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North American ginseng protects against muscle damage and reduces neutrophil infiltration after an acute bout of downhill running in rats Mehrbod Estaki and Earl G. Noble

Abstract: Eccentric muscle contractions such as those experienced during downhill running are associated with inflammation, delayed-onset of muscle soreness, myofiber damage, and various functional deficits. North American ginseng (Panax quinquefolius L.) has been reported to possess anti-inflammatory properties and thus may offset some of this exercise-induced damage. Hence, we tested the hypothesis that intervention with North American ginseng would reduce eccentric exercise-induced muscle damage and inflammation. Male Wistar rats were fed (300 mg/(kg·day)–1) of either an alcohol (AL) or aqueous (AQ) extract of North American ginseng for 14 days before a single bout of downhill running and were compared with matching nonexercised (C) groups. Plasma creatine kinase levels were significantly reduced in both ginseng treated groups compared with the C group that received a water placebo (p < 0.002). Further, the AQ but not AL group also showed attenuated morphological signs of damage (hemotoxylin and eosin) as well as reduced levels of infiltrating neutrophils (HIS48) in the soleus muscle (p < 0.001). In summary, supplementation with an AQ but not AL extract of North American ginseng was able to reduce eccentric exercise-induced muscle damage and inflammation. Key words: Panax quinquefolius, myofiber, inflammation, exercise, creatine kinase. Résumé : Les contractions musculaires pliométriques telles qu’observées a` la course sur une pente négative sont associées a` l’inflammation, a` la douleur musculaire d'apparition retardée, aux lésions myofibrillaires et a` divers déficits fonctionnels. D’après des études, le ginseng nord-américain (Panax quinquefolius L.) aurait des propriétés anti-inflammatoires pouvant contrer des dommages suscités par l’exercice physique. Nous avons donc testé l’hypothèse selon laquelle l’apport de ginseng nord-américain atténue les dommages musculaires causés par les contractions pliométriques et l’inflammation. On procure a` des rats Wistar mâles durant 14 jours un extrait alcoolisé (« AL ») ou aqueux (« AQ ») de ginseng nord-américain a` raison de 300 mg/(kg∙jour)−1, et ce, avant une séance de course en pente négative et on les compare a` des rats appariés n’ayant effectué aucun exercice. La concentration plasmatique de créatine kinase diminue significativement dans les deux groupes ayant reçu le ginseng nord-américain comparativement au groupe de contrôle ayant reçu un placebo a` l’eau (P < 0,002). De plus, le groupe AQ, mais pas le groupe AL, présente moins de signes morphologiques de dommage (coloration a` l'hématoxyline et a` l'éosine) et une moins grande concentration de neutrophiles infiltrants (HIS48) dans le muscle soléaire (P < 0,001). En bref, la supplémentation en extrait AQ de ginseng nord-américain, mais pas en extrait AL, diminue l’inflammation et les dommages musculaires causés par les contractions pliométriques. [Traduit par la Rédaction] Mots-clés : Panax quinquefolius, myofibre, inflammation, exercice physique, créatine kinase.

Introduction Ginseng, a perennial from the Araliaceae family, has been used in traditional Chinese medicine for thousands of years and remains today as one of the world’s most popular medicinal herbs (Choi 2008). The 2 most common species of ginseng are Asian ginseng (Panax ginseng C.A. Meyer) and North America ginseng (Panax quinquefolus L.) and they are associated with a wide range of therapeutic claims (Xiang et al. 2008). The word Panax originates from the root’s traditional use as a panacea or “cure all”. Ginseng extracts are commonly used as an adaptogen and as an ergogenic aid, though scientific data to support such claims are lacking. Studies in humans have shown ginseng to be ineffective in improving various parameters of exercise, such as heart rate, oxygen uptake, endurance, lactate threshold, or perceived rate of exertion (Engels and Wirth 1997; Kulaputana et al. 2007; Biondo et al. 2008b; Ping et al. 2011). Interventions with either Asian or North

American ginseng, however, have consistently been shown to reduce circulating creatine kinase (CK) levels after a single bout of strenuous exercise (Cabral de Oliveira et al. 2001; Hsu et al. 2005; Jung et al. 2011). CK leakage into the blood such as that seen after a bout of downhill running is often used as a marker of increased sarcolemma permeability, which is a common feature of damaged muscle cells (Armstrong et al. 1983). In addition to CK leakage, structural damage within muscles is associated with focal cellular disruption, local inflammation, and presence of leukocytes immediately after exercise. Several mechanisms have been postulated to account for the muscle damage observed after eccentric exercise (Proske and Morgan 2001). One of these is a loss of calcium homeostasis, subsequent to disruption of t-tubular and sarcoplasmic reticular function, leading to elevated intracellular calcium levels and activation of proteolytic pathways (Warren et al. 1993; Balnave and Allen 1995). Additionally, muscle cells release myokines, which mediate the

Received 13 August 2014. Accepted 1 October 2014. M. Estaki. School of Kinesiology, The University of Western Ontario, London, ON N6A 3K7, Canada. E.G. Noble. School of Kinesiology, The University of Western Ontario, London, ON N6A 3K7, Canada; Lawson Health Science Research Institute, The University of Western Ontario, London, ON N6A 3K7, Canada. Corresponding author: Earl Noble (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 40: 1–6 (2015) dx.doi.org/10.1139/apnm-2014-0331

Published at www.nrcresearchpress.com/apnm on 10 October 2014.

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infiltration of neutrophils to sites of injury (Suzuki et al. 2002; Smith et al. 2008). Once attracted to these sites, neutrophils release proteolytic enzymes and reactive oxygen species, which initiate a series of pro-apoptotic events in a bid to remove damaged cells (Tidball 2005). These cytotoxic molecules, however, can also inadvertently damage healthy neighboring cells, imposing additional unwanted damage and delayed recovery time (Cannon et al. 1990, 1991; Kyriakides et al. 1999). Limiting the rise in intracellular calcium and/or suppressing neutrophil activity may therefore be beneficial in restricting muscle damage and reducing recovery time in response to strenuous exercise. The finding that ginseng supplementation reduces postexercise CK levels suggests that ginseng may protect working muscles against structural damage and the accompanying inflammation. The morphological changes and localization of neutrophil activity associated with ginseng-derived muscle protection has not been previously examined. The primary aim of this experiment, therefore, was to evaluate the effects of North American ginseng on skeletal muscle integrity and neutrophil infiltration in response to a single bout of unaccustomed eccentrically biased exercise. Different processes used in preparation of ginseng extracts lead to distinct bioprofiles that may have particular pharmacological effects. In this experiment we use ginseng compounds prepared by the 2 most common methods with either an alcohol (AL) or aqueous extraction (AQ) process. We hypothesized that prior daily supplementation with either an AL or AQ extract of North American ginseng would reduce neutrophil infiltration within damaged muscle cells, reduce structural damage and sarcolemma permeability, leading to decreased circulating CK levels.

Materials and methods Animals and ethics approval This study was approved by the University of Western Ontario Council on Animals Care and was carried out in accordance with The Guidelines of the Canadian Council on Animal Care (available from www.ccac.ca). Adult 8-week-old male Wistar rats were obtained from Charles River (St. Constant, Que., Canada) and housed in pairs in standard rat cages. They were maintained at a constant temperature and humidity with a 12-h light/12-h dark cycle. Animals had access to water and standard food chow ad libitum. Ginseng extracts The ginseng extracts used in this experiment were provided by Dr. Edmund Lui from The University of Western Ontario. The total ginsenosides content from the AL and AQ extracts, as determined by HPLC analysis, were 28.25% and 13.87% dry weight of extract, respectively. The complete preparation, extraction protocol, and analysis of the extracts have been previously described elsewhere (Azike et al. 2011). Experimental design Forty animals were randomly assigned to 1 of 3 experimental groups. Two groups (n = 10) were given 300 mg/kg of either an AL or AQ ginseng extract (dissolved in water) by oral gavage, while a third group (placebo (P); n = 20), was given water only (via gavage). Ginseng extracts or water placebo were administered daily, at the onset of the dark cycle, for 14 days. On the fifteenth day, after an overnight fast, each group was randomly divided into 2 subgroups of either exercise (EX) or nonexercise control (C). The EX groups performed 1 bout of exercise on a rodent treadmill during their active cycle while the C groups were kept in the same room as the treadmill during the runs. Animals were returned to their cages after exercise and subsequently sacrificed 24 h later for blood and tissue collection.

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Appl. Physiol. Nutr. Metab. Vol. 40, 2015

Exercise protocol All animals were oriented to the rodent treadmill by undergoing two 15-min familiarization sessions at 15 m/min (0% grade), 5 and 3 days before the exercise bout. This light familiarization protocol was chosen to prepare the animals for the exercise procedure while avoiding training adaptations. The exercise bout consisted of 60 min of downhill running at a –14% incline at a speed of 24 m/min. Jets of air on the haunches and tactile stimulation (hand touching) were used to encourage running. All animals completed the running protocol. Blood and tissue collection Blood was collected from the abdominal aorta in EDTA-coated syringes while animals were under anesthesia (65 mg/kg pentobarbital sodium); animals were then sacrificed by cardiac excision. Blood was kept on ice for 30 min and then plasma was separated by centrifugation (4000 r/min (1600g), 4 °C) for 15 min. The plasma layer was collected into micro-centrifuge tubes and stored at –70 °C until later analysis. Immediately following cardiac excision, the soleii (Sol) were removed from both legs and rinsed with ice-cold saline. For histochemical evaluation, a portion of 1 Sol was mounted on cork using an optimal cutting temperature compound and then frozen in isopentane precooled in liquid nitrogen. The remaining Sol was placed in a storage tube and frozen in liquid nitrogen. All samples were stored at –70 °C until analysis. Plasma CK analysis CK activity was analyzed spectrophotometrically in plasma samples using a commercially available kit (BioAssay Systems, Calif., USA). Histochemical analysis Cross sections (9-␮m thickness) of frozen Sol muscle were cut using a cryostat (Leica CM350) cooled to –19 °C. Sections were mounted on positively charged glass slides, dried at room temperature for 30 min, then stored at –20 °C until use. Haematoxylin and eosin (H&E) staining Frozen sections were brought to room temperature then stained with H&E using an automatic stainer (Leica Autostainer XL). A representative whole-muscle cross-section image from each group is presented in Supplementary Fig. S11. Slides were coded and subsequently examined under a light microscope (Zeiss, Axiovert S 100) at 200× magnification. Five fields, 1 from each quadrant and 1 from the center, were randomly selected from each section and graded by 3 blinded investigators. Criteria used to determine muscular damage were adopted from Armstrong et al. (1983) and were as follows: pale cytoplasm, centrally located nuclei, appearance of mononuclear cells and edema as evident by widened interstitial space, and loss of polygonal shape. Each field was given a grade between 1 and 5 on the basis of percentage of cells showing signs of damage: 1 = no cells, 2 = up to 25%, 3 = up to 50%, 4 = up to 75%, and 5 = all cells; the average score for all 5 fields were used as the final score for that sample. HIS48 Muscle sections were immunostained for HIS48, a marker of neutrophils as per methods described by Enns and Tiidus (2008). Slides were brought to room temperature and fixed in cold acetone (4 °C) for 10 min then submerged in 0.6% hydrogen peroxide in absolute methanol for 10 min to inactivate endogenous peroxidases. Slides were then washed in 15 mmol/L phosphate-buffered saline (PBS, pH 7.6) and nonspecific sites were blocked for 30 min using 5% normal goat serum in PBS containing 5% nonfat dry milk powder. After a quick rinse with PBS, primary antibody (BD Bio-

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/apnm-2014-0331. Published by NRC Research Press

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Estaki and Noble

sciences, Mississauga, Ont., Canada) was applied at a 1:50 dilution with 2% normal goat serum for 3 h at room temperature in a humidity chamber. Sections used as negative controls for each set of slides were incubated with dilution solution only, for the same amount of time. Slides were washed as before and developed using a commercially available kit (LSAB-2, DAKO Canada) containing a universal biotinylated secondary antibody and streptavidinconjugated horseradish peroxidase. Slides were then visualized using diaminobenzidine chromagen with nickel (Vector Laboratories). Finally, coverslips were mounted and sealed using nail polish. Five fields were randomly selected from each section (2 sections per sample) as described above and positively stained cells were counted manually. The average count of positively stained cells from the 2 sections (which represented about 1000 myofibers per sample) was calculated. Statistical analysis The Shapiro–Wilk normality test was used, where possible, to confirm normal distribution of groups and a Kolmogorov–Smirnov test was used in subgroups with lower n values. All data were subjected to 2-way ANOVA using GraphPad Prism (version 6.01) statistical analysis software. Holm–Sidak post hoc tests were used to locate significant differences between groups once a main difference was detected. All data are expressed as means ± SD and a p value of