involvement of phosphatidylinositol 3-kinase and insulin-like growth factor-i in yxlst-mediated...
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Brain Research 1006 (2004) 198–206
Research report
Involvement of phosphatidylinositol 3-kinase and insulin-like growth
factor-I in YXLST-mediated neuroprotection
Xin Yana,b, Hua Shenb, Marian Zahariac, Jun Wangb, Delia Wolf d, Feng Lie,Garrick D. Leef, Wei Caoe,*
aThe Institute of Chongqing Traditional Chinese Medicine, Chongqing, ChinabAmerican New Medicine Institute, New York, NY 10107, USA
cUniversity of Sherbrooke, Sherbrooke, Quebec, CanadadHarvard Medical School, Boston, MA, USA
eDepartment of Ophthalmology, University of Oklahoma, Dean A. McGee Eye Institute, 608 Stanton L. Young Boulevard, Oklahoma, OK 73104, USAfNational Institute of Health, National Institute on Aging, Baltimore, MD, USA
Accepted 7 January 2004
Abstract
In the present study, we examine the neuroprotective role of the external Qi of YXLST in cultured retinal neurons. Primary retinal
neuronal cultures were grown from retinas of 0–2-day-old Sprague–Dawley rats. Cultures were treated directly with external Qi of YXLST
30 min prior to H2O2 exposure in most experiments. Cell viability was measured by 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium
bromide (MTT) assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling TUNEL assay, and by
DNA laddering analysis. Northern blot analysis was performed to examine the level of insulin-like growth factor-I (IGF-I) gene expression.
Phosphatidylinositol 3-kinase (PI3K) assay was performed to study the PI3K activity. The results showed that treatment of external Qi of
YXLST significantly attenuated neuronal death that was induced by 24-h exposure to hydrogen peroxide, and greatly inhibited hydrogen
peroxide-induced apoptosis. External Qi of YXLST also upregulated IGF-I gene expression and increased PI3K activity. These observations
indicate that external Qi-mediated IGF-I expression and PI3K signaling could be one of the mechanisms in neuroprotection by YXLST.
D 2004 Elsevier B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Neuroprotection
Keywords: Neuroprotection; Insulin-like growth factor-I; Phosphatidylinositol 3-kinase; Yan Xin Life Sciences Technology; External Qi
1. Introduction trained practitioner [25]. The external Qi concept has been
‘‘Qi’’ is described as basic element of human vital
energy in classic Chinese literatures. The underlying theory
of Traditional Chinese Medicine is fully based on balanc-
ing Qi according to the theory of Yin–Yang and five
elements, which have been used for more than 3000 years
[29]. Medical application of Qi for health and healing
consists of internal and external Qi. Internal Qi refers to Qi
inside the human body and the external Qi refers to Qi
projected outside a human body by a highly talented/
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2004.01.068
* Corresponding author. Tel.: +1-405-271-3370; fax: +1-405-271-372.
E-mail address: [email protected] (W. Cao).
implanted in Chinese medical textbooks as curriculum in
medical schools in China. External Qi therapy has also
been under managed by Chinese health authority as one of
medical therapies, and this technique is widely appreciated
in Asian countries, and has been rapidly growing in
popularity in North American [30]. Although no sound
theoretical framework in modern science is yet available
for explaining the mechanism of the external Qi effects, a
body of experimental research on this subject is accumu-
lated and certain special attributes of external Qi have been
summarized, speculated upon, and characterized phenom-
enologically [16,20,42,44,50]. Gu and Lin [10] reported an
unusual infrared radiation time spectrum from external Qi
emitted by Lin. Feng et al. [8] observed a bi-directional
X. Yan et al. / Brain Research 1006 (2004) 198–206 199
effect of external Qi on the growth and annihilation of
bacteria. Yan et al. [42] have demonstrated that external Qi
produced significant structural changes in water and aque-
ous solutions, altered the phase behavior of dipalmitoyl
phosphatidyl liposomes, and enabled the growth of Fab
protein crystals. These data showed objective phenomena
resulting from external Qi and the potential of this ancient
technology system in material process. More recently, Yan
et al. [44] have demonstrated, in the presence of external
Qi, strong responses from thermoluminescent dosimeters,
and the strong waves in aqueous solution structure as
probed with laser Raman spectroscopy and alterations in
the half-life of 241Am as probed with both gamma-ray
spectroscope and a solid-state nuclear track detector. Fur-
thermore, it has been demonstrated that external Qi can
restore PI3K activity within a time window of 3 days of
the initial emission to an outdated enzyme sample that had
lost activity [45]. It has also shown that external Qi
supported long-term survival of mouse hybridoma cells
[46]. All these indicate that external Qi may alter molec-
ular events and the uniqueness of external Qi requires
further study.
Phosphatidylinositol 3-kinase (PI3K) is one of the most
important regulatory proteins that is involved in different
signaling pathways and controlling of key functions of the
cell [9,24]. PI3K is now considered as one of the main
intracellular factors responsible for the transmission of
anti-apoptotic signal and controlling the growth of cells
[4,13]. Over-expression of PI3K in cells is accompanied
by a strongly marked anti-apoptotic effect and causes a
significant increase in cell survival under the influence of
radiation [6]. On the contrary, PI3K specific inhibitors
cause increased apoptosis and decreased cell growth [3].
In addition, several experimental evidences demonstrated
that PI3K is involved in the control of aging [22] through
activation of anti-apoptotic pathway and especially protein
kinase B (PKB) [17]. It has been demonstrated that
external Qi of Yan Xin Life Sciences and Technologies
(YXLST) can modulate PI3K enzyme activity [42,44]. It
has also been reported that XY99-5038, a product of
external Qi of YXLST, can prolong the survival of
neurons [47] and inhibit hydrogen peroxide-induced apo-
ptosis [43], and induce insulin-like growth factor-I (IGF-I)
gene expression detected by cDNA microarray analysis
[48,49].
Previous investigations of external Qi of YXLST have
demonstrated that external Qi treatment can alter the rate
of decay of radioactive isotopes [35], the polarization
plane of a linearly polarized laser beam [36], the laser
Raman spectrum and UV absorption of water [37], the
phase behavior of phospholipids in liposomes [38], and the
requisite conditions for gas phase chemical synthesis [39].
In the present study, we use cultured retinal neurons as a
model system to examine the protective effect of external
Qi of YXLST on neurons against hydrogen peroxide-
induced cell death. Our data demonstrated that external
Qi of YXLST prevented cell loss, increased cell viability,
inhibited apoptosis, activated PI3K pathway and up-regu-
lated IGF-I gene expression.
2. Materials and methods
2.1. Materials
Primary retinal neurons were cultured as described
previously [43,47]. Fetal bovine serum, gentamicin sulfate,
L-Glutamine, creatine, poly-D-lysine, sodium dodecyl sul-
fate (SDS), N,N-dimethyl formamide (DMF), MTT (3,(4,5-
dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide),
hydrogen peroxide were purchased from Sigma (St. Louis,
MO). DMEM/F12 and Tii-Medium were purchased from
Gibco-BRL (Grand Island, NY) and JRH Biosciences
(Lenexa, KS), respectively. Tissue culture plastic ware
and 12 mm diameter coverslips were from Fisher Scientific
(Pittsburgh, PA). Apoptosis detection kits was purchased
from Oncor (Gaithersburg, MD).
2.2. External Qi of YXLST treatment
The treatment of external Qi of YXLST involved in the
emitting external Qi from Dr. Yan Xin toward the neuronal
culture for 10 min in each session. The number of treatment
sessions and schedules varied in each set of the experiments.
The retinal neuron cultures were randomly assigned. A
randomized dual-blind design was used to examine the
effect of external Qi on cultured neurons. The term ‘‘Du-
al-blind’’ follows a suggestion by Caspi et al. [5]. The
research assistants who performed all assays were not aware
of which conditions the cultures had been in (randomly
selected code letters were used as labels for each condition
in the analyses). The external Qi provider (Dr. Yan Xin) was
not involved in any cell collection or assays or data
analyses. The data were analyzed by assessor who was
blinded to the experimental condition (randomly selected
code letters were used as labels for each condition in the
analyses). The final evaluation was performed by project
coordinator who was not marked.
The cultures were prepared before an experiment was
started. The cultures were randomly assigned as external
Qi treatment and non-treatment. The cultures assigned for
external Qi treatment were put in a designated room 30 ft
from tissue culture room by project coordinator. The door
was locked so no one could enter into the room while
external Qi was being emitted. The treatment of external
Qi of YXLST involved in the emitting external Qi from
Dr. Yan Xin toward the neuronal culture for 10 min in
each session. After the emission of external Qi, the
cultures assigned for external Qi treatment were taken
back to the incubator in the tissue culture room by project
coordinator. The number of treatment sessions and sched-
ules varied in each set of the experiments. Non-external Qi
Fig. 1. External Qi of YXLST attenuates H2O2-induced cytotoxicity in
cultured retinal neurons. (A) Control. (B) Cultured retinal neurons were
treated with 100 AM H2O2 for 24 h showing significant cell loss. (C)
Cultures were pretreated with external Qi of YXLST for 10 min
preventing cell loss induced by 100 AM H2O2 for 24 h. (D)
Neuroprotective effect of external Qi of YXLST on H2O2-induced
cytotoxicity, as determined by MTT assay. Cultures were pretreated with
external Qi of YXLST for 10 min before exposure to 100 AM H2O2 for
24 h. *p<0.05 versus the same dose of H2O2 exposure without external Qi
pretreatment (meanFS.D., n=9).
X. Yan et al. / Brain Research 1006 (2004) 198–206200
treated groups had undergone a sham-operated procedure
in the tissue culture room where the incubators were
located.
2.3. Primary culture of retinal neurons
As described previously [43,47], timed pregnant
Sprague–Dawley rats were ordered each week and the
retinas of 10–15 pups, 0–2 days old, were removed with
the aid of a dissecting microscope under sterile conditions in
a tissue culture hood. The retinas were suspended in 25 ml
of DMED-F12 plus 10% fetal calf serum in a plastic bag and
mechanically dissociated for 2 min using a Stomacher set on
low power. The suspension was first filtered through a 230
Am sieve, which was then rinsed once more time with
medium, and the combined filtrates were passed through a
140 Am sieve followed by a rinse with undiluted fetal calf
serum. The filtered suspension was centrifuged at 1000�g
in a clinical centrifuge for 5 min, the supernatant decanted,
and the cell pellets resuspended in 25 ml of media using a
sterile 5 ml pipette. The concentration of cells was deter-
mined with a cell counter or hemocytometer and the
suspension diluted with medium to 1�105 cells per ml.
The cells (1 ml per well) were plated in 24 well tissue
culture plates on 12 mm coverslips that had been pre-treated
overnight with poly-D-lysine (10 Ag/ml). The cells were
maintained in either DMEM-F12 with 2% fetal calf serum
for the first 3–4 days and then in synthetic serum-free media
afterward. The cultures were used in experiments 10 days
after plating.
2.4. MTT assay
MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazoli-
um bromide) (Sigma) was dissolved at a concentration of 5
mg/ml in PBS. Lysing buffer was prepared as follows:
20% w/v of SDS (Sigma) was dissolved at 37 jC in a
solution of 50% of each DMF (N,N-dimethyl formamide)
(Sigma) and deionized water. Twenty-five microliters of
the 5 mg/ml stock solution of MTT was added to each well
and, after 2 h of incubation at 37 jC, 100 Al of the lysing
buffer was added. After an overnight incubation at 37 jC,absorbance of the samples was read at 562 nm using a
microtiter plate ELISA reader.
2.5. TUNEL assay
Detection of apoptosis using the TdT-mediated digox-
igenin-dUTP nick-end labeling (TUNEL) method was
carried out with a commercially available in situ apoptosis
detection kit as described previously [43,47]. Staining for
the TUNEL assay was performed according to the manu-
facturer’s protocol. Cells were stained with diaminobenzi-
dine as the substrate for the kit’s horseradish peroxidase.
TUNEL-positive cells were identified with a Nikon Eclipse
800 microscope, and images were captured by a digital
camera and stored in a computer. The percentage of
apoptotic cells was calculated by dividing TUNEL positive
cells by the total number of cells visualized by Nomarski
optics in the same field. Three digitized images of similar
total cell numbers were selected from each coverslip for
counting and averaging, and were considered as one
independent experiment. Three independent experiments
were then averaged.
2.6. DNA fragmentation
DNA laddering was carried out as follows. The cells
were homogenized in 1 ml of extraction buffer (50 mM
Tris–HCl pH8, 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml
of freshly prepared proteinase K) using a Tissue Tearork,
Biospec Products. Each sample was placed on ice for 20
min and then centrifuged at 15,000�g for 10 min. After
centrifugation, the supernatant from each sample was
extracted with Phenol/Chloroform until the white precipi-
tate was no longer visible in the aqueous fraction. This
usually took 3–6 extractions. The genomic DNA was then
precipitated overnight at �20 jC with 0.1 volume of 3 M
sodium acetate pH5.2 and 2.5 volumes of 100% ethanol.
The samples were then centrifuged at 5000�g for 20 min
and the resulting pellets were re-suspended in 100 Al of TEbuffer pH 8.0. RNase A was then added to a final
concentration of 20 Ag/ml and the samples were incubated
at 37 jC for 2 h. Finally, 3–5 Al of each sample were run
on a 2% agarose gel at 40 V for 2 h.
search 1006 (2004) 198–206 201
2.7. PI3K activity assay
Enzyme assays were carried out essentially as described
previously [12,26]. Briefly, assays were performed directly
on total cells in 50 Al of the reaction mixture containing 0.2
mg/ml PI-4,5-P2, 50 AM ATP, 0.2 ACi [g32 P]ATP, 5 mM
MgCl2, and 10 mM HEPES buffer (pH 7.5). The reactions
were performed for 15 min at room temperature and stopped
by the addition of 100 Al of 1 N HCl followed by 200 Al ofchloroform–methanol (1:1, v/v). Lipids were extracted in
cold room and resolved on oxalate-coated TLC plates (silica
gel 60) with a solvent system of 2-propanol/2 M acetic acid
X. Yan et al. / Brain Re
Fig. 2. External Qi of YXLST inhibits H2O2-induced apoptosis. (A) Nomaski im
Nomarski image of cultured retinal neurons treated only with external Qi of YXLS
Qi of YXLST for 10 min before exposure to 100 AM H2O2 for 24 h. (E) Percentag
neurons as determined by the TUNEL assay. *p<0.05 versus the same H2O2 expo
YXLST prevents DNA fragmentation induced by H2O2. Lane 1: DNA molecular
H2O2 for 24 h. Lane 4: Cells treated with external Qi of YXLST for 10 min. Lane 5
H2O2 for 24 h.
(65:35, v/v). The TLC plates were prepared by placing in
1% (w/v) potassium oxalate in 50% methanol (v/v) and
baked in an oven at 100 jC for 1 h before use. TLC plates
were exposed to X-ray film overnight at �70 jC and
radioactive lipids were scraped and quantified by liquid
scintillation counting.
2.8. RNA extraction and northern blot analysis
Cultured rat retinal neurons were lysed and homogenized
in a 5.5 M guandinium thiocyanate solution (5.5 M guani-
dinium thiocyanate, 25 mM sodium citrate, 0.5% sodium
age of H2O2 untreated control; (B) H2O2 treated (100 AM, 24 h) cells; (C)
T; (D) Nomarski image of cultured retinal neurons pretreated with external
e inhibition of H2O2-apoptosis by external Qi of YXLST in cultured retinal
sure without external Qi pretreatment (meanFS.D., n=6). (F) External Q of
weight markers. Lane 2: Untreated cells. Lane 3: Cells treated with 100 AM: Cells pretreated with external Qi of YXLST for 10 min before exposure to
X. Yan et al. / Brain Research 1006 (2004) 198–206202
lauryl sarcosine, pH 7.0) and total RNAwas isolated using a
CsTFA (cesium trifluoroacetate, Pharmacia, Piscataway, NJ)
gradient method. Total RNA (20 Ag of each sample) was
electrophoresed on 1% agarose formaldehyde gels and
downward wick transferred in 20� SSC to a nylon mem-
brane. Blots were UV irradiated to immobilize RNA and
then prehybridized for 4 h in a hybridization solution
containing: 50% formamide, 5� dehardt’s solution, 5�SSPE, 200 Ag/ml denatured salmon sperm DNA, and 5%
SDS at 50 jC. Random primed 32P labeled cDNA IGF-I
probe were added to the hybridization buffer and hybridized
at 50 jC overnight. Blots were then washed twice in 2�SSC, 0.1%SDS at room temperature for 5 min and twice in
0.1� SSC, 0.1%SDS at 65 jC for 10 min. Rat IGF-I cDNA
probe with 376 base pair (bp) is a gift of Dr. W. Lowe et al.
[18]. After the post-hybridization wash, blots were exposed
to phoshor screen. In all Northern blots, two major bands of
IGF-I mRNA were detected, one at 7.5 kb, the other at 1.2
kb. Data were digitized from the major band, 7.5 kb band,
and analyzed. Hard copies of blots were obtained by
exposing blots to Hyper Film. Blots were reprobed with
18 s rRNA probe, and data of 18 s rRNA served as a control
for RNA loading. 18 s rRNAwith 1.1 kb cDNAwas a gift of
Dr. D. Schlessinger.
2.9. Statistical analysis
Data were analyzed by means of analysis of variance
(ANOVA), and further assessed by post hoc tests. Statistical
differences reaching p<0.05 were accepted as significant.
Fig. 3. Increase in PI3K activity by external Qi of YXLST. (A) Cultures
were treated with external Qi of YXLST for 10 min, and samples were
collected from different time period after the treatment. TLC autoradio-
gram shows a dramatic increase in PI3K activity by external Qi of
YXLST. (B) Comparison of PI3K activity induced by external Qi of
YXLST as a function of treatment time. *p<0.05 versus control group
(meanFS.D., n=3).
3. Result
3.1. Increase in cell viability by external Qi of YXLST
Morphologies of primary cultured retinal neurons in
response to different treatments are shown in Fig. 1. The
control culture shows that the most of the cultured cells
were rounded neuron-like cells with long processes (Fig.
1A). All of those cells can be identified by specific
retinal neuron markers [43,47]. Fig. 1B shows that the
most cells were lost dentritic processes and cell bodies
had had significant shrinkage after exposure to 100 AMhydrogen peroxide for 24 h. Pretreatment of external Qi
of YXLST for 10 min almost completely prevented
H2O2-induced morphological distortion (Fig. 1C). Signif-
icant decrease in cell viability as measured by MTT assay
was seen after the exposure to 100 AM H2O2 for 24 h,
and external Qi treatment inhibited cytotoxicity induced
by H2O2 (Fig. 1D).
3.2. Inhibition of apoptosis by external Qi of YXLST
TUNEL assay was performed to determine whether
external Qi of XYLST inhibits apoptotic cell death induced
by H2O2. A few positive staining cells were noted in control
cultures (Fig. 2A), whereas cultures treated with 100 AMH2O2 for 24 h had large numbers of cells undergoing
apoptosis (Fig. 2B). However, the cells received 10-min
treatment of XYLST external Qi prior to H2O2 (100 AM)
exposure led to a dramatic decrease in the numbers of
apoptotic cells (Fig. 2D). A few TUNEL positive cells were
noted in the group pretreated with external Qi of XYLST
without exposure to H2O2 (Fig. 2C). The percentage of
TUNEL-positive cells (Fig. 2E) in control cultures or
external Qi treated cultures without H2O2 exposure from
three independent experiments was 3%. H2O2-treated cul-
tures without external Qi treatment exhibited 48% positive
cells, whereas with external Qi treatment the numbers of
TUNEL positive cells in H2O2-treated cultures were only
5%. This inhibition of apoptosis by external Qi of XYLST
was also evidenced in DNA fragmentation study showing a
complete prevention of DNA fragmentation induced by
H2O2 (Fig. 2F).
search 1006 (2004) 198–206 203
3.3. Increase in PI3K activity by external Qi of YXLST
The PI3K cascade has been shown to provide neuro-
protection to stressed neuronal cells [21]. In this study we
first measured the PI3K activity in cultured retinal neurons
in response to the treatment of external Qi of XYLST.
Dramatic increase in PI3K activity was seen at 30 min
after 10 min of external Qi treatment. This increase
reached a peak at 1 h after the treatment (Fig. 3A), and
slowly declined after 3 and 6 h of treatment. The PI3K
activity 24 h after external Qi treatment remained higher
than that of control. The fold-increase in PI3K activity by
external Qi treatment was shown in Fig. 3B. PI3K activity
was significantly increased to 3.5 folds 30 min after
external Qi treatment in comparison to control level. This
increase reached 6-folds at 1 h after the treatment, and
remained 3-folds at 24 h after the treatment. To determine
whether PI3K remains activated, retinal neuron cultures
were treated with either H2O2 alone or pretreatment of
external Qi for 10 min prior exposure to H2O2. As shown
in Fig. 4A, addition of H2O2 alone to culture medium for 24
X. Yan et al. / Brain Re
Fig. 4. Effect of external Qi on PI3K activity of neurons exposed to H2O2.
(A) Cultured retinal neurons were pretreated with external Qi of YXLST for
10 min before 100 AM H2O2 or PBS was added to the medium for an
additional 24 h, or treated with 100 AM H2O2 alone for 24 h. Cultures
without treatment served as controls. (B) Data from three independent
experiments were averaged and presented relative fold to the control level
(meanFS.D., n=3).
Fig. 5. Up-regulation of IGF-I gene expression by external Qi of YXLST.
Cultures were treated with external Qi of YXLST for 10 min, and samples
were collected from different time period after the treatment. (A) A major
IGF-I transcript at 7.5 kb was detected in all lanes. (B) Densities from this
major band were calculated and compared as a function of treatment time.
*p<0.05 versus control group (meanFS.D., n=3).
h only slightly reduced PI3K activity compared to basal level
of PI3K activity. Similar to Fig. 3, PI3K activity was
increased at 24 h in the culture receiving 10 min of external
Qi treatment without exposure to H2O2. This increase,
however, was still evident in the culture pretreated with
external Qi for 10 min before exposure to H2O2. The
difference between Qi-treated group and Qi+H2O2 group
was statistically insignificant ( p>0.2188).
3.4. Regulation of IGF-I gene expression by external Qi of
YXLST
Retinal neuronal cultures were exposed to external Qi of
YXLST for 10min. Samples were collected at 0.5, 1, 3, 6, and
24 h after the treatment. IGF-I gene expression was exam-
ined. As shown in Fig. 5A, a major IGF-I transcript at 7.5 kb
was detected in all lanes. Quantitative data from three
separate experiments (Fig. 5B) reveal that up-regulation of
IGF-I mRNA expression started significantly at 1 h after
external Qi treatment, while this up-regulation was margin-
X. Yan et al. / Brain Research 1006 (2004) 198–206204
ally seen at 0.5 h after the treatment. The up-regulation
reached a plateau by 6 h, and maintained similar level at 24
h after the treatment. Fig. 6 shows the effect of external Qi on
IGF-I gene expression in cultured neurons exposed to H2O2.
Cultured retinal neurons were pretreated with external Qi of
YXLST for 10 min before 100 AM H2O2 or phosphate-
buffered saline (PBS) was added to the medium for an
additional 24 h, or treated with 100 AM H2O2 alone for 24
h. Cultures without external Qi or H2O2 treatment served as
controls. Addition of H2O2 alone to culture medium for 24
h down-regulated IGF-I gene expression compared to control
level of the expression. Similar to Fig. 5, up-regulation of
IGF-I gene expression was seen at 24 h in the culture
receiving external Qi treatment without exposure to H2O2.
This up-regulation, however, was still evident in the culture
pretreated with external Qi before exposure to H2O2. The
difference between Qi-treated group and Qi+H2O2 group in
IGF-I gene expression was statistically insignificant
( p>0.3336).
Fig. 6. Effect of external Qi on IGF-I gene expression in cultured neurons
exposed to H2O2. (A) Cultured retinal neurons were pretreated with
external Qi of YXLST for 10 min before 100 AM H2O2 or PBS was added
to the medium for an additional 24 h, or treated with 100 AM H2O2 alone
for 24 h. Cultures without treatment served as controls. (B) Data from three
independent experiments were averaged and presented relative fold to the
control level (meanFS.D., n=3).
4. Discussion
Historically, various extraordinary therapeutic effects of
traditional Chinese medicine (TCM) were explained using
Qi or external Qi that is described frequently in classic
Chinese literature [25,31] and in the literatures of TCM
[32,33]. Numerous health-related applications of external
Qi of YXLST have been reported in recent years
[11,15,19,23]. The effects or existence of external Qi of
YXLST can be physically confirmed using modern meth-
odologies/instruments [35,40,41,45,50]. A body of sub-
stantial experimental studies on YXLST effects has
accumulated. It has been reported that external Qi of
YXLST produces significant structural changes in water
and aqueous solutions, alters the phase behavior of dipal-
mitoyl phosphatidyl choline liposome, and enables the
growth of Fab protein crystals [42,44], and improves
large-scale industrial productions of antibiotics [16]. It
has been demonstrated that external Qi of YXLST can
modulate PI3K enzyme activity [42,45]. It has also been
reported that XY99-5038, a product of external Qi of
YXLST, can prolong the survival of neurons [47] and
inhibit H2O2-induced apoptosis [43], and induce IGF-I
gene expression detected by cDNA microarray analysis
[48,49]. These results provide intriguing scientific corrob-
oration that external Qi of YXLST can affect physical
substances and objects at various levels from cellular,
molecular, to the nuclear level. We, in the present study,
used H2O2 as a toxic stimulus to examine the neuro-
protective role of external Qi of YXLST. Our data from
present study clearly demonstrated that direct treatment
with external Qi of YXLST prevented cell damage, in-
creased cell viability and inhibited apoptosis induced by
H2O2 in cultured retinal neurons. These observations
support our previous reports that YXLST external Qi-
treated product can protect against H2O2-induced cell death
[43] and prolong neuronal survival [47] via external Qi of
YXLST. Evidence has accumulated that apoptosis of
neurons is involved in the pathogenesis of a variety of
human diseases, including AIDS, neurodegenerative dis-
orders, myelodysplastic syndromes, ischemic injury and
toxin-induced diseases [34]. Therefore, specific therapies
designed to inhibit apoptosis of cells could form the basis
for the treatment of those human degenerative diseases.
The results from our study suggest that YXLST external
Qi could be useful for the development of therapeutic
strategy in the treatment of neurodegenerative disorders.
Neuron survival appears to depend on the constant
supply of survival signals. Cells will undergo apoptosis in
culture if exogenous survival factors are depleted or if too
few survival signals are received enough from the environ-
ment. PI3K plays an important role in the signaling of cell
survival and the inhibition of apoptosis [4,13]. PI3K is a
very active enzyme that is a central component of many
signal transduction pathways involved in regulating key
activities of cells [9,24]. PI3K activity can be stimulated
X. Yan et al. / Brain Research 1006 (2004) 198–206 205
through ligand binding of variety of receptors such as the
insulin receptor, IGF-I receptor and among others [7], and is
intrinsically regulated via the interaction between the sub-
units of PI3K. We have found that the increase in PI3K
reached a peak at 1 h after 10 min of external Qi treatment,
and then gradually declined and remained 2-fold increase at
24 h after external Qi treatment. Although in the present
study, measurements of the PI3K activities were up to 24 h,
the result is in agreement with previous observation that
YXLST effect on PI3K was significant reduced in a time-
dependent manner [42,45], suggesting that the effect of
external Qi on PI3K activity is time-limited. It is clear that
PI3K activity was dramatically increased by direct treatment
of external Qi of YXLST and remained significant high in
H2O2-treated neurons. This is consistent with the observa-
tion that the activity of outdated PI3K can be restored to the
level of freshly prepared enzyme after the treatment with
external Qi of YXLST [42,45]. The increase in PI3K
activity in cultured neurons by external Qi of YXLST
may contribute to the YXLST external Qi-mediated neuro-
protection. It has been reported that there is an extra broad
peak in Raman spectrum of water following exposure to
external Qi of YXLST [44] and an enhancing effect of
external Qi on the crystallization of proteins [42]. The effect
of external Qi of YXLST on PI3K may be direct. Further
studies are needed to investigate the mechanism(s) under-
lying YXLST external Qi mediated increase of PI3K activity
in neurons.
IGF-I is a 70-amino-acid peptide that belongs to a family
of polypeptide trophic factors sharing a high degree of
sequence homology with insulin [27]. Evidence strongly
suggests that IGF-I is synthesized by most tissues of the
body and functions as a paracrine or autocrine agent in a
tissue-specific manner [14]. IGF-I promotes the survival,
differentiation of neurons [1,2], and stimulates neurite
outgrowth and DNA synthesis [28]. Up-regulation of IGF-
I gene expression by external Qi of YXLST is clearly shown
in the present study. Similar to the YXLST effect on PI3K
activity, the effect of external Qi of YXLST on IGF-I gene
expression is time-dependent. This is also somewhat anal-
ogous to the observations that the protective effect of
YXLST product was significantly reduced in cells that
had been damaged from prolonged H2O2 exposure in
contrast to cells that had been exposed for a shorter duration
[43], although previous reports indicated that YXLST
effects can last for months [11,15,23]. Interestingly, YXLST
external Qi can block H2O2-induced down-regulation of
IGF-I gene expression and PI3K activity remains high in
YXLST external Qi treated samples with H2O2 exposure.
These further suggest a neuroprotective role of YXLST
external Qi in H2O2-induced cell death.
In summary, these studies indicate that biological effects
of external Qi of YXLST can be measured using multidis-
ciplinary methodologies. External Qi-mediated IGF-I ex-
pression and PI3K signaling could be involved in the
neuroprotection against oxidative stress. These suggest that
YXLST could be useful in the treatment of neurodegener-
ative disorders. The mechanism(s) underlying the YXLST-
mediated neuroprotection remains unclear.
Acknowledgements
This study was supported by Yan Xin Foundation, and an
unrestricted grant from Research to Prevent Blindness to the
Department of Ophthalmology and by NIH grants
EYEY12190 and EY014427. Authors thank Dr. Alexis
Traynor-Kaplan for her kind help and critical comments in
the preparation of this manuscript.
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