration in susceptibility to insults potentially inflicted by downhill running.

Oxidative stress increases when the cellular production of oxidizing species rises or the efficacy of antioxidant defenses falls. Oxidative stress negatively affects muscle contractile properties, and oxidative modification of Ca2+-ATPase via carbonylation reduces the contractile force of muscle (27,39). Moreover, oxidative stress in muscle severely disturbs muscle activity during exercise but does not affect basal spontaneous activity (24). The exercise-induced increases in MDA levels in plasma and the gastrocnemius muscle, and that of carbonylated protein in the soleus muscle, were significantly suppressed by catechin intake (Tables 1 and 2), suggesting that suppression of oxidative stress by catechins contributes to the observed recovery of muscle contractile force and endurance after downhill running.

Downhill running induces muscle damage and triggers a series of inflammatory responses (35). In the present study, catechin ingestion significantly alleviated downhill exercise-induced accumulation of inflammatory cytokines in the gastrocnemius muscle (Table 2). In addition, inflammatory cytokine levels were lower in the muscles of mice that had earlier ingested catechins than that in controls; the levels of mRNAs encoding inflammatory cytokines were in agreement with these findings (Table 2). Although transcriptional regulation of inflammatory cytokines by catechins during a period of exercise has not been proven, catechins may inhibit activation of nuclear factor-κB (NF-κB), which is induced by eccentric exercise (20). NF-κB critically regulates the expression of several inflammatory cytokines including TNF-α and MCP-1 (3,22), and catechins attenuate the activation of NF-κB (5,40). Thus, catechins may attenuate exercise-induced NF-κB activation. In turn, the levels of mRNAs encoding molecules mediating inflammation would fall.

The observed attenuation of inflammation may also be explained by a fall in the activity of MPO; this enzyme is synthesized by neutrophils, and the activity thereof is associated with development of both oxidative stress and inflammation. Neutrophil infiltrations of injured muscle sites can endure for even 48 h after injury (3,26). Both neutrophils and macrophages produce ROS and hypochlorous acid via the action of MPO (4,38). A recent study found that the lipopolysaccharide-induced loss of muscle force production was attenuated in neutrophil-deleted soleus muscle; when the muscle was reloaded after hindlimb unloading, the proportion of injured muscle fibers was lower and MPO activity was reduced, compared with those of neutrophil-undeleted muscle (15). This suggests that both neutrophils and MPO per se compromise recovery of muscle force production. In the present study, muscle samples from mice that had previously ingested catechins after downhill running had lower levels of MPO and MCP-1, and reduced expression levels of mRNAs encoding Ly6G and CD68 (markers of neutrophils and macrophages, respectively; Tables 2 and 3). Therefore, catechin ingestion reduces the extent of several inflammatory responses, including cytokine production and neutrophil and macrophage infiltration, and this reduction in inflammation may hasten recovery from loss in physical performance.

In addition, recent reports have shown that brain IL-1β concentrations significantly increased during exercise-induced fatigue and that manipulation of brain IL-1β activity via intracerebroventricular injection of an IL-1 receptor antagonist hastened recovery of voluntary wheel-running activity after completion of an episode of downhill running (10,11); these results suggest that brain IL-1β plays an important role in the response to fatigue in terms of physical performance. Given that catechins can pass through the blood–brain barrier to become absorbed by and distributed within the brain (21,32) and that catechin ingestion attenuated the downhill running-induced increase in muscle IL-1β (Table 2), IL-1β production in brain might also be alleviated by catechin ingestion; this would enhance voluntary wheel-running activity after downhill running. However, further study on modulation of brain inflammation by catechins is required.

In the current study, chronic (3 wk) ingestion of catechins had several beneficial effects in mice. In our previous study, single oral administration of catechins did not alter muscle force, oxidative stress, or inflammation after downhill running in senescence-accelerated prone mice (17); this lack of effect might reflect a decreased rate of catechin absorption. The absorption rate of epigallocatechin gallate after single oral administration to mice is as low as <13% (25). In contrast, plasma levels of catechins are reported to be increased after chronic consumption in humans (16). These results suggest that repeated intake of catechins is required to garner their beneficial effects.

In conclusion, we have shown that catechin consumption alleviates some oxidative stress and inflammation in mouse muscles after an episode of downhill running and promotes recovery of physical performance. These findings have important implications and will help to establish human nutritional strategies that hasten recovery of postexercise physical performance.

No funding for this work was received from any of the following institutions: National Institutes of Health, Wellcome Trust, Howard Hughes Medical Institute, and others.

The authors thank Satoko Soga and Yukiko Horigane for their valuable support in conducting the experiment.

The authors declare no conflict of interest.

The results of the this study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

CONTRACTILE FORCE; GREEN TEA; INFLAMMATION; OXIDATIVE STRESS; WHEEL-RUNNING ACTIVITY

© 2013 American College of Sports Medicine

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