Intern. J.

Neuroscience, 114:529–537, 2004

Copyright  Taylor & Francis Inc.
ISSN: 0020-7454 / 1543-5245 online
DOI: 10.1080/00207450490422849

ENDOCRINE AND IMMUNE EFFECTS

OF QI-TRAINING

MYEONG SOO LEE

Professional Graduate School of Oriental Medicine and
Center for Integrative Medicine, Institute of Medical Science
Wonkwang University
Iksan, Republic of Korea

CHANG-WON KANG
Department of Physiology, College of Veterinary Medicine
Chunbuk National University
Jeonju, Republic of Korea

HOON RYU
Department of Neurology
University of Massachusetts Medical School
Worcester, MA, USA

SUN-ROCK MOON
Center for Integrative Medicine
Institute of Medical Science, Wonkwang University
Iksan, Republic of Korea
and
Department of Radiation Oncology
Wonkwang University School of Medicine
Iksan, Republic of Korea

Received 24 July 2003.

This study was supported by Wonkwang University in 2003.
Address correspondence to Sun-Rock Moon, MD, PhD, Department of Radiation Oncology,
Wonk-wang University School of Medicine, Iksan 570-711, Republic of Korea. E-mail:
sunrmoon@wonkwang.ac.kr, integmed@hitel. net.

529
530 M. S. Lee et al.

Psychoneuroimmunological approaches have shown that Qi-training in-

fluences the holistic health state in men. We found that Qi-training increased
the plasma level of growth hormone (GH) and insulin-like growth factor
(IGF)-I. In addition, the respiratory function and adhesion capacity of
neutrophil was increased after Qi-training. In an in vitro analysis, GH
primed and increased the respiratory function and adhesion of neutro-
phils through tyrosin kinase passway. Taken together, in vivo and in vitro
data suggest that the beneficial effects of Qi-training on immunological
functions are mediated via neuroendocrine responses.
Keywords adhesion, growth hormone, IGF-I, neutrophils, qigong, super-
oxide generation

Qi-training (Qigong) is oriental, traditional psychosomatic training,

not only for the development of physical balance through isotonic,
isometric slow motions but also for psychological stabilization through
meditation (Eisenberg, 1985). Among various types of interventions,
Qi-training and its beneficial effects have received increasing atten-
tion, due to the link between psychological stabilization and im-
mune function (Ryu et al., 1995; Lee et al., 1998, 2000, 2003). It is
possible that the beneficial effects of Qi-training on psychological
and immunological functions, including cardiovascular function, may
be mediated via neuroendocrine responses. Although there are a
number of positive findings in psychophysiological and immuno-
logical functions, there have been few systematic investigations of
the precise neuroendocrine mechanisms by which Qi-training may
modulate immunological functions.
Therefore, the present study was designed to examine the effect
of Qi-training on the plasma levels of GH and IGF-I in young males.
We also intended to investigate the immunological effects of Qi-
training by neutrophils’ superoxide generation, and adhesion capacity.
To investigate the possible mechanism of the neuroendocrine-im-
mune pathway, we studied the effects of GH on cellular signaling
by using GH with enhanced O2– generation and adhesive capacity in
human neutrophils.

MATERIALS AND METHODS

Participants

All subjects were recruited on a voluntary basis from three cities in

Korea. Prior to the study, volunteers completed medical history ques-
 Endocrine and Immune Effects of Qi-Training 531

tionnaires and reported experience of other complementary therapy.

Respondents who had experienced other relaxation training, thera-
peutic touch, acupuncture or massage therapy, and reported a history
of medical treatment for neurohormonal and immunological disease
were eliminated from the experiment. The study received institu-
tional approval from the Human Investigation Committee and admin-
istrative approval from the facility before subjects were approached
to obtain written consent. All subjects agreed to participate in the
study and signed an informed consent form from the Human Subjects
Review Board in Wonkwang University Hospital and School of
Medicine.
Thirty-four men met the criteria and agreed to participate. They
were divided into four experimental procedures. Sixteen healthy young
men (mean age = 29) agreed to participate in a study of neuroendo-
crine effects of Qi-training, and nine healthy young men (mean age
= 26) participated in a study of the immunological effects. Nine
men volunteered to draw the blood sample for in vitro experiment
of growth hormone on neutrophil responses.

Polymorphonuclear Leukocytes (PMN) Isolation

Human venous blood (10 ml) was collected from healthy adult vol-
unteers into heparinized tubes. To obtain neutrophils from periph-
eral blood, the heparinized blood was centrifuged for 10 min at 250
× g to remove platelet-rich plasma. After 2% dextran sedimentation
of erythrocytes for 30 min, neutrophils were isolated under sterile
conditions by density gradient centrifugation on Ficoll-Paque cush-
ions in conical tubes. The tubes were centrifuged at 400 × g for 30
min in swing-out buckets at room temperature. The contaminating
erythrocytes were lysed with a hypotonic solution containing NH4Cl-
EDTA, then washed twice. The cells were gently resuspended in
magnesium-free Hanks’ balanced salt solution (HBSS) containing
1.6 mM CaCl2 ; then the cell number and viability were determined.
The entire procedure was conducted in sterile conditions at room
temperature. The final cell preparation comprised at least 97% neu-
trophils and <0.2% monocytes, as assessed by Wright-Giemsa dif-
ferential staining. The viability of neutrophils was >98%, as deter-
mined by trypan blue exclusion.
532 M. S. Lee et al.

Priming of PMN
PMN were kept in a serum-free suspension during the course of any
treatment. Each milliliter of the treatment mixture contained 1 × 106
PMN and 0.1 ml of various concentrations (10 to 1000 ng/ml) of
recombinant human growth hormone (rGH) (LG Biotech, Taejon,
Republic of Korea) in HBSS. Cells were incubated at 37°C in a
CO2 incubator for 1 h. After incubation, the cells were harvested
and resuspended in a Veronal buffer to measure the respiratory burst
activity.

Treatment of Reagent
To study the effect of tyrosin kinase activity on priming, PMN (1 ×
106/ml) were kept in 1 ml of serum-free suspension and were left
untreated or treated with genestein (10 µM). Cells were incubated at
37°C with 5% CO2 for 30 min. After incubation, the cells were
harvested, washed, and resuspended in a medium to add another
reagent.

Materials
HBSS and phosphate-buffered saline (PBS) were prepared by con-
ventional methods and sterilized. HBSS contained 5 mM D-glu-
cose. A single batch of opsonized zymosan (used for all experi-
ments) was prepared by incubating 10 mg of Zymosan-A particles
(highly glycosylated fragments of yeast cell walls) (Sigma, St. Louis,
MO, USA) with 1 ml of fresh human plasma for 30 min at 37ºC.
The suspension was centrifuged at 400×g for 2 min, and the pellet
was washed twice in PBS before resuspension in PBS at a final
concentration of 10 mg/ml. Aliquots (l ml) were stored frozen and
used only once after thawing. Counting by hemocytometer revealed
that this preparation contained approximately 106 zymosan particles/
microliter.

Measurement of Cell Adhesion

Neutrophil adhesion to 96-well tissue culture plate was evaluated as
described by Nagata et al. (1995). Briefly, nonadherent cells were
 Endocrine and Immune Effects of Qi-Training 533

decanted immediately following the incubation with the reagent. The

reaction wells were rigorously washed three times with 37°C HBSS.
A total of 200 µl of 0.5% crystal violet in 12% neutral formaldehyde
solution and 10% ethanol were then added to each well for 1 h to fix
and stain cells. The samples were thoroughly washed with water and
air-dried for 30 min. Crystal violet was extracted by the addition of
1% SDS, and absorbency was measured at 570 nm. Adhesion was
expressed as increased absorbency at 570 nm.

Measurement of Superoxide Anion (O2–) Production

O2– was measured by chemiluminescence assays. Ten microliters of
lucigenin (10, 10’-dimethyl-9,9’-acridinum) (Sigma) (0.3 mM final
concentration), as a chemiluminescence source, was added to each
tube containing 1 × 106 cell in a 300 µ volume of Veronal buffer
(containing BSA, glucose, Ca2+ and Mg2+). The tubes were immedi-
ately placed in a lightproof and thermostatic chamber of a six-channel
Biolumant LB9505 (Berthold, Bad Wildbad, Germany) and stabi-
lized for 20 min. For the enhancement of O2– production, the trig-
gering agent, N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP)
(220 nM final concentration) (Sigma), was added to the tubes. The
number of resultant photons from lucigenin-dependent chemilumi-
nescence was recorded in counts per minute (cpm) for 60 min.

Experimental Procedure and Blood Sampling

ChunDoSunBup Qi-training, which has previously been reported by
Ryu et al. (1995), was performed by the subjects for 1 h and was
directed by a master. For 1 h of Qi-training, subjects rested for 10
min before Qi-training and three types of exercises (sound (Chunmoon)
reciting for 15 min, slow motions for 15 min, and meditation for 20
min) were performed (ChunDoSunBup Inst., 1992). The peripheral
blood was obtained by venipuncture using heparinized (10 U/ml)
syringes from the median cubital vein. Blood was drawn before Qi-
training [Pre, that is 10 min before Qi-training] and immediately
after the Qi-training [Post, within 10 min (+10 min)]. The samples
were immediately treated to separate neutrophils through the above-
mentioned methods.
534 M. S. Lee et al.

TABLE 1. Effects of Qi-training on hormones

Time

Pre Post

GH 1.26 ± 0.37 2.18 ± 0.56**

IGF-I 245.6 ± 13.9 287.0 ± 23.1*

Note. Values are expressed as mean ± SEM. Pre: before Qi-training;

Post: immediately after Qi-training.
*Significantly different from Pre at p < .05 and **p < .01.

Statistical Analysis
We used SPSS for windows (version 7.5) for statistical analysis.
The results are presented as means ± SEM. Paired t-tests were used
to compare values measured at different times.

RESULTS

Qi-Training Increased the Secretion

of GH and IGF-1

Changes in GH and IGF-1 level are presented in Table 1. The plasma

GH level was increased after Qi-training compared to pre Qi-train-
ing (p < .01). The plasma level of IGF-1 was significantly increased
after Qi-training (p < .05).

TABLE 2. Effects of Qi-training on neutrophils function

Time

Pre Post

Superoxide generation 4.40 ± 0.97 5.83 ± 0.63**

Adhesion 0.19 ± 0.01 0.23 ± 0.02*

Note. Values are expressed as mean ± SEM. Pre: before Qi-training; Post:
immediately after Qi-training.
*Significantly different from Pre at p < .05 and **p < .01.
 Endocrine and Immune Effects of Qi-Training 535

Qi-Training Increased Neutrophils Superoxide

Generation and Adhesion Capacity
The responsiveness of neutrophils isolated from the peripheral blood
of subjects to opsonized zymosan was changed by Qi-training
(Table 2). Superoxide generation was significantly enhanced after
Qi-training compared to before Qi-training (p < .01). The adhesive
capacity of neutrophils was significantly enhanced after Qi-training
(p < .05).

Exogenous GH-Modulated Neutrophils

Superoxide Generation
We determined the dose-dependent priming of human neutrophil by
GH. As shown in Figure 1, GH significantly increased the secretion
of O2– by human neutrophils, which had been triggered with fMLP.
The priming of human neutrophils for increased O2– production by
GH was dose-dependent. Significant priming of human neutrophils
by GH was observed at 10 ng/ml (p < .05), 100 ng/ml (p < .01), and

FIGURE 1. Dose dependent effect of GH and genestein (10 µM) on O 2– production in

human neutrophils. *, significantly different from 0 dose of GH at p < .05 and **, p <
.01.
536 M. S. Lee et al.

250 ng/ml (p < .05). According to the dose-response curve, we

selected one submaxinal dose as 250 ng/ml (p < .05).
To verify the link between GH-receptor mediated tyrosine kinase
activity, we examined the inhibitory effect of genestein on the in-
creases of intracellular O2– production. A pre-treatment with genestein
resulted in decreases of the relative fluorescence intensity of intra-
cellular O2– production in human neutrophils, even when the cells
were primed by GH.

Exogenous GH Increases Neutrophils Adhesion

In the first series of experiments the time- and dose-dependent ef-
fects of GH on neutrophil adhesion were determined. The time course
for the direct adherence of neutrophils to the plastic substratum of
cell cultureware was assessed with or without GH. The maximal
adherence of neutrophils was observed at 30 min for both control
and GH-treated samples. GH augmented the neutrophil adhesion in
a bell-shaped dose-response fashion, with the peak effect observed
at 100 ng/ml (Figure 2). Then we examined the effect of genestein,
a typical tyrosine kinase inhibitor, on GH-stimulated neutrophil ad-
hesion. This result shows that 10 µM genestein decreased the adher-
ence of neutrophils by itself and blocked the effect of GH.

DISCUSSION

These results suggest that Qi-training might modulate the secretion

of growth factors and hormone in men. In addition, respiratory function
of neutrophil was rapidly increased in the isolated neutrophils after
Qi-training. Qi-training was sufficient to elevate the capacity of cir-
culating neutrophils to produce superoxide anion (O2–) and adhesive
capacity upon stimulation in aged men. In an in vitro analysis, GH
primed and increased the superoxide generation and adhesion of
neutrophils through the tyrosin kinase passway. Taken together, in
vivo and in vitro data suggest that Qi-training influences the secre-
tion of GH, IGF-I, and neutrophils function, which may enhance the
immunological response against infection and inflammation through
the modulation of an innate defense function. It is possible that the
 Endocrine and Immune Effects of Qi-Training 537

FIGURE 2. Dose-dependent effect of GH on the adhesion of neutrophils and inhibi-

tory effect of genestein (10 µM) on the adhesion of GH-stimulated neutrophils. **, sig-
nificantly different from 0 dose of GH at p < .01 and #, significantly different from the
value of GH (100 ng/ml)-stimulated cell, p < .05.

beneficial effects of Qi-training on psychological and immunologi-

cal functions are mediated via neuroendocrine responses.

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