Immunological

Communication

Biosci. Biotech. Res. Comm. 9(4): 596-602 (2016)

Comparative immune regulatory effects of

Cordyceps

militaris

Flammulina velutipes

extracts along with

glutamine on rat’s body after exhaustive exercise

Xuewen Tian

1,2

, Qipeng Song

1,2

, Ming He

, Yingli Lu

, Yancheng Liu

and

Lianshi Feng

3,1

School of Kinesiology, Shanghai University of Sport, Shanghai 200438

Shandong Institute of Sport Science, Jinan, Shandong 250102

Biology Center, China Institute of Sport Science, Beijing 100061, P.R. China

ABSTRACT

Exhaustive exercise can impair immune balance in the rat’s body due to change of Th1- and Th2-related cytokines,

and some natural products can regulate immune banlance. The aim of this study is to compare the immunoregula-

tion action of Cordyceps militaris cordycepin, Flammulina velutipes polysaccharide, and glutamine on rats that were

subjected to an intense running session by analyzing Th1- and Th2-related cytokines and transcription factors. All

rats were subjected to the exercise protocol except for a subgroup rat that was used as the negative control group.

Blood samples were collected for the evaluation of biochemical parameters. The protein expression of cytokines and

transcription factors were examined by ELISA and Western blot. Meanwhile, the mRNA expression of the cytokines

and transcription factors in the spleen of rats was assessed via real-time PCR Furthermore, the ratio of Th1/Th2 was

determined via  ow cytometry. The blood analysis results show that cordycepin was more effective than polysaccha-

ride and glutamine in regulating all kind of indexs. Three immunomodulator could adjust the expression of the tran-

scription factors and cytokines to normal levels, whereas Flammulina velutipes polysaccharide and glutamine could

only induce several of these transcription factors or cytokines. Thus, this study implies that the action of cordycepin

had the most signi cant effect on the immunoregulation of rats that were subjected to exhaustive exercise.

KEY WORDS: CORDYCEPIN;

FLAMMULINA VELUTIPES

POLYSACCHARIDE; GLUTAMINE; EXHAUSTIVE EXERCISE; IMMUNOREGULATION

596

ARTICLE INFORMATION:

*Corresponding Author: 52169180@qq.com

Received 26

Oct, 2016

Accepted after revision 12

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Xuewen Tian et al.

The dual effect of exercise on the immune system has

been previously studied in great detail, and many epi-

demiological studies have demonstrated that moderate

exercise improves the immune function and increases

resistance to infections. On the other hand, exhaus-

tive exercise had opposite effects(Dos Santos, et al.,

2009, Nieman, 2007), most of which have been asso-

ciated with an increased incidence of respiratory tract

infections(Nieman, 1997). The cytokine from the T cell

has a critical function in the development of immune

responses against invading pathogens. The cellular basis

for this heterogeneity in cytokine is due to the presence

of two distinct cytokine-producing CD4+ T helper and

CD8+ T cytotoxic cell phenotypes. The CD4+ T helper

has been designated as Th1 and Th2 cells based on their

distinct cytokine pro le(Mosmann, et al., 1986). Th1

cells secrete IFN-, IL-2, and tumor necrosis factor-,

and are responsible for defense against intracellular

pathogens, whereas T h2 predominantly secrete IL-4,

IL-5, and IL-13, and are responsible for defense against

extracellular pathogens (Schoenborn, et al., 2007, Seder,

et al., 1994, Kutukculer, et al., 2016).

The imbalance of the Th1/Th2 ratio, which is attrib-

uted to exhaustive exercise, can cause an imbalance

in the immune function of athletes. Thus, researchers

should use special drugs to recover the Th1/Th2 ratio of

athletes that are prone to exhaustion.

In terms of effector T-cell differentiation, Th2 cells

development was the  rst process to be linked to the

actions of a cytokine. IL-4 was recognized early on to

promote the development of Th2 cells subset. Later, this

activity was shown to operate through the actions of

STAT6. Th cells stimulated with interleukin-4 (IL-4) and

antigen, through the T-cell receptor (TCR), upregulate

GATA3 transcription. The GATA3 protein induces herita-

ble remodelling of the IL-4 locus, which is characteristic

of fully differentiated Th2 cells. Th cells activated under

Th1-inducing conditions are exposed to IFN- signal-

ling during TCR engagement, leading to the activation

of signal transducer and activator of transcription 1

(STAT1). T-bet was identi ed as a Th1 cells speci c fac-

tor that can induce the production of IFN- by develop-

ing Th2 cells. T-bet seems to be expressed in developing

and committed Th1 cells (Murphy, et al., 2002, Tian, et

al., 2015 and Kutukculer, et al., 2016).

Thus, the change of Th1 and Th 2 number can through

the levels of these transcription factors. Cordyceps mili-

taris is a traditional Chinese medicinal mushroom that

has been shown to have a variety of bene ts on human

health, such as antitumor, antimutagenic, and hypo-

glycemic effects (Thomadaki, et al., 2008). Cordycepin

from Cordy ceps militaris is involved in many phar-

macological activities, which include immunological

stimulation, anticancer, antivirus, and anti-infection

activities(Nakamura, et al., 2005). Recently, the level of

several cytokines from Th1 and Th2 has been reported

to all increase after mouse splenocytes were exposed

to puri ed cordycepin (Jeong, et al., 2012, Tian, et al.,

2015 and Kutukc uler, et al., 2016 ).

Flammulina velutipes is as an edible and medical

resource, and many of its bioactive molecules have been

identi ed. Polys accharides are the best known and the

most potent Flammulina velutipes, from which sub-

stances with antitumor and immunomodulating proper-

ties can be derived(Wasser, 2002). In addition, studies

have shown a decrease in plasm a gluta mine concentra-

tion after exhaustive exercise in humans and animals,

(Castell, 2003). These small decre ases in the plasma glu-

tamine concentration are suf cient to promote immu-

nosuppression ( Hiscock, et al., 2002 Dos Santos, et al.,

2009 and Harriss, et al., 2011 and Tian, et al., 2015).

The objective of the present study want to use some

sensitive biochemical criterions that can response

immune injury and recover after injury to compare that

cordycepin, polysaccharide from  ammulina velutipes,

and glutamine, which are natural medicine with the pos-

sible of immunomodulatory effect on rat’s body after a

high-intensity running session.

MATERIAL AND METHODS

CONSTRUCTION OF EXHAUSTIVE ANIMAL

MODEL

A total of 50 eight-week-old Sprague Dawley (SD)

(160–200 g) rats were purchased from Shandong Uni-

versity. The rats were routinely screened for common

rat pathogens. Immediately after arrival, the rats were

weighed and randomly divided into  ve subgroups (n =

10) as follows: control group (C; the group was not sub-

jected to the exercise protocol); exhau stive exercise (EE;

after exercise, all rats were perfused with 2 mL of ster-

ile saline); cordycepin (CM; after exercise, all rats were

perfused with 2 mL of aqueous solution of cordycepin

based on a dosage of 400 mg/kg weight); Flamm ulina

velutipes (FV; after exercise, all rats were perfused with

2 mL of aqueous solution of Flammulina velutipes poly-

saccharide based on a dosage of 150 mg/kg weight) and

glutamine (G; after exercise, all rats were perfused with

2 mL of aqueous solution of glutamine based on a dos-

age of 100 mg/kg weight). Except for the C subgroup, all

rats were accustomed to running on a rodent treadmill

based on the exercise protocol, as shown in Table 1. On

the tenth week, the exercise was stopped. All animals

were killed by decap itation after having recovered on

the tenth week. All animal procedures were approved by

the Sports Science Research Center of Shandong Prov-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARATIVE IMMUNE REGULATORY EFFECTS OF

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EXTRACTS 597

Xuewen Tian et al.

ince Animal Investigational Committee, and were per-

formed in accordance with the Guide for the Care and

Use of Laboratory Animals published by the Ministry of

Health of People’s Republic of China and IJSM’s ethical

standards document(Harriss, et al., 2011), This study has

been approved by the ethics committee of Sports Science

Research Center of Shandong Province.

HEMAT OLOGICAL INVESTIGATIONS

Blood samples were collected from the killed rats for

hematological investigations. The param eters investi-

gated were leucocyte count, red blood cell count, hemo-

globin content, hematocrit value (PCV), creatine kinase,

and urea nitrogen(Silva, et al., 2007, Spiess, et al., 1998,

Tilak, et al., 2007).

QRT-PCR

Total RNA was isolated from less than 100 mg of splee n

tissue by using a TRI-zol Reagent (TaKaRa) according

to the manufacturer’s instructions. cDNA was obtained

according to Wang’s method(Wang, et al., 2011). The

cDNA product was stored at -20 °C until use. The oli-

gon ucleotides for real-time PCR (Table 2) were designed

using the Primer Premier 5.0 software (PREMIER Biosoft

International). BLAST analysis was then performed

against other organism genome sequence for speci city

con dence (http://www.ncbi.nlm.nih.gov/BLAST/). The

Mfold web server was used to avoid positioning on risky

secondary structures. Primer speci city was analyzed

before qRT-PCR.

The reaction mix ( nal volume of 20 μL) consisted

of 10 μL of SYBR Ex Taq II, 0.4 μL of ROX Reference

Dye, 0.8 μL of each primer ( nal concentration of 250

nM), 7 μL of ddH

O, and 1 μL of cDNA (dilution factor

of 1/10). The thermocycling program consisted of two

phases, which include one cycle of 94 °C for 3 min, 40

cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30

s. After completion of these cycles, the melting-curve

data were obtained to verify PCR speci city, contami-

nation, and the absence of primer dimers. Each sample

was tested in triplicates in an ABI 7300 real-time PCR

apparatus (Applied Biosystem). The relative expression

levels of MTs were normalized against the GAPDH gene

as an internal standard. The relative quantity levels of

MT mRNA expression were obtained using the formula

−ΔΔCt

ELISA ASSAYS

The tested rats were killed by breaking their necks on

the tenth week after a week of rest. Their serum were

collected from whole blood and immediately frozen at

-80 °C for ELISA assays. IL-4 and IFN- were meas-

ured using ELISA kits according to the manufacturers’

instructions (Dakewe Biotech Co. Ltd., China). Microti-

tration plates w ere coated overnight with 100 μL of a

10 μg/mL solution of anti-IL-4 or IFN- fragments in a

carbonate buffer (pH 9.6). After blocking and washing,

100 μL of the tissue supernatant was incubated for 1 h

at room temperature. The plates were washed and incu-

bated with peroxidase-linked anti-IL-4 or IFN- frag-

ments for 1 h at room temperature. After washing, 3,

3’, 5, 5’-tetramethylbenzidine (TMB) was added to the

plates for color development. The reaction was stopped

after 15 min by adding 20 μL of a 1:20 sulfuric acid

solution. The abs orbance at 450 nm was measured using

an ELISA plate reader. Duplicate readings were taken for

all samples, and the means were calculated. The cut-off

was determined using the values from the control group.

WESTERN BLOT

Western blot analysis tissue were lysed with sample

buffer [62.5 mmol Tris-HCl, pH 6.8, 2% sodium dodecyl

sulfate (SDS), 10% glycerol, 50 mmol dithiothreitol, and

0.1% bromophenol blue] and heated at 100°C for 5 – 10

min before being loaded and separated on 10% SDS-pol-

yacrylamide gels. The proteins were electrotransferred to

a nitrocellulose membrane in transfer buffer containing

48 mmol Tris-HCl, 39 mmol glycine, 0.037% SDS, and

20% methanol at 4°C for 1 h. Non-speci c binding to

the membrane was blocked for 1 h at room temperature

with blocking buffer [5% non-fat milk in Tris-buffered

saline (TBS), and 0.1% Tween 20]. The membranes were

incubated for 16 h at 4°C with various primary antibod-

ies (STAT6, STAT1, GATA3, T-bet and Actin) in blocking

buffer at the dilutions speci ed by the manufacturers,

followed by incubation with horseradish peroxidase–

conjugated secondary antibody for 1 h. Membranes were

then washed, and visualized using the enhanced chemi-

luminescence system.

FLUORESCENT LABELING AND FLOW

CYTO METRY

Whole blood (200 μL ) was directly pipetted into a 12×75

mm  uorescen ce-activated cell sorting tube containing

20 μL of monoclonal antibodies for the T-helper surface

antigen CCR5 and CCR 8 (CD4-PerCP, Bec ton Dickinson,

San Diego, CA), and was then incubated at room tem-

perature in the dark for 10 min. About 0.5 mL of 1% par-

aformaldehyde was then added for 8 min to stabilize the

monoclonal antibody-surface antigen complex. RBCs

were lysed using 3 mL of a 1× uorescence-activated cell

sorting lysing solution (Becton Dickinson) for 8 min.

After centrifugation at 1,930 rpm for 5 min, the super-

natant was aspirated. A 1×permeab ilizing solution (500

598 COMPARATIVE IMMUNE REGULATORY EFFECTS OF

CORDYCEPS MILITARIS

FLAMMULINA VELUTIPES

EXTRACTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Xuewen Tian et al.

μL, Becton Dickinson) was added into the pellet, and the

solution was incubated for 10 min at room temperature

in the dark. After washing with 3 mL of the buffer (1%

bovine serum albumin, 0.1% NaN

, 1×PBS), cytokine-

speci c antibodies (20 μL, CCR5-FITC, CCR8-PE, Becton

Dickinson) were added to the cells and incubated for 30

min at room temperature in the dark. After one  nal

wash, the cells were resuspended in 1% paraformalde-

hyde (500 μ L) and stored at 4 °C until  ow cytometry

analysis. The cells were obtained using a Beckman-

Coulter EPICS XL  ow cytometer (Miami, FL), and the

data were analyzed using the CellQuest software. The

percentages of Th1 and Th2 cytokine-producing cells

were identi ed as the number of CCR5- and CCR8-posi-

tive cells present, respectively, in the total population of

CD4+ T-helper cells(Chanova, et al., 2016) A minimum

of 5,000 CD4+ cells was counted from each sample.

STATISTICAL ANALYSIS

All results are presented as mean ± SD. The data were

evaluated using the SPSS software (SPSS company,

USA). Statistical differences were determined using Stu-

dent’s t-test with a signi cance level set at P <0.05 or

P <0.01.

RESULTS

CHANGES OF THE HEMATOLOGIC

PARAMETERS

The blood parameters mentioned in the above se ction

between the C and EE subgroups show a signi cant dif-

ference (P<0.05 or P<0.01). The results of the EE sub-

gro up show an imbalance in the immune function of

the rats. CV subgroup could signi cantly elevate RBC

quantity, Hb concentration, and PC V to normal levels (C

subgroup) when compared with FV or G subgroups after

the tenth week (P<0.05 or P<0.01). However, these three

blood parameters between FV and G subgroups also had

an obvious increasing, as shown in Table 3. The leuco-

cyte counts were 4.97±0.27×109 and 4.77±1.84×109/mL

in the CM and G subgroups, respectively. These results

suggest that the regulating ability of cordycepin and

glutamine on leucocyte restoration was lower than that

of Flammulina velutipes polysaccharides, which elevated

the leucocyte quantity to normal levels (6.20±0.85×109/

mL) on the tenth week. The blood parameters’ analysis

demonstrated that cordycepin was more effective than

Flammulina velutipes polysaccharides and glutamine

in alleviating the condition of the rats from exhaus-

tive exercise because estimating the fatigue of athletes

is usually determined by detect i ng serum urea nitrogen

(BUN) and creatine kinase(CK) (indicators of muscle

damage)(Haralambie, 1973, Shi, 2005).

As data of Table 3 show, although the concentration

of urea nitrogen and creatine kina se for CV, FV and G

subgroups that all decreased when compared to the EE

subgroup, whereas Flammulina velutipes polysaccharide

(7.78±0. 27mmol/L) or glutamine (7.99±0.50mmol/L)

is effective than cordycepin (8.99±0.15 mmol/L) in

decreasing BUN (P<0.0 5 or P<0.01), but cordycepin

(166.00±10.84U/L) is more effective than Flammulina

velutipes polysaccharide (231.00±17.87U/L) or glu-

tamine (206.33±18.96U/L) in decreasing CK (P<0.05 or

P<0.01). These results show that three immunomodula-

tor could repair the damaged muscle tissue, as well as

reduce the release of creatine kinase from cells. Their

treatment effects for tissue damage were very ideal, but

the more effective treatment was cordycepin.

CHANGE OF TH1- AND TH2-RELATED

CYTOKINES AND TRANSCRIPTION FACTORS

The production of cytokines associated with Th1 and Th2

cells in the serum was evaluated via ELISA. As shown in

Table 3, we observed that level IFN- did icrease after

running if the rats were treated with cordycepin, Flam-

mulina velutipes polysaccharide and glutamine. Addi-

tionally, CM subgroups signi cantly increased the level

of IL-4 compared to the EE subgroup.

The mRNA expression o f T h1- and Th2-related tran-

scription factors and cytokines in t he spleen of rats

was determined by real-time PCR assay (Fig. 1). After

a week of rest, the expressions of IL-4, GATA3, T-Bet,

IFN-, STAT1, and STAT6 in the EE subgroup remained

higher than those in the C subgroup, especially STAT1

and STAT6. However, the expression levels of STAT1 and

STAT6 in the CM, FV, and G subgroups were already

close to those of the C subgroup. The mRNA levels of

T-bet and GATA-3 in the CM, FV, and G subgroups also

signi cantly decreased. But the mRNA concentrations

of IFN- and IL-4 in the FV and G subgroups remained

close to or higher than those of the EE subgroup, respec-

tively. These results sugg est that cordycepin was more

effective in regulating Th1- and Th2-related transcrip-

tion factors than Fl ammulina velutipes polysaccharide

and glutamine.

The level of the transcription factors expression asso-

ciated with Th1 and Th2 cells in the spleen was evalu-

ated via western blot, show in Fig. 2. After a week of

rest, the protein expressions of STAT6 and T-bet sig-

ni cantly increased, whereas the protein expressions of

STAT1 and GATA3 were decreased rema rkablely in the

EE subgroup. CM subgroup can reduce the expressions

of STAT6 and T-bet, in addition to elevate the expres-

sions of STAT1 and GATA3. Four types of transcription

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS COMPARATIVE IMMUNE REGULATORY EFFECTS OF

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EXTRACTS 599

Xuewen Tian et al.

factors were mostly close to C subgroup. However, only

the protein levels of STAT6 signi cantly decreased in

FV and G subgroups, another level of the transcription

factors did not change toward nomal levels(C subgroup).

CHANGE OF TH1 AND TH2 CELLS BALANCES

The amounts of CCR5 and CCR8 could be used to esti-

mate the number of Th1 and Th2 cells because CCR5

and CCR8 are characteristic of T h1 and Th2 cells,

respectively(Kutukculer, et al., 2016). In the present

study, Th1- and Th2-related cytokines, namely, CCR5

and CCR8, in the whole blood of rats were analyzed

via  ow cytometry. The CCR5 and CCR8 results for the

 ve subgroups are summarized in Table 5, and were

calculated to assess the regulating effect of cordycepin,

Flammulina velutipes polysaccharides and glutamine.

Under the effect of cordycepin, the ratio of CCR5/CCR8

(0.155±0.043) was mostly close to the normal level

(0.163±0.032) than those of Flammulina velutipes poly-

saccharides and glutamine that also down-regulated the

ratio of CCR5/CCR8.

DISUSSION

Over the past two decades, many accumulated evidence

have demonstrated the diverse responses of T1 and T2

cells to exhaustive exercise or long-term training at

moderate and high intensities(Zhao, et al., 2012). Stud-

ies on the immune-regulating effect of natural prepara-

tions such as Herbkines, WooKiEum, Bouum-Myunyuk-

Dan, Cool-Cool, Pelargonium sidoides, and s o on have

been previously reported(Luna, et al., 2011, Shin, et al.,

2004). Cordycepin, Flammu lina velutipes, and glutam ine

were also used in regulating the immune system(Ike, et

al., 2012, Jeong, et al., 2012, Pohlenz, et al., 2012). In

the present study, the regulating ability of cordycepin,

Flammulina velutipes polysaccharide, and glutamine on

the immune system was compared and analyzed after

the rats showed signs of immunity imbalance caused by

exhaustive exercise.

The quick recovery of the immune imbalance of ath-

letes caused by exhaustive exercise is a common strat-

egy to prevent upper respiratory tract infections (URTIs)

after the athletes have terminated their short-term

intense training. In this study, the concentration level of

serum urea nitrogen and creatine kinase in the EE sub-

group remained higher than those of the C subgroup on

the tenth week. The nitrogen and creatine kinase results

show that the fatigue level of rats in the EE subgroup

remained high because of exhaustive exercise (Lee, et

al., 2010). Cordycepin, Flammulina velutipes polysac-

charides, and glutamine could regulate the concentra-

tion of urea nitrogen and creatine kinase, as shown from

the results. Thus, cordycepin was more effective than

Flammulina velutipes polysaccharides and glutamine in

alleviating fatigue. Similarly, cordycepin also showed a

remarkable ability in regulating RBC, Hb, and PCV. The

induction of Flammulina velutipes polysaccharides on

leukocytes was more remarkable than that of cordycepin

and glutamine.

The JAK-STAT pathway is the major signaling path-

way that regulates Th1 and Th2 differentiation and

functions (Tian, et al., 2015). IFN- reduction was sup-

pressed by down-regulating the STAT1/STAT4/T-bet

pathway, which is critical for Th1 differentiation, as

well as the GATA3/STAT6 pathway, which is essential

for Th2 differentiation(Jia, et al., 2011, Murphy, et al.,

2002). Our results con rm that cordycepin was more

effective in regulating Th1- and Th2-related transcrip-

tion factors than Flammulina velutipes polysaccharides

and glutamin e. Thus, cordycepin could quickly regulate

Th1- and Th2-related transcription factors to normal

levels.

Studies have suggested that cordycepin puri ed

from Cordyceps militaris could up-regulate the level of

Th1 cytokine, IL-12, and Th2 cytokines, IL-4 and IL-

10(Jeong, et al., 2012). The production of cytokines in

rats with appropriate complementary glutamine was

enhanced, and symptoms of upper respiratory tract

infection decreased(Dos Santos, et al., 2009). However,

the CM, FV, and G subgroups could all adjust the pro-

tein levels to normal level. But our results suggest that

cordycepin can more effective restore immune balance

that the levels of IL-4, GATA3, T-Bet, IFN-, STAT1, and

STAT6 were mostly close to C subgroup.

In addition, we also assessed the change in the num-

ber of Th1 and Th2 cells in the whole blood of rat via

 ow cytometry. Flow cytometric detection is a functional

assay that measures the ability of speci c immune cells

to express type 1 and type 2 cytokines after polyclonal

stimulation with mitogens(Jung, et al., 1993, Pala, et al.,

2000, Prussin, 1997). Two functionally distinct T-helper

lymphocyte subsets are distinguished by their signature

cytokines as follows: CCR5 for Th1 lymphocytes and

CCR8 for Th2 lymphocytes(Kutukculer, et al., 2016). We

con rmed that cordycepin quickly adjusted the ratio

of CCR5/CCR8

into a normal level (C subgroup level) a

week after the rats ended their exercise. A similar effect

was also observed in Flammulina velutipes polysaccha-

rides and glutamine.

Based on the above analysis, we propose that

cordycepin functions as a better modulator of immune

imbalance than Flammulina velutipes polysaccharides

and glutamine in rats that were subjected to exhaustive

exercise by regulating Th1- and Th2-related cytokines

and transcription factors. However, the regulation

pathway of T helper cell differentiation induced by

600 COMPARATIVE IMMUNE REGULATORY EFFECTS OF

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Xuewen Tian et al.

cordycepin and the ef ciency of the two or more food

mixing for immunoregulation requires further research.

ACKNOWLEDGEMENTS

This work was supported by grants from the Natu-

ral Scienti c Foundation of Shandong Province,

China (ZR2010CQ031 and ZR2014CM046), graduates’

innovation project of Shanghai University of Sport

(yjscx2015020) ,the Science Foundation of China Insti-

tute of Sport Science (CISS)(No.16-18 and No.16-24)

and Shanghai Key Lab of Human Performance, Shang-

hai University of Sport (grant no. 11DZ2261100).

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