Allergic and photoallergic contact dermatitis from ketoprofen: results of (photo) patch testing and follow-up of 42 patients.
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):159-66. doi: 10.1111/j.07.01296.x.Allergic and photoallergic contact dermatitis from ketoprofen: results of (photo) patch testing and follow-up of 42 patients.1, , , .1Department of Dermatology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.AbstractBACKGROUND: Photoallergic contact dermatitis from topical ketoprofen (KP), a nonsteroidal anti-inflammatory agent, is a well-known side effect.OBJECTIVES: To investigate photo-contact allergic reactions to KP and other nonsteroidal anti-inflammatory drugs (NSAIDs), sunscreens, and fragrance components as well as the presence of prolonged photosensitivity related to it.PATIENTS/METHODS: From June 1993 to June 2007, 42 patients were patch tested and photopatch tested with the ingredients of a KP preparation and other relevant substances. A questionnaire was performed in order to determine the importance of prolon 40/42 did respond.RESULTS: 38 patients showed photo-contact reaction, 1 photoaggravated reaction, and 3 contact allergic (CA) reaction to KP. Simultaneous photo-contact allergic reactions were frequently observed not only to structurally related but also to non-structurally related NSAIDs and sunscreens. Simultaneous CA to fragrance components was common. 1/3 of the patients reported prolonged photosensitivity, i.e. from 1 up to 14 years after having stopped KP application.CONCLUSIONS: The history is often not a good guidance to determine KP-related (photo) allergic contact dermatitis and the severe clinical symptoms sometimes require hospitalization, and/or systemic corticosteroids. As for the association between KP and sunscreen intolerance (being 1 of the possible causal factors for recurrent dermatitis), routine standard photopatch testing with KP might be indicated.PMID:
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Research article
Xiao-Qing-Long-Tang shows preventive effect of asthma in an allergic asthma mouse model through neurotrophin regulation
Ren-Shiu Chang, Shulhn-Der Wang, Yu-Chin Wang, Li-Jen Lin, Shung-Te Kao* and Jiu-Yao Wang*
Corresponding authors:
Graduate Institute of Chinese Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402, Taiwan
School of Post-Baccalaureate Chinese Medicine, College of Chinese Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan
School of Chinese Medicine, College of Chinese Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan
Department of Chinese Medicine, China Medical University Hospital, No. 2 Yude Road, Taichung, 40447, Taiwan
Department of Pediatrics, College of Medicine, National Cheng Kung University, No. 138, Sheng-Li Road, Tainan 70428, Taiwan
Department of Chinese Medicine, Tainan Sin-Lau Hospital, No. 57, Sec. 1, Dongmen Rd, Tainan 70142, Taiwan
For all author emails, please .
BMC Complementary and Alternative Medicine 2013, 13:220&
doi:10.82-13-220
The electronic version of this article is the complete one and can be found online at:
Received:7 March 2013
Accepted:6 September 2013
Published:8 September 2013
& 2013 Chang et al.; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Background
This study investigates the effect of Xiao-Qing-Long-Tang (XQLT) on neurotrophin in
an established mouse model of Dermatophagoides pteronyssinus (Der p)-induced acute
allergic asthma and in a LA4 cell line model of lung adenoma. The effects of XQLT
on the regulation of nerve growth factor (NGF) and brain-derived neurotrophic factor
(BDNF), airway hyper-responsiveness (AHR) and immunoglobulin E were measured.
LA4 cells were stimulated with 100 μg/ml Der p 24 h and the supernatant was collected
for ELISA analysis. Der p-stimulated LA4 cells with either XQLT pre-treatment or XQLT
co-treatment were used to evaluate the XQLT effect on neurotrophin.
Balb/c mice were sensitized on days 0 and 7 with a base-tail injection of 50 μg Dermatophagoides
pteronyssinus (Der p) that was emulsified in 50 μl incomplete Freund’s adjuvant (IFA).
On day 14, mice received an intra-tracheal challenge of 50 μl Der p (2 mg/ml). XQLT
(1g/Kg) was administered orally to mice either on days 2, 4, 6, 8, 10 and 12 as a
preventive strategy or on day 15 as a therapeutic strategy.
XQLT inhibited expression of those NGF, BDNF and thymus-and activation-regulated cytokine
(TARC) in LA4 cells that were subjected to a Der p allergen. Both preventive and therapeutic
treatments with XQLT in mice reduced AHR. Preventive treatment with XQLT markedly
decreased NGF in broncho-alveolar lavage fluids (BALF) and BDNF in serum, whereas
therapeutic treatment reduced only serum BDNF level. The reduced NGF levels corresponded
to a decrease in AHR by XQLT treatment. Reduced BALF NGF and TARC and serum BDNF levels
may have been responsible for decreased eosinophil infiltration into lung tissue.
Immunohistochemistry showed that p75NTR and TrkA levels were reduced in the lungs
of mice under both XQLT treatment protocols, and this reduction may have been correlated
with the prevention of the asthmatic reaction by XQLT.
Conclusion
XQLT alleviated allergic inflammation including AHR, IgE elevation and eosinophil
infiltration in Der p stimulated mice by regulating neurotrophin and reducing TARC.
These results revealed the potential pharmacological targets on which the XQLT decotion
exerts preventive and therapeutic effects in an allergic asthma mouse model.
Keywords: A Xiao-Qing-Long-Tang (XQLT); Nerve growth factor (NGF); Brain-derived neurotrophic factor (BDNF); p75 neurotrophin receptor (p75NTR)Background
The pathology of allergic asthma is characterized by clear eosinophil infiltration
in the airways and the elevation of systemic IgE levels, mediated by type 2 T-helper
(Th2) cells and cytokines. Airway remodeling, observed in severe form of allergic
asthma, is a consequence of repetitive airway inflammation and Th2-type reaction.
Although the imbalance of Th1/Th2 immune response is well known and has been used
to elucidate the immune-pathogenesis of allergy and other autoimmune diseases, this
relevant theory is incomprehensive and satisfactory clinical applications are lacking.
Traditional Chinese Medicine (TCM) decotion such as Xiao-Qing-Long-Tang (XQLT) is
frequently used in Asia for the clinical treatment of bronchial asthma and allergic
rhinitis [,], XQLT, in particular, has been found to have beneficial effects in relieving Th2-based
reactions in the airways of animal models of allergic asthma [-]. Despite its effects on immunological regulation, the molecular mechanism and pharmacological
action of XQLT remain unclear. XQLT is highly popular in TCM and complementary medicine,
because it has no major side effects. Therefore, the therapeutic targets and ethno-pharmacological
action of the decotion must be thoroughly examined before it can be widely used for
the prevention and treatment of asthma.
Members of the neurotrophin family have been shown not only to act as growth factors
in the nervous system, but also to act as pro-inflammatory factors in the immune system.
Airway hyper-responsiveness (AHR) can be mediated through substance P, neurokinin
A, and other members of the neurotrophin family, suggesting that neural hyper-innervation
of the airways may be responsible for AHR []. Nerve growth factor (NGF) which is a member of the neurotrophin family is one product
of activated Th2 cells [,]. Moreover, our recent research demonstrated that a major allergen of house dust mite,
Dermatophagoides pteronyssinus Group 2 (Der p 2) could induce NGF production and reactive oxygen species in the
airway, as well as allergic inflammation after direct intra-tracheal instillation
into the lungs of mice [].
NGF and the brain-derived neurotrophic factor (BDNF) are survival and activation factors
of eosinophil in patients with allergic bronchial asthma []. NGF and BDNF are expressed in multiple cells, including epithelial cells, active
immune cells, and neural cells. In allergic asthma, the tissue that is primarily responsible
for allergen presentation is the bronchiolar epithelium. These epithelial cells present
allergens and induce allergy pathways that involve multiple events, including dendritic
cell activation and chemokine secretion [,]. Moreover, NGF and BDNF have been observed at elevated concentration in patients
with allergic diseases. Although BDNF has not yet been implicated in early allergic
reactions as NGF, its role in allergic airway dysfunction has been found to be important
[]. BDNF is now known to be directly involved in airway smooth muscle hyperplasia and
hypertrophy by interacting with tyrosine kinase B (TrkB), but not with p75 neurotrophin
receptor (p75NTR), and through the secretion of metalloproteinase-9 (MMP-9) [,]. BDNF is also known responsible for neuronal plasticity in brain and lung. Neuronal
plasticity is also a key factor in airway remodeling and airway hyper-responsiveness.
p75NTR is required for BDNF in regulating depression or anxiety in brain function,
but it is not a necessary factor in smooth muscle hypertrophy which result in airway
remodeling [,]. p75NTR is a low-affinity receptor of all factors of the neurotrophin family, and
allergic inflammation and eosinophil infiltration have been eliminated in p75NTR-knockout
mice [,]. p75NTR is known for inducing NF-κB activation that has been demonstrated to be a
major transcriptional factor in the Th2-type immune response [,]. NGF may also affect dendritic cells (DCs) through p75NTR [].
This paper presents our findings that XQLT inhibited the production of the members
of the neurotrophin family in a mouse model of allergic asthma, alleviating AHR and
the allergic inflammation of the airway. LA4 is a bronchial epithelial cell line of
murine lung origin and produces NGF in response to Der p allergen []. XQLT has been found to inhibit NGF and BDNF and p75NTR expression in LA4 cells.
These results identified the potential pharmacological targets of the XQLT decotion
that might exert its preventive and therapeutic effects in a mouse model of allergic
TCM preparation: Xiao-Qing-Long-Tang (XQLT)
XQLT extract powder was kindly provided by KO-DA Pharmaceutical Co. (Taoyuan, Taiwan,
R.O.C.). All of the eight herbs listed in description below were originally grown
in mainland China and collected by the KO-DA Pharmaceutical Co. from professional
herbal growers. The voucher specimens have been deposited in the publicly available
herbarium of KO-DA Pharmaceutical Co. Those eight herbs were authenticated by Professor
Shih-Chang Lee, China Medical University, Taiwan. The XQLT extract was prepared as
described in a previous study []. Briefly, eight herbal ingredients were mixed by proportion which is shown as number
that is in the brackets behind each scientific name of herbal. They were Pinellaiae tuber (6.0, root of Pineliaternata breitenbach), Ephedrae herba (3.0, stem of Ephedra sinica Stapf), Schizandrae fructus (3.0, a fruit of Schizandra chinensis Baill.), Cinnamonomi cortex (3.0, cortex of Cinnamomum cassia Blume), Paeoniae radix (3.0, root of Paeonia lactiora Pall.), Asariherba cum radice (3.0, whole plant of Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa), Glycyrrhizae radix (2.0, root of Glycyrrhiza uralensis Fisch. Et. DC), and Zingiberis siccatum rhizoma (1.0, steamed root of Zingiber officinale Roscoe). The mixture was extracted sequentially with 17.5 L and 12.5 L boiling water each
time for 1 h. The extracted liquid was mixed and filtered. After filtration, the dregs
of the decotion were removed. The filtered liquid was lyophilized then crushed into
a thin powder. The yield of dried extract from starting crude material was 661.8 g
(26.4%, w/w). The dried extract was subsequently used for all experiments in this
research. The batch number of XQLT extract is .
The XQLT mixture was suspended in distilled water to a fixed concentration for being
orally administered to the mice through feeding needle and swallowing. The feeding
volume was adjusted within 0.2~0.3 ml to avoid mice of pain. For in vitro use, the XQLT mixture was dissolved in distilled water, after which the solution
was centrifuged at 7500 rpm for 30 min. Following filtration, the aqueous extract
was lyophilized and weighed. This XQLT extract was re-dissolved in pyrogen-free isotonic
saline (YF Chemical, Taipei, Taiwan) and filtered through a 0.22-μm filter (Microgen,
Laguna Hills, CA, USA). A sample of the filtered pyrogen-free solution was lyophilized
and weighed. The final concentration of the last filtered pyrogen-free solution was
estimated by the sample. The filtered pyrogen-free solution was stored at -20°C until
Production of neurotrophin in cell lines following Der p stimulation
LA4 (murine lung adenoma) cell line was purchased from the Bioresource Collection
and Research Center (BCRC, Hsinchu, T BCRC60239 derived from ATCC: CCL-196).
The LA4 cells were cultured in Ham’s F12 medium (Gibco), which contained 2.5 mM L-glutamine,
15% fetal bovine serum (FBS), and 0.1% gentamicin, in an incubator at 37°C with 5%
CO2. Group XC comprised LA4 cells that were treated with XQLT alone for 24 h. Group XP
comprised LA4 cells that were treated with XQLT for 1 h, then the XQLT was washed
out, subsequent to 24 h Der p stimulation. Group XD comprised LA4 cells which were
simultaneously treated with XQLT and Der p for 24 h. Group D comprised LA4 cells which
were treated with Der p alone for 24 h. Group N comprised entirely untreated LA4 cells.
1 mg/ml XQLT and 100 μg/ml Der p concentrations set following a pre-titration trial
(data not shown) were administered to the cells in all groups. After the treatment
of all cell groups for 24 h, supernatants and total cell protein were collected for
analysis. All the protocols were schematic in Figure&A. LA4 cells were all seeded at concentration of 1×106 cells/ ml.
XQLT inhibited NGF, BDNF, TARC and p75NTR expression in LA4 cells stimulated with
Der p. Pre-treatment (XP) and co-treatment (XD) with XQLT in Der p-stimulated cells
and merely XQLT treated cells (XC) were used to assess the effects of XQLT on neurotrophin
and associated receptor. (A) The supernatant was collected and analyzed by ELISA to obtain NGF, BDNF and TARC
levels. Student’s t test was performed versus Group D. Schematic of cell experiment
groups is also shown under those ELISA data. (B) Whole cell extracts were used for western blotting with anti-p75NTR antibody or anti-NGF
antibody (commercial antibody also recognizes pro-NGF). Protein expression level was
assessed as western band density detected by densitometry and was depicted as proportional
bar plots (negative group level set as 1). Student’s t test was performed versus Group
D. All the experiments in (A) and (B) were repeated for at least three times. The values shown are means ± SD. * means
p & 0.05, ** means p & 0.01, *** means p & 0.005 in (A) and (B).
Western blot analysis of NGF and p75NTR expression in cell lines
Cells (1~2 × 106 cells/ml) were lysed using a Triton X-100-based lysis buffer that contained 1% Triton
X-100, 150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM EDTA, 5 mM NaN3, 10 mM NaF, and 10 mM sodium pyrophosphate. Cell extracts were separated using SDS-PAGE,
then transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA). After
blocking, the blots were developed using rabbit polyclonal anti-p75NTR antibody or
rabbit polyclonal anti-NGF antibody. The blots were then hybridized using HRP-conjugated
goat anti-rabbit IgG (Calbiochem, San Diego, CA, USA) and developed with a chemiluminescence
kit (Western Lightning Chemiluminescence Reagent PLUS; PerkinElmer Life Sciences Inc.,
Boston, MA, USA). The western band density that corresponded to the p75NTR or NGF
or pro-NGF or β-actin was determined using an image analysis system. The detected
density was representation of expression level of each protein. The density of p75NTR,
NGF or pro-NGF was calculated versus density of β-actin and result was shown as proportion.
The proportion was plotted as bar graph with the value of group N set to be 1. Single
Antibody: anti-p75NTR {Abcam Inc. ab8874}; anti-NGF {NGF (M-20), SANTA CRUZ BIOTECHNOLOGY,
INC. sc-549}.
Acute asthma model set-up
Specific pathogen-free, 6~8 week-old female BALB/c mice from the Laboratory Animal
Center of National Cheng Kung University were used in this study. The mice were housed
in microisolator cages (Laboratory Products, Maywood, NJ, USA) and provided with sterile
food and water ad libitum. All care and treatment of the experimental animals followed the guidelines set by
the National Science Council of the Republic of China. The Institutional Animal Care
and Use Committee (IACUC) of the China Medical University (Permit Number: 100-139-N)
approved the protocol.
On days 0 and 7, groups of mice were subcutaneously injected at the base of their
tails with a 50 μl emulsion that contained 50 μg of Der p in incomplete Freund’s adjuvant
(IFA; Difco, Detroit, MI, USA). Fourteen days later, the mice were lightly anesthetized
with an intra-peritoneal (i.p.) injection of 60 μg/kg body weight of sodium pentobarbital
(Nembutal, Abbott Laboratories, North Chicago, IL, USA). The animals received intra-tracheal
(i.t.) instillation with 50 μl of Der p (2 mg/ml) for the allergen challenge (AC),
after which they were held in an upright position for 1 min, so that they could resume
normal breathing. Figure&A schematically depicts the complete protocol for XQLT treatment. The XQLT dose that
was used in this study was based on the authors’ previous study [] and the pilot study before this research. In Group D, the mouse model for an acute,
Der p allergen-induced asthmatic attack was as follows: initial sensitization with
Der p on Day 0, a Der p booster on Day 7, and an i.t. Der p AC on Day 14. The animals
were sacrificed on Day 16. To evaluate the effects of XQLT on this model, mice in
the therapeutic protocol (Group T) were given 1 g/kg BW of XQLT once at 24 h after
AC, and mice in the preventive protocol (Group P) were given 1 g/kg BW of XQLT six
times, every other day from Day 2 onward, with the last treatment administered 48
h before AC. Mice in Group XC (control) were administered 1 g/kg BW of XQLT every
other day from Day 2 onward without Der p sensitization or AC. Group N (naive group)
comprised animals without Der p sensitization, challenge or XQLT treatment. They were
included in the experiments for comparison. Each group, associated with one experimental
condition, comprised six mice.
Respiratory flow resistance results showed inhibition of AHR by XQLT in a mouse model
of Der p-induced acute asthma. (A) Schematic of mice experiment groups. (B) Respiratory flow resistance was measured by intubation 2 d after Der p challenge.
The standard curve chart displays resistance values under methacholine challenge ranging
from 0 mg/ml to 4 mg/ml. Values represent the means ±SD (n = 6 mice for each group,
all experiments repeated for at least three times). Student's t test was performed
versus Group D (* means p & 0.01; ** means p & 0.005). Student’s t test was also performed
between Group P and Group T (# means p & 0.05).
Lyophilized house dust mites (Dermatopha Der p) were purchased
from Allergon (Engelholm, Sweden). Crude Der p preparation was extracted with ether.
After dialysis with deionized water, the Der p extract was lyophilized and stored
at -70°C until use. LPS concentration of the Der p preparations was 1.96EU/mg of Der
p (Limulus a E-T Sigma-Aldrich).
Invasive measurement of airway resistance
The lung function of mice that had been anesthetized with sodium pentobarbital (60
μg/kg BW) was invasively analyzed. An 18-gauge stainless-steel cannula was inserted
into the trachea of each mouse, which was placed on the FlexiVent system (Scireq(R), Montreal, QC, Canada) for forced oscillation measurements after tracheostomy and
consecutive examinations of total lung capacity (TLC). According to manufacturer’s
instruction and previous studies [,], a single-compartment model of respiratory mechanics was used to evaluate lung function
and the responses of the airway to methacholine (0 mg/ml to 4 mg/ml) 48 h after the
Der p challenge. Total respiratory system resistance (Rrs) was measured using a snapshot
perturbation maneuver. Methacholine was aerosolized for ventilation with an ultrasonic
nebulizer for 10 s, and 12 snapshot perturbations were performed.
Collection of serum and broncho-alveolar lavage fluid (BALF)
The following procedures were based on previous study [,] with slightly modification. The mice were sacrificed by administering a sodium pentobarbital
overdose (20 mg/ml) following Der p challenge. After sacrifice, BALF was collected
by flushing the lung with two separate normal saline through the trachea, around 1
ml of BALF was recovered. Cells were recovered from BALF by centrifugation at 200
× g for 5 min at 4°C, then washed in red blood cell lysis solution, and finally diluted
with RPMI-1640 medium (GIBCO/BRL, Life Technologies, Inc., Gaithersburg, MD, USA).
The total leukocyte content of the BALF was determined using a cytometer to be 1 ×
105 cells/ml. Blood was collected either through the axillary artery or directly from
the heart. The collected blood was left to stand for 1 h at room temperature to clot.
Centrifugation at 14,000 rpm removed the clotted matter to obtain the serum.
Total and Der p-specific IgE/IgG1/IgG2a concentrations in the BALF and serum
IgE, IgG1, and IgG2a concentrations in the BALF and serum were measured using an ELISA
kit (Bethyl Laboratories, Inc). The wells of a 96-well ELISA plate (Model No. 445101,
NUNC) were coated with 100 μl of affinity-purified mouse antibody in 50 mM carbonate-bicarbonate
buffer (pH 9.6). The plate was incubated at room temperature (20°C–25°C) for 1 h.
After the antibody solution was removed, 200 μl of blocking solution that contained
50 mM Tris, 0.14 M NaCl, and 1% bovine serum albumin (BSA) (pH 8.0) was placed in
each well and incubated at room temperature for 30 min. The plates were washed five
times with PBST (0.05% Tween 20). The dilutions of BALF or serum with sample/conjugate
diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20) were added to the wells.
After being sealed with adhesive tape, the plates were incubated at room temperature
for 1 h and again washed five times. After 100 μl of diluted horseradish peroxidase
(HRP)-conjugated antibody was added to each well, the plates were again incubated
at room temperature (20°C–25°C) for 1 h. Thereafter, the plates were again washed
five times, and 100 μl of tetramethylbenzidine (TMB) substrate solution was added
to each well. The plates were then kept in the dark at room temperature for 15 min
for fluorescence developing. The enzyme reaction was stopped by adding 100 μl of stop
solution (0.18 M H2SO4). Absorbance was measured at a wavelength of 450 nm on an ELISA plate reader.
Antibody ELISA: Mouse IgE ELISA Quantitation Set, Bethyl Laboratories, Inc. E90-115;
Mouse IgG1 ELISA Quantitation Set, Bethyl Laboratories, Inc. E90-105; Mouse IgG2 ELISA
Quantitation Set, Bethyl Laboratories, Inc. E90-107;
IFN-γ/ IL-5/IL-13/TGF-β1/TARC/NGF/BDNF concentrations in BALF, serum, and cell line
supernatants
The concentrations of NGF and BDNF in the BALF, serum, and cell culture supernatant
were measured using the appropriate ELISA kits (NGF & BDNF Emax Immuno-Assay Systems),
according to the manufacturers’ instructions (Promega, Madison, WI, USA). The concentrations
of IFN-γ, IL-5, TGF-β1, TARC and IL-13 in the BALF, serum, and cell culture supernatants
were measured using an ELISA kit according to the manufacturer’s instructions.
Cytokine ELISA: NGF Emax(R) ImmunoAssay System, Promega G7630; BDNF Emax(R) ImmunoAssay System, Promega G7610; mouse IFN-gamma {R&D Systems, Inc. DuoSet ELISA
DY485}; mouse IL-5 {R&D Systems, Inc. DuoSet ELISA DY405}; mouse TGF-beta1 {R&D Systems,
Inc. DuoSet ELISA DY1679}; mouse IL-13 {R&D Systems, Inc. DuoSet ELISA DY413}.
Lymphocyte/macrophage/neutrophil/eosinophil percentages in the BALF
BALF cells were spun down onto a glass slide at 360 rpm for 8 min by cytospinning.
The slides were then dried and stained by the hematoxylin and eosin (H&E) or eosinophil-specific
staining methods (Eosinophil-Mast Cell Stain Kit, CEM-1-IFU; ScyTek Laboratories,
Inc., Utah, USA). More than 200 cells were counted under a photomicroscope and the
percentages of lymphocytes, macrophages, neutrophils, and eosinophils were thereby
determined.
Immunohistochemistry
The entire lung was removed and embedded in paraffin for slicing. The paraffin lung
slices were mounted on glass slides. The paraffin was then depleted at 60°C. Each
slice was then sequentially treated with th xylene, ethanol,
3% H2O2 (80% methanol) (v/v), and 0.01 M sodium citrate buffer (pH 6.0, 95°C). After 10%
non-fat milk was used to block the cooled slice, a rabbit polyclonal anti-p75NTR antibody
or a rabbit polyclonal anti-TrkA antibody (Abcam, Cambridge, UK) were used for immunostaining
(4°C, overnight). Anti-Rabbit IgG antibody (FITC or Phyc Abcam,
Cambridge, UK) was used as a secondary antibody to develop fluorescence. The developed
slice was observed under a light microscope. The area density of fluorescence was
analyzed using an image analysis system. The fluorescence density results are shown
as a bar graph. Single Antibody: anti-p75NTR {Abcam Inc. ab8874}; anti-TrkA antibodies
{Abcam Inc. ab76291}.
Statistical analysis
The data are presented as mean ± standard deviation. Statistical comparisons were
performed using Student’s t test analysis, with significance set at P & 0.05 or as
indicated in each figure legend (two-sided test). All the statistical differences
are indicated in the figure legends.
XQLT inhibited NGF, BDNF, and p75NTR expression in LA4 cell line following Der p stimulation
An in vitro model of mouse lung adenoma cells (LA4 cell line) was used to study the possible
mechanism of XQLT. Schematic of cell experimental groups is shown in Figure&A. LA4 cells stimulated with Der p produced three times more NGF than un-stimulated
cells did (“D” and “N” in Figure&A). Cells with XQLT treatment alone (XC) exhibited decreased NGF expression compared
to untreated cell (N). Treatment with XQLT alone inhibited the expression of NGF in
LA4 cells without causing toxicity (by MTT assay, data not shown). Pre-treatment with
XQLT (“XP” in Figure&A) decreased NGF expression more than did co-treatment with XQLT (“XD” in Figure&A). A single dose (determined in a pre-titration trial, whose results are not shown)
of XQLT as a pre-treatment appeared to suffice for the inhibition Der p-induced NGF
expression (“XP” in Figure&A). LA4 cells exhibited high baseline levels of BDNF production. Der p stimulation
slightly elevated LA4 BDNF levels, whereas XQLT clearly reduced them (“XP” and “XD”
in Figure&A). The levels of thymus-and activation-regulated cytokine (TARC), which is the cytokine
that recruits eosinophil and Th2 inflammatory cells in the early phase of an allergic
reaction, were also reduced in XQLT-treated LA4 cells (“XP” and “XD” in Figure&A).
p75NTR knockout mice are known to have a lower response to allergic stimulation []. XQLT also inhibited the expression of p75NTR at the protein level in LA4 (“XP”,
“XD” and “XC” in Figure&B). Pre- and co-treatment (XP and XD) with XQLT indistinguishably reduce p75NTR levels.
XQLT treatment (Figure&B) also inhibited the expression of long-form pro-NGF, which was induced by Der p.
A decrease in pro-NGF level might be responsible for the observed decrease in NGF
level in the cell culture supernatant.
XQLT had a regulatory effect on neurotrophin and TARC from epithelial cells that were
stimulated by Der p. An established acute asthmatic mouse model was then used to study
how XQLT would affect neurotrophin in the asthma reaction.
Measurements of respiratory flow resistance showed inhibition of AHR by XQLT in Der
p-induced acute asthma
Figure&A schematically depicts the experimental groups of mice. Figure&B reveals that the Der p-challenged mice that had been preventively administered XQLT
orally (Group P) had a significantly lower respiratory resistance than did the Der
p-challenged mice that had not been treated with XQLT (Group D; p & 0.01 marked by
* symbol). The preventive strategy also decreased the respiratory flow resistance
significantly more than did the therapeutic strategy (Group T; p & 0.05 marked by
# symbol). Mice treated with XQLT oral administration only (Group XC) had airway resistance
similar to the airway resistance of naive mice (Group N). These results suggested
that XQLT positively inhibited Der p-induced AHR in mice. The airway resistance of
Group XC was not increased. Although XQLT has been found to inhibit AHR in Der p stimulated
mice, the supported data was based on the indirect Penh method []. In this research, AHR values were measured directly via intubation in a mouse model
of Der p-challenged acute asthma and could be more precise.
BALF cytokine profile and eosinophil infiltration in the lung of the acute asthma
mouse model
Both IL-5 and IL-13 are known to be involved in the recruitment of eosinophil. However,
the data did not show that XQLT had any clear effect on IL-5 or IL-13 (Figure&A). XQLT did not have any significant effects on IFN-γ levels in this Der p-induced
acute asthma mice model (Figure&A). However, the thymus-and activation-regulated cytokine (TARC), which is known to
be involved in the Th2-cell attraction response and may be regulated by NGF [], was greatly suppressed by both strategies of XQLT administration (Group P and Group
T). If these relationships between NGF, BDNF and TARC were taken together, XQLT might
regulate neurotrophin and TARC for showing preventive effect on asthmatic processes.
To understand the possible pathways affected by XQLT, further research was conducted
to find the target receptor of XQLT effect.
BALF cytokine profile and eosinophil infiltration in the lung of a mouse model of
acute asthma. (A) BALF was collected for ELISA analysis to evaluate IL-5, IL-13, IFN-γ, TARC, and TGF-β1
levels. TARC levels were clearly decreased by XQLT (Group P and T). Values shown are
the mean ± SD (Student’s t test versus Group D) of the six mice in each group. All
experiments were repeated for at least three times. * means p & 0.05; *** means p & 0.01
(B) Eosinophil infiltration in the lungs of mice in acute asthma model. Lung sections
were prepared for a special stain to identify eosinophil ( as arrow pointed)
and structure cells (deep blue). Eosinophil counts were calculated from 30 vision
fields and listed at the bottom-left of the images in the bar plot. Values shown are
the mean ± SD (Student’s t test versus Group D; * means p & 0.05; *** means p & 0.01)
(n = 6 mice for each group). All experiments were repeated for at least three times.
The data obtained using specific eosinophil/mast cell staining kits revealed that
Der p stimulation induced the infiltration of eosinophil ( as arrow
pointed in Figure&B) into the lungs. Both preventive (Group P) and therapeutic strategies (Group T)
reduced eosinophil infiltration (bottom-left bar graph in Figure&B).
Effects of XQLT on total and Der p-specific IgE, IgG1, and IgG2a levels in serum and
BALF in Der p-induced acute asthma mice model
Monitoring immune-pharmacological and physiological effects is important when a decotion
is used for study in an asthma animal model. Our analysis of Der p-induced antibodies
revealed that XQLT (Group P and Group T) tended to decrease serum total IgE, IgG1,
and IgG2a levels, particularly in mice that had been subject to preventive strategy
(Figure&A). Der p-specific IgE levels were not significantly altered by XQLT, but Der p-specific
IgG1 and IgG2a levels were decreased by XQLT, especially in the preventive strategy
group (Group P in Figure&A). XQLT also reduced total cell infiltration in BALF (Group P and Group T in Figure&B). Mice treated with XQLT alone (Group XC) had antibody quantities or cell infiltration
similar to those in na?ve mice (Group N). The results suggested that repeated XQLT
treatments could decrease allergic inflammation in a mouse model of Der p-challenged
acute asthma.
Effects of XQLT on serum total and Der p-specific IgE, IgG1, and IgG2a levels in a
mouse model of Der p-induced acute asthma. (A) XQLT administration, especially as a preventive strategy, decreased serum total IgE,
IgG1, and IgG2a levels. Der p-specific IgG1 and IgG2a levels were also decreased by
the preventive XQLT strategy. * means p & 0.05; *** means p & 0.01 (B) Total cell infiltration in the lungs was also decreased by the action of XQLT. Values
represent the means ± SD (n = 6 mice for each group, all experiments repeated for
* means p & 0.05; ** means P & 0.01; *** means p & 0.005). Student’s
t test was performed versus Group D in (A) and (B).
XQLT inhibited NGF and BDNF levels in Der p-induced acute asthma
Der p stimulation evidently increased NGF levels in the serum and BALF (Figure&A). The preventive strategy (Group P) clearly reduced NGF levels in BALF, whereas
the effect of the therapeutic strategy (Group T) was uncertain (Figure&A). Those results showed similar trends in compared with the trends of AHR measurements
shown in Figure&B. Our results suggested that XQLT might down-regulate the AHR response of asthma
by regulating NGF, a factor in the early phase of asthma [,]. Serum NGF was not significantly affected in either the therapeutic or the preventive
group (Figure&A). The mechanism by which XQLT down-regulated NGF levels in the BALF without affecting
serum NGF levels will be investigated further. The therapeutic strategy appeared not
to cause significant differences of BALF NGF.
XQLT decreased NGF and BDNF levels in a mouse model of acute asthma. (A) NGF and (B) BDNF levels in the serum and BALF were measured after sacrificing the mice. Values
shown are the mean ± SD (Student’s t test versus Group D) of the six mice in each
group. The preventive XQLT strategy (P) decreased NGF and BDNF levels the most, particularly
in the BALF and serum. * means p & 0.05; ** means P & 0.01; *** means p & 0.005.
In contrast, both the preventive and therapeutic strategies reduced BDNF levels in
the serum more than in the BALF (Figure&B). BDNF has been found to play a role in the late phase of asthma [], and has been reported to be involved in the infiltration of eosinophils, which are
known for their persistent activity in asthma, into the lungs [].
XQLT reduces Der p-induced p75NTR and TrkA expression in lungs
p75NTR knockout mice are known to have a lower response to allergic stimulation []. Immunofluorescence was shown as Total Area density in bar graph of Figure&. Der p stimulation induced high level of p75NTR expression in the lung (Group D).
Data herein revealed that XQLT reduced Der p-stimulated p75NTR in the lungs of acute
asthmatic mice model. XQLT might affect the asthmatic reaction by regulating p75NTR
expression (Figure&A). The preventive and therapeutic oral strategies showed inhibitive effects of minor
statistical difference on p75NTR, revealing that other inhibition mechanisms must
exist (Group P and Group T in Figure&A). The inhibition of p75NTR and BALF NGF by XQLT might together explain how XQLT
prevents the Der p-induced asthmatic reaction. TrkA is also involved in NGF-related
allergic reaction. Our data also showed that Der p-stimulated mice to which XQLT was
orally administered expressed less TrkA in the lungs (Group P and Group T in Figure&B) than did the Der p-stimulated mice (Group D).
XQLT decreased Der p-induced p75NTR and TrkA expression in the lungs. The Der p-challenged lung was used to be sliced for immunofluorescence staining with
an anti-p75NTR antibody or an anti-TrkA antibody. (A) p75NTR (B) TrkA. The symbol {I} represented antibody isotype control. Fluorescence was measured
by image analysis system and shown as total area density in the bar plot. Fluorescence
area density was average fluorescence density values from 36-50 vision fields. Values
shown are the mean ± SD (Student’s t test versus Group D) (n = 6 mice for each group).
All experiments were repeated for at least three times. * mean p & 0.05; ** means
Discussion
XQLT has been reported to inhibit allergic reactions in mice models of both acute
and chronic Der p-induced asthma [,]. In this study, the action of XQLT was found to inhibit allergic reactions which
were correlated with the allergic asthma response. IgE levels (Figure&A) and the extents of eosinophil and leukocyte infiltration (Figure&B and Figure&B) were reduced, causing the XQLT-induced down-regulation of any prolonged allergic
reaction in the Der p-induced acute asthma model. Hence, in this mouse model, XQLT
might affect the Th2 response, which is actively involved in allergic reactions. Yet
IgG2a which is considered to be Th1 reaction product will be controversial because
XQLT also decreased IgG2a. Der p induced more IgG1 than IgG2a that indeed indicated
a Th2 favored reaction. It seemed that XQLT might also have an effect on Th1 reaction.
By further analyzing cytokine such as IL-12 or IL-4 may reveal the nature of this
controversy. Der p also induced obvious TGF-β1 which is known to induce Treg cell
that inhibits T helper cell reaction including both Th1 and Th2 []. XQLT did not inhibit TGF-β1 (Figure&A) in this research. Former research also indicated that XQLT induced CD4-CD8+ T cell
or CD4-CD8- T cell [] that usually represents phenotype of Treg cell. By analyzing IL-10 expression [] and subset of infiltrated cell with different phenotype in BALF in the future will
show how XQLT affect these factors thus regulating T helper reaction.
Yamada et al. [] reported that XQLT treatment was effective in their mouse model of ovalbumin-induced
acute asthma. After an ovalbumin challenge and the XQLT oral administration, elevated
NGF level in the BALF has been observed []. Since NGF and other neurotrophin have been reported to be factors that are involved
in asthmatic reactions, XQLT may influence allergic reactions in asthma by regulating
neurotrophin.
In this study, a mouse model of Der p challenge-induced acute asthma was used to show
that XQLT inhibited the release of NGF (Figure&A) and p75NTR receptor expression (Figure&A) in the lungs. The preventive XQLT strategy inhibited NGF expression more than the
therapeutic XQLT strategy (Figure&A). Therefore, XQLT down-regulated Th2 responses and inhibited allergic reactions.
AHR, which can clearly indicate acute asthma attacks and is directly affected by NGF,
was also reduced by XQLT (Figure&B). The preventive XQLT strategy also inhibited the expression of p75NTR receptor
more than did the therapeutic XQLT strategy. Although serial doses of XQLT were in
the preventive strategy (Figure&A), a single dose of XQLT in vitro, administered as a pre-treatment, was sufficient
to inhibit Der p-induced NGF production (Figure&A; XP). Pre-treatment of LA4 with XQLT (Figure&A; XP) reduced both NGF and TARC levels, which finding was consistent with the in vivo data. Western blot assays revealed that pre-treating LA4 cells with XQLT inhibited
p75NTR receptor and pro-NGF expression in vitro (Figure&B). Hence, XQLT was predicted to have a preventive effect on the acute asthma.
Both in vivo and in vitro data revealed that XQLT clearly reduced BDNF levels. Pre-treatment with a single
dose of XQLT in vitro reduced BDNF levels (Figure&A; XP) more than did preventive XQLT administration in vivo (Figure&B; Group P). BDNF has been reported to be a constitutive factor in the lungs and plays
an important role in lung development []. The effects of BDNF are most apparent in the late phase of asthma, rather than in
the acute phase [], and BDNF has also been reported to affect the infiltration of eosinophils into the
lungs []. BDNF also has an important role in smooth muscle hypertrophy which contributes to
persistent late phase of asthma [,]. XQLT inhibited TARC (Figures&A and A), which has been shown to be a factor that attracts eosinophil in early phase of
immunological reactions [-]. XQLT also reduced BALF NGF (Figure&A) which has been shown to be a survival factor of eosinophil in local compartments
such as alveoli and bronchiole [,]. These results may elucidate the effects of XQLT on eosinophil, and help to explain
how XQLT performs a prolonged regulatory function in latent asthma. XQLT targets both
eosinophil and neurotrophin in allergic asthma, and this targeting is likely to support
the preventive value of XQLT. By regulating BALF NGF, TARC and serum BDNF levels,
XQLT may control allergic inflammation and eosinophil infiltration in both the early
and the late phases of asthma.
The exact mechanisms of action of the components of XQLT decotion remain to be elucidated.
By fractionation of XQLT with chromatographic methods, such as those involving silica
gel columns, can further elucidate the mechanisms of XQLT, including the pharmacodynamics
and interactions. This fractionation research of XQLT will enable treatment with XQLT
to be more precisely monitored to increase the effectiveness of treatment of asthma
patients. Nagai et al. [] reported that XQLT treatment was effective in their ovalbumin-induced acute asthmatic
mouse model and might provide a hint for studying the XQLT mechanism. In their research,
one of the XQLT ingredients, Pinellaiae ternata, played an important role in decreasing
OVA-specific IgE. Lee et al. [] and Shin et al. [] also provided data that Pinellaiae ternata might play a definite role in decreasing
Th2 reaction in asthmatic animal models. Pinellaiae ternata is in highest proportion
of XQLT components. It is worthy to know how Pinellaiae ternata will affect the neurotrophin
in an asthma mouse model.
Our data revealed that XQLT influenced members of the neurotrophin family. According
to internal medical principles and acupuncture practices in TCM, XQLT can be used
to resolve symptoms or diseases that are associated with qi malfunctions of the bladder meridian, which used to be thought to be related to functions
of the autonomic nervous system. By interfering with the neurotrophin, XQLT and its
derivative decotions may act on the nervous system and thereby potentially regulate
spinal or brain functions. Neurotrophin also appear to have a different role in the
immune system, such as autoimmunity. One of our future goals is to extend the use
of XQLT and its derivative decotions in the treatment of neurotrophin-related diseases
of immune malfunction.
Conclusion
In conclusion, XQLT regulates neurotrophin in a Der p stimulated cell line model and
in an asthmatic mouse model. The effects of XQLT on neurotrophin may cause down-regulation
of asthma reaction including AHR and eosinophil infiltration.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
R-SC designed the study and performed the statistical analysis. S-DW and L-JL provided
and discussed the animal model design, they also helped in surveying results of animal
study. Y-CW helped statistical analysis and performed those techniques such as ELISA,
western and cell culture. R-SC also participated in the sequence alignment and drafted
the manuscript. S-TK and J-YW modified the design of study and sequence alignment
of manuscript, they also made final approval to submit this manuscript. All authors
read and approved the final manuscript.
Acknowledgment
This work was supported by grants from the National Science Council, Taiwan (NSC 101-2320-B-039-056-MY2,
China Medical University (CMU99-EW-07) and Tainan Sin-Lau Hospital, Taiwan (R.O.C.).
Ted Knoy is appreciated for his assistance in editing English.
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