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Introduction
Propyl gallate (PG) is a well-known synthetic antioxidant
on the US Food and Drug Administration list (Figure 1). It is
a derivative of gallic acid, which is one of the active
ingredients of Paeoniae Radix, a commonly used traditional
Chinese medicine (TCM). Besides its antioxidant activity, PG
has many pharmacological effects such as free radical
elimination[1,2], inhibiting platelet
aggregation[3,4], anti-inflammatory
effects[5] and anti-tumor
activities[6]. It is used widely to nourish blood, activate circulation, alleviate pain, regulate
menstruation and treat liver disease and cancer. However,
until now the research on PG has been focused on its
curative effects at the holistic level. There are no detailed
studies on the mechanism and dynamics of action at the
molecular level, which limits the further clinical use of PG.
Tumor necrosis factor-a (TNF-a) is produced mainly by
activated macrophages and monocytes. Mature TNF-a
mole-cules are released as 17 kDa monomers, under physiological
conditions, and the monomers associate non-covalently to
form a homotrimer that is biologically
active[7,8]. TNF-a is one of the most pleiotropic cytokines; besides its antitumor
activity in vitro and
in vivo, it plays an important role in
many physiological and pathophysiological responses, such
as inflammatory reactions, thrombosis, cardiac failure,
infection, immune regulation, antiviral responses and
endo-toxic shock, as well as in the pathogenesis of certain
autoimmune diseases[9-13].
The biological activities of TNF-a are mediated by
binding to 2 distinct membrane TNF receptors (TNFR), TNFR-I
(p55, ~55 kDa) and TNFR-II (p75, ~75 kDa). Although both
TNFR-I and TNFR-II bind TNF-a with high affinity, it has
been generally believed that most of the cellular TNF
responses are dominated by TNFR-I. Membrane TNFR (mTNFR) can be cleaved proteolytically to
release soluble forms of the receptors (sTNFR). Though sTNFR
lacks an intracellular domain, it binds free TNF-a with high affinity,
competing with mTNFR[14,15].
Though their physiological roles are not fully understood,
sTNFR might regulate the function of TNF
in vivo through 2 different mechanisms: (i) by eliminating mTNFR and thereby
reducing cellular reactivity to TNF, sTNFR may protect the
cell from TNF pathological injury; and (ii) whereas sTNFR
inhibits the activity of TNF by binding and neutralizing the
cytokines at high concentrations, it may also enhance
TNF activity at low concentrations by stabilizing TNF trimer
molecules and prolonging their availability for binding mTNFR,
thereby acting as an
immunoregulator[16,17].
Because sTNFR is present constitutively in serum
derived from receptor shedding after cellular activation by
stimuli such as TNF-a production[18], sTNFR
serum level is considered a reliable indicator of
TNF-a system activation[19]. Moreover,
TNF-a and sTNFR are considered to be key disease molecules and therapeutic
targets[9,20]. Because the pharmacological functions of PG and the
biological activity of TNF-a are probably correlative, the
TNF-a-mTNFR-sTNFR system may be a potential target of PG
in vivo. In order to find out if this is the case, in the present
paper, an affinity biosensor, the cuvette-based IAsys
biosensor, was used to study the effects of PG on the
interaction of TNF-a with sTNFR-I.
Materials and methods
Chemicals N-hydroxysuccinimide (NHS),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), ethanolamine and
PG were purchased from Sigma-Aldrich (St Louis, MO, USA).
TNF-a and sTNFR-I were purchased from PeproTech EC
(London, UK). Carboxymethyl dextran (CMD) dual-well
cuvettes were purchased from Labsystems Affinity Sensors
(Cambridge, UK). Phosphate-buffered saline Tween-20
(PBST, pH 7.4) was composed of 10 mmol/L
Na2HPO4/NaH2PO
4, 138 mmol/L NaCl, 2.7 mmol/L KCl and 0.05%
Tween-20. All solutions were made by using deionized water.
All reagents were of analytical grade and were used without
further purification.
Apparatus All analyses were carried out using an IAsys
Plus optical biosensor (Labsystems Affinity Sensors,
Cambridge, UK), which is based on resonant mirror technology. This instrument employs a dual-well stirred
cuvette (pre-derivatized with CMD) and monitors the
interaction between a pair of biomolecules inside the reaction
cuvette[21-23]. One of the biomolecules, the ligand, is
immobilized to the sensing surface of the cuvette. Its binding partner,
the ligate, is then injected into the cuvette. Changes in
refractive index and thickness on the sensing surface occur
upon binding (association) of ligate to the immobilized ligand.
The IAsys biosensor detects these changes continuously,
producing a plot of responses that are measured in arc
seconds[24-27]. Following each interaction, the surface of the
cuvette is regenerated to remove bound ligate, and another
ligate can then be introduced to the sensing surface. The
IAsys biosensor has been applied to many kinds of studies
on molecular recognition, affinity, kinetics, solute
concentrations and multi-molecular interactions. It is especially
useful for capturing measurements quickly and in real time,
and can eliminate the need for radiolabels or other chemical
tags. Compared to traditional methods, numerous additional
steps are saved.
sTNFR-I immobilization To examine the binding of
TNF-a to sTNFR-I, the receptor protein was immobilized onto the
cuvette surface through its residual amine groups via amide
with CMD. The IAsys instrument parameters were set at
100% for stirring, and 0.3 s for the sampling interval. The
running buffer was PBST. Data acquisition then began and
baseline data was gathered for several minutes. The surface
of the cuvette was activated with 40 µL of a 1:1 (v/v)
EDC/NHS mixture (0.4 mol/L EDC and 0.1 mol/L NHS) for 7 min.
The function of EDC/NHS is to activate and promote
the formation of covalent linkages by forming the
N-hydroxysuccinimide ester. The carboxyl on the CMD bound NHS under the
action of EDC and created active ester intermediates that bound
the primary amines of sTNFR-I to make the amide bonds.
Thus, sTNFR-I can be covalently immobilized on the CMD.
The cuvette was subsequently washed with PBST and
10 mmol/L acetate (pH 5.5). After gathering baseline data in
the acetate buffer, 10 µL diluted sTNFR-I was added to the
cuvette to begin the electrostatic uptake of protein and
covalent coupling. After a sufficient reaction time, 15 min or
so, unreacted NHS esters were blocked by 40 µL 1 mol/L
ethanolamine (pH 8.5) and the baseline was stabilized by
washing with PBST.
At the same time, another cuvette channel was used as
the blank control, without anchoring of sTNFR-I. The
acetate buffer was injected instead of the sTNFR-I solution.
Binding detection The sTNFR-I modified and
unmodified cuvettes were both rinsed 3 times with
40 µL PBST, and 10 µL TNF-a (0.01 mg/mL) was then added into each of the
cuvettes to equilibrate for 15 min, before they were washed
with PBST. The resonant angle response was recorded. The
chip surface was then regenerated by adding 10 mmol/L HCl,
until the response signal returned to baseline to proceed
with another binding cycle. To evaluate the affinity binding
of TNF-a and sTNFR-I, a wide range of TNF-a
concentrations (from 0.625 µg/mL to
20 µg/mL) were used.
Effects of PG on binding of TNF-a and
sTNFR-I After 3 times of washes with 40 µL PBST, 10 µL
TNF-a (0.01 mg/mL), preincubated with different concentrations of PG,
was added into the sTNFR-I-modified cuvette. Binding between
TNF-a and sTNFR-I was detected as described above.
Results
sTNFR-I immobilization on the surface of
cuvettes As described in the "sTNFR-I immobilization" section, the
sTNFR-I molecules were immobilized onto the surface of the
IAsys cuvette by the ester exchange reaction of NHS
to COOH groups on CMD. Figure 2 shows the response sensorgram
of the IAsys biosensor to EDC/NHS activation as well as to
the consequent sTNFR-I immobilization (solid line). Phase
(1) is the initial baseline obtained when the surface of the
cuvette was exposed to the PBST; phase (2) is the addition
of EDC/NHS mixture to the sensor chip; phase (3) is the
buffer washing to remove unreacted EDC/NHS mixture; phase
(4) is the washing with acetate buffer; phase (5) is the
addition of sTNFR-I; phase (6) is the dissociation course by
washing with PBST; phase (7) is the blocking of non-coupled
activated CMD sites with ethanolamine; phase (8) is the
baseline stabilization. Arrow (9) is the resonant angle shift
caused by the immobilization of sTNFR-I onto the cuvette
surface. The broken line indicates the reference channel
signal in order to follow non-specific binding on the
biosensor surface.
The amount of immobilized sTNFR-I was calculated by
subtracting the baseline level after phase (4) from that after
phase (8), described in Figure 2 as arrow (9), which is
approximately 450.61 arc seconds. The sensitivity of the CMD
cuvette is 163 arc seconds/ng per mm2. Thus the density of
immobilized sTNFR-I is 2.76 ng/mm2. Dual-well IAsys
cuvettes have a sensor area of
4 mm2, so the total amount of sTNFR-I immobilized was 11.04 ng.
Detection of interaction between sTNFR-I and
TNF-a Figure 3 shows the comparison of
TNF-a binding to the sTNFR-I modified cuvette surface (solid line) and the
unmodified surface (broken line). The results showed that
there was no specific binding on the unmodified cuvette
surface except for non-specific absorption, which was easily
washed away. This indicates that TNF-a bound sTNFR-I on
the cuvette surface specifically.
The dissociation equilibrium constant
(KD) of binding of TNF-a and sTNFR-I were determined by using the FASTfit
program, which is specifically designed for biomolecular
interaction analysis using the IAsys biosensor. The
KD value was
5.05×10-7 mol/L, while the
KD of TNF-a/mTNFR-I binding has been reported as about
1×10-9 mol/L[28]. It can be
inferred that though sTNFR-I can bind TNF-a with high
affinity, its binding ability is lower than mTNFR-I. The
results indicate that sTNFR-I binding affinity for
TNF-a is decreased, which is probably attributable to the
denaturation that occurred when sTNFR-I was produced.
Effects of PG on the interaction between
TNF-a with sTNFR-I Figure 4 shows the experimental results obtained
at a PG concentration of 500 nmol/L. With the presence of
PG, the initial velocity of TNF-a/sTNFR-I binding increased
and the time taken to approach equilibrium decreased, which
indicated that PG was able to enhance the binding of
TNF-a to sTNFR-I.
The concentration-dependent effects of PG on the
binding of sTNFR-I and TNF-a were studied. The PG
concentrations varied from 5 nmol/L to 500 nmol/L, and the results are
summarized in Figure 5. The increase in amplitude of the
initial velocity of TNF-a/sTNFR-I binding, as well as the
decrease in amplitude of the time taken to
approach equilibrium, both increased as PG concentration increased, which
indicated that PG enhanced TNF-a/sTNFR-I binding in a
dose-dependent manner.
Discussion
Recent researches showed that the pathogenesis of the
traditional Chinese medicine syndromes is the result of
perturbation of cytokine networks, and the mechanism of TCM
is to regulate and correct the perturbation of cytokine
networks. It has been observed for many years that Chinese
prescriptions can regulate the gene expression level of
cytokines or their receptors. But for the initial step of the
cytokine action, the binding to the corresponding receptor,
there is few reports mainly due to the lack of appropriate
methods. In the present study, an affinity biosensor, the
IAsys biosensor, was used to explore the interaction of the
cytokine and its receptor. The involvement of
TNF-a in the pathogenesis of various diseases makes it an obvious and
attractive therapeutical target. The effects of PG on the
interaction between TNF-a and sTNFR-I were examined by
using the IAsys biosensor in an in vitro assay. The
experimental results showed that PG enhanced TNF-a/sTNFR-I
binding in a dose-dependent manner. Though the
mechanisms of PG are not clear and the roles of
TNF-a and sTNFR-I are debated, it can be concluded that the binding between
TNF-a and sTNFR-I is another target that PG can act on
in vivo. Researches showed that serum level of sTNFR-I are
often increased in a variety of conditions which are
characterized by an antecedent increase in TNF-a, such as sepsis,
inflammatory responses, autoimmune diseases, human
immuno-deficiency virus infection, transplant rejection and
malignant tumor[29-32], which indicated that sTNFR-I
production seems to neutralize and reduce the toxicity associated
with the elevated serum TNF-a level. PG can enhance the
cellular protection of sTNFR-I through increasing
neutralization to TNF-a, which is in agreement with the
experimental and clinical results
reported[33]. Thus it may sound theoretically that PG is helpful for therapy or assistant therapy in
TNF-a corresponding diseases, which may leads to the new
therapeutic application of PG. These are the first results
obtained using affinity biosensors for studying the
interactions of these molecules. This finding offers a new clue to
the study of the mechanism of action of PG. The IAsys
biosensor could be used widely as a reliable tool for similar
studies. With respect to most of the other available
methodologies to study biomolecular interactions, there are many
advantages of this new method, such as real time
measure-ment, high sensitivity, high selectivity and high veracity, as
well as being easy to handle and eliminating the need for
labeled compounds. It has tremendous application
prospects in studies on the mechanisms of TCM in
vitro. It will be more compellent when data on bioactivity, such as from
cell experiments, were acquired to support the results from
those from biosensor technology. Further studies in this
area are in progress.
References
1 Reddan JR, Giblin FJ, Sevilla M, Vanita P, Dorothy CD, Victor
RL, et al. Propyl gallate is a superoxide dismutase mimic and
protects cultured lens epithelial cells from
H2O2 insult. Exp Eye Res 2003; 76: 49-59.
2 Shanker G, Aschner M. Methylmercury-induced reactive oxygen
species formation in neonatal cerebral astrocytic cultures is
attenuated by antioxidants. Brain Res Mol Brain Res 2003; 110:
85-91.
3 Panganamala RV, Miller JS, Gwebu ET, Sharma HM, Cornwell
DG. Differential inhibitory effects of vitamin E and other
antioxidants on prostaglandin synthetase, platelet aggregation and
lipoxidase. Prostaglandins 1977; 14: 261-71.
4 Chen KJ. Antithrombosis of propyl gallate. Chin Prescrip Drug
2003; 9: 38-9.
5 Franzone JS, Natale T, Cirillo R. Influence of propyl gallate and
2-mercapto-propionyl glycine on the development of acute
inflammatory reactions and on biosynthesis of
PGE2. Bull Soc Ital Biol Sper 1980; 56: 2539-45.
6 Lee SMY, Li MLY, Tse YC, Leung SCL, Lee MMS, Tsui SKW,
et al. Paeoniae Radix, a Chinese herbal extract, inhibits hepatoma
cells growth by inducing apoptosis in a p53 independent pathway.
Life Sci 2002; 71: 2267-77.
7 Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B.
An endotoxin-induced serum factor that causes necrosis of tumors.
Proc Natl Acad Sci USA 1975; 72: 3666-70.
8 Smith RA, Baglioni C. The active form of tumor necrosis factor
is a trimer. J Biol Chem 1987; 262: 6951-4.
9 Taylor PC, Williams RO, Feldmann M. Tumour
necrosis factor a as a therapeutic target for immune-mediated inflammatory
diseases. Curr Opin Biotechnol 2004, 15: 557-63.
10 Piguet PF, Vesin C, Chen DK. Activation of platelet caspases by
TNF and its consequences for kinetics. Cytokine 2002; 18:
222-30.
11 Guarntz D, Cowling RT, Vaiki N, Frikovsky E, Moore CD,
Greenberg BH. IL-1b and TNF-a upregulate angiotensin II type
1 (AT1) receptors on cardiac fibroblasts and are associated with
increased AT1 density in the post-MI heart. J Mol Cell Cardiol
2005; 38: 505-15.
12 Zimmermann VS, Bondanza A, Rovere-Querini P, Colombo B,
TNF receptors and their interaction with hTNF-a. Acta Biochim
Biophys Sin 1999; 31: 25-30.
29 Carmen P, Antonio A, Manuel F. In vitro anti-inflammatory
activity of Phlebodium decumanum. Modulation of tumor
necrosis factor and soluble TNF receptors. Inter Immunopharm 2003;
3: 1293-9.
30 Le Y. Clinical implication of soluble tumor necrosis factor
receptors of infection diseases. Clin Med J Chin 2004; 11(2):
184-5.
31 Klaus P. Biological functions of tumor necrosis factor cytokines
and their receptors. Cytokine Growth Factor Rev 2003; 14:
185-91.
32 Monika H, Thomas P, Janet MM. Diurnal and sleep-wake
dependent variations of soluble TNF- and IL-2 receptors in healthy
volunteers. Brain Behavior Immunity 2004; 18: 361-7.
33 Jiang YR, Yin HJ, Chen KJ. Current status of research on paeoniae
radix 801. Chin J Integ Tradit West Med 2004; 24: 760-3.
Sacchi A, Fascio U, et al. Characterisation of
functional biotinylated TNF-á targeted to the membrane of apoptotic melanoma cells. J
Immunol Methods 2003; 276: 79-87.
13 Mocellin S, Rossi CR, Pilati P, Nitti D. Tumor necrosis factor,
cancer and anticancer therapy. Cytokine Growth Factor
Rev 2005; 16: 35-53.
14 Mwangi SM, Stabel J, Lee EK, Kehrli ME, Taylor MJ.
Expression and characterization of a recombinant soluble form of bovine
tumor necrosis factor receptor type I. Vet
Immunol Immunopathol 2000; 77: 233-41.
15 Bao L, Lindgren JU, Zhu Y, Ljunggren HG, Zhu J. Exogenous
soluble tumor necrosis factor receptor type I ameliorates murine
experimental autoimmune neuritis. Neurobiol Dis 2003; 12:
73-81.
16 Pennica D, Kohr WJ, Fendly BM, Shire SJ, Raab HE, Borchardt
PE, et al. Characterization of a recombinant extracellular
domain of the type 1 tumor necrosis factor receptor: evidence
for tumor necrosis factor a induced receptor aggregation.
Biochemistry 1992; 31: 1134-41.
17 Aderka D, Engelmann H, Maor Y, Brakebusch C, Wallach D.
Stabilization of the bioactivity of tumor necrosis factor by its
soluble receptors. J Exp Med 1992; 175: 323-9.
18 Beutler B, van Huffel C. Unraveling function in the TNF ligand
and receptor families. Science 1994; 264: 667-8.
19 Leist M, Gantner F, Jilg S, Wendel A. Activation of the 55 kDa
TNF receptor is necessary and sufficient for TNF-induced liver
failure, hepatocyte apoptosis, and nitrite release. J Immunol
1995; 154: 1307-16.
20 Punzon C, Alcaide A, Fresno M.
In vitro anti-inflammatory activity of
Phlebodium decumanum. Modulation of tumor necrosis
factor and soluble TNF receptors. Int Immunopharmacol 2003;
3: 1293-9.
21 Gambari R. Biospecific interaction analysis (BIA) as a tool for
the design and development of gene transcription modifiers. Curr
Med Chem 2001; 1: 277-91.
22 Baker KN, Rendall MH, Patel A, Boyd P, Hoare M, Freedman
RB, et al. Rapid monitoring of recombinant protein products: a
comparison of different technologies. Trends Biotechnol 2002;
20: 149-56.
23 Wilson WD. Analyzing biomolecular interactions. Science 2002;
295: 2103-5.
24 Buckle PE, Davies RJ, Kinning T, Yeung D, Edwards PR, Pollard
KD. The resonant mirror: a novel optical sensor for direct
sensing of biomolecular interactions: Part II. Applications.
Biosens Bioelectron 1993; 8: 355-68.
25 Karlsson R, Michaelsson A, Mattsson L. Kinetic analysis
of monoclonal antibody-antigen interactions with a new biosensor based
analytical system. J Immunol Methods 1991; 145: 229-40.
26 Dmitriev DA, Massino YS, Segal OL. Kinetic analysis of
interactions between bispecific monoclonal antibodies and
immobilized antigens using a resonant mirror biosensor. J Immunol
Methods 2003; 280: 183-202.
27 Bertucci C, Cimitan S, Menotti L. Optical biosensor analysis
in studying herpes simplex virus glycoprotein D binding to target nectin1
receptor. J Pharm Biomed Anal 2003; 32: 697-706.
28 Fang J, Wang DB, Chen CQ. Expression in BHK cells of
two human
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