allergic rhinitis ( ar ) is an allergic inflammation of the nasal airways and is characterized by sneezing , nasal congestion , nasal itching , and rhinorrhea , in any combination .
ar affects approximately 60 million people in the united states and the prevalence is about 1030% in adults and nearly 40% in children [ 13 ] . a nationwide questionnaire survey conducted with 42,886 koreans using the international study of asthma and allergies in childhood ( issac ) questionnaire demonstrated that 12-month prevalence of 612 and 1215 years of age with ar were 28.8% and 29.1% , respectively .
patients with ar present an inflammatory ige - mediated response characterized by allergen type 2 helper t cells ( th2 ) immunological pattern with mast cells and eosinophil activation and release of inflammatory mediators in response to exposure to allergens [ 57 ] .
allergens are any substance , most often eaten or inhaled , that can be found in a variety of sources , such as dust , pollen , and pet dander
. such allergens ( antigens ) induce eosinophil recruitment into the tissues and increase serum ige level .
in allergic reactions , mast cells become active when an allergen binds to ige [ 9 , 10 ] . in addition , not only mast cells but also b cells play an important role in the pathogenesis of ar [ 1116 ] .
allergen type 2 helper t cells ( th2 ) play a major role in the regulation of ige antibody producing b cells , mast cells , and eosinophils .
moreover , th2 secrete il-4 , il-5 , il-6 , and il-13 and regulate b cell and eosinophil mediated responses , whereas th1 produce interferon .
th1/th2 cells are associated with a series of immune and inflammatory diseases , such as bacterial and viral infectious diseases [ 17 , 18 ] .
t cell - mediated immunity is an important process of elaborating antigen ( ag ) specific t lymphocytes to remove viral , bacterial , or parasitic infections or malignant cells .
cytotoxic cd8 + t cells are t lymphocytes that kill infected , damaged , or malignant cells bearing the ag , while cd4 + t helper cells generate cytokines that can be directly poisonous to the target cells or can stimulate other t cell effector functions and b cell antibody production and mobilize powerful inflammatory mechanisms .
the traditional herbal medicine hyeonggaeyeongyo - tang ( hyt ) is known to treat otitis media , sinusitis , tonsillitis , and a variety of otolaryngology symptoms . in japan and china ,
hyt is known as keigai - rengyo - to or jing jie lian qiao tang , respectively .
antihistamines and steroids are the most common medications used to treat ar . however , long - term use of drug generates resistance or side effects requiring the development of new drug .
therefore , in this study , we investigated the effects of hyt on allergic responses in ovalbumin- ( ova- ) induced ar mice model .
we also measured the levels of cytokines in the serum and the level of leukocytes in the blood .
hyt used in this study contains thirteen different herbal medicines : schizonepeta spica 0.47  g , forsythiae fructus 0.47  g , saposhnikoviae radix 0.93  g , angelicae gigantis radix 0.93  g , cnidii rhizoma 0.93  g , paeoniae radix alba 0.93  g , bupleuri radix 0.47  g , aurantii fructus 0.93  g , scutellariae radix 0.93  g , gardeniae fructus 0.93  g , angelicae dahuricae radix 0.93  g , platycodi radix 0.93  g , and glycyrrhizae radix 0.67  g. they were prepared by hanpoong pharm ( jeonju , republic of korea ) following good manufacturing practice ( gmp ) procedure .
dried hyt powder was stored in aliquots at 80c until further analysis ( table 1 ) .
male balb / c mice ( aged 6 weeks ) were purchased from orient ( seoul , republic of korea ) .
the average body weight of mice was 26  g. the animals were housed in air - conditioned room with a 12  h light / dark cycle .
all procedures performed on the mice were approved by the animal care center of kyung hee university ( approval number khuasp ( se)-12 - 048 ) .
the mice were divided into five groups ( n = 8) : group 1 , normal saline ; group 2 , ova + saline ; group 3 , ova + hyt ( 101  mg / kg ) ; group 4 , ova + hyt ( 202  mg / kg ) ; group 5 , ova + hyt ( 404  mg / kg ) . the experimental procedures for allergic sensitization and challenge are summarized in figure 1 . for establishment of ova - induced ar model , male 6-week - old balb / c mice were intraperitoneally sensitized with 25  ug of ova twice a week for three weeks . after three weeks , ova group of mice were sensitized by intranasal instillation of 500  ug ova in 30  ul pbs twice a week for one week .
finally , mice were fed with hyt together with ova sensitization for 2 weeks . at the end of experiment ,
each section was stained with hematoxylin and eosin ( h & e ) for inflammatory cells or with toluidine blue ( t.b ) for mast cells counts and examined under light microscopy ( olympus ) .
mast cells and inflammatory cells were counted in 10 parts of high power fields ( hpf ) at 40x , 400x , and 1000x magnification .
expression of cd4 + lymphocytes was detected by immunohistochemical analysis using the anti - cd4 + antibody .
after a microwave treatment , the sections were treated with 3% hydrogen peroxide in pbs for 15  min to inhibit endogenous peroxidase activity of blood cells . following hydrogen peroxide treatment ,
the nasal sections were incubated with 5% bovine serum albumin ( bsa ) in pbs , contained a blocking reagent for 1 hour at room temperature .
nasal sections were incubated with mouse monoclonal cd4 + antibody overnight at 4c and subsequently incubated with secondary biotinylated anti - rabbit igg for 1  h at room temperature .
sections were treated with avidin - biotin hrp complex ( vectastain abc kit , vector labs , ca , usa ) for 30  min at 4c and stained with diaminobenzidine tetrachloride ( dab ) as the substrate .
the slides were mounted with an aqueous mounting solution ( dako , glostrup , denmark ) and cover - slipped .
all the sections were analyzed using an olympus microscope and images were captured using a digital video camera .
blood serum was analyzed by the bio - plex multiplex cytokine assay ( bio - rad laboratories , hercules , ca , usa ) according to the manufacturer 's instructions .
the assay was read using a luminex 100 ( austin , tx ) and analyzed using a bio - plex manager software .
the mean concentration of cytokines ( il-4 , il-13 , and lif ) in supernatants from ova - stimulated cells over the unstimulated cells ( background ) was then calculated .
the blood was placed in vacutainer tm tubes containing edta ( bd science , usa ) .
anticoagulated blood was processed to determine hematological parameters ( wbc , lymphocytes , monocytes , eosinophils , basophils , and neutrophils ) in a hemavet 950 hematology analyzer ( drew scientific , inc .
, oxford ) in accordance with manufacturer ' recommendation . suspension of spleen from normal mice under aseptic condition was prepared by homogenization in rpmi-1640 medium ( containing 10% fbs , 1% ab , and 0.05  mm mercaptoethanol ) .
the contaminating red blood cells were removed by using red blood cell lysis buffer ( sigma , usa ) .
mice splenocytes ( 1  10 cells / well ) were plated in 96-well culture plates and incubated for 24  h. cells were treated with con a ( 2  ug / ml ) or hyt ( 10 , 50 , 100 , 200 , 500 , 1000 , and 2000  ug / ml ) .
after 24  h incubation , 10  ul of wst solution was added to each well of the plate , and the plates were incubated in the dark at 37c for another 2  h. optical density was measured at 450  nm using an elisa plate reader ( versamax , molecular devices , ca , usa ) .
all experiment results were expressed as the means  standard deviations ( sd ) or means  sem ( n = 8) of at least three separate tests .
student 's t - test was used for single variable comparisons , and a p value < 0.05 was considered statistically significant .
we found that normal group shows higher weight as compared to other groups ( figure 2(a ) ) .
we also found that hyt did not show any toxicity maintaining body weight ( figure 2(a ) ) .
to determine whether hyt reduces infiltration of inflammatory cells into nasal cavity , we performed h & e staining on the nasal mucosa samples .
the respective numbers of inflammatory cells in the nasal mucosa in ar mice model were shown to be higher than those in the normal mice .
hyt decreased such infiltration of inflammatory cells into nasal cavity ( figure 3(a ) ) .
the respective numbers of mast cells in the nasal mucosa in ar mice were shown to be higher than those in the normal mice .
hyt decreased such infiltration of mast cells into nasal cavity ( figure 4(a ) ) .
lowest hyt concentration showed the highest effect to reduce infiltration of mast cells into nasal mucosa .
cd4 + t helper cells generate cytokines that can stimulate other t cell effector functions and b cell antibody production .
we also performed immunocytochemistry to measure intracellular level of cd4 + ( total t cells ) .
we found that ova increased numbers of cd4 + ( total t cells ) , while hyt decreased them in nasal cavity ( figure 5 ) .
therefore , we measured the levels of cytokines in the blood samples by multiplex cytokine assay .
we found that ova increases the levels of il-4 , il-13 , and lif , while hyt inhibits such increase ( figures 6(a ) , 6(b ) , and 6(c ) ) .
it is known that allergic diseases activate eosinophils , neutrophils , monocytes , basophils , and lymphocytes [ 24 , 25 ] . to investigate whether hyt suppresses inflammatory phenomenon , we measured leukocytes levels in cardiovascular blood samples using hemavet 950 hematology analyzer .
we found that ova increases the levels of eosinophils , neutrophils , monocytes , basophils , lymphocytes , and wbc , while hyt inhibits such increase ( figures 7(a ) , 7(b ) , 7(c ) , 7(d ) , 7(e ) , and 7(f ) ) .
finally , we measured the effect of hyt on splenocyte viability . for that purpose , we treated splenocyte with various concentrations of hyt ( 10 , 50 , 100 , 200 , 500 , 1000 , and 2000  ug / ml ) and measured cell viability using wst assay .
we found that con a increases cell viability , while hyt decreases it ( figure 8) .
in this study , we investigated the effect of hyt on allergic responses in ova - induced ar mice . increase of infiltration of inflammatory and mast cells into nasal cavity is known in ova - induced ar mice .
immunohistochemical study showed thathyt reduced increased number of cd4 + t cells induced by ova .
moreover , induction of various cytokines due to ar was also suppressed by hyt in mice .
the activation of eosinophils releases a variety of chemicals to cause inflammation and tissue injuries [ 26 , 27 ] . a variety of cytokines are released by inflammatory stimulation [ 2830 ] .
we found that ova increases the levels of il-4 , il-13 , and lif , while hyt inhibits such increase .
we also found that ova increases the levels of eosinophils , neutrophils , monocytes , basophils , lymphocytes , and wbc , while hyt inhibits such increase .
ar , also known as hay fever , affects approximately 20 percent of people of all ages .
the risk of developing ar is much higher in people with asthma or eczema and in people who have a family history of asthma or rhinitis .
ar can begin at any age , although most people first develop symptoms in childhood or young adulthood .
the symptoms are often at their worst in children and in people in their 30s and 40s .
the severity of symptoms tends to vary throughout life ; many people experience periods when they have no symptoms at all .
the treatment of ar includes reducing exposure to allergens and other triggers , in combination with medication therapy .
nasal glucocorticoids ( steroids ) delivered by a nasal spray are the first - line treatment for the symptoms of ar .
hyt is known to treat otitis media , sinusitis , tonsillitis , and a variety of otolaryngology symptoms . in this study
our present study clearly demonstrates that hyt suppresses the progression of ar induced by ova .
 allergic rhinitis ( ar ) is an allergic inflammation of the nasal airways . 
 the prevalence of ar is increasing worldwide . 
 we investigated whether hyeonggaeyeongyo - tang ( hyt ) is effective to suppress the progression of ar induced by ovalbumin ( ova ) . 
 male balb / c mice were used for this study . 
 allergic rhinitis was induced by ova . 
 treatment with hyt was assessed to study the effect of hyt on allergic rhinitis in mice . 
 histological analysis , immunohistochemistry , multiplex cytokine assay , blood analysis , and cell viability assay were performed to verify inhibitory effect of hyt on allergic rhinitis . 
 hyt did not show any toxicity maintaining body weight . 
 food intake was steady without variation in mice . 
 hyt reduced infiltration of inflammatory cells and mast cells into nasal cavity . 
 hyt reduced the levels of cytokines and leukocytes in the blood . 
 hyt decreased the splenocyte cell viability . 
 antihistamines and steroids are the most common medications used to treat allergic rhinitis . however , long - term use of drug generates resistance or side effects requiring the development of new drug . 
 our present study clearly demonstrates that hyt suppresses the progression of allergic rhinitis induced by ova . 
 this suggests that hyt might be a useful drug for the treatment of allergic rhinitis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion