Reduced stem cell aging in exercised human skeletal muscle is enhanced by ginsenoside Rg1

Ethical approval

The study was approved by the Institutional Review Board of University of Taipei, Taipei, Taiwan (IRB-2017–41). All experimental procedures were conducted in accordance with the Declaration of Helsinki [29]. Participants were given full explanation of the purpose, experimental procedure, and the potential risks of participation. Written informed consent was received prior to the commencement of the study.

Participants

Participants with cardiovascular, pulmonary, metabolic, bone or joint problem, smoking habit, muscle and joint injuries during the past 6 months were excluded. Eighteen young men were enrolled to participate in this study with 6 dropouts due to time conflict. A total of 12 men (age: 22 ± 2 years; height: 173 ± 4 cm; weight: 72 ± 8 kg) with an experience of weight training (at least 2 years, ≥ 3 times/week) completed this study.

Study design

To assess the muscle response after an acute bout of squat exercise under PLA- and Rg1-supplemented conditions, a randomized, double-blind placebo-controlled, counter-balanced crossover study design was conducted with a 3-week washout period. Following the 12-h overnight fast, participants were randomized to the PLA- and Rg1-trials in a counter-balanced order and the supplements were orally delivered 1 h before squat exercise. Capsules containing PLA (cornstarch) or ginsenoside Rg1 were orally delivered to participants via a drink (Herbalife Formula One shake, California, USA) 1 h before the squat exercise in the morning. This drink at a protein dose of 0.12 g·kg^-1·body weight was supplied for the purpose of providing sufficient nitrogen and carbon for enhancing muscle protein synthesis [30]. Participants could not distinguish the difference between PLA and Rg1 supplements during oral delivery. A Rg1 dose of 5 mg was used in this study (NuLiv Science, Inc., Brea, CA, USA), based on a previous report [21].

Rating of Perceived Exertion (RPE) was self-reported immediately after the exercise [31]. Each participant completed the 1 repetition maximum (1-RM) assessment, as a maximum muscle strength assessment, three weeks prior to squat exercise. Muscle biopsies were performed at baseline and 24 h after squat exercise. The baseline muscle biopsy was performed > 10 days before squat exercise. Participants were informed to stop any form of their own training activity for a week before the baseline biopsy and the exercise challenge. Post-exercise muscle biopsy was performed 24 h after the squat exercise (25 h after PLA or Rg1 supplementation) to allow a short recovery.

Maximum leg strength

Maximum muscle strength was determined as 1-RM based on a previous protocol [15]. After a warm-up exercise consisted of dynamic stretching exercises, participants conducted a specific warm-up included a set of 10 repetitions with 50% of an estimated 1-RM (according to perceived capacity), a set of 5 repetitions with 75% of the estimated 1-RM, and a final set of 1 repetition with 90–95% of the estimated 1-RM. After a 5-min rest period, participants completed 3 to 5 attempts with progressively heavier weights (∼5%), interspersed with 3–5 min rest intervals, until a 1-RM was achieved. Participants were instructed to adopt a shoulder width stance in keeping with their normal squat stance, descend in a controlled manner, avoid bouncing at the bottom position, maintain as near a vertical torso as possible, and feet always flat on the ground.

Squat exercise

A qualified trainer instructed the exercise protocol for all participants within a weight training room to ensure the consistency of the challenge. Each participant was required to perform a back-squat exercise using a barbell and a power half squat rack. Participants were advised to lower the barbell until their knees reach 90°, by having the gluts touching the adjustable multi-angle gym bench. Each participant completed his own set of warm-up followed by a structured set of 8 repetitions (50% 1-RM) of barbell back squat. The squat exercise consisted of 6 sets of 8 repetitions (70% 1-RM) with a 90-s rest interval between sets. Maximum weight lifted in relation to body weight ranged from 0.97 to 1.38 kg/kg body weight.

Rates of perceived exertion and pain

RPE was self-reported immediately after the completion of the exercise. Participants reported the RPE by observing a numerical scale, ranging from 1 “rest” to 10 “maximal effort”. The RPE scale was also based on repetitions in reserve (RIR) for resistance exercise [32]. Before (Pre) and after (Post, 24 h, 48 h, 72 h) the exercise protocol, perceived muscle soreness/pain were assessed with the visual analog scale (VAS), using a continuous 10-cm scale anchored by two verbal descriptors labeled from the left (no pain) to the right (worst possible pain) [33].

Muscle biopsy

The muscle biopsy procedure was conducted by an experienced physician using a 14-gauge Temno disposable cutting needle (REFT149, CareFusion, Vernon Hills, IL U.S.A) inserted into the vastus lateralis at 3 cm depth and ~20 cm proximal to kneecap. Two biopsied muscle samples were collected at each time point. Local anesthesia (2% lidocaine hydrochloride) was administered prior to the procedure. The baseline biopsy was collected from the right leg, more than 10 days before the exercise challenge. After collection, muscle tissue was immediately placed into 20 ml glass vial containing 10% formalin and then embedded into paraffin wax block. This formalin-fixed paraffin wax-embedded (FFPE) tissues were then cut using serial sectioning protocol and mount on glass slides for staining.

Immunohistochemistry (IHC) staining

IHC analysis as a semi-quantitative method was conducted by a pathologist at the Taipei Institute of Pathology (Taipei, Taiwan). The XT UltraView DAB v3 and BenchMark XT IHC/ISH Staining Module protocol (Ventana Medical Systems, AR, USA) were used to detect expression of monoclonal antibodies p16^INK4A (1:200, ab108349; Cambridge, MA, USA), ß-Gal (1:150, NBP2-45731; Novus Biologicals, USA), and CD34 (Ventana Medical Systems, USA) in muscle serial sections. The binding of primary antibody to a specific antigen was located by enzyme labelled secondary antibodies. The complex was then visualized with hydrogen peroxide substrate and 3,3′-diaminobenzidinetetrahydrochloride (DAB) chromogen, which produced brown precipitates. Pale to dark blue coloration represents cell nuclei, whereas brown coloration represents positively stained antigens.

For p16^INK4a detection, slides were deparaffinized, washed twice for 5 min in TBS plus 0.025% Triton X-100 and blocked in 10% normal serum with 1% BSA in TBS for 2 h at room temperature. Slides were drained before applying p16^INK4a antibody diluted in TBS with 1% BSA and incubated overnight at 4°C. After two 5-min rinses with TBS 0.025% Triton, the slides were incubated in 0.3% H₂O₂ in TBS for 15 min. Enzyme-conjugated anti-rabbit secondary antibody was applied to the slide, diluted in TBS with 1% BSA, and incubated for 1 h at room temperature. After a 5-min rinse in tap water, the slides were counterstained with hematoxylin.

For ß-Gal detection, muscle paraffin sections (2 μm thick) were deparaffinized and rehydrated with xylene and ethanol (100%, 95%, 70% 50% and deionized water). Slides were boiled in 10 mM sodium citrate buffer (pH 6.0), cooled on bench top (30 min), and immersed in distilled water (5 min). Tissue sections were quenched with 3.0% hydrogen peroxidase in methanol for 15 min, washed in distilled water (5 min), washed twice with permeabilization buffer containing 1% animal serum and 0.4% Triton X-100 in Phosphate-buffered saline (PBS-T), and incubated with 5% animal serum in PBS-T for 30 min at room temperature. Primary antibody (diluted in 1% animal serum in PBS) was added, incubated at room temperature for 1–2 h, and overnight incubation at 4°C in a humidified chamber. Sections were washed twice with 1% serum in PBS-T for 10 min before adding anti-mouse secondary antibody to each section. The sections were incubated at room temperature for 1 h before washing twice with 1% serum PBS-T for 10 min each. DAB working solution was prepared and applied to tissue that causes chromogenic reaction. Sections developed brown color with positive reaction. Slides were then immersed in deionized water twice for 2 min then were counterstained with hematoxylin. Pale to dark blue coloration represents cell nuclei, whereas brown coloration represents positively stained antigens.

The samples were washed and then incubated with the anti-rabbit secondary antibody for 10 min. After color development with 3,3′-diaminobenzidine at room temperature for 10 min, the sections were counterstained with hematoxylin for 15 min, dehydrated, and mounted according to the standard protocol.

Image analysis

The muscle cross-sectional area on the slides were reviewed and captured at 10× magnification (BX50 Olympus microscope, Tokyo, Japan) using MShot Image Analysis System v1.0. The analyses were conducted using manual inspection (Image J, National Institute of Health, USA) of 6 visual fields that contains the most compact and complete muscle fibers (> 50). Positively stained markers were quantified via cells expressing brown coloration as antigen. Positive marker criteria include: (a) muscle cell must be whole and intact; (b) the positive marker must have brown (antigen) stained coloration; (c) the positive marker must be intact/attached with muscle cell; (d) colocalization of positive marker must meet criteria for both markers fulfilling (a), (b), and (c) concurrently. Total positive signals (p16^INK4A+, CD34⁺, ß-Gal⁺) and colocalization number of positive signals of p16^INK4A+/CD34⁺ of the images were measured. Positive cell counts were normalized to fibers number.

Quantitative PCR

RNA was extracted from ~15 mg of biopsied muscle using the RNeasy kit (QIAGEN 74104) after a 60-s homogenization in QIAzol Lysis Reagent (QIAGEN, Hilden, Germany, 79306). One microgram of RNA in a total volume of 20 μl was reverse-transcribed to cDNA using iScript cDNA Synthesis Kit (#170–8890) (Bio-Rad, Hercules, CA, USA). Real-time PCR was performed using CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), PrimePCR™ Probe Assay (Bio-Rad, Hercules, CA, USA) and iQ Supermix kit (#170–8862) (Bio-Rad, Hercules, CA, USA). The cycling parameters were: 95°C for 3 min, then 50 cycles at 95°C for 10 s and 58°C for 30 s. Gene expression, normalized to the geometric mean of a housekeeping genes (RPP30), was quantified using the 2−(ΔCt) method and expressed as fold difference relative to the RPP30. The primers and probes used to amplify target were supplied from Bio-Rad PrimePCR™ Probe Assay: p16^INK4a (or CDKN2A) (Assay ID: qHsaCEP0057827), CD34 (Assay ID: qHsaCIP0026476), GLB1 (Assay ID: qHsaCEP0057625), MPO (Assay ID: qHsaCEP0049167), and RPP30 (Assay ID: qHsaCEP0052683). Test-retest reliability values for p16^INK4a mRNA and MPO mRNA are r = 0.87 and r = 0.94, respectively.

Statistical analysis

All results were presented as mean ± standard error (SE). Type 1 error equal or less than 5% for comparing mean difference was considered significant. Two-way ANOVA with repeated measure was used to determine the main effect and interactive effects of time (Pre and Post) and supplement (PLA and Rg1). The % change after exercise from baseline between PLA- and Rg1-trials were analyzed using paired t-test. Cohen’s d effects sizes were calculated and interpreted as small (d = 0.2), medium (d = 0.5), and large (d = 0.8) [34]. Pearson’s correlation was used to determine the magnitude of association between variables. Correlations were evaluated as follows: trivial (0.00–0.09), small (0.10–0.29), moderate (0.30–0.49), large (0.50–0.69), very large (0.70–0.89), nearly perfect (0.90–0.99), and perfect (1.00) [35].