  contact dermatitis ( cd ) in the framework of occupational diseases remains prevalent among workers worldwide , impacting their quality of life and workability .
the employees most affected by cd are hairdressers , healthcare workers , and metal workers   because they are continuously exposed to harmful environments when working . as a result
, employees with cd tend to use anti - inflammatory and immunomodulatory agents such as corticosteroids repeatedly .
corticosteroids are effective and powerful agents for cd , but their doses should be restricted because of their adverse side effects .
herbal medicines have recently emerged in the framework of complementary and alternative medicines ( cam ) for corticosteroids because they have relatively lower cost and safety .
are frequently used as traditional or folk medicines for patients with abdominal pain , dysmenorrhoea , uterine haemorrhage , and inflammation in china , japan , and korea .
a. argyi , chinese mugwort , is herbaceous perennial plant known in japanese as gaiyou and in chinese as aiye .
a. princeps , japanese mugwort , is a perennial and very vigorous plant known as yomogi in japanese .
recently , the leaves of a. argyi and a. princeps and their components have been shown to have antitumour [ 58 ] , antifungal , anticoagulant , antidiabetic , and anti - inflammatory [ 12 , 13 ] activity . based on these findings
, we examined the effects of artemisia leaf extract ( ale ) on inflamed tissues in mice with cd and anti - inflammatory activities in raw 264.7 cells .
specifically , the effects of ale on histopathological changes including ear swelling , epidermal hyperplasia , immune cell infiltration , and cytokine production such as interferon- ( ifn- ) gamma (  ) , tumour necrosis factor- ( tnf- ) alpha (  ) , interleukin- ( il- ) 6 , and il-10 in ear tissues were assessed in mice with cd induced by topical application of 1-fluoro-2,4-dinitrobenzene ( dnfb ) .
the anti - inflammatory effects on production levels of prostaglandin e2 ( pge2 ) and nitric oxide ( no ) and the expression levels of cyclooxygenase 2 ( cox-2 ) and inducible nitric oxide synthase ( inos ) were investigated in raw 264.7 cells .
artemisia leaf was purchased from gwangmyungdang ( ulsan , korea ) . the artemisia leaf consisted of a mixture of a. argyi and a. princeps and was authenticated by professor jung - hoon kim , one of the authors of this study .
twenty grams of artemisia leaf was immersed in 500  ml of methyl alcohol and sonicated for 15  min , after which they were extracted for 24  h. following extraction , the supernatant was transferred and the artemisia leaf was again extracted with 500  ml of methanol for 24  h. the two extracts were then combined and filtered through whatman no .
20 filter paper , after which they were condensed using a rotary evaporator ( eyela , tokyo , japan ) .
the evaporated extract was subsequently dried using a lyophilizer ( labconco , kansas city , mo , usa ) , which yielded 1.04  g of freeze - dried powder ( yield , 5.21% ) .
mh2013 - 040 ) were deposited in the herbarium located in the school of korean medicine , pusan national university .
six - week - old male balb / c mice were obtained from samtako ( incheon , korea ) .
all mice used in this experiment were housed in the cages under specific conditions , including a 12  h light / dark cycle and specific pathogen - free conditions .
in addition , mice were provided with free access to standard rodent feed and water .
we conducted all animal experiments according to institutional guidelines and all experimental procedures were approved by our animal care committee ( pnu-2012 - 0140 ) .
cd was induced using our standard method as previously described . briefly , 0.1% dnfb ( 50  l ) in vehicle composed of acetone and olive oil ( 4  :  1 , aoo ) was applied onto the shaved back of mice for three successive days ( sensitization ) .
mice were then treated by application of 0.2% dnfb ( 30  l ) in vehicle onto the backside of their ears every two days . for topical treatment with drugs , dexamethasone ( dex ) and ale were dissolved in ethanol , filtered using a syringe filter ( 0.45  m ) , and finally diluted in vehicle ( aoo  :  ethanol , 4  :  1 ) .
ale ( 30 or 300  g / ear ) was topically applied onto the backside of ears for 7 days .
a total of 36 mice were used in this study ( nor group and dex group , 6 mice ; ctl group and ale group , 8 mice ) .
mice were sacrificed with co2 , after which ear pieces ( 5  mm in diameter ) obtained via dermal punch were weighed using a microbalance and the thicknesses of both ears were measured with digimatic calipers ( mitutoyo , kanagawa , japan ) at the same time .
obtained tissues were fixed in 4% formalin for 24  h and then dehydrated using ethyl alcohol .
ear tissues ( 4  m ) were subsequently resected , after which sections were stained with haematoxylin - eosin ( h / e ) and observed using a light microscope ( 50x ) . to evaluate hyperplasia in the epidermis and infiltration of immune cells , five nonoverlapping fields per slide
the height from the basal lamina to the top of the stratum granulosum was quantified to evaluate the epidermal thickness .
total immune cell numbers were quantified by counting immune cells in the same size counting grid .
cytokine levels in ear tissues were evaluated using a mouse inflammation cytometric bead array ( cba ) kit ( bd biosciences , san jose , ca , usa ) .
briefly , to obtain tissue lysates , resected inflamed tissues were lysed using protein extraction solution ( intron bio , daejeon , korea ) and a homogenizer ( next advance , ny , usa ) .
next , 50  g of lysates was used to evaluate the levels of tnf- , ifn- , il-6 , and il-10 .
raw 264.7 cells , the immortalized murine macrophage cell line , were cultured using dmem ( hyclone , logan , ut , usa ) containing foetal bovine serum ( 10% , fbs ) and antibiotics ( 1% , penicillin - streptomycin ) .
cells were seeded in 96-well plates at a density of 1  10 cells / well and then incubated overnight .
next , cells were treated with the indicated concentrations of ale for 4  h , after which they were stimulated with 1  g / ml of lipopolysaccharide ( lps ) for 20  h. following stimulation , 100  l of supernatants was mixed with 100  l of griess reagent ( 2% sulphanilamide in 10% h3po4 and 0.2% of n-(1-naphthyl)ethylenediamine in distilled water ) and then incubated at room temperature for 10  min .
the absorbance at 540 nm was subsequently measured using a spectrophotometer ( tecan , mnnedorf , switzerland ) .
the production of pge2 was measured by enzyme - linked immunosorbent assay ( elisa ) using pge2 elisa assay kit ( enzo life science , farmingdale , ny , usa ) .
the culture supernatants were then collected , after which the optical densities were measured using a spectrophotometer ( tecan , mnnedorf , switzerland ) at a wavelength of 405  nm .
briefly , ale and lps treated raw 264.7 cells were harvested , lysed , and homogenized as previously described . the protein concentration of each cell lysate
primary antibodies against inos ( 482728 , merck millipore , darmstadt , germany , 1:1000 ) , cox-2 ( sc-19999 , santa cruz biotechnology , santa cruz , ca , usa , 1:1000 ) , -actin ( santa cruz biotechnology , santa cruz , ca , usa ) , and horseradish - conjugated secondary antibody ( enzo life sciences , farmingdale , ny , usa , 1:3000 ) were used to detect specific protein levels .
in addition , a west - q chemiluminescent substrate kit ( gendepot , barker , tx , usa ) and a las 4000 mini ( ge healthcare , piscataway , nj , usa ) were used to visualize the antigen - antibody complex .
the mann - whitney u test was used for data obtained from in vivo experiments ; student 's t - test was used for data obtained from in vitro experiments , and prism 5 for window version 5.01 ( graphpad software inc .
at the end of experiment , the thicknesses and weights of both ears were evaluated . in ctl group , repeated treatment with dnfb elevated the levels of ear thickness and weight more than three times relative to the normal group .
30 and 300  g / ear of ale treatment effectively inhibited enlargement of ear thickness ( figure 2(a ) ) and 300  g / ear of ale inhibited ear weight gain significantly ( figure 2(b ) ) .
the effects of ale on epidermal hyperplasia , one of the major features of cd , and immune cell infiltration were investigated .
marked increases in epidermal thickness ( yellow bars ) and infiltration of immune cells into inflamed tissues were observed in the nontreated cd mice .
in addition , a large pustule ( filled arrow ) and vesicles were also observed ( figure 3(a ) ) .
treatment with ale effectively inhibited epidermal hyperplasia and reduced immune cell infiltration compared to the ctl mice ( figure 3(b ) ) .
we also checked the effects of ale on cytokine productions in inflamed tissues . in our results , elevated levels of tnf- , ifn- , and il-6 production were observed in ctl mice ( figure 4 ) .
treatment with 300  g / ear of ale significantly lowered the production levels of tnf- , ifn- , and il-6 , respectively .
the il-10 production levels were not affected by induction of cd or treatment with ale , and only the dex treated group showed lower production of il-10 than the normal and control groups ( figure 4 ) .
expression of inos was elevated in the lps treated ctl group compared to the normal group .
treatment with more than 25  g / ml of ale lowered inos expression in a concentration dependent manner ( figure 5(a ) ) .
in addition , treatment with lps elevated no production level more than nine times relative to neither lps nor ale treated group .
this increase in no production was effectively inhibited by ale in a dose dependent fashion ( figure 5(b ) ) .
the lps stimulated raw 264.7 cells showed marked increases in cox-2 , representative cox enzyme , and treatment with ale inhibited cox-2 expression ( figure 6(a ) ) .
in addition , the production of pge2 , one of the major final metabolites , was markedly induced by lps stimulation .
treatment with 50  mg / ml of ale inhibited pge2 production significantly ( figure 6(b ) ) .

 artemisia species have traditionally been utilized for amelioration of diseases including malaria , hepatitis , parasites , and various cancers worldwide .
the anti - inflammatory effects of a. princeps in antigen - stimulated t cells and regulatory t cells have been reported by chang et al . .
additionally , the major active components of artemisia species are sesquiterpenoids and flavonoids such as eupatilin and jaceosidin , which are extracted from most herbs in artemisia species , and these components have been reported to have anti - inflammatory effects in mice .
based on the anti - inflammatory and immunomodulatory effects of artemisia species , we investigated whether ale can reduce inflammatory reactions in an animal model of cd to ameliorate its symptoms .
the skin of cd patients tends to thicken because of chronic and repeated inflammatory reactions , which are closely related to epidermal and dermal hyperplasia . in our experiment ,
repeated application of dnfb induced ear swelling , which was effectively inhibited by topical application of ale ( figure 2 ) . given that the degree of ear swelling is recognized as an index of inflammatory reaction , these results imply that ale can exert anti - inflammatory action in animal models of cd . in our animal model of cd ,
repeated application of dnfb induced hyperplasia in the epidermis and massive infiltration of immune cells to the epidermis and into the connective tissues .
in addition , spongiotic changes , vesicles , and pustules were seen in the control group ( figure 3(a)(b ) ) .
topical application of ale effectively inhibited hyperplasia in the epidermis and infiltration of immune cells .
in addition , the area of spongiotic changes and vesicles was diminished and pustules were rarely seen in the ale and dex group ( figure 3 ) .
these findings indicate that ale can act as an anti - inflammatory agent in cd to reduce immune cell infiltration , resulting in inhibition of spongiotic changes , as well as pustule and vesicle formation . tnf- and ifn-
, hallmarks of th1 skewing reaction , can stimulate keratinocytes , which subsequently proliferate , resulting in epidermal hyperplasia .
these two cytokines are also closely related to infiltration of immune cells into the inflamed tissues . in the inflammatory cascade of cd ,
activated keratinocytes can release tnf- and il-6 , which are growth promoting cytokines , resulting in accelerated immune cell infiltration and prolonged lifespan of immune cells .
il-6 has also been reported to accelerate proliferation and migration of keratinocytes , leading to skin disorders , including psoriasis . in this study ,
topical treatment with ale effectively reduced production levels of tnf- , ifn- , and il-6 in ear tissues ( figure 4 ) . when combined with previous results ,
these findings imply that ale can inhibit hyperplasia in the epidermis and infiltration of immune cells via regulation of proinflammatory cytokines , finally leading to inhibition of enlargement of skin thickness .
histopathological analyses revealed that many types of inflammatory cells , including macrophages , infiltrate into the dermis and epidermis in cd .
macrophages also play an important role by releasing inflammatory mediators such as no , prostaglandins , and cytokines during initiation of cd . for these reasons
, we also investigated anti - inflammatory effects of ale in vitro using a macrophage cell line , raw 264.7 .
treatment with up to 100  g / ml of ale had no effect on the viability of raw 264.7 cells ( data not shown ) .
ale significantly inhibited inos expression and no production in a dose dependent manner ( figure 5 ) .
in addition , ale inhibited cox-2 expression and pge2 production induced by lps stimulation ( figure 6 ) .
these findings indicate that ale inhibits inflammatory response in macrophages via regulation of no and pge2 production .
as shown in figures 5 and 6 , ale strongly inhibited inos expression and no production , while it had little effect on pge2 production or cox-2 expression .
it is well known that intracellular signaling pathways such as erk , jnk , and p38 and transcription factors such as creb and nf-b play a central role in activation and inflammatory mediator production in macrophages .
we checked the effects of ale on expression levels of signaling pathways and the nf-b pathway .
treatment with ale prevented phosphorylation of p38 but had no effect on the nf-b pathway ( data not shown ) . considering these results , the anti - inflammatory effects of ale may be closely related to the p38 signaling pathway , and the nf-b pathway may not participate in the anti - inflammatory mechanisms of ale .
kim et al . reported immune - stimulatory effects of hot water extracts of the leaves of a. princeps .
specifically , they found that extracts of a. princeps elevated production levels of no and tnf- , which is inconsistent with our results .
many previous studies of a. princeps have shown anti - inflammatory or antiallergic effects , as well as antioxidative effects .
in addition , components isolated from a. princeps such as eupatilin and jaceosidin have been reported to have inhibitory effects against ige - induced hypersensitivity and carrageenan - induced inflammation , respectively [ 16 , 22 ] .
however , kim et al . did not describe why their findings were inconsistent with those of previous results .
in addition , water extracts of plant materials should also be checked for microbial contamination before use because they are more easily contaminated than methanol or ethanol extracts .
overall , the results of this study suggest that the anti - inflammatory effects of ale in cd mice are closely related to regulation of the activation of immune cells , especially macrophages and cytokine production in inflamed tissues .
in addition , ale effectively lowered the production levels of tnf- , ifn- , and il-6 in inflamed tissues .
these anti - inflammatory actions prevented epidermal hyperplasia and immune cell infiltration . finally , ale effectively inhibited ear swelling induced by dnfb .
these results imply that ale can be used for the treatment of patients with cd as a complement and alternative medicine ( cam ) to corticosteroids .
 the leaves of artemisia argyi lev . 
 et vant . and a. princeps pamp . 
 are well known medicinal herbs used to treat patients in china , japan , and korea with skin problems such as eczema and itching , as well as abdominal pain and dysmenorrhoea . 
 we investigated the anti - inflammatory effects of artemisia leaf extract ( ale ) using cd mice and raw 264.7 cells . 
 the effects of ale on histopathological changes and cytokine production in ear tissues were assessed in mice with cd induced by 1-fluoro-2,4-dinitrobenzene ( dnfb ) . moreover 
 , the anti - inflammatory effects on production levels of prostaglandin e2 ( pge2 ) and nitric oxide ( no ) and expression levels of cyclooxygenase 2 ( cox-2 ) and inducible nitric oxide synthase ( inos ) were investigated in raw 264.7 cells . 
 topical application of ale effectively prevented ear swelling induced by repeated dnfb application . 
 ale prevented epidermal hyperplasia and infiltration of immune cells and lowered the production of interferon- ( ifn- ) gamma (  ) , tumour necrosis factor- ( tnf- ) alpha (  ) , and interleukin- ( il- ) 6 in inflamed tissues . in addition , ale inhibited expression of cox-2 and inos and production of no and pge2 in raw 264.7 cells . 
 these results indicate that artemisia leaf can be used as a therapeutic agent for inflammatory skin diseases and that its anti - inflammatory effects are closely related to the inhibition of inflammatory mediator release from macrophages and inflammatory cytokine production in inflamed tissues . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion