Hindawi Publishing Corporation

BioMed Research International

Volume 2016, Article ID 5387258, 12 pages
[Link]

Research Article
Effects of 8-Week Hatha Yoga Training on Metabolic and
Inflammatory Markers in Healthy, Female Chinese Subjects:
A Randomized Clinical Trial

Neng Chen,1 Xianghou Xia,2 Liqiang Qin,1 Li Luo,3 Shufen Han,1

Guiping Wang,4 Ru Zhang,1 and Zhongxiao Wan1,5
1
Department of Nutrition and Food Hygiene, School of Public Health, Soochow University, 199 Renai Road, Suzhou 215123, China
2
Department of Breast Surgery, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China
3
School of Physical Education and Sports Science, Soochow University, Suzhou 215006, China
4
Laboratory Animal Center, Soochow University, 199 Renai Road, Suzhou 215123, China
5
Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Disease, Soochow University, 199 Renai Road,
Suzhou 215123, China

Correspondence should be addressed to Zhongxiao Wan; zhxwan@[Link]

Received 23 January 2016; Accepted 30 June 2016

Academic Editor: Edward J. Ryan

Copyright © 2016 Neng Chen et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We aimed to determine the effects of an 8 wk Hatha yoga training on blood glucose, insulin, lipid profiles, endothelial microparticles
(EMPs), and inflammatory status in healthy, lean, and female Chinese subjects. A total of 30 healthy, female Chinese subjects were
recruited and randomized into control or yoga practice group. The yoga practice included 8 wks of yoga practice (2 times/wk) for a
total of 16 times. Fasting blood samples were collected before and after yoga training. Plasma was isolated for the measurement
of lipid profiles, glucose, insulin, EMPs, and inflammatory cytokines. Whole blood was cultured ex vivo and stimulated with
lipopolysaccharide (LPS) and Pam3Cys-SK4. Peripheral blood mononuclear cells (PBMCs) were isolated for the measurement of
TLR2 and TLR4 protein expression. Yoga practice significantly reduced plasma cholesterol, LDL-cholesterol, insulin levels, and
CD31+/CD42b− EMPs. Cultured whole blood from the yoga group has reduced proinflammatory cytokines secretion both at
unstimulated condition and when stimulated with Pam3Cys-SK4; this might be associated with reduced TLR2 protein expression
in PBMCs after yoga training. Hatha yoga practice in healthy Chinese female subjects could improve hallmarks related to MetS; thus
it can be considered as an ancillary intervention in the primary MetS prevention for the healthy population. This trial is registered
with ChiCTR-IOR-14005747.

1. Introduction the glucose signal in healthy subjects. Bhattacharya et al. [6]

found that yoga practice can improve the antioxidant status
Yoga is a mind-body therapy that has become increasingly of the healthy individual.
popular worldwide. Accumulating evidence suggests that Endothelial microparticles (EMPs) are complex vesicular
yoga meditation could improve risk factors associated with structures shed from endothelial cells in response to stimuli
metabolic syndrome (MetS) such as obesity, disordered lipid such as inflammatory activation [7]. They are now considered
profile [1], and insulin resistance [2–4]. However, most of as novel biomarkers of endothelial activation and damage that
these studies are conducted in clinical populations [1–4] are increased in overweight/obese individuals at risk for MetS
and there are surprisingly few studies examining how yoga [8, 9]. Evidence suggests that EMPs change with alterations
training affects MetS’ related risk factors in healthy subjects in physical activity (PA) [10–12]. For example, reduced
[5, 6]. In regard to this, Manjunatha et al. [5] reported that 5 daily PA by taking <5,000 steps/day with a total of 5 days
days of yoga asanas increased the sensitivity of the 𝛽 cells to resulted in elevated CD31+/CD42b− EMPs in recreationally
2 BioMed Research International

active men [12]. Similarly, enforced physical inactivity by antibody against TLR2 (cat. number 12276) was from Cell
subjecting healthy men to 7 days of dry immersion also led to Signaling Technologies (MA, USA). TLR4 antibody (cat.
increased circulating CD31+/CD41− EMPs [10]. In contrast, 6 number MAB1478) was from R&D Systems (NE, USA). All
months of supervised aerobic exercise training with moderate other chemicals were purchased from Sigma (MO, USA)
intensity could improve circulating EMPs levels as evidenced unless otherwise noted.
by decreased CD31+/CD42− EMPs in sedentary African
American adults [11]. As yoga practice altered the blood flow 2.2. Trial Design and Changes after Trial Commencement.
velocity and consequently shear stress at the vascular wall This investigation reports a single-arm parallel-randomized
[13], presumably it could affect EMPs. However, currently, controlled trial comparing the effects of 8 weeks of yoga
there is no evidence whether yoga practice could affect EMPs, intervention on metabolic and inflammatory markers in
especially in Chinese subjects.
healthy female subjects. Ethical approval was obtained
Inflammation is one of the key mechanisms involved in from the Human Research and Ethical Committee of the
the pathogenesis of MetS [14]. Presently evidence examining Soochow University and all participants provided signed
effects of yoga on inflammatory processes is limited. Existing informed consent. All methods were performed following the
evidence suggests that yoga could positively affect circulating approved guidelines and regulations. This trial was registered
inflammatory markers in heart failure patients [15, 16], breast in the Chinese Clinical Trial Registry with the number
cancer survivors [17, 18], and patients with chronic inflamma- ChiCTR-IOR-14005747 on December 27, 2014. No changes
tory diseases and overweight/obese subjects [19]. Meanwhile, to the methodology occurred following trial commencement.
mind-body interventions that include some elements of yoga The data were reported according to the CONSORT state-
can reduce inflammatory signaling through NF-𝜅B pathway ment [26].
[17, 20, 21]. Toll-like receptors (TLRs), especially TLR2 and
TLR4, play critical roles in innate immunity and may be 2.3. Participants, Eligibility Criteria, and Settings. This study
involved in the link between physical activity, inflammation, was conducted at School of Public Health, Soochow Uni-
and MetS [22–24]. However, it remains unclear whether yoga versity, Jiangsu Province, China. Participants were recruited
practice could affect circulating TLR2 and/or TLR4 response from the Campus of Soochow University via poster advertise-
in healthy subjects. ment. The study inclusion criteria included age 18–25 years
Hatha yoga is the most commonly practiced worldwide. old; BMI > 18.5 and <23.9 kg/m2 ; the blood glucose, triacyl-
The key components of Hatha yoga are stretching exercises glycerol, cholesterol, HDL-C, LDL-C, systolic blood pressure
and physical postures, breath control, and concentration and (SBP), and diastolic blood pressure (DBP) being within the
thinking techniques designed to promote the well-being of normal ranges; and self-reported regular menstrual cycles
the subjects both physically and emotionally [25]. With the (i.e., cycle 24–36 days long and at least 10 cycles in the
above points in mind, the primary aim of the present study previous 12 months). The exclusion criteria included subjects
is to determine the effects of an 8 wk Hatha yoga practice having history for using of pharmacologic contraceptives
on metabolic markers including blood glucose, insulin, lipid (past 6 months) and history of breast cancer, heart dis-
profiles, blood pressure, and EMPs in healthy, lean, and eases, diabetes mellitus, or other serious medical conditions
female Chinese subjects; the secondary aim is to determine and subjects suffering from musculoskeletal conditions that
the effects of Hatha yoga practice on inflammatory markers in would prevent participation in a yoga training.
the above subjects via measuring circulating cytokine levels,
LPS, and Pam3Cys-SK4 (Pam) stimulated cytokines secretion 2.4. Interventions. A total of 30 female subjects were
in whole blood culture ex vivo, as well as TLR2 and TLR4 recruited and randomized into control or yoga practice
protein expression in PBMCs. group. Participants in the yoga group were then asked to
attend supervised Hatha yoga sessions 2 times per week
2. Materials and Methods over the 8 wks of the study. Yoga classes were offered on
Monday and Thursday every week (from 6 p.m. to 7 p.m.).
2.1. Materials. RPMI-1640, LPS (cat. number L6529-1) and Each class has a total of 60 minutes and had the following
900 nm Latex beads carboxylate modified polyester (CLB9) components: breathing exercise (6 mins); loosening exercise
were from Sigma (MO, USA). A custom human Adipokine (i.e., corn tree pose) (10 mins); standing poses (i.e., warrior
Panel 2 (cat. number HADK2MAG-61K) containing primary pose and mountain pose) (8 mins); supine poses (i.e., bridge
and detection antibodies for interleukin- (IL-) 6, IL-8, IL- pose and dolphin plank pose) (8 mins); prone poses (i.e., hare
1𝛽, monocyte chemoattractant protein- (MCP-) 1, tumor pose and locust pose) (8 mins); sitting poses (i.e., staff pose
necrosis factor- (TNF-) 𝛼, and insulin was purchased from and hero pose) (8 mins); relaxation/corpse pose (6 mins);
Merck Millipore (MA, USA). Pam (cat. number tlrl-pms) and seated meditation (6 mins). Approximately 32 minutes is
was from InvivoGen (CA, USA). Human IL-6 (cat. number spent in active poses. The classes were held in a yoga training
DY206), IL-1𝛽 (cat. number DY201), and TNF-𝛼 (cat. number room and taught by a registered, specialized yoga instructor.
DY210) ELISA kit (DuoSet) was from R&D Systems (NE, The yoga practice was specifically designed for this study;
USA). Antibodies against CD42b-PE (cat. number 555473), however the yoga classes were not observed by study staff.
CD31-FITC (cat. number 555445), and CD62E-APC (cat. Subjects were also instructed to maintain their usual physical
number 551144) were from BD Biosciences (NJ, USA). An activity and dietary habits for the study.
BioMed Research International 3

2.5. Primary and Secondary Outcomes. On day 1 of the study frozen plasma samples were thawed at room temperature
and 2 days after the whole yoga practice, subjects reported for 20 minutes and centrifuged at 1500 g for 15 minutes.
to the laboratory after an overnight fast; a baseline and a The top two-thirds of plasma were then further centrifuged
final blood sample (10 mL), respectively, were obtained by at 1500 g for another 15 minutes to obtain platelet-poor
venipuncture from an antecubital vein and collected into plasma. The top 100 𝜇L of platelet-poor plasma was then
EDTA tubes. Blood (9 mL) was centrifuged at 1500 g for incubated with fluorochrome labeled antibodies specific for
10 mins at 4∘ C and plasma was immediately frozen at −80∘ C PE-CD42b, FITC-CD31, and APC-CD62E for 20 minutes
for subsequent batch analyses of plasma cytokines, clini- in the dark at 4∘ C. Samples were then fixed with 93 𝜇L of
cal biomarkers (i.e., insulin, glucose, triacylglycerol (TG), 2% paraformaldehyde and diluted up to 500 𝜇L with sterile,
HDL-cholesterol, LDL-cholesterol, and total cholesterol), and 0.2 𝜇M filtered PBS and analyzed on a FC500 Beckman
endothelial microparticles. About 1 mL blood was utilized Coulter (CA, USA). A microparticle size gate was determined
for whole blood culture. The height, body weight, SBP, and using 900 nm Latex beads carboxylate modified polyester.
DBP of the subjects were measured by trained research Unstained and fluorescence minus one controls were used
assistants following standardized procedures using calibrated to differentiate between true events and background/debris.
equipment. EMPs were identified as CD62E+ and CD31+/CD42b− events
The primary outcome measure for this trial was plasma within the microparticle size gate.
insulin level, while secondary outcomes were (1) other clin-
ical biomarkers (i.e., glucose, TG, HDL-cholesterol, LDL- 2.11. Whole Blood Culture. Whole blood was diluted 1 : 10
cholesterol, and total cholesterol); (2) EMPs; and (3) plasma with serum-free RPMI-1640 medium (penicillin 100 U/mL,
cytokines and cytokines from culture whole blood ex vivo. streptomycin 100 𝜇g/mL) (i.e., 540 𝜇L whole blood diluted
There were no changes to outcomes following trial com- in 4.86 mL RPMI-1640 medium), plated in duplicate on 24-
mencement. well plates at a final volume of 600 𝜇L, and cultured at
37∘ C in a humidified incubator (5% CO2 ) as described by
2.6. Sample Size Calculation. The sample size was based on Wan et al. [28]. Samples were stimulated with the TLR4
(1) published findings from other research groups who have agonist LPS (1, 10 ng/mL) and TLR2 agonist Pam3Cys-SK4
reported the beneficial effects of yoga with similar sample size [29] (1, 10 ng/mL) and supernatants were harvested after
[5, 6, 15] and (2) calculations assuming two-tailed 𝛼 = 0.05 24 h via centrifuge at 2000 g for 15 min at 4∘ C. Samples
and 1-𝛽 = 90% to detect a 10% difference for the plasma insulin were then stored at −80∘ C before batch analysis of TNF-𝛼,
levels, which is the primary outcome of the present study. IL-6, and IL-1𝛽 via ELISA according to the manufacturer’s
instructions. Biological replicates were analyzed, with the
2.7. Randomization and Blinding. Following recruitment ran- average coefficient of variation (CV) for each cytokine being
domization was carried out via computer-generated random <5%.
numbers with unrestricted equal participant allocation (1 : 1)
by one research investigator, who is independent of the yoga 2.12. PBMCs Isolation. PBMCs were isolated by gradient
intervention and data analysis. Participants were not blinded density centrifugation of peripheral blood using Ficoll-Paque
to the study. Plus as described previously by our laboratory [28]. Briefly,
5 mL of blood was layered onto 5 mL of Ficoll-Paque Plus in
2.8. Plasma Clinical Metabolic Biomarkers Measurement. a sterile 15 mL tube and was centrifuged for 15 min at 800 g
Clinical biomarkers including glucose, TG, HDL-cholesterol, and at 20∘ C. The layer of PBMCs was recovered and washed
LDL-cholesterol, and total cholesterol were measured on three times with sterile PBS for 10 min at 250 g at room
an automatic analyzer (Hitachi 7600, Tokyo, Japan). The temperature. Isolated PBMCs were then stored at −80∘ C until
homeostasis model assessment of insulin resistance (HOMA- further protein expression analysis by western blotting.
IR) was calculated using the following equation: HOMA-IR =
fasting insulin (FIns, 𝜇IU/mL) × fasting blood glucose (FBG, 2.13. Western Blotting. Proteins from isolated PBMCs were
mmol/L)/22.5. extracted. The protein expression of TLR2 and TLR4 was
determined by western blotting following the methods
2.9. Plasma Cytokines and Insulin Measurement. Plasma published by our laboratory previously [30]. Signals were
cytokines including IL-6, IL-8, IL-1𝛽, MCP-1, TNF-𝛼, and visualized using Immobilon western chemiluminescent HRP
plasma insulin were measured from EDTA plasma using substrate and bands were quantified by densitometry. Beta
Luminex technology according to the kit manufacturer’s actin was used as an internal control.
instructions. The detection limits for IL-6, IL-8, IL-1𝛽, MCP-
1, TNF-𝛼, and insulin were 0.2, 0.3, 0.4, 1.2, 0.3, and 3.8 pg/mL, 2.14. Statistical Analysis. All data are presented as mean
respectively. Plasma IL-6 and IL-1𝛽 levels were below the ± standard error of the mean (SEM). Statistical analyses
detection limit of the assay in our study. The average CV for were performed with SPSS version 15.0 for Windows (IL,
duplicates in the assay is <6%. USA). Data were analyzed for normality and homogeneity
before statistical test. Two-way ANOVA was utilized for
2.10. Endothelial Microparticles (EMPs) Measurement. Circu- comparisons between groups. Tukey’s Honestly Significant
lating EMPs were measured in platelet-poor plasma by flow Difference (HSD) was applied for post hoc comparisons.
cytometry following the method of Jenkins et al. [27]. In brief, Statistical significance was set at 𝑝 < 0.05.
4 BioMed Research International

Consort 2010 flow diagram

Enrollment
Assessed for eligibility (n = 32)

Excluded (n = 2)
(i) Not meeting inclusion criteria (n = 2)
(ii) Declined to participate (n = 0)
(iii) Other reasons (n = 0)

Randomized (n = 30)

Allocation

Allocated to yoga group (n = 15)

Allocated to control group (n = 15)
(i) Received yoga intervention (n = 15)
(i) Received no intervention (n = 15)
(ii) Did not receive yoga intervention (n = 0)

Follow-up

Lost to follow-up (give reasons) (n = 0) Lost to follow-up (give reasons) (n = 0)

Discontinued intervention (give reasons) (n = 0) Discontinued intervention (give reasons) (n = 0)

Analysis

Analysed (n = 15) Analysed (n = 15)

(i) Excluded from analysis (give reasons) (n = 0) (i) Excluded from analysis (give reasons) (n = 0)

Figure 1: CONSORT flowchart.

3. Results 3.4. No Effect of Yoga Practice on Circulating Proinflammatory

Cytokines. As shown in Table 2, there were no significant
3.1. Participants’ Flow and Participation Rate. The CON- effects of yoga practice on levels of plasma proinflammatory
SORT flowchart of subject recruitment and intervention was cytokines (IL-8, MCP-1, and TNF-𝛼) measured in the fasted
shown in Figure 1. From March 2015 to June 2015, all recruited state.
subjects completed the whole yoga practice with no dropout.
There were no harmful effects observed by the yoga practice.
3.5. Yoga Practice Resulted in Decreased Proinflammatory
Cytokine Response. At baseline level, yoga group demon-
3.2. Yoga Practice Decreased Plasma Insulin, Total, and LDL-
strated elevated IL-6 secretion in supernatant from cultured
Cholesterol Level. A total of 8 wks yoga practice resulted
whole blood at unstimulated condition (Figure 3(a)). Yoga
in significant reduction in plasma insulin, total cholesterol,
group had reduced secretion of IL-6, TNF-𝛼, and IL-1𝛽
and LDL-C levels compared to preyoga practice; meanwhile,
HOMA-IR from yoga group is reduced compared to both levels after yoga training (Figure 3). Furthermore, when
yoga groups at baseline level and control group after inter- cultured blood was challenged with Pam at both 1 ng/mL and
vention, while there is no difference for glucose, TG, HDL- 10 ng/mL, a well-known agonist of TLR-2 receptor [29], yoga
C, SBP, DBP, body weight, and BMI before and after yoga practice group also demonstrated damped cytokines secre-
practice between groups (Table 1). tion including IL-6, TNF-𝛼, and IL-1𝛽 levels compared to
preyoga condition and control group (Figure 4). Meanwhile,
3.3. Yoga Practice Reduced Circulating CD31+/CD42b− EMPs. at baseline level, yoga group has reduced TNF-𝛼 secretion
As shown in Figure 2, there was a significant reduction in compared to control group when stimulated with LPS (at
circulating CD31+/CD42b− EMPs after yoga intervention both 1 ng/mL and 10 ng/mL); this trend was maintained after
compared to yoga group at baseline level and control group yoga training (Figure 5(b)). There is no difference for IL-6 and
(Figures 2(a) and 2(b)), while yoga practice had no effect on IL-1𝛽 secretion when stimulated with LPS (Figures 5(a) and
CD62E+ EMPs (Figures 2(c) and 2(d)). 5(c)).
BioMed Research International 5

Table 1: Comparison of metabolic characteristics between groups before and after yoga intervention.

Control Yoga
Pre Post Pre Post
Insulin (mIU) 6.17 ± 0.60 5.55 ± 0.75 6.58 ± 0.98 4.06 ± 0.87∗
Glucose (mM) 4.59 ± 0.07 4.51 ± 0.08 4.59 ± 0.13 4.48 ± 0.1
HOMA-IR 1.26 ± 0.12 1.13 ± 0.17 1.36 ± 0.21 0.75 ± 0.18∗,#
TG (mM) 0.60 ± 0.06 0.60 ± 0.04 0.66 ± 0.03 0.68 ± 0.09
Cholesterol (mM) 3.90 ± 0.18 3.64 ± 0.15 4.13 ± 0.12 3.75 ± 0.15∗
LDL-C (mM) 1.93 ± 0.15 1.76 ± 0.13 2.14 ± 0.11 1.81 ± 0.13∗
HDL-C (mM) 1.69 ± 0.07 1.68 ± 0.05 1.67 ± 0.05 1.58 ± 0.05
SBP (mmHg) 108.0 ± 2.7 105.9 ± 1.5 106.8 ± 2.1 102.5 ± 2.3
DBP (mmHg) 76.15 ± 1.8 72.62 ± 1.8 74.77 ± 2.4 71.83 ± 2.00
Body weight (kg) 54.08 ± 1.65 53.81 ± 1.68 53.35 ± 1.53 52.71 ± 1.57
BMI (kg/m2 ) 20.68 ± 0.46 20.18 ± 0.46 20.55 ± 0.52 20.49 ± 0.52
TG: triacylglycerol; LDL-C: low density lipoprotein-cholesterol; HDL-C: high density lipoprotein-cholesterol; SBP: systolic blood pressure; DBP: diastolic blood
pressure; and BMI: body mass index
Data are expressed as mean ± SEM. ∗ Compared with preintervention baseline level; # compared with control group after intervention.

Table 2: Plasma cytokines measured in the fasted state before and after yoga intervention.

Control Yoga
Pre Post Pre Post
IL-8 (pg/mL) 6.17 ± 0.61 5.13 ± 0.52 5.64 ± 0.56 5.03 ± 0.34
MCP-1 (pg/mL) 159.70 ± 20.86 148.95 ± 20.00 145.71 ± 19.95 149.52 ± 14.44
TNF-𝛼 (pg/mL) 2.15 ± 0.30 2.36 ± 0.38 1.66 ± 0.19 1.96 ± 0.30
Data are expressed as mean ± SEM.

3.6. Yoga Practice Resulted in Decreased TLR2 Protein Expres- practice (2 times/wk) could reduce total cholesterol and LDL-
sion in PBMCs. As shown in Figure 6, there is no difference C level, indicating that Hatha yoga practice is an effective
for TLR2 protein expression between groups at baseline way for reducing risk factors associated with disordered lipid
level; yoga practice resulted in significant reduction in TLR2 profiles even in healthy subjects. Randomized trials [35] and
protein expression in PBMCs, while there is no difference meta-analyses [36] have consistently demonstrated a modest
for TLR4 protein expression between groups before and after but consistent reduction in blood pressure following yoga
yoga practice. practice. However, we observed no alterations in SBP and
DBP after 8 wks of yoga practice. This might be related to
4. Discussion multiple factors. First, different yoga practice type, length,
and frequency might affect its effects on blood pressure.
The main findings of the present study are that (1) 8 wks Second, the subjects in our present study are healthy; thus it
of Hatha yoga practice in healthy Chinese female subjects might be hard to observe reductions in blood pressure.
reduced plasma insulin, cholesterol levels, and circulating Yoga has been increasingly accepted as a cost-effective
CD31+/CD42b− EMPs and that (2) cultured whole blood therapeutic strategy for T2DM patients [2, 37]. Evidence
from yoga practice group had reduced proinflammatory in regard to how yoga practice affects plasma insulin level
cytokines secretion at unstimulated condition, as well as remains inconsistent. Hunter et al. [13] reported that Bikram
when stimulated with a TLR2 agonist, and this might be yoga, which is one of the most popular forms of hot yoga,
associated with reduced TLR2 protein expression after yoga resulted in reduction in plasma insulin and HOMA-IR only
training. in older adults (53 ± 2 yrs). Vizcaino [38] demonstrated
The most significant risk factors for MetS include dys- that 6 wks of Hatha yoga (3 times/wk) has no effect on
lipidemia, hypertension, and physical inactivity [31]. Yoga fasting insulin level in patients with T2DM. In contrast,
practice improved lipid profiles in clinical patients with Manjunatha et al. [5] reported that yoga practice reduced
cardiovascular diseases [32, 33] and hypertension [34]. In serum insulin level in healthy subjects, while the majority
particular, Bijlani et al. [34] reported that the TG-lowering of them were male. Our present study further confirmed
effects of yoga were more prominent in subjects with hyper- that in healthy female subjects 8 wks of Hatha yoga could
cholesterolemia [34]. Therefore, when assessing yoga’s effects significantly reduce plasma insulin level and consequently
on improving lipid profiles, it is important to consider HOMA-IR.
participants’ health conditions. Our present study confirmed Elevation of EMPs is rapidly being accepted as an alter-
that, in healthy, female Chinese subjects, 8 wks of Hatha yoga nate surrogate marker of CVDs and endothelial function
6 BioMed Research International

104

FL2-H:: CD42b-PE
12 103

10 102
CD31+/CD42b− EMPs/𝜇L plasma

101
∗#
8
100
100 101 102 103 104
6 FL1-H:: CD31-FITC
104
4

FL2-H:: CD42b-PE
103

2 102

101
0
Control Yoga
100
Pre 100 101 102 103 104
Post FL1-H:: CD31-FITC
(a) (b)
4
10
50
103
FL2-H:: CD31-PE

102
40

101
CD62E+ EMPs/𝜇L plasma

30 100
100 101 102 103 104
FL4-H:: CD62-APC
20 104

103
FL2-H:: CD31-PE

10
102

101
0
Control Yoga
100
Pre 100 101 102 103 104
Post FL4-H:: CD62-APC
(c) (d)

Figure 2: Yoga practice reduced circulating CD31+/CD42b− EMPs levels. Circulating EMPs were measured in platelet-poor plasma by flow
cytometry with fluorochrome labeled antibodies specific for PE-CD42b, FITC-CD31, and APC-CD62E. EMPs were identified as CD62E+
and CD31+/CD42b− events with a diameter <1 𝜇M. (a) Fasting CD31+/CD42b− EMPs were reduced postyoga practice compared to preyoga
training condition. (b) Representative fluorescence-activated cell sorter dot plots of CD31+/CD42b− of a subjects before (top) and after
(bottom) yoga practice. (c) No difference for CD62E+ EMPs between groups. (d) Representative fluorescence-activated cell sorter dot plots of
CD62E+ of a subjects before (top) and after (bottom) yoga practice. Data are presented as mean + SEM (𝑁 = 15). ∗ 𝑝 < 0.05 versus preyoga
training condition within the same group in (a); # 𝑝 < 0.05 versus control group at baseline level.
BioMed Research International 7

300 80

250 ∗
60

TNF-𝛼 secretion (pg/mL)

200
IL-6 secretion (pg/mL)

150 40

100

20
50

∗# ∗#
0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(a) (b)
50

40
IL-1𝛽 secretion (pg/mL)

30

20

10

0
Control Yoga

Pre
Post
(c)

Figure 3: Reduced secretion of IL-6, TNF-𝛼, and IL-1𝛽 from cultured whole blood ex vivo after yoga training. Whole blood was collected at
baseline and after yoga; then blood was diluted and cultured at 24-well plates under identical culture conditions. Supernatants were centrifuged
and collected at 24 hr for the measurement of IL-6, TNF-𝛼, and IL-1𝛽 secretion via ELISA. There is significant reduction of IL-6 (a), TNF-𝛼
(b), and IL-1𝛽 (c) secretion after yoga compared to preyoga condition. Data are presented as mean + SEM (𝑁 = 15). ∗ 𝑝 < 0.05 versus preyoga
training condition within the same treatment; # 𝑝 < 0.05 versus control group at baseline level.

[39]. CD62E+ EMPs generally reflect endothelial activation days of dry water immersion [10] is associated with increased
or inflammation whereas CD31+/CD42b− EMPs are released concentrations of CD31+/CD42b− EMPs and CD31+/CD41−
upon endothelial cell apoptosis [7]. Recent evidence has EMPs, respectively. Our study is the very first to reveal
confirmed that moderate-intensity endurance training could that 8 wks of Hatha yoga could significantly reduce plasma
reduce circulating EMP levels [11, 40, 41]. In contrast, physical CD31+/CD42b− EMPs in healthy subjects. High concen-
inactivity via reducing daily PA [12] or subjecting subjects to 7 trations of EMPs are associated with a proinflammatory
8 BioMed Research International

800 Pam: 1 ng/mL 1000 Pam: 10 ng/mL

800
600
IL-6 secretion (pg/mL)

IL-6 secretion (pg/mL)

600
400
400 ∗#
∗#
200
200

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(a)
400 Pam: 1 ng/mL 600 Pam: 10 ng/mL

500
300
TNF-𝛼 secretion (pg/mL)

TNF-𝛼 secretion (pg/mL)

400

200 300 ∗#

∗#
200
100
100

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(b)
160 Pam: 1 ng/mL 250 Pam: 10 ng/mL
140
200
120
IL-1𝛽 secretion (pg/mL)
IL-1𝛽 secretion (pg/mL)

100 150
80 ∗#
∗#
60 100

40
50
20

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(c)

Figure 4: Attenuated Pam3Cys-SK4 (1 and 10 ng/mL) induced IL-6, TNF-𝛼, and IL-1𝛽 secretion from ex vivo whole blood cultures after
yoga practice. Whole blood was collected at baseline and after yoga; then blood was diluted, cultured at 24-well plates, and stimulated with
Pam under identical culture conditions. Supernatants were centrifuged and collected at 24 hr for the measurement of IL-6, TNF-𝛼, and IL-1𝛽
secretion via ELISA. Yoga training led to blunted IL-6 (a), TNF-𝛼 (b), and IL-1𝛽 (c) secretion upon Pam stimulation at both 1 and 10 ng/mL.
Data are presented as mean + SEM (𝑁 = 15). ∗ 𝑝 < 0.05 versus preyoga practice condition within the same treatment; # 𝑝 < 0.05 versus control
group at baseline level.
BioMed Research International 9

1000 LPS: 1 ng/mL 1000 LPS: 10 ng/mL

800 800

IL-6 secretion (pg/mL)

IL-6 secretion (pg/mL)
600 600

400 400

200 200

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(a)
1000 1000
LPS: 1 ng/mL LPS: 10 ng/mL

800 800
TNF-𝛼 secretion (pg/mL)

TNF-𝛼 secretion (pg/mL)

600 600
∗#
∗#
400 ∗ 400

200 200

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(b)
250 LPS: 1 ng/mL
300 LPS: 10 ng/mL

250
200
IL-1𝛽 secretion (pg/mL)

IL-1𝛽 secretion (pg/mL)

200
150
150
100
100

50
50

0 0
Control Yoga Control Yoga

Pre Pre
Post Post
(c)

Figure 5: Reduction in TNF-𝛼 secretion from yoga group compared to control group at baseline and when stimulated with LPS. IL-6 (a),
TNF-𝛼 (b), and IL-1𝛽 (c) secretion from groups. ∗ 𝑝 < 0.05 versus control group at baseline level; # 𝑝 < 0.05 versus control group postyoga
practice condition.
10 BioMed Research International

Control Yoga
Pre Post Pre Post

TLR2

TLR4

Actin

1.4 1.4

1.2 1.2

1.0 1.0
TLR2 protein content (A.U.)

TLR4 protein content (A.U.)

0.8 ∗# 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
Control Yoga Control Yoga

Pre Pre
Post Post
(a) (b)

Figure 6: Reduction in TLR2 protein expression from PBMCs after yoga practice. PBMCs were isolated at baseline level and after yoga
training, and the protein expression of TLR2 and TLR4 was measured via western blotting. Yoga practice resulted in reduction in TLR2
protein expression with no effect on TLR4. Western blotting images are given at the top of the quantified data. ∗ 𝑝 < 0.05 versus preyoga
training group; # 𝑝 < 0.05 versus control group at baseline level.

and antiangiogenic status in the vascular system [42]; thus secretion has been observed after yoga training. The whole
reduction of CD31+/CD42b− EMPs after yoga suggested that blood culture method is based on an optimal dilution of the
yoga might improve vascular function via affecting EMPs blood cells in medium and no unphysiological cell separa-
levels. Furthermore, Jenkins et al. [27] reported that an acute tion is involved; thus it represents a physiologically much
reduction in shear stress via disturbed blood flow increased more relevant environment for the cells. Our findings could
local concentrations of CD31+/CD42b− and CD62E+ EMPs suggest that yoga practice may reduce the inflammatory
in the human forearm. Our result could also suggest that, status at the whole blood culture level. It is possible that a
unlike pathological stress, physiological stress like yoga may longer-term yoga practice than the present study design is
decrease EMPs release. This might be one of the mechanisms required to reduce circulating proinflammatory cytokines in
through which yoga intervention exerts its cardiac and vas- healthy subjects. Furthermore, yoga group also demonstrated
cular protective effects. However, further studies are required reduced IL-6, TNF-𝛼, and IL-1𝛽 secretion following TLR2
to confirm this hypothesis. agonist stimulation but not TLR4; this was also associated
Improved circulating inflammatory markers after yoga with reduced TLR2 protein expression in PBMCs after yoga
practice have been observed in clinical patients with heart intervention. Collectively, it is suggested that yoga practice
failure [15, 16], breast cancer [17, 18], chronic inflammatory could result in blunted TLR2 response. We are yet to deter-
diseases, and overweight/obese subjects [19]. In our present mine whether a yoga-induced blunting of TLR2 response
study, although yoga practice had no effect on circulating IL- represents a positive change for the health status in the long
8, TNF-𝛼, and MCP-1 levels in healthy subjects, via whole run. Considering that chronic inflammation is one of the key
blood culture ex vivo, reduction in IL-6, TNF-𝛼, and IL-1𝛽 mechanisms involved in the pathogenesis of MetS [14], in the
BioMed Research International 11

long term, a decrease in TLR2 response may exert a beneficial Liqiang Qin), and the University Science Research Project of
effect because it decreases the inflammatory capacity of Jiangsu Province (Grant no. 14KJD330002).
inflammatory cells, consequently suppressing whole body
chronic inflammation. Compared with the reported effects References
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