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Sirt1 pathway
Accepted Manuscript The effect of exercise, resveratrol or their combination on Sarcopenia in aged rats via regulation of AMPK/Sirt1 pathway
Zhi-Yin Liao, Jin-Liang Chen, Ming-Han Xiao, Yue Sun, Yu-Xing Zhao, Die Pu, An-Kang Lv, Mei-Li Wang, Jing Zhou, Shi-Yu Zhu, Ke-Xiang Zhao, Qian Xiao PII: DOI: Reference:
S0531-5565(17)30417-5 doi: 10.1016/j.exger.2017.08.032 EXG 10139
To appear in:
Experimental Gerontology
Received date: Revised date: Accepted date:
31 May 2017 15 August 2017 23 August 2017
Please cite this article as: Zhi-Yin Liao, Jin-Liang Chen, Ming-Han Xiao, Yue Sun, YuXing Zhao, Die Pu, An-Kang Lv, Mei-Li Wang, Jing Zhou, Shi-Yu Zhu, Ke-Xiang Zhao, Qian Xiao , The effect of exercise, resveratrol or their combination on Sarcopenia in aged rats via regulation of AMPK/Sirt1 pathway, Experimental Gerontology (2017), doi: 10.1016/j.exger.2017.08.032
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ACCEPTED MANUSCRIPT The Effect of Exercise, Resveratrol or their combination on Sarcopenia in Aged Rats via regulation of AMPK/Sirt1 pathway
Zhi-Yin Liao1 #, Jin-Liang Chen1 #, Ming-Han Xiao2, Yue Sun1, Yu-Xing Zhao1, Die
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Pu1, An-Kang Lv1, Mei-Li Wang1, Jing Zhou1, Shi-Yu Zhu1, Ke-Xiang Zhao1, Qian
Department of Geriatrics, the First Affiliated Hospital of Chongqing Medical
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Xiao*
Department of Cardiology, the First Affiliated Hospital of Chongqing Medical
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2
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University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China.
Zhi-Yin Liao and Jin-Liang Chen contributed equally to this work.
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#
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University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China.
*Correspondence to Qian Xiao, Department of Geriatrics, the First Affiliated Hospital of Chongqing Medical University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China. E-Mail: [email protected]; Tel.: +86 89011632; Fax: +86 89011632.
ACCEPTED MANUSCRIPT ABSTRACT Sarcopenia is an age-related syndrome characterized by progressive loss of muscle mass and function. Exercise is an important strategy to prolong life and increase muscle mass, and resveratrol has been shown a variety beneficial effects on skeletal
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muscle. In the present study, we investigated the potential efficacy of using short-term
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exercise (six weeks), resveratrol (150 mg/kg/day), or combined exercise + resveratrol
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(150 mg/kg/day) on gastrocnemius muscle mass, grip strength, cross-sectional area and microscopic morphology in aged rats, and explored the potential mechanism at
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the apoptosis level. Six months old SD rats were used as young control group and 24
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months old SD rats were adopted as aged group. After six weeks intervention, the data provide evidence that exercise, resveratrol or their combination significantly increase
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the relative grip strength and muscle mass in aged rats (P<0.05). Electron microscopy
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discovered a significant increase in sarcomere length, I-band and H-zone in aged rats (P<0.05), and exercise, resveratrol or their combination significantly reduced the
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increasement (P<0.05). Moreover, light microscopy revealed a significant increase on
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Feret’s diameter and cross-sectional area (CSA) in aged rats (P<0.05), but exercise and resveratrol did not show significant effects on them (P＞0.05). Furthermore, exercise, resveratrol or their combination significantly increased the expression of p-AMPK and SIRT1, decreased the expression of acetyl P53 and Bax/Bcl-2 ratio in aged rats (P<0.05). These findings show that aged rats show significant changes in gastrocnemius muscle morphology and ultrastructure, and the protective effects of exercise, resveratrol and their combination are probably associated with anti-apoptotic
ACCEPTED MANUSCRIPT signaling pathways through activation of AMPK/Sirt1.
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Keywords Sarcopenia, Gastrocnemius muscle, Exercise, Resveratrol, Apoptosis
ACCEPTED MANUSCRIPT 1. Introduction Sarcopenia is an age-related syndrome characterized by progressive loss of muscle mass and function (Rosenberg, 2011), sarcopenia may affect as much as 15% of the population aged over 65 years and approximately 50% of individuals aged over
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80 years. This loss of muscle substantially reduces the quality of life and physical
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activity of older people (Cosqueric et al., 2006). There is irrefutable evidence that
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exercise prolongs life expectancy and reduces the risk of chronic diseases in rodents and humans (Chakravarty et al., 2008, Stessman et al., 2009). In fact, exercise is one
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of the most important strategies to prevent muscle loss, prolong life and improve the
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health of the elderly (Sanchis-Gomar et al., 2011). The positive effects of exercise are directly associated with beneficial changes in function and structure of sarcopenia.
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Resveratrol (3,4',5-trihydroxystilb), a polyphenol compound which is primarily
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present in the skin of red grapes has aroused considerable attention for its anti-aging effects and life extension properties (Howitz et al., 2003). Resveratrol has shown
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beneficial effects on skeletal muscles both in vitro and in vivo studies. It has been
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shown to inhibit protein degradation and attenuate skeletal muscle atrophy in vitro studies (Wyke et al., 2004, Wang et al., 2014). In vivo studies, some reported resveratrol supplementation improved muscle mass and muscle function in sarcopenia in aged animals (Bennett et al., 2013, Joseph et al., 2013), while others failed to show similar benefits in muscle mass and muscle function (Jackson et al., 2011, Ballak et al., 2015), these contrasting results seem to be mainly attributed to differences in treatment dose and time.
ACCEPTED MANUSCRIPT Resveratrol has been shown to mimic the metabolic effects of calorie restriction via activation of AMPK/Sirt1 signaling (Tennen et al., 2012), and exercise has also been proved to activate AMPK/Sirt1 signaling (Huang et al., 2016). Sirt1 plays variety roles in regulating physiological functions, including inflammation (Ghanim et
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al., 2010), longevity (Kobayashi et al., 2005), mitochondrial biogenesis (Lagouge et
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al., 2006), oxidative stress and antioxidation (Hasegawa et al., 2008). Furthermore,
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previous studies show that Sirt1 also plays a key role in animal fat deposition and muscle development(Bai et al., 2006). Apoptosis is involved in the regulation of many
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intracellular physiological and pathological conditions, we examined if resveratrol
gastrocnemius muscle of aged rats.
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and exercise altered the apoptotic signaling through activation of AMPK/Sirt1 in the
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Previous study reported the combination of resveratrol and exercise improved the
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skeletal muscle function in middle-aged mice (Kan et al., 2016). To date, although the beneficial effects of exercise and resveratrol have been established, the molecular
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mechanisms still remain unclear. In the present, we hypothesized that six weeks of
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exercise, resveratrol supplement or their combination would improve the skeletal muscle mass and muscle function in aged-rats, and reduce the apoptosis in muscle mass, and that combined treatment would exert an additive protective effect in aged rats.
ACCEPTED MANUSCRIPT 2. Material and methods 2.1 Animals 50 male Sprague-Dawley (SD) rats, 10 young adult and 40 aged rats (6 and 25 months old, respectively), were purchased from the animal center of Chongqing
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Medical University. All animals were housed five per cage in a temperature-controlled
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room (22±2℃) with a 12h light/dark cycle (lights on 8 a.m.-8 p.m.), and given free
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access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Chongqing Medical University, and all animal
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experiments were performed in accordance with the National Institutes of Health
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guidelines for the care and use of laboratory animals. All animal studies complied with the ARRIVE guidelines.
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The rats (n=50) were randomly selected and allocated to five groups: (1) old
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control group (OC group, n=10), (2) old-exercise-treated group (OE group, n=10); (3) old-resveratrol-treated group (OR group, n=10); (4) old-exercise combined
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resveratrol-treated group (OER group, n=10); (5) young control group (YC group,
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n=10). The old rats were acclimatized for 7 months prior to the intervention and young rats were acclimatized for 3 months. At the end of the 6-week treatment, all rats were sacrificed, and the gastrocnemius muscles were separated, weighed, frozen in liquid nitrogen, and stored at -80C for further analysis.
ACCEPTED MANUSCRIPT 2.2 Movement Scheme The rats were exercised on an animal treadmill (Zhongshi Inc.) for 7 days a week, beginning with adaptation in the first week of training, followed by formal training there after (Bedford et al., 1979, Siu et al., 2004), with some modifications to produce
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a specific movement scheme, as shown in Table 1.
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2.3 Dosage regimen
Prior to the intervention, the OR and OER rats were separated into individual
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cages. Resveratrol (150 mg/kg/d ,99%, Jianfeng Inc.) was produced as a
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beef-flavoured feed and administered according to each rat’s body weight, mixing in the corresponding weight of resveratrol feed with their normal food pellets. The
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animals received resveratrol treatment for 6 weeks.
2.4 Grip strength test
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Grip strength was measured using an electronic grip strength meter
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(Cat.47200,Ugo Basil). After the animals were placed on the fence, the rats were slowly grasped at the base of their tails to determine the highest grip strength. The measurements were repeated 3 times, and the maximal readings were recorded. The grip strength was measured every week.
ACCEPTED MANUSCRIPT 2.5 Transmission Electron Microscopy Gastrocnemius muscle tissue was fixed overnight in 5% glutaraldehyde fixative at 4C and then randomly cut into five to six tissue blocks (1mm3) in the same area between groups. All samples were subsequently processed for transmission electron
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microscopy (TEM) at the School of Life Sciences at Chongqing Medical University.
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Ultrathin sections were made by technicians as previously reported (Yang et al., 2008).
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The sections were then viewed with a TEM (Hitachi-7500, Japan) by another technician who was blinded to the experiment. From each section, ten fields of images
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were randomly captured at a magnification of 15,000×.
2.6 Western Blotting
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Skeletal muscle tissues were homogenized in RIPA buffer (Beyotime Inc.)
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supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). The protein concentrations were determined using the BCA Protein
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Assay Kit (Beyotime Inc.). Equal amounts of protein from different samples were
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loaded onto 10% SDS polyacrylamide gels and separated by electrophoresis at 100 V for 120 min. The proteins were transferred onto PVDF membranes (Millipore) and subsequently blocked using 5% non-fat milk or 5% bovine serum albumin (BSA) for 1 hour at room temperature. The membranes were incubated with different primary antibodies against AMPK (1:1000, CST), p-AMPK (1:1000, CST), Sirt1 (1:1000, Abcam), p53 (1:1000, Abcam), bax (1:1000, CST), bcl-2 (1:1000, CST), GAPDH (1:1000, Zhongshan Inc.) at 4℃ overnight. The next day, the membranes were
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three
times
with
TBST
and
incubated
with
a
horseradish
peroxidase-conjugated anti-mouse (1:5000, Zhongshan Inc.) or anti-rabbit (1:5000, Zhongshan Inc.) secondary antibody for 1 hour at 37 ℃. . The membranes were subsequently washed three times with TBST, and the protein bands were detected
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using an enhanced chemiluminescence substrate (Thermo Scientific), followed by
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using Bio-1D software (Vilber Lourmat, France).
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exposure to X-ray film (Vilber Lourmat, France). The band intensities were quantified
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2.7 Real-time PCR
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All gene primers were synthesized in Beijing Invitrogen and listed in Table 2. RNA was extracted using TRNzol total RNA extraction reagent (Tiangen Inc.), RNA
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concentration and purity were measured by using a spectrophotometer (NanoDrop®
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ND-2000, Thermo scientific), 1 μg RNA was added into 1 fold volume of formaldehyde. The final concentration of the dye was 10 μg / ml. After denatured by
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heating to 70 ° C for 5 minutes, the sample was loaded in a plastic hole at a pressure
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of 5 - 6 V / cm to a bromophenol blue indicator at least 2-3 cm. cDNA reverse transcription was performed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). The target gene and internal parameters of each sample were respectively subjected to Real-time PCR reaction, and three wells were detected in each sample. The data were analyzed by 2 - △ △ CT method.
ACCEPTED MANUSCRIPT 2.8 Immunohistochemistry Gastrocnemius muscle tissue was fixed at 4° C in paraformaldehyde fixative, and all samples were paraffin-embedded and sliced at the School of Life Sciences at Chongqing Medical University. After xylene dewaxing, the samples were dehydrated
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in ascending graded concentrations of ethanol, followed by haematoxylin and eosin
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(H & E) staining for general histological examination.
2.9 Statistical Analysis
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All statistical analysis was carried out using SPSS 21.0. The results are presented
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as mean ± SD (standard deviation). After confirming a normal distribution with the Shapiro-Wilk test and equal variances of the data, relative grip strength were analyzed
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using the two-way repeated measures ANOVA, and data from different treatment
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groups at each measurement time were compared using a multivariate ANOVA. Other data among multiple groups were compared using the one-way ANOVA followed by
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Turkey’s post hoc tests. P <0.05 was considered statistically significant.
3. Results
3.1 The effects of exercise, resveratrol and their combination on body weight, grip strength and muscle mass. As shown in Fig. 1A, at the beginning of the experiment, compared with the YC group, the aged rats among different groups were significantly heavier than the young rats, indicating that age was a key factor affecting body weight in rats. No significant
ACCEPTED MANUSCRIPT difference was found in weight among the aged rats in different groups. During interventions, the weight of the rats in the OR, OE and OER groups decreased, and there was a significant difference between the OE group and the OC group at the end of week five. At the end of week six, a significant difference was observed between
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the OR group and the OE group. Exercise, but not resveratrol, significantly reduced
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the weight of aged rats (P<0.05), the combination of exercise and resveratrol did not
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show an additive effect on weight loss.
According to previous report(Kim et al., 2016), in order to eliminate the effects
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of weight on grip strength, relative grip strength (grip strength / weight) was adopted
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in our research. As shown in Fig. 1B, during the experiment, the relative grip strength in YC group was significantly higher than that in the aged groups (P<0.05). During
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the first five weeks, there was no significant difference of relative grip strength among
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the OE, OR, OER and OC groups (P＞0.05). At the end of week six, the relative grip strength of the rats in the OE, OR and OER groups was significantly higher than that
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in the OC group (p <0.05), suggesting that exercise, resveratrol or their combination
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could increase the grip strength of aged rats. However, no significant difference was found among the OE, OR and OER groups (P＞0.05), suggesting that there was no synergistic effect of combined intervention on increasing rat grip strength. In the present study, Edstrom's SI (Sarcopenia Index) value was used to determine whether the model of sarcopenia was successful. The SI value is defined as the mass of the target skeletal muscle (mg) / subject weight (g) and used as an evaluation index(Edstrom and Ulfhake, 2005). When the SI value of the aged group
ACCEPTED MANUSCRIPT was significantly lower than that of the young group and the difference was greater than 2 times SD, sarcopenia was indicated(Baumgartner et al., 1998). As shown in Fig. 1C, compared with the YC group, the gastrocnemius SI value of OC rats was significantly lower (P<0.05) and decreased by 2 times SD, indicating that
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25-month-old SD rats can be used as sarcopenia model rats. Compared with the OC
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group, the SI value of rats in the OE and OER groups significantly increased
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(P<0.05), while the increasement of SI value in the OR group did not show a statistical significance (P＞0.05), indicating that exercise or exercise combined with
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resveratrol could increase the muscle mass in aged rats.
3.2 Effects of exercise, resveratrol or their combination on myofibrils.
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Electron microscopy revealed that the myofibrils in the aged group of rats were
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sparsely arranged, in which were accompanied with fat infiltration, mitochondrial swelling and other changes (Fig. 2A). The lengths of the sarcomere, A-band, I-band
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and H-zone were measured in the present study, consistent with previous studies, as
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presented in Fig. 2B, compared with the YC group, the sarcomere length and I-band length were significantly increased in the OC group (P<0.05), whereas length of A-band and length of H-zone did not change (P＞0.05). Compared with the OC group, the sarcomere length, I-band length and H-zone length in the OE, OR and OER groups decreased significantly (P<0.05). There was no significant difference in A-band length between the groups. Besides, the sarcomere length in the OER group was significantly reduced compared with the OE and OR group (P<0.05), suggesting
ACCEPTED MANUSCRIPT that the combined intervention of exercise and resveratrol reduced the sarcomere length as a synergistic effect. Collectively, these results suggest that the myofibrils in aged rats were increased in sarcomere, I-band and H-band lengths. Exercise and resveratrol can improve the ultrastructural changes of myofibrils. Exercise and
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resveratrol combination show a synergistic effect on decreasing sarcomere length in
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aged rats, but not on I-band length and H-band length.
cross-sectional area and perimeter.
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3.3 Effects of exercise, resveratrol or their combination on Feret’s diameter,
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To assess the perimeter, cross-sectional area (CSA) and Feret’s diameter of single muscle fibres, H&E staining was conducted. As presented in Fig. 3A-B, compared
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with the YC group, it can be seen that age induced an increase in the perimeter, CSA
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and Feret’s diameter of single muscle fibres. Of note, only Feret’s diameter and CSA increasement reached statistical significance (P<0.05). Furthermore, compared with
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the OC group, OR group increased CSA significantly (P<0.05), while OE, OR or
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their combination did not show any significant increasement in the perimeter and Feret’s diameter (P＞0.05).
3.4 Effect of exercise, resveratrol or their combination on AMPK, Sirt1, p53, Bax and Bcl-2 protein expression To find out the possible molecular mechanisms of exercise, resveratrol or their combination on gastrocnemius muscle in rats, western blotting were conducted to
ACCEPTED MANUSCRIPT evaluate the protein expression of AMPK, p-AMPK, Sirt1, P53, Bax and Bcl-2. As demonstrated in Fig. 4A-B, compared with the YC group, the expression of p-AMPK, Sirt1 and Bcl-2 were significantly decreased and the expression of P53 and Bax were significantly increased in the OC group (P<0.05). Compared with the OC group, OE,
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OR and OER group increased significantly p-AMPK and Bcl-2 expression (P<0.05),
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and decreased significantly Bax expression (P<0.05). Of note, OE group did not
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significantly increase Sirt1 expression and decrease P53 expression (P＞0.05), but OR and OER group significantly increase Sirt1 expression and decrease P53
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expression compared with the OC group (P<0.05). Furthermore, the OER group
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increased significantly p-AMKP, Sirt1, Bcl-2 expression and decreased significantly expression of P53 and Bax compared with the OE and OR group (P<0.05), indicating
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that exercise and resveratrol combination may have a synergistic effect on molecular
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signaling expression. These results suggest that both individual exercise or resveratrol intervention can increase the expression of p-AMPK and Sirt1, thereby reducing the
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acetylation of p53 protein, inhibiting downstream Bax, and increasing downstream
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Bcl-2 to inhibit apoptosis. Moreover, the combination of exercise and resveratrol showed a synergistic anti-apoptotic effect.
3.5 Effect of exercise, resveratrol or their combination on mRNA expression of p53, Bax and Bcl-2 We next examined the mRNA expression of P53, Bax and Bcl-2 in all groups of rats. As shown in Fig. 5, consistent with our findings in western blotting, compared
ACCEPTED MANUSCRIPT with the YC group, the mRNA expression of Bax was significantly increased and the expression of Bcl-2 was significantly decreased in the OC group (P<0.05). Furthermore, exercise, resveratrol and their combination were found to significantly decrease the expression of Bax and increase the expression of Bcl-2 compared with
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the OC group (P<0.05) (Fig. 5B-C). Of note, the results of mRNA expression of P53
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were not in accordance with the protein expression of P53 in the present study. As
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presented in Fig. 5A, compared with the YC group, the P53 mRNA expression in the OC group was significantly decreased (P<0.05), and compared with the OC group,
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exercise or exercise combined resveratrol interventions significantly increased the
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mRNA expression of P53 (P<0.05). These conflicts between protein expression and mRNA expression could be explained by the protein post translational modification,
4. Discussion
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like deacetylation.
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The life expectancy of SD rats ranges from 30 to 36 months, 6-month-old rats
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are equivalent to 18-year-old human beings, and 24-month-old rats are equivalent to 60-year-old humans. In the present study, 25-month-old SD rats were adopted as aged rats, and the SI value of the aged group was significantly lower than that of the young group and the difference was greater than 2 times standard deviation, which indicates that the aged group could be regarded as model of sarcopenia. The clinical diagnosis of sarcopenia includes two aspects: skeletal muscle mass decline, and skeletal muscle weakness (Chen et al., 2016). In the present study, we adopt the SI value and the
ACCEPTED MANUSCRIPT relative grip strength to evaluate the skeletal muscle mass and function. Our data show that resveratrol, exercise or their combination significantly increase the relative grip strength, which indicates all interventions could ameliorate the skeletal muscle function. While for muscle mass, exercise and exercise combined resveratrol group
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significantly increased the SI value, there is a tendency for SI value to be elevated by
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dietary resveratrol supplementation, however the result does not reach statistical
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significance. Previous studies have shown that exercise can improve muscle loss by hindering apoptosis (Lenk et al., 2010), improving mitochondrial function (Zampieri
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et al., 2016), improving antioxidant capacity (Brioche and Lemoine-Morel, 2016),
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improving the level of inflammatory factors (Buford et al., 2009), and activating multiple pathways to promote protein synthesis and inhibit protein breakdown
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(Osorio-Fuentealba et al., 2013, Bacurau et al., 2016, Ju et al., 2016, Fan et al., 2017).
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Among these mechanisms, the inhibition of apoptosis is a recent research hotspot, however, the molecular mechanism of exercise-induced apoptosis remains unclear.
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The effect of resveratrol on skeletal muscle in aged rats seems to be
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dose-dependent. According to the previous studies, 12.5 mg/kg/d and 50 mg/kg/d are too low to effect muscle mass and muscle function (Jackson et al., 2010, Jackson et al., 2011), while 125mg/kg/d resveratrol significantly attenuate the muscle wasting and increase the muscle function (Bennett et al., 2013). Resveratrol has antioxidant and anti-free radical properties, improves immune system activity, opposes thrombosis, provides cardiovascular and neurological protection, and exerts other pharmacological effects. Previous studies have shown that resveratrol increases the endogenous
ACCEPTED MANUSCRIPT antioxidant factor and reduces the peroxide loading in skeletal muscle cells, thereby reducing oxidative damage associated with muscle loading, unloading and ageing (Jackson et al., 2010, Ryan et al., 2010). Several in vitro studies have shown that resveratrol plays a role in inhibiting skeletal muscle fibrin degradation and improving
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atrophy (Wyke et al., 2004, Russell et al., 2006, Wyke and Tisdale, 2006, Alamdari et
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al., 2012), and previous studies have shown that resveratrol can improve the recovery
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of muscle wasting atrophy (Bennett et al., 2013). However, the effect of resveratrol on muscle function and its potential mechanism have not been established.
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With aging, the most obvious changes in muscle morphology is the reduction in
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the number of muscle fibres which ultimately induces the muscle volume reduction. What’s more, the capillaries in the muscle tissue decreased and the connective tissue
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increased, which induce the dysfunction of muscle fibres. Myofibrils are the
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contraction units of skeletal muscle fibres, comprising thick muscle filaments (myosin) that form the dark band and fine filaments (actin) that form the light band. With aging,
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the myofibrils and muscle fibres are changed accordingly. To further clarify the
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underlying mechanisms leading to the sarcopenia, we evaluate the micro and ultrastructure of the gastrocnemius muscle. The performance of the CSA, the perimeter, the Feret's diameter, the sarcomere length, the I-band length and the H-band length were detected. Consistent with the previous study (Sayed et al., 2016), the lengths of the sarcomere, I-band and H-band are significantly increased with aging, no significant change was found in A-band length, it indicates the age-related contractile function loss in skeletal muscle. Exercise, resveratrol or their combination
ACCEPTED MANUSCRIPT ameliorate the muscle function are closely related to the decrease of lengths of the sarcomere, I-band and H-band. The CSA and the Feret's diameter in the aged group were significantly increased, this increase of muscle fibres size in aged rats could be a compensatory mechanism for reduction of the fibre number.
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Different mechanisms contributing to sarcopenia have been reported, include
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hormonal changes, inflammation, apoptosis, oxidative stress, etc. In the present study,
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we found exercise and resveratrol significantly increase the expression of AMPK and Sirt1, decrease the expression of acetylated p53, and increase the ratio expression of
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Bcl-2/Bas, suggesting that inhibition of apoptosis by resveratrol or exercise may be
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associated with the activation of the AMPK/Sirt1 pathway. Exercise and resveratrol is the activator of Sirt1. Sirt1 can inhibit p53 activity through deacetylation, and reduce
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apoptosis induction by reducing p53 effect on downstream target gene activation
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(Zhang et al., 2014). P53 is a tumour suppressor gene, with more than 50% of p53 mutations detected in all malignancies (Romer et al., 2006). p53-encoded proteins are
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transcription factors that control cell cycle initiation. Many signals control the levels
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of p53 protein in healthy cells. If the cells are damaged and cannot be repaired, p53 will participate in the progression the cell cycle, eventually leading to cell death (Cakir et al., 2011, Fujita and Ishikawa, 2011, Kung and Murphy, 2016). Bcl-2 can prevent apoptosis-forming factors such as cytochrome C from mitochondria release, which
has
an
anti-apoptotic
effect,
Bax
interacts
with
mitochondrial
voltage-dependent ion channels, mediates cytochrome C release, and exerts apoptosis-related effects. P53 increases the expression of Bax, decreases the
ACCEPTED MANUSCRIPT expression of Bcl-2, and promotes apoptosis (Basu and Haldar, 1998).
5. Conclusions In summary, it seems apparent from our results that exercise could improve the
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muscle mass and strength of aged rats and improve the ultrastructure of aged
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myofibrils. Although resveratrol does not increase the quality of skeletal muscle, it
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could still improve the grip strength and ultrastructure of myofibrils and muscle fibres. Our results reveal that both exercise and resveratrol treatment might activate the
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AMPK/Sirt1 pathway to inhibit apoptosis, improving muscle quality and preventing
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sarcopenia. Furthermore, we find that exercise and resveratrol combined therapy may
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have a synergistic effect on improving sarcopenia.
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Acknowledgments This work was financially supported by grants from Chongqing Health and Family Planning Commission (No. 2015MSXM016); grants from
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Chongqing Development and Reform Commission (No. [2013]1420). This study was
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also supported by National Key Clinical Specialties Construction Program of China (No. [2013]544).
Conflict of Interest The authors declare that there are no conflicts of interest.
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Yang, S., Li, C., Zhang, W., Wang, W., Tang, Y., 2008. Sex differences in the white matter and myelinated nerve fibers of Long-Evans rats. Brain Res. 1216, 16-23.
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ACCEPTED MANUSCRIPT mitochondrial calcium uniporter expression levels and affects mitochondria dynamics. Physiol Rep. 4. Zhang, Z.N., Chung, S.K., Xu, Z., Xu, Y., 2014. Oct4 maintains the pluripotency of human embryonic stem cells by inactivating p53 through Sirt1-mediated
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deacetylation. Stem cells. 32, 157-165.
ACCEPTED MANUSCRIPT Figure captions Fig. 1 Effects of exercise, resveratrol and their combination on body weight, relative grip strength and muscle mass. (A): Body weight of rats in different groups at different time. (B): Relative grip strength of rats in different groups at different time.
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(C): The SI value of gastrocnemius in different groups. Data were analyzed using a
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two-way ANOVA of repeated measures followed by Turkey’s post hoc tests (B), and a
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one-way ANOVA followed by Turkey’s post hoc tests (A, C). a P< 0.05 OC vs. OE, b P< 0.05 OC vs. OR, c P< 0.05 OC vs. OER, d P< 0.05 OR vs. OE, * P < 0.05 vs. YC,
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# P < 0.05 vs. OC. Results are expressed as the mean±SD; n = 10 animals/group.
Fig. 2 Effects of exercise, resveratrol and their combination on gastrocnemius
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myofibrils. (A): Representative electron micrographs of longitudinal sections of
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gastrocnemius myofibers from different groups. Magnification ×15,000. Scale bars represent 1 μm. n = 3 animals/group. (B): Morphometric analysis of sarcomere length,
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A-band, I-band and H-zone in five groups of rats. All data were analyzed using a
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one-way ANOVA followed by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P< 0.05 vs. OE, d P< 0.05 vs. OR, # P< 0.05 vs. OER. Results are expressed as the mean±SD.
Fig. 3 Photomicrographs of paraffin sections of cross sections of gastrocnemius muscle fibers in five groups. (A): Transverse sections of gastrocnemius muscle stained with hematoxylin and eosin. Magnification ×200. Scale bars represent 100 μm.
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Fig. 4 Effects of exercise, resveratrol and their combination on metabolic signaling in the gastrocnemius muscle. (A): Western blotting of AMPK, p-AMPK, SIRT1, Acetyl
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P53, Bax and Bcl2 protein levels in rat gastrocnemius muscle. GAPDH was used as
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the loading control. (B): Quantitative analysis of AMPK, p-AMPK, SIRT1, Acetyl P53, Bax and Bcl2 levels. All data were analyzed using a one-way ANOVA followed
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P< 0.05 vs. OR, # P < 0.05 vs. OER. Results are expressed as the mean±SD. n = 6
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Fig. 5 Relative mRNA expression of p53、Bax and Bcl-2 (A-C). All data were analyzed using a one-way ANOVA followed by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P < 0.05 vs. OE, d P< 0.05 vs. OR, # P < 0.05 vs. OER. Results are expressed as the mean±SD. n = 6 animals/group.
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Time (min)
1（Adaptive training）
5
5
2
15
15
3
20
25
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Sequence 5’-3’ (forward)
Sequence 5’-3’ (reverse)
AGATGTTCCGAGAGCTGAATGAG
TTTTTTATGGCGGGACGTAGA
Bax
GGTGGTTGCCCTTTTCTACTTTGC
GCTCCCGGAGGAAGTCCAGTG
Bcl2
GGGCTACGAGTGGGATACTGGAG
CGGGCGTTCGGTTGCTCT
GAPDH
TTCCTACCCCCAATGTATCCG
CCACCCTGTTGCTGTAGCCATA
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ACCEPTED MANUSCRIPT Highlights ·Exercise, resveratrol or combination increased muscle mass and function in aged rats.
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·Exercise and resveratrol ameliorated ultrastructure of muscle fibrils in aged rats.
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·Exercise and resveratrol prevented muscle apoptosis via AMPK/Sirt1 activation.
Zhi-Yin Liao, Jin-Liang Chen, Ming-Han Xiao, Yue Sun, Yu-Xing Zhao, Die Pu, An-Kang Lv, Mei-Li Wang, Jing Zhou, Shi-Yu Zhu, Ke-Xiang Zhao, Qian Xiao PII: DOI: Reference:
S0531-5565(17)30417-5 doi: 10.1016/j.exger.2017.08.032 EXG 10139
To appear in:
Experimental Gerontology
Received date: Revised date: Accepted date:
31 May 2017 15 August 2017 23 August 2017
Please cite this article as: Zhi-Yin Liao, Jin-Liang Chen, Ming-Han Xiao, Yue Sun, YuXing Zhao, Die Pu, An-Kang Lv, Mei-Li Wang, Jing Zhou, Shi-Yu Zhu, Ke-Xiang Zhao, Qian Xiao , The effect of exercise, resveratrol or their combination on Sarcopenia in aged rats via regulation of AMPK/Sirt1 pathway, Experimental Gerontology (2017), doi: 10.1016/j.exger.2017.08.032
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ACCEPTED MANUSCRIPT The Effect of Exercise, Resveratrol or their combination on Sarcopenia in Aged Rats via regulation of AMPK/Sirt1 pathway
Zhi-Yin Liao1 #, Jin-Liang Chen1 #, Ming-Han Xiao2, Yue Sun1, Yu-Xing Zhao1, Die
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Pu1, An-Kang Lv1, Mei-Li Wang1, Jing Zhou1, Shi-Yu Zhu1, Ke-Xiang Zhao1, Qian
Department of Geriatrics, the First Affiliated Hospital of Chongqing Medical
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Xiao*
Department of Cardiology, the First Affiliated Hospital of Chongqing Medical
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2
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University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China.
Zhi-Yin Liao and Jin-Liang Chen contributed equally to this work.
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#
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University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China.
*Correspondence to Qian Xiao, Department of Geriatrics, the First Affiliated Hospital of Chongqing Medical University, Friendship Road 1, Yuan Jiagang, 400016, Chongqing, China. E-Mail: [email protected]; Tel.: +86 89011632; Fax: +86 89011632.
ACCEPTED MANUSCRIPT ABSTRACT Sarcopenia is an age-related syndrome characterized by progressive loss of muscle mass and function. Exercise is an important strategy to prolong life and increase muscle mass, and resveratrol has been shown a variety beneficial effects on skeletal
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muscle. In the present study, we investigated the potential efficacy of using short-term
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exercise (six weeks), resveratrol (150 mg/kg/day), or combined exercise + resveratrol
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(150 mg/kg/day) on gastrocnemius muscle mass, grip strength, cross-sectional area and microscopic morphology in aged rats, and explored the potential mechanism at
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the apoptosis level. Six months old SD rats were used as young control group and 24
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months old SD rats were adopted as aged group. After six weeks intervention, the data provide evidence that exercise, resveratrol or their combination significantly increase
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the relative grip strength and muscle mass in aged rats (P<0.05). Electron microscopy
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discovered a significant increase in sarcomere length, I-band and H-zone in aged rats (P<0.05), and exercise, resveratrol or their combination significantly reduced the
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increasement (P<0.05). Moreover, light microscopy revealed a significant increase on
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Feret’s diameter and cross-sectional area (CSA) in aged rats (P<0.05), but exercise and resveratrol did not show significant effects on them (P＞0.05). Furthermore, exercise, resveratrol or their combination significantly increased the expression of p-AMPK and SIRT1, decreased the expression of acetyl P53 and Bax/Bcl-2 ratio in aged rats (P<0.05). These findings show that aged rats show significant changes in gastrocnemius muscle morphology and ultrastructure, and the protective effects of exercise, resveratrol and their combination are probably associated with anti-apoptotic
ACCEPTED MANUSCRIPT signaling pathways through activation of AMPK/Sirt1.
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Keywords Sarcopenia, Gastrocnemius muscle, Exercise, Resveratrol, Apoptosis
ACCEPTED MANUSCRIPT 1. Introduction Sarcopenia is an age-related syndrome characterized by progressive loss of muscle mass and function (Rosenberg, 2011), sarcopenia may affect as much as 15% of the population aged over 65 years and approximately 50% of individuals aged over
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80 years. This loss of muscle substantially reduces the quality of life and physical
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activity of older people (Cosqueric et al., 2006). There is irrefutable evidence that
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exercise prolongs life expectancy and reduces the risk of chronic diseases in rodents and humans (Chakravarty et al., 2008, Stessman et al., 2009). In fact, exercise is one
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of the most important strategies to prevent muscle loss, prolong life and improve the
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health of the elderly (Sanchis-Gomar et al., 2011). The positive effects of exercise are directly associated with beneficial changes in function and structure of sarcopenia.
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Resveratrol (3,4',5-trihydroxystilb), a polyphenol compound which is primarily
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present in the skin of red grapes has aroused considerable attention for its anti-aging effects and life extension properties (Howitz et al., 2003). Resveratrol has shown
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beneficial effects on skeletal muscles both in vitro and in vivo studies. It has been
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shown to inhibit protein degradation and attenuate skeletal muscle atrophy in vitro studies (Wyke et al., 2004, Wang et al., 2014). In vivo studies, some reported resveratrol supplementation improved muscle mass and muscle function in sarcopenia in aged animals (Bennett et al., 2013, Joseph et al., 2013), while others failed to show similar benefits in muscle mass and muscle function (Jackson et al., 2011, Ballak et al., 2015), these contrasting results seem to be mainly attributed to differences in treatment dose and time.
ACCEPTED MANUSCRIPT Resveratrol has been shown to mimic the metabolic effects of calorie restriction via activation of AMPK/Sirt1 signaling (Tennen et al., 2012), and exercise has also been proved to activate AMPK/Sirt1 signaling (Huang et al., 2016). Sirt1 plays variety roles in regulating physiological functions, including inflammation (Ghanim et
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al., 2010), longevity (Kobayashi et al., 2005), mitochondrial biogenesis (Lagouge et
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al., 2006), oxidative stress and antioxidation (Hasegawa et al., 2008). Furthermore,
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previous studies show that Sirt1 also plays a key role in animal fat deposition and muscle development(Bai et al., 2006). Apoptosis is involved in the regulation of many
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intracellular physiological and pathological conditions, we examined if resveratrol
gastrocnemius muscle of aged rats.
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and exercise altered the apoptotic signaling through activation of AMPK/Sirt1 in the
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Previous study reported the combination of resveratrol and exercise improved the
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skeletal muscle function in middle-aged mice (Kan et al., 2016). To date, although the beneficial effects of exercise and resveratrol have been established, the molecular
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mechanisms still remain unclear. In the present, we hypothesized that six weeks of
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exercise, resveratrol supplement or their combination would improve the skeletal muscle mass and muscle function in aged-rats, and reduce the apoptosis in muscle mass, and that combined treatment would exert an additive protective effect in aged rats.
ACCEPTED MANUSCRIPT 2. Material and methods 2.1 Animals 50 male Sprague-Dawley (SD) rats, 10 young adult and 40 aged rats (6 and 25 months old, respectively), were purchased from the animal center of Chongqing
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Medical University. All animals were housed five per cage in a temperature-controlled
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room (22±2℃) with a 12h light/dark cycle (lights on 8 a.m.-8 p.m.), and given free
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access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of Chongqing Medical University, and all animal
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experiments were performed in accordance with the National Institutes of Health
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guidelines for the care and use of laboratory animals. All animal studies complied with the ARRIVE guidelines.
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The rats (n=50) were randomly selected and allocated to five groups: (1) old
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control group (OC group, n=10), (2) old-exercise-treated group (OE group, n=10); (3) old-resveratrol-treated group (OR group, n=10); (4) old-exercise combined
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resveratrol-treated group (OER group, n=10); (5) young control group (YC group,
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n=10). The old rats were acclimatized for 7 months prior to the intervention and young rats were acclimatized for 3 months. At the end of the 6-week treatment, all rats were sacrificed, and the gastrocnemius muscles were separated, weighed, frozen in liquid nitrogen, and stored at -80C for further analysis.
ACCEPTED MANUSCRIPT 2.2 Movement Scheme The rats were exercised on an animal treadmill (Zhongshi Inc.) for 7 days a week, beginning with adaptation in the first week of training, followed by formal training there after (Bedford et al., 1979, Siu et al., 2004), with some modifications to produce
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a specific movement scheme, as shown in Table 1.
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2.3 Dosage regimen
Prior to the intervention, the OR and OER rats were separated into individual
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cages. Resveratrol (150 mg/kg/d ,99%, Jianfeng Inc.) was produced as a
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beef-flavoured feed and administered according to each rat’s body weight, mixing in the corresponding weight of resveratrol feed with their normal food pellets. The
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2.4 Grip strength test
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Grip strength was measured using an electronic grip strength meter
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(Cat.47200,Ugo Basil). After the animals were placed on the fence, the rats were slowly grasped at the base of their tails to determine the highest grip strength. The measurements were repeated 3 times, and the maximal readings were recorded. The grip strength was measured every week.
ACCEPTED MANUSCRIPT 2.5 Transmission Electron Microscopy Gastrocnemius muscle tissue was fixed overnight in 5% glutaraldehyde fixative at 4C and then randomly cut into five to six tissue blocks (1mm3) in the same area between groups. All samples were subsequently processed for transmission electron
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microscopy (TEM) at the School of Life Sciences at Chongqing Medical University.
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Ultrathin sections were made by technicians as previously reported (Yang et al., 2008).
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The sections were then viewed with a TEM (Hitachi-7500, Japan) by another technician who was blinded to the experiment. From each section, ten fields of images
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2.6 Western Blotting
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Skeletal muscle tissues were homogenized in RIPA buffer (Beyotime Inc.)
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supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). The protein concentrations were determined using the BCA Protein
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Assay Kit (Beyotime Inc.). Equal amounts of protein from different samples were
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loaded onto 10% SDS polyacrylamide gels and separated by electrophoresis at 100 V for 120 min. The proteins were transferred onto PVDF membranes (Millipore) and subsequently blocked using 5% non-fat milk or 5% bovine serum albumin (BSA) for 1 hour at room temperature. The membranes were incubated with different primary antibodies against AMPK (1:1000, CST), p-AMPK (1:1000, CST), Sirt1 (1:1000, Abcam), p53 (1:1000, Abcam), bax (1:1000, CST), bcl-2 (1:1000, CST), GAPDH (1:1000, Zhongshan Inc.) at 4℃ overnight. The next day, the membranes were
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TBST
and
incubated
with
a
horseradish
peroxidase-conjugated anti-mouse (1:5000, Zhongshan Inc.) or anti-rabbit (1:5000, Zhongshan Inc.) secondary antibody for 1 hour at 37 ℃. . The membranes were subsequently washed three times with TBST, and the protein bands were detected
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using an enhanced chemiluminescence substrate (Thermo Scientific), followed by
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using Bio-1D software (Vilber Lourmat, France).
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exposure to X-ray film (Vilber Lourmat, France). The band intensities were quantified
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2.7 Real-time PCR
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All gene primers were synthesized in Beijing Invitrogen and listed in Table 2. RNA was extracted using TRNzol total RNA extraction reagent (Tiangen Inc.), RNA
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concentration and purity were measured by using a spectrophotometer (NanoDrop®
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ND-2000, Thermo scientific), 1 μg RNA was added into 1 fold volume of formaldehyde. The final concentration of the dye was 10 μg / ml. After denatured by
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heating to 70 ° C for 5 minutes, the sample was loaded in a plastic hole at a pressure
AC
of 5 - 6 V / cm to a bromophenol blue indicator at least 2-3 cm. cDNA reverse transcription was performed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). The target gene and internal parameters of each sample were respectively subjected to Real-time PCR reaction, and three wells were detected in each sample. The data were analyzed by 2 - △ △ CT method.
ACCEPTED MANUSCRIPT 2.8 Immunohistochemistry Gastrocnemius muscle tissue was fixed at 4° C in paraformaldehyde fixative, and all samples were paraffin-embedded and sliced at the School of Life Sciences at Chongqing Medical University. After xylene dewaxing, the samples were dehydrated
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in ascending graded concentrations of ethanol, followed by haematoxylin and eosin
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(H & E) staining for general histological examination.
2.9 Statistical Analysis
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All statistical analysis was carried out using SPSS 21.0. The results are presented
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as mean ± SD (standard deviation). After confirming a normal distribution with the Shapiro-Wilk test and equal variances of the data, relative grip strength were analyzed
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using the two-way repeated measures ANOVA, and data from different treatment
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groups at each measurement time were compared using a multivariate ANOVA. Other data among multiple groups were compared using the one-way ANOVA followed by
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Turkey’s post hoc tests. P <0.05 was considered statistically significant.
3. Results
3.1 The effects of exercise, resveratrol and their combination on body weight, grip strength and muscle mass. As shown in Fig. 1A, at the beginning of the experiment, compared with the YC group, the aged rats among different groups were significantly heavier than the young rats, indicating that age was a key factor affecting body weight in rats. No significant
ACCEPTED MANUSCRIPT difference was found in weight among the aged rats in different groups. During interventions, the weight of the rats in the OR, OE and OER groups decreased, and there was a significant difference between the OE group and the OC group at the end of week five. At the end of week six, a significant difference was observed between
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the OR group and the OE group. Exercise, but not resveratrol, significantly reduced
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the weight of aged rats (P<0.05), the combination of exercise and resveratrol did not
SC
show an additive effect on weight loss.
According to previous report(Kim et al., 2016), in order to eliminate the effects
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of weight on grip strength, relative grip strength (grip strength / weight) was adopted
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in our research. As shown in Fig. 1B, during the experiment, the relative grip strength in YC group was significantly higher than that in the aged groups (P<0.05). During
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the first five weeks, there was no significant difference of relative grip strength among
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the OE, OR, OER and OC groups (P＞0.05). At the end of week six, the relative grip strength of the rats in the OE, OR and OER groups was significantly higher than that
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in the OC group (p <0.05), suggesting that exercise, resveratrol or their combination
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could increase the grip strength of aged rats. However, no significant difference was found among the OE, OR and OER groups (P＞0.05), suggesting that there was no synergistic effect of combined intervention on increasing rat grip strength. In the present study, Edstrom's SI (Sarcopenia Index) value was used to determine whether the model of sarcopenia was successful. The SI value is defined as the mass of the target skeletal muscle (mg) / subject weight (g) and used as an evaluation index(Edstrom and Ulfhake, 2005). When the SI value of the aged group
ACCEPTED MANUSCRIPT was significantly lower than that of the young group and the difference was greater than 2 times SD, sarcopenia was indicated(Baumgartner et al., 1998). As shown in Fig. 1C, compared with the YC group, the gastrocnemius SI value of OC rats was significantly lower (P<0.05) and decreased by 2 times SD, indicating that
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25-month-old SD rats can be used as sarcopenia model rats. Compared with the OC
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group, the SI value of rats in the OE and OER groups significantly increased
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(P<0.05), while the increasement of SI value in the OR group did not show a statistical significance (P＞0.05), indicating that exercise or exercise combined with
MA
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resveratrol could increase the muscle mass in aged rats.
3.2 Effects of exercise, resveratrol or their combination on myofibrils.
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Electron microscopy revealed that the myofibrils in the aged group of rats were
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sparsely arranged, in which were accompanied with fat infiltration, mitochondrial swelling and other changes (Fig. 2A). The lengths of the sarcomere, A-band, I-band
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and H-zone were measured in the present study, consistent with previous studies, as
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presented in Fig. 2B, compared with the YC group, the sarcomere length and I-band length were significantly increased in the OC group (P<0.05), whereas length of A-band and length of H-zone did not change (P＞0.05). Compared with the OC group, the sarcomere length, I-band length and H-zone length in the OE, OR and OER groups decreased significantly (P<0.05). There was no significant difference in A-band length between the groups. Besides, the sarcomere length in the OER group was significantly reduced compared with the OE and OR group (P<0.05), suggesting
ACCEPTED MANUSCRIPT that the combined intervention of exercise and resveratrol reduced the sarcomere length as a synergistic effect. Collectively, these results suggest that the myofibrils in aged rats were increased in sarcomere, I-band and H-band lengths. Exercise and resveratrol can improve the ultrastructural changes of myofibrils. Exercise and
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resveratrol combination show a synergistic effect on decreasing sarcomere length in
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aged rats, but not on I-band length and H-band length.
cross-sectional area and perimeter.
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3.3 Effects of exercise, resveratrol or their combination on Feret’s diameter,
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To assess the perimeter, cross-sectional area (CSA) and Feret’s diameter of single muscle fibres, H&E staining was conducted. As presented in Fig. 3A-B, compared
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with the YC group, it can be seen that age induced an increase in the perimeter, CSA
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and Feret’s diameter of single muscle fibres. Of note, only Feret’s diameter and CSA increasement reached statistical significance (P<0.05). Furthermore, compared with
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the OC group, OR group increased CSA significantly (P<0.05), while OE, OR or
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their combination did not show any significant increasement in the perimeter and Feret’s diameter (P＞0.05).
3.4 Effect of exercise, resveratrol or their combination on AMPK, Sirt1, p53, Bax and Bcl-2 protein expression To find out the possible molecular mechanisms of exercise, resveratrol or their combination on gastrocnemius muscle in rats, western blotting were conducted to
ACCEPTED MANUSCRIPT evaluate the protein expression of AMPK, p-AMPK, Sirt1, P53, Bax and Bcl-2. As demonstrated in Fig. 4A-B, compared with the YC group, the expression of p-AMPK, Sirt1 and Bcl-2 were significantly decreased and the expression of P53 and Bax were significantly increased in the OC group (P<0.05). Compared with the OC group, OE,
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OR and OER group increased significantly p-AMPK and Bcl-2 expression (P<0.05),
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and decreased significantly Bax expression (P<0.05). Of note, OE group did not
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significantly increase Sirt1 expression and decrease P53 expression (P＞0.05), but OR and OER group significantly increase Sirt1 expression and decrease P53
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expression compared with the OC group (P<0.05). Furthermore, the OER group
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increased significantly p-AMKP, Sirt1, Bcl-2 expression and decreased significantly expression of P53 and Bax compared with the OE and OR group (P<0.05), indicating
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that exercise and resveratrol combination may have a synergistic effect on molecular
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signaling expression. These results suggest that both individual exercise or resveratrol intervention can increase the expression of p-AMPK and Sirt1, thereby reducing the
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acetylation of p53 protein, inhibiting downstream Bax, and increasing downstream
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Bcl-2 to inhibit apoptosis. Moreover, the combination of exercise and resveratrol showed a synergistic anti-apoptotic effect.
3.5 Effect of exercise, resveratrol or their combination on mRNA expression of p53, Bax and Bcl-2 We next examined the mRNA expression of P53, Bax and Bcl-2 in all groups of rats. As shown in Fig. 5, consistent with our findings in western blotting, compared
ACCEPTED MANUSCRIPT with the YC group, the mRNA expression of Bax was significantly increased and the expression of Bcl-2 was significantly decreased in the OC group (P<0.05). Furthermore, exercise, resveratrol and their combination were found to significantly decrease the expression of Bax and increase the expression of Bcl-2 compared with
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the OC group (P<0.05) (Fig. 5B-C). Of note, the results of mRNA expression of P53
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were not in accordance with the protein expression of P53 in the present study. As
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presented in Fig. 5A, compared with the YC group, the P53 mRNA expression in the OC group was significantly decreased (P<0.05), and compared with the OC group,
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exercise or exercise combined resveratrol interventions significantly increased the
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mRNA expression of P53 (P<0.05). These conflicts between protein expression and mRNA expression could be explained by the protein post translational modification,
4. Discussion
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like deacetylation.
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The life expectancy of SD rats ranges from 30 to 36 months, 6-month-old rats
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are equivalent to 18-year-old human beings, and 24-month-old rats are equivalent to 60-year-old humans. In the present study, 25-month-old SD rats were adopted as aged rats, and the SI value of the aged group was significantly lower than that of the young group and the difference was greater than 2 times standard deviation, which indicates that the aged group could be regarded as model of sarcopenia. The clinical diagnosis of sarcopenia includes two aspects: skeletal muscle mass decline, and skeletal muscle weakness (Chen et al., 2016). In the present study, we adopt the SI value and the
ACCEPTED MANUSCRIPT relative grip strength to evaluate the skeletal muscle mass and function. Our data show that resveratrol, exercise or their combination significantly increase the relative grip strength, which indicates all interventions could ameliorate the skeletal muscle function. While for muscle mass, exercise and exercise combined resveratrol group
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significantly increased the SI value, there is a tendency for SI value to be elevated by
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dietary resveratrol supplementation, however the result does not reach statistical
SC
significance. Previous studies have shown that exercise can improve muscle loss by hindering apoptosis (Lenk et al., 2010), improving mitochondrial function (Zampieri
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et al., 2016), improving antioxidant capacity (Brioche and Lemoine-Morel, 2016),
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improving the level of inflammatory factors (Buford et al., 2009), and activating multiple pathways to promote protein synthesis and inhibit protein breakdown
D
(Osorio-Fuentealba et al., 2013, Bacurau et al., 2016, Ju et al., 2016, Fan et al., 2017).
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Among these mechanisms, the inhibition of apoptosis is a recent research hotspot, however, the molecular mechanism of exercise-induced apoptosis remains unclear.
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The effect of resveratrol on skeletal muscle in aged rats seems to be
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dose-dependent. According to the previous studies, 12.5 mg/kg/d and 50 mg/kg/d are too low to effect muscle mass and muscle function (Jackson et al., 2010, Jackson et al., 2011), while 125mg/kg/d resveratrol significantly attenuate the muscle wasting and increase the muscle function (Bennett et al., 2013). Resveratrol has antioxidant and anti-free radical properties, improves immune system activity, opposes thrombosis, provides cardiovascular and neurological protection, and exerts other pharmacological effects. Previous studies have shown that resveratrol increases the endogenous
ACCEPTED MANUSCRIPT antioxidant factor and reduces the peroxide loading in skeletal muscle cells, thereby reducing oxidative damage associated with muscle loading, unloading and ageing (Jackson et al., 2010, Ryan et al., 2010). Several in vitro studies have shown that resveratrol plays a role in inhibiting skeletal muscle fibrin degradation and improving
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atrophy (Wyke et al., 2004, Russell et al., 2006, Wyke and Tisdale, 2006, Alamdari et
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al., 2012), and previous studies have shown that resveratrol can improve the recovery
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of muscle wasting atrophy (Bennett et al., 2013). However, the effect of resveratrol on muscle function and its potential mechanism have not been established.
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With aging, the most obvious changes in muscle morphology is the reduction in
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the number of muscle fibres which ultimately induces the muscle volume reduction. What’s more, the capillaries in the muscle tissue decreased and the connective tissue
D
increased, which induce the dysfunction of muscle fibres. Myofibrils are the
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contraction units of skeletal muscle fibres, comprising thick muscle filaments (myosin) that form the dark band and fine filaments (actin) that form the light band. With aging,
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the myofibrils and muscle fibres are changed accordingly. To further clarify the
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underlying mechanisms leading to the sarcopenia, we evaluate the micro and ultrastructure of the gastrocnemius muscle. The performance of the CSA, the perimeter, the Feret's diameter, the sarcomere length, the I-band length and the H-band length were detected. Consistent with the previous study (Sayed et al., 2016), the lengths of the sarcomere, I-band and H-band are significantly increased with aging, no significant change was found in A-band length, it indicates the age-related contractile function loss in skeletal muscle. Exercise, resveratrol or their combination
ACCEPTED MANUSCRIPT ameliorate the muscle function are closely related to the decrease of lengths of the sarcomere, I-band and H-band. The CSA and the Feret's diameter in the aged group were significantly increased, this increase of muscle fibres size in aged rats could be a compensatory mechanism for reduction of the fibre number.
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Different mechanisms contributing to sarcopenia have been reported, include
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hormonal changes, inflammation, apoptosis, oxidative stress, etc. In the present study,
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we found exercise and resveratrol significantly increase the expression of AMPK and Sirt1, decrease the expression of acetylated p53, and increase the ratio expression of
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Bcl-2/Bas, suggesting that inhibition of apoptosis by resveratrol or exercise may be
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associated with the activation of the AMPK/Sirt1 pathway. Exercise and resveratrol is the activator of Sirt1. Sirt1 can inhibit p53 activity through deacetylation, and reduce
D
apoptosis induction by reducing p53 effect on downstream target gene activation
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(Zhang et al., 2014). P53 is a tumour suppressor gene, with more than 50% of p53 mutations detected in all malignancies (Romer et al., 2006). p53-encoded proteins are
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transcription factors that control cell cycle initiation. Many signals control the levels
AC
of p53 protein in healthy cells. If the cells are damaged and cannot be repaired, p53 will participate in the progression the cell cycle, eventually leading to cell death (Cakir et al., 2011, Fujita and Ishikawa, 2011, Kung and Murphy, 2016). Bcl-2 can prevent apoptosis-forming factors such as cytochrome C from mitochondria release, which
has
an
anti-apoptotic
effect,
Bax
interacts
with
mitochondrial
voltage-dependent ion channels, mediates cytochrome C release, and exerts apoptosis-related effects. P53 increases the expression of Bax, decreases the
ACCEPTED MANUSCRIPT expression of Bcl-2, and promotes apoptosis (Basu and Haldar, 1998).
5. Conclusions In summary, it seems apparent from our results that exercise could improve the
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muscle mass and strength of aged rats and improve the ultrastructure of aged
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myofibrils. Although resveratrol does not increase the quality of skeletal muscle, it
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could still improve the grip strength and ultrastructure of myofibrils and muscle fibres. Our results reveal that both exercise and resveratrol treatment might activate the
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AMPK/Sirt1 pathway to inhibit apoptosis, improving muscle quality and preventing
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sarcopenia. Furthermore, we find that exercise and resveratrol combined therapy may
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have a synergistic effect on improving sarcopenia.
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Acknowledgments This work was financially supported by grants from Chongqing Health and Family Planning Commission (No. 2015MSXM016); grants from
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Chongqing Development and Reform Commission (No. [2013]1420). This study was
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also supported by National Key Clinical Specialties Construction Program of China (No. [2013]544).
Conflict of Interest The authors declare that there are no conflicts of interest.
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Zampieri, S., Mammucari, C., Romanello, V., Barberi, L., Pietrangelo, L., Fusella, A., Mosole, S., Gherardi, G., Hofer, C., Lofler, S., Sarabon, N., Cvecka, J., Krenn, M., Carraro, U., Kern, H., Protasi, F., Musaro, A., Sandri, M., Rizzuto, R., 2016. Physical exercise in aging human skeletal muscle increases
ACCEPTED MANUSCRIPT mitochondrial calcium uniporter expression levels and affects mitochondria dynamics. Physiol Rep. 4. Zhang, Z.N., Chung, S.K., Xu, Z., Xu, Y., 2014. Oct4 maintains the pluripotency of human embryonic stem cells by inactivating p53 through Sirt1-mediated
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deacetylation. Stem cells. 32, 157-165.
ACCEPTED MANUSCRIPT Figure captions Fig. 1 Effects of exercise, resveratrol and their combination on body weight, relative grip strength and muscle mass. (A): Body weight of rats in different groups at different time. (B): Relative grip strength of rats in different groups at different time.
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(C): The SI value of gastrocnemius in different groups. Data were analyzed using a
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two-way ANOVA of repeated measures followed by Turkey’s post hoc tests (B), and a
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one-way ANOVA followed by Turkey’s post hoc tests (A, C). a P< 0.05 OC vs. OE, b P< 0.05 OC vs. OR, c P< 0.05 OC vs. OER, d P< 0.05 OR vs. OE, * P < 0.05 vs. YC,
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# P < 0.05 vs. OC. Results are expressed as the mean±SD; n = 10 animals/group.
Fig. 2 Effects of exercise, resveratrol and their combination on gastrocnemius
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myofibrils. (A): Representative electron micrographs of longitudinal sections of
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gastrocnemius myofibers from different groups. Magnification ×15,000. Scale bars represent 1 μm. n = 3 animals/group. (B): Morphometric analysis of sarcomere length,
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A-band, I-band and H-zone in five groups of rats. All data were analyzed using a
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one-way ANOVA followed by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P< 0.05 vs. OE, d P< 0.05 vs. OR, # P< 0.05 vs. OER. Results are expressed as the mean±SD.
Fig. 3 Photomicrographs of paraffin sections of cross sections of gastrocnemius muscle fibers in five groups. (A): Transverse sections of gastrocnemius muscle stained with hematoxylin and eosin. Magnification ×200. Scale bars represent 100 μm.
ACCEPTED MANUSCRIPT n = 3 animals/group. (B): Quantification of morphometrical measurements of the perimeter, Feret's diameter and CSA of the muscle fibers. All data were analyzed using a one-way ANOVA followed by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P< 0.05 vs. OE, d P< 0.05 vs. OR, # P< 0.05 vs. OER. Results are
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expressed as the mean±SD.
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Fig. 4 Effects of exercise, resveratrol and their combination on metabolic signaling in the gastrocnemius muscle. (A): Western blotting of AMPK, p-AMPK, SIRT1, Acetyl
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P53, Bax and Bcl2 protein levels in rat gastrocnemius muscle. GAPDH was used as
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the loading control. (B): Quantitative analysis of AMPK, p-AMPK, SIRT1, Acetyl P53, Bax and Bcl2 levels. All data were analyzed using a one-way ANOVA followed
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by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P < 0.05 vs. OE, d
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animals/group.
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P< 0.05 vs. OR, # P < 0.05 vs. OER. Results are expressed as the mean±SD. n = 6
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Fig. 5 Relative mRNA expression of p53、Bax and Bcl-2 (A-C). All data were analyzed using a one-way ANOVA followed by Turkey’s post hoc tests. a P< 0.05 vs. YC, b P< 0.05 vs. OC, c P < 0.05 vs. OE, d P< 0.05 vs. OR, # P < 0.05 vs. OER. Results are expressed as the mean±SD. n = 6 animals/group.
ACCEPTED MANUSCRIPT Table 1. Animals movement scheme. Speed (m/min)
Time (min)
1（Adaptive training）
5
5
2
15
15
3
20
25
4
25
5
25
6
30
7
30
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Week
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ACCEPTED MANUSCRIPT Table 2. Primers used for the RT-PCR analysis. Gene name
Sequence 5’-3’ (forward)
Sequence 5’-3’ (reverse)
AGATGTTCCGAGAGCTGAATGAG
TTTTTTATGGCGGGACGTAGA
Bax
GGTGGTTGCCCTTTTCTACTTTGC
GCTCCCGGAGGAAGTCCAGTG
Bcl2
GGGCTACGAGTGGGATACTGGAG
CGGGCGTTCGGTTGCTCT
GAPDH
TTCCTACCCCCAATGTATCCG
CCACCCTGTTGCTGTAGCCATA
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ACCEPTED MANUSCRIPT Highlights ·Exercise, resveratrol or combination increased muscle mass and function in aged rats.
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·Exercise and resveratrol ameliorated ultrastructure of muscle fibrils in aged rats.
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·Exercise and resveratrol prevented muscle apoptosis via AMPK/Sirt1 activation.