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Clinical Biochemistry 37 (2004) 138–145
Effect of Agkistroden blomhoffi (mamushi) on the proliferation
of human fibroblasts
Qing Yang,a,b Guoguang Yang,b,c Jianying Zhang,b Noriyoshi Masuoka,d Yun Z. Riffle,b
Zhigang Wang,b Hideo Ebinuma,e and Hiroyuki Kodamab,*
aYale University, New Haven, CT 06520, USAbDepartment of Chemistry, Kochi University Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan
cDepartment of Orthopaedic Surgery, Musculoskeletal Research Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USAdDepartment of Biochemistry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
eTohtoshu Seizo Co. LTD., 2-17-1, Nagishi, Taitoku, Tokyo 110-0003, Japan
Received 7 July 2003; received in revised form 15 October 2003; accepted 15 October 2003
Abstract
Objectives: The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on human patellar
tendon cells in vitro, to pharmacologically explain the natural medicine’s healing effect on tendon, bone and muscle injuries.
Design and methods: Human patellar tendon fibroblasts (HPTF) were incubated in media containing different concentrations of mamushi
aqueous extract. Cell proliferation was studied by microscopic observations and total protein, actin, collagen I, and cyclooxygenase-2 (Cox 2)
expressions.
Results: Mamushi aqueous extract enhanced HPTF proliferation when its concentration was lower than 333 Ag/ml. Cells cultured in
manushi-containing medium showed developed intercellular structure and increased protein production. However, mamushi extract higher
than 500 Ag/ml oppressed cell growth. At 667 Ag/ml, mamushi induced Cox 2 production, a sign of cytotoxicity.
Conclusion: A. blomhoffi aqueous extract was found to directly stimulate the proliferation and protein production, particularly collagen I
synthesis, of HPTF in a dose-dependent manner.
D 2003 The Canadian Society of Clinical Chemists. All rights reserved.
Keywords: Agkistroden blomhoffi (mamushi); Human patellar tendon fibroblasts (HPTF); Cell proliferation; Type I collagen; Actin
Introduction [2]. Ecdysis of snake suggests that they have a regenera-
Agkistroden blomhoffi (mamushi), a venomous snake,
has been well known for its tonic effect and used as a
source of stamina in ancient China [1]. Mamushi pit viper
has been taken orally in Asia to raise male vigor and
strength. Among the earliest recorded use of snakes in
Chinese medicine is the application of snake slough for the
treatment of superficial diseases, including skin eruptions,
eye infections or opacities, sore throat, and hemorrhoids
0009-9120/$ - see front matter D 2003 The Canadian Society of Clinical Chemis
doi:10.1016/j.clinbiochem.2003.10.012
Abbreviations: HPTF, human patellar fibroblast; PBS, phosphate buffer
solution; CLAP, chymostatin, leupeptin, antipain and pepstatin A; FBS,
fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
Cox 2, cyclooxygenase-2; Mamushi, Agkistroden blomhoffi (mamushi).
* Corresponding author. Fax: +81-888-655937.
E-mail address: [email protected] (H. Kodama).
tive quality for treating chronic skin problems. Snake
gallbladder and bile are used to improve visual acuity,
whooping cough, rheumatic pain, high fever, infantile
convulsion, hemiplegia, hemorrhoids, gum bleeding, and
skin infections [3,4]. Whole bodies, parts and derivatives
like organs, tissues, secretions and feces of snakes are the
main items used for kidney functions [5]. They are
believed to release the excess fire accumulated in the
body, regulate the balance between air and water, espe-
cially improving the circulation of fluids [5].
Snakes are also frequently used in treatments involving
repair of bone, muscle and tendon tissues. Using agkistro-
don-containing formula to treat 118 cases of rheumatoid
arthritis in China, 16 showed complete recovery, 58 showed
good improvement and 39 cases showed some relief, with a
total efficiency of 95.64% [6]. Agkistrodon acutus is found
ts. All rights reserved.
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145 139
in various prescriptions for muscle spasms [7,8]. A wound-
healing cream containing agkistrodon acutus was found to
increase the permeability in skin capillary circulation and the
skin’s ability to prevent the entrance of etiological micro-
organisms. This cream is used for the prevention and
treatment of skin diseases such as fungal invasion of hands,
feet, body and head, psoriasis, scabies, furuncle, erysipelas,
eczema and dermatitis. Its effects include inhibiting bacteria,
reducing inflammation, soothing pain, relieving swelling and
itching, and removing damaged skin [9,10].
However, because these prescriptions were produced in
centuries by trial and error and their effects were con-
firmed only through repeated clinical applications, it is
unclear how the snake genus pharmacologically influences
the tissue to prompt healings. Possibly, the impact of
agkistrodon on the circulation and immune systems has
improved nutrition supply and immunity of the injured
site. Nevertheless, especially in the case of pasting med-
icine in contact with the injured tissue, agkistrodon is
likely to have direct stimulations on tissue regeneration.
Currently, no scientific research has been done on this
subject.
The present research focuses on the direct cellular-level
effect of agkistrodon on tendon cells. It tests whether
agkistrodon in the form of aqueous extract has the ability
to stimulate the proliferation and protein production, partic-
ularly type I collagen synthesis, of human patellar tendon
fibroblast cells. It also pays attention to whether different
concentrations of agkistrodon can induce cytotoxicity during
culture.
Methods
Materials
A. blomhoffi (mamushi) were obtained from Tohtoshu
Seizo Co. Ltd. (Tokyo, Japan). All chemicals and laboratory
materials were purchased from Sigma Co. (St. Louis, MO)
or Gibco BRL Inc., (Grand Island, NY) unless otherwise
stated.
Human patellar tendon fibroblast cells (HPTF) were
prepared from a patient who underwent reconstruction of
the anterior cruciate ligaments using the patellar tendon as
a healthy autograft [11]. Immediately after surgical remov-
al, the fresh sample for primary explants culture was rinsed
twice with sterilized PBS and once with 1% penicillin–
streptomycin–neomycin antibiotic mixture in PBS. The
fatty tissues and blood vessels attached to the explants
were removed and placed in serum-free Dulbecco’s Mod-
ified Eagle Medium (DMEM). The explants were cut into
small pieces (1 � 1 mm) under sterile conditions, then
placed in trypsin–EDTA solution for 5 min. Ten percent
fetal bovine serum (FBS) in DMEM was used to neutralize
the effect of trypsin. The pieces of explants were placed in
35-mm culture dishes and cultured with 10% FBS–
DMEM. After the fibroblasts had migrated from the
explants tissue and reached confluence, the fibroblasts
were trypsinized for 3 min at 37jC. Trypsin activity was
neutralized with five-time volume of 10% FBS–DMEM.
The trypsinized fibroblasts were centrifuged at 1500 rpm
for 5 min, washed with DMEM and resuspended in fresh
10% FBS–DMEM. Cell density was determined and the
cells were seeded into a 25-cm2 culture flask at a density
of 2 � 105 cells per flask.
Preparation of A. blomhoffi (mamushi) aqueous extract
The traditional method for the clinical preparation of
Chinese medicine treatment was employed. Briefly, ground
powder of whole body mamushi (1 g) was added into a flask
with 100 ml of phosphate buffer solution (PBS: 8 g NaCl,
0.2 g KCl, 0.14 g Na2HPO4, 0.24 g KH2PO4 in one liter
H2O; pH 7.3) and stirred at room temperature for 24 h.
Subsequently, residue precipitation was filtered off and put
into water for secondary extraction. The aqueous extracts
were mixed and evaporated to dryness under reduced
pressure with a rotary evaporator at 40jC. The dried residue
was dissolved in PBS and 10 mg/ml of mamushi aqueous
extract was used for cell culture.
Cell culture
Initially, human patellar tendon fibroblast cells (HPTF)
were cultured in 250-ml tissue culture flask (Becton Dick-
inson, England) without mamushi aqueous extract using 15
ml DMEM with 10% FBS and antibiotics (1% penicillin and
streptomycin) at 37jC with 5% CO2 and 95% air. The cells
showed complete adhesion to the bottom of the flask after 2
days. The medium was changed on the third day. On the
seventh day of culture, the cells were harvested with 3 ml
Trypsin-EDTA (Gibco, USA) and diluted with 15 ml of
fresh medium.
Newly harvested HPTF cells were split into six-well
tissue culture plate for subculture under the influence of
mamushi aqueous extract. Each dish contained 2 ml of
harvested cells and 0 to 200 Al of 10 mg/ml of mamushi
aqueous extract with corresponding amount of DMEM so
that the total volume of culture medium was 3 ml with a
plating cell density 10 � 104 cells/ml. For the control
group, instead of mamushi extract 200 Al of PBS and 2.8
ml of DMEM were added since the aqueous extract
contained considerable amount of PBS. Three sets of six
samples with final mamushi aqueous extract concentrations
0, 83, 167, 333, 500 and 667 Ag/ml of medium were
obtained.
The cells were cultured in the conditions described
above for 21 days. The growth of cells was monitored
under a light microscope every 12 h and the living cells
were measured using MTT method [12]. Visualization of
HPTF cells in different culture conditions was performed by
light microscope. The cells were fixed in 2.5% paraformal-
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145140
dehyde and 2% glutaraldehyde in PBS for 30 min and
microscopic pictures were taken on the 4th, 7th, 10th and
14th day.
Actin staining of HPTF cells
After 10 days of culture, the HPTF cells were washed
twice with PBS at room temperature and fixed with 3.7%
paraformaldehyde–PBS for 10 min. Subsequently, the cells
were washed three times with PBS for 5 min each, per-
meabilized with 0.25% Triton X-100/PBS for 5 min, then
washed the cells three times with PBS for 5 min each. The
cells were incubated with Rhodamine-phalloidin (0.165 AMin PBS) at room temperature for 30 min, then washed twice
with PBS. The stained cells were mounted for microscopy
using PBS and observed by a Nikon Diaphot inverting
fluorescent microscopy with filters DM 430 (EX380-425
and BA 510) [13].
Harvest cells and protein extraction
After 21 days of culture, the cells in each dish were
harvested with 0.5 ml Tripsin. After the cells completely
detached from the bottom of the dish, the mixture was
diluted with 2.5 ml of DMEM and transferred to 10-ml
tubes to be centrifuged at 1000 rpm for 5 min. The
Fig. 1. The effect of A. blomhoffi (mamushi) on HPTF cells growth over the 21
covered by HPTF. The cells were treated as described in Methods and the values
supernatant was discarded, and the cells were rinsed once
with 1 � PBS. Each tube was added 200 Al of lysis buffer–CLAP solution (lysis buffer: 0.187 g HEPE, 0.4235 g NaCl,
0.001 g MgCl2 and 0.19 g EGTA dissolved in 50 ml PBS;
CLAP solution: 4 Al each of chymostatin, leupeptin, anti-
pain and pepstatin A in 100 Al PBS; lysis buffer/CLAP
solution: 100 Al CLAP solution added to 6.6 ml lysis
buffer). To assure complete rupture of the cells, the tubes
were stored in �20jC for 12 h. Lysed cells for each
mamushi aqueous extract or control treatment were pipetted
into an ependorf tube.
Total protein concentration was quantified using the
BCA protein assay kit (Fisher Scientific, USA), which
measured the light absorbance at 562 nm verses a standard
curve on a microplate reader. Four 15 Al of 1:5 diluted
samples were drawn for each treatment.
Western blot analysis for type I collagen and
Cyclooxygenase-2 (Cox 2)
The protein was obtained as described above and the
sample volume that would contain 15 Ag total protein was
calculated according to the protein concentration. The
samples, each with 15 Al loading buffer (Loading buffer:
2.4 ml of 1 M Tris–HCl; pH 6.8, 3 ml of 20% SDS, 3 ml
of 10% glycerol, 1.6 ml h-mercaptoethanol and 6 mg
-day culture period, expressed here as the cell numbers in the culture dish
are mean F SD of three samples.
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145 141
bromophenol blue), were then boiled at 100jC for 3 min
and subjected to gel electrophoresis with pre-prepared
10% sodium dodecyl sulfate-polyacrylamide gel (SDS-
PAGE) for 110 min at 125 V [14,15]. Electrophoresed
proteins were transferred onto Imobilon-P membrane (Nip-
pon Millipore Ltd.) using a semidry blotting apparatus
(Sartorius, USA) for 60 min at 2.0 mA/cm2. The mem-
brane was rinsed with deionized water, placed into 5% fat-
free milk, 1% FBS and 1 � PBS-Tween to shake
overnight.
To ensure that equal amount of total protein was loaded
to the membrane, GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) was detected using rat-anti-human GAPDH
antibody (ICN Biochemicals, USA) followed by peroxi-
Fig. 2. HPTF cells on the 10th day of culture as affected by the A. blomhoffi (ma
Methods and the pictures were taken at magnification 10� under light microscop
dase-conjugated rabbit-anti-rat IgG antibody (E.Y. Labora-
tories, Inc., USA). Next, Western blotting membranes were
prepared in the same method for detection of type I collagen
and Cox 2 (Cyclooxygenase-2), a dioxygen and peroxida-
tive enzyme acting as the inflammatory factor of cells [16]
which was tested here for the cytotoxicity of mamushi
extract. Type I collagen was detected using goat-anti-human
collagen I antibody (ICN Biochemicals, USA) followed by
peroxidase-conjugated rabbit-anti-goat IgG antibody (E.Y.
Laboratories, Inc., USA). Cox 2 was detected using mouse-
anti-human Cox 2 antibody (ICN Biochemicals, USA) and
goat-anti-mouse IgG antibody (E.Y. Laboratories, Inc.,
USA). Molecular weights of the proteins were determined
using prestained molecular weight standards (14,300–
mushi) concentration in the medium. The cells were treated as described in
e.
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145142
200,000 molecular weight range; GIBCO BRL). The lanes
were scanned and the intensity of the protein bands was
analyzed using NIH Image software [14].
Statistical analysis
The results were obtained from three separate experi-
ments. The calculation for total protein concentration from
optical density was performed on SPFT max Pro program
(Molecular Devices, Co., USA). Other statistical analyses
were carried out on Microsoft Excel. All quantitative data
reported here are expressed as means of samples for each
treatment with or without mamushi aqueous extract. Statis-
tical analyses also included a Student t test, with signifi-
cance established at P V 0.05.
Fig. 3. Effect of A. blomhoffi (mamushi) on actin synthesis of HPTF cells cultured w
by rhodamine-phalloidin and pictures taken at magnification 10� under flouresce
Results
Growth of human patellar tendon fibroblast cells
Fig. 1 showed the growth of HPTF cells over the 21-day
culture period. The control group cultured without mamushi
aqueous extract expressed steady increase and was confluent
by the fifth day. At concentrations of 167 and 333 Ag/ml,
mamushi aqueous extract accelerated cell growth over the
control group (P < 0.05). The best concentration of
mamushi aqueous extract in the present investigation was
333 Ag/ml (P < 0.01). On the other hand, at higher
concentrations, mamushi aqueous extract had suppressing
effect on HPTF cells. Cell growth started to slow down but
still maintained a growth rate better than that of the cells in
ith or without mamushi aqueous extract for 10 days. The cells were stained
nt microscope.
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145 143
control group. The suppressing effect was most evident at
the highest concentration 667 Ag/ml.
As shown in Fig. 2, HPTF cells grown in different
culture mediums expressed significant difference in prolif-
eration by the 10th day of culture. In the control dish, the
cells held high density and well-developed intercellular
collagen networks. Cell density increased as the concentra-
tion of mamushi aqueous extract in the medium increased
up to 500 Ag/ml. But decreased when the concentration of
mamushi aqueous extract was higher than 500 Ag/ml. Well-
developed matrix connections were observed in the
mamushi aqueous extract concentration of 333 Ag/ml (D)
after 14 days of culture. Occasionally, over extended matrix
networks were present in mamushi aqueous extract medium.
However, generally, shrinkage of the cell body and large
number of dead cells, which are indicated by the bright
spots, were evident (E and F). Precipitations from mamushi
aqueous extract were also observed at higher concentrations.
The cell growth was often unbalanced in higher concen-
trations of mamushi aqueous extract medium, having
crowded and completely empty places on the dishes.
Actin secretion
Fig. 3 showed the effect of mamushi aqueous extract on
the synthesis and secretion of actin by HPTF cells. On the
tenth day of culture, stronger red fluorescent protein ex-
pression was found in the cells grown in the medium with
mamushi aqueous extract, indicating high actin protein
expression. Mamushi aqueous extract did not change cell
morphology, but the cells grown in the medium with
mamushi aqueous extract exhibited a highly organized
stress fiber apparatus. The stress fiber strength of the cells
Fig. 4. Protein production of human patellar tendon fibroblast cells at the end of
protein/ml). The values are mean F SD of four samples.
increased as the concentration of mamushi aqueous extract
increased from 0 to 333 Ag/ml. When concentration of
mamushi was higher than 500 Ag/ml, the stress fiber was
shorter and weaker.
Total protein expression
Fig. 4 showed the concentration of total protein produced
by HPTF cells at the end of 21-day culture period. When
mamushi aqueous extract was added to the medium, protein
production in the tendon cells was increased. However, at
the highest concentration of mamushi aqueous extract, 667
Ag/ml, the protein production was reduced to 15% of that in
control medium (P < 0.01). On other hand, the cells grown
in the medium with lower concentration of mamushi aque-
ous extract synthesized and secreted more proteins than that
of the cells in control medium (P < 0.05 for concentrations
333 and 500 Ag/ml). The highest protein production was
observed in the cells grown in the medium with 333 Ag/ml
aqueous extract of mamushi (P < 0.01).
Collagen I and Cox 2 expressions
Western blot analysis results on Fig. 5 showed the
influence of HPTF grown in the medium with different
concentrations of mamushi aqueous extract. GAPDH
showed an equal protein expression of the cells grown in
all dishes (A). Type I collagen expressed an increasing trend
accompanying the increase of mamushi concentration in
medium with the peak at 333 Ag/ml (B). Under most
treatments, the cells presented very pale expression for
Cox 2 protein; slightly intensified band showed at the
highest mamushi concentration, 667 Ag/ml (C).
21-day culture period, expressed as the concentration of total protein (Ag
Fig. 5. Western blot analysis for GAPDH, type I collagen and Cox 2 protein
expression in human patellar tendon fibroblast cells at the end of 21-day
culture period as affected by the concentration of A. blomhoffi (mamushi) in
medium. (A) GAPDH, (B) Collagen type I and (C) Cox 2. The result was
one of three independent experiments.
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145144
Discussion
From quantitative and morphological observation on the
human patellar tendon fibroblast cells during the 3-week
culture period, this experiment suggests that low concen-
trations of mamushi aqueous extract accelerated the prolif-
eration of tendon cells. On the other hand, at a higher
concentration of 667 Ag/ml, addition of mamushi to culture
medium had suppressive effect on the cell proliferation and
differentiation. The results of this experiment also suggests
that lower concentration supplement of mamushi aqueous
extract increase the total amount of protein produced in
HPTF cells and stimulated the synthesis of actin and type I
collagen, two important proteins synthesized by fibroblast
cells, particularly tendon fibroblasts.
Tendons and ligaments are strong, ropelike structures
composed of dense connective tissue. All connective tissues
consist of cells surrounded by extracellular matrix. In
tendons and ligaments, the primary cell type is the fibro-
blast, and the main extracellular matrix component is
collagen. The extracellular matrix also contains noncollag-
enous proteins, including a class of large, aggregating
molecules called proteoglycans. The primary function of
collagen is to lend tensile strength to the tendon or ligament,
whereas the proteoglycans serve mainly to resist compres-
sive forces.
Healing of ligaments and tendons can be separated into
four phases—hemorrhage, inflammation, proliferation and
remodeling or maturation [17]. The hemorrhagic phase is
characterized by the formation of a blood clot. Platelets
become trapped in the clot, and subsequently release
biochemical molecules called growth factors that attract
white blood cells to the wound. Macrophages, a specialized
type of white blood cell that destroys necrotic tissue,
predominate the inflammatory phase. As a result, fibroblasts
and other cells enter the wounded area. The proliferative
phase is marked by an increase in the number of fibroblasts,
which begin to synthesize both collagenous and noncollag-
enous proteins. Capillary buds also begin to form at this
time. The fourth and final phase, remodeling and matura-
tion, involves a gradual decrease in the number of cells in
the wound, as well as an increase in collagen fibril diameter
and collagen cross-links. Both of these events have been
linked to an increase in the strength of healed tendons and
ligaments.
Our experiment has shown that mamushi aqueous extract
stimulated human patellar tendon fibroblast cells growth and
accelerated protein synthesis at lower concentrations. These
results suggest that dry mamushi body powder used in
traditional Chinese medicine might supply an adequate
environment for tissue regeneration and thus are the key
factors in wound repair for tendon and muscles.
Evidently from the depletion of cell density, shrinkage
of cell body, low total protein concentration and Cox 2
expression, the highest concentration of mamushi aqueous
extract used in this experiment, 667 Ag/ml, has caused
Q. Yang et al. / Clinical Biochemistry 37 (2004) 138–145 145
some cytotoxicity to HPTF cells during in vitro culture.
Probably, some chemical components of mamushi body
powder dissolved in the aqueous extract reached the
maximum limit of safe concentration and damaged the
cells. We have determined several amino acids in the
organs and tissues of mamushi [18]. Furthermore, Noguchi
et al. [19] have shown that cystathionine and taurine
contents in water extract of whole body of A. blomhoffi
are much higher when compared with those in tuna, eel,
beef, Ginseng radix and Astragail radix. Cystathionine is
well known as an important intermediate in the transsulfu-
ration pathway from methionine to cysteine in mammalian
tissues. Wisniewski et al. [20] have reported that human
brain contains a high concentration of cystathionine. In
other mammalian species, the concentration of L-cystathio-
nine in the pool of free amino acid is higher in brain than in
any other tissue [21]. Some researchers have also demon-
strated the presence of large amount of cystathionine in
diseased human brain [22]. Whether the toxicity of
mamushi on HPTF cells culture came from cystathionine
and/or taurine are not clear. However, Hwang and Wang
[23] has reported that the toxic effect of cadmium was
significantly reduced when the rats fed diet with supple-
ment of taurine.
It then seems to be a contradictory phenomenon that the
production of type I collagen was the strongest at 333 Ag/ml
mamushi aqueous extract concentration. One possible ex-
planation is that mamushi in the medium became a stimulus
for the cells to generate the specific proteins. This stimula-
tion was too trivial to detect at high concentrations, so the
protein productions were close to those expressed by the
cells cultured in control medium. High mamushi aqueous
extract concentration may inhibit the proliferation of the
cells.
This research introduced some insights to the subject of
the effect of A. blomhoffi (mamushi) on human tendon cells.
Mamushi not only enhanced total protein production signif-
icantly but also stimulated type I collagen synthesis, in-
creasing the portion of collagen in total protein. Usually
traditional prescriptions combine 3 to 10 herbal and mineral
medicines; although only 1 or 2 are responsible for the
central effect, the supplemental ingredients are also impor-
tant in achieve the goal of remedy. Therefore, some aspect
of mamushi may only be present in combination with other
medicines, which may simultaneously lessen its cytotoxic-
ity. Typical length of clinical treatment for tendon, bone and
muscle injuries with mamushi ranges from a week to a
couple months. Some long-term effects of mamushi may not
have been revealed in the short culture period of this
experiment. In addition, the concentration of mamushi
may change once the medicine is taken into the body
because of protein-binding compounds. The concentrations
used in this research were only a standard in vitro. Whether
the effect of A. blomhoffi (mamushi) will change in vivo is
not known. Farther investigation is planed to examine these
possibilities.
Acknowledgment
We thank Mr. Toshikazu Mariyama, President of
Tohtoshu Seizo Co. Ltd, for his gift of mamushi.
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