periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues , leading to alveolar bone loss in severe clinical cases .
prostaglandin e2 ( pge2 ) , interleukin- ( il-)6 , and il-8 are known to play important roles in inflammatory responses and tissue degradation .
pge2 has several functions in vasodilation , the enhancement of vascular permeability and pain , and the induction of osteoclastogenesis and is believed to play important roles in inflammatory responses and alveolar bone resorption in periodontal disease .
il-8 acts as a chemoattractant for neutrophils   that produce proteases such as cathepsin , elastase , and matrix metalloproteinase- ( mmp-)8 , leading to tissue degradation .
recently , we reported that several kampo medicines , shosaikoto , hangeshashinto , and orento , suppress lipopolysaccharide- ( lps- ) induced pge2 production by hgfs and suggested that these kampo medicines have anti - inflammatory effects in periodontal disease .
another kampo medicine , kakkonto ( tj-1 ) , has been clinically used for various diseases such as the common cold , coryza , the initial stage of febrile diseases , and inflammatory diseases .
there are several reports that kakkonto shows antiallergic effects [ 8 , 9 ] and antiviral effects [ 1013 ] in animal and in vitro experimental models . for anti - inflammatory effects
therefore , we considered the possibility that kakkonto decreases pge2 production by human gingival fibroblasts ( hgfs ) and has anti - inflammatory effects with respect to periodontal disease .
moreover , lps - treated hgfs produce inflammatory chemical mediators such as pge2 and inflammatory cytokines such as il-6 and il-8 [ 2 , 15 , 16 ] . moreover , because hgfs have sustained production of pge2 , il-6 , and il-8   in the presence of lps , these mediators and cytokines in periodontal tissues are thought to be derived from hgfs .
therefore , we believe that examining the effects of drugs on hgfs , as well as on monocytes and macrophages , is important in the study of periodontal disease . in the present study , we examined the effect of kakkonto on lps - induced pge2 , il-6 , and il-8 production using this in vitro model .
kakkonto was purchased from tsumura & co. ( tokyo , japan ; lot number : d23122 ) , and its components are listed in table 1 .
kakkonto was suspended in dulbecco 's modified eagle 's medium ( d - mem , sigma , st .
louis , mo , usa ) containing 10% heat - inactivated fetal calf serum , 100 units / ml penicillin , and 100  mg / ml streptomycin ( culture medium ) and was rotated at 4c overnight .
then , the suspension was centrifuged and the supernatant was filtrated through a 0.45  m - pore membrane .
lps from porphyromonas gingivalis 381 was provided by professor nobuhiro hanada ( school of dental medicine , tsurumi university , japan ) .
hgfs were prepared as described previously . in brief , hgfs were prepared from free gingiva during the extraction of an impacted tooth with the informed consent of the subjects who consulted matsumoto dental university hospital .
the free gingival tissues were cut into pieces and seeded onto 24-well plates ( agc techno glass co. , chiba , japan ) .
hgfs were maintained in culture medium at 37c in a humidified atmosphere of 5% co. for passage , hgfs were trypsinized , suspended , and plated into new cultures in a 1  :  3 dilution ratio .
this study was approved by the ethical committee of matsumoto dental university ( number 0063 ) .
the numbers of cells were measured using wst-8 ( cell counting kit-8 ; dojindo , kumamoto , japan ) according to the manufacturer 's instructions . in brief , hgfs ( 10,000 cells / well ) were seeded in 96-well plates ( agc techno glass co. , chiba , japan ) and incubated in serum - containing medium at 37c overnight . then , the cells were treated with various concentrations of kakkonto ( 0 , 0.5 , 1 , 2 , 5 , and 10  mg / ml ) in the absence or presence of lps ( 10  ng / ml ) for 24  h ( 200  l each well ) in quadruplicate for each sample .
then , the media were removed by aspiration and the cells were treated with 100  l of mixture of wst-8 with culture medium for 2  h at 37c in co incubator .
optical density was measured ( measured wavelength at 450  nm and reference wavelength at 655  nm ) using an imark microplate reader ( bio - rad , hercules , ca , usa ) , and the mean background value was subtracted from each value .
hgfs ( 10,000 cells / well ) were seeded in 96-well plates and incubated in serum - containing medium at 37c overnight .
then , the cells were treated with various concentrations of kakkonto ( 0 , 0.01 , 0.03 , 0.1 , 0.3 , and 1  mg / ml ) in the absence or presence of lps ( 10  ng / ml ) for 24  h ( 200  l each well ) in triplicate for each sample .
after the culture supernatants were collected , viable cell numbers were measured using wst-8 as described above .
the concentrations of pge2 , il-6 , and il-8 in the culture supernatants were measured by elisa according to the manufacturer 's instructions ( pge2 , cayman chemical , ann arbor , mi , usa ; il-6 and il-8 , biosource international inc . ,
camarillo , ca , usa ) and were adjusted by the number of viable cells .
data are represented as ng or pg per 10,000 cells ( mean  sd , n = 3 ) .
the effects of kakkonto on the activities of cyclooxygenase ( cox)-1 and cox-2 were analyzed using a cox inhibitor screening assay kit ( cayman chemical , ann arbor , mi , usa ) according to the manufacturer 's instructions .
cox activities were evaluated by the measurement of prostaglandin produced from arachidonic acid by cox-1 or cox-2 .
these values were normalized to a relative value of 100% for cells without lps or kakkonto treatments , and are represented as means  sd ( n = 3 ) .
hgfs were cultured in 60  mm dishes and treated with combinations of lps and kakkonto for the indicated times .
then , cells were washed twice with tris - buffered saline , transferred into microcentrifuge tubes , and centrifuged at 6,000  g for 5  min at 4c .
supernatants were aspirated and cells were lysed on ice in lysis buffer ( 50  mm tris - hcl , ph 7.4 , 1% nonidet p-40 , 0.25% sodium deoxycholate , 150  mm nacl , 1  mm ethyleneglycol bis(2-aminoethylether ) tetraacetic acid ( egta ) , 1  mm sodium orthovanadate , 10  mm sodium fluoride , 1  mm phenylmethylsulfonyl fluoride , 10  g / ml aprotinin , 5  g / ml leupeptin , and 1  g / ml pepstatin ) for 30  min at 4c .
then , samples were centrifuged at 12,000  gfor 15  min at 4c , and supernatants were collected .
the protein concentration was measured using a bca protein assay reagent kit ( pierce chemical co. , rockford , il , usa ) .
the samples ( 10  g of protein ) were fractionated in a polyacrylamide gel under reducing conditions and transferred onto a polyvinylidene difluoride ( pvdf ) membrane ( hybond - p ; ge healthcare , uppsala , sweden ) .
the membranes were blocked with 5% ovalbumin for 1  h at room temperature and incubated with primary antibody for an additional 1  h. the membranes were further incubated with horseradish peroxidase - conjugated secondary antibodies for 1  h at room temperature .
antibodies against cox-2 ( sc-1745 , 1  :  500 dilution ) , cytoplasmic phospholipase a2 ( cpla2 , sc-438 , 1  :  200 dilution ) , annexin1 ( sc-11387 , 1  :  1,000 dilution ) , and actin ( sc-1616 , 1  :  1,000 dilution ) , which detects a broad range of actin isoforms , were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .
antibodies against extracellular signal - regulated kinase ( erk ; p44/42 map kinase antibody , 1  :  1,000 dilution ) and phosphorylated erk ( phospho - p44/42 mapk ( thr202/tyr204 ) ( e10 ) monoclonal antibody , 1  :  2,000 dilution ) were from cell signaling technology ( danvers , ma , usa ) .
horseradish peroxidase - conjugated anti - goat igg ( sc-2020 , 1  :  20,000 dilution ) was from santa cruz , and anti - rabbit igg ( 1  :  20,000 dilution ) and anti - mouse igg ( 1  :  20,000 dilution ) were from dakocytomation ( glostrup , denmark )
. differences between groups were evaluated by the two - tailed pairwise comparison test with a pooled variance , followed by correction with the holm method ( total 16 null hypotheses ; 5 null hypotheses without kakkonto versus with kakkonto in the absence of lps , 5 null hypotheses without kakkonto versus with kakkonto in the presence of lps , and 6 null hypotheses without lps versus with lps ) ( figures 1 and 2 ) .
differences between the control group and experimental groups were evaluated by a two - tailed dunnett 's test ( figure 3 ) .
dunnett 's test was performed using the  glht  function in the  multcomp  package .
the viability of hgfs was approximately 90% at up to 1  mg / ml of kakkonto for a 24  h treatment in the absence or presence of lps ( figure 1 ) .
the viabilities were approximately 70% and 20% at 5  mg / ml and 10 
therefore , we used kakkonto at the concentrations of up to 1  mg / ml in further experiments .
we examined whether kakkonto affects the production of pge2 and inflammatory cytokines ( il-6 and il-8 ) by hgfs . because kakkonto affects cell viability , the concentrations of pge2 , il-6 , and il-8 needed to be adjusted according to viable cell number . when hgfs were treated with 10  ng / ml of lps , hgfs produced large amounts of pge2 , il-6 , and il-8 .
indomethacin decreased lps - induced pge2 production in a concentration - dependent manner but slightly decreased lps - induced il-6 and il-8 production ( data not shown ) .
kakkonto significantly decreased pge2 production in a concentration - dependent manner ( figure 2(a ) ) . in the absence of lps
in contrast , kakkonto increased lps - induced il-6 and il-8 production ( figures 2(b ) and 2(c ) ) . in the absence of lps , up to 0.1 
mg / ml of kakkonto did not affect il-6 and il-8 production , but above 0.3  mg / ml of kakkonto , their concentrations were increased ( figures 2(b ) and 2(c ) ) .
similar results were obtained using human skin fibroblast tig-103 cells ( data not shown ) .
because pge2 production is regulated by cox enzymes and suppressed by acid nsaids such as aspirin and diclofenac sodium , which inhibit cox activities , we examined whether kakkonto inhibits cox-1 and cox-2 activities .
kakkonto decreased cox-1 activity to approximately 70% at 1  mg / ml but did not affect cox-2 activity ( figure 3 ) .
cpla2 is the most upstream enzyme in the arachidonic acid cascade and releases arachidonic acid from plasma membranes .
kakkonto did not alter cpla2 expression in the absence or presence of lps ( figure 4 ) .
however , kakkonto did not alter lps - induced cox-2 expression ( figure 4 ) .
annexin1 , also named lipocortin1 , is an anti - inflammatory mediator produced by glucocorticoids that inhibit cpla2 activity [ 19 , 20 ] .
however , neither lps nor kakkonto showed an effect on annexin1 expression ( figure 4 ) .
cpla2 is reported to be directly phosphorylated at ser505 by erk , resulting in cpla2 activation [ 21 , 22 ] .
erk phosphorylation was enhanced at 0.5  h after lps treatment and thereafter was attenuated .
one  mg / ml of kakkonto suppressed lps - induced erk phosphorylation at 0.5  h to 2  h ( figure 5 ) .
in the present study , we examined the effect of kakkonto on lps - induced pge2 , il-6 , and il-8 production by hgfs .
kakkonto concentration dependently decreased lps - induced pge2 production but did not affect pge2 production without lps treatment , similar to shosaikoto , hangeshashinto , and orento [ 57 ] .
moreover , kakkonto suppressed lps - induced erk phosphorylation . in contrast , kakkonto increased lps - induced il-6 and il-8 production .
it is widely known that pge2 leads to inflammatory responses such as vasodilation , enhanced vascular permeability , and pain generation .
acid non - steroidal anti - inflammatory drugs nsaids show anti - inflammatory effects by suppression of pge2 production , even though they do not affect il-6 and il-8 production .
our findings showing that kakkonto decreases lps - induced pge2 production suggest that kakkonto also has anti - inflammatory effects in periodontal disease and that its effects are mainly mediated by suppression of pge2 production even though kakkonto increased lps - induced il-6 and il-8 production .
previously , we demonstrated that orento inhibits lps - induced erk phosphorylation and cpla2 activation , leading to the suppression of pge2 production in hgfs .
therefore , we consider that kakkonto decreased lps - induced pge2 production through the suppression of erk phosphorylation in hgfs . although kakkonto increased cox-2 expression in the absence of lps , kakkonto did not alter pge2 production .
we consider a likely reason to be the suppression of cpla2 activation through the inhibition of erk phosphorylation and/or the suppression of cox-1 activity .
our results showed that kakkonto increased lps - induced il-6 and il-8 production by hgfs .
previously , we reported that the activation of the protein kinase a ( pka ) pathway by adrenaline or aminophylline increases lps - induced il-6 and il-8 production in hgfs   and that h-89 , a pka inhibitor , decreases lps - induced il-6 and il-8 production [ 23 , 24 ]
in general , steroidal anti - inflammatory drugs ( saids ) suppress the expression of cpla2 , cox-2 , and inflammatory cytokines ( such as il-6 and il-8 ) and induce the expression of annexin1 .
however , kakkonto did not affect cpla2 , annexin1 , or lps - induced cox-2 expression , and it increased il-6 and il-8 production .
this therefore suggests that the mechanism by which kakkonto decreases pge2 production is different from that of saids .
many studies have demonstrated that nsaid administration prevents gingival inflammation   and several clinical studies have indicated that the concentration of pge2 in gingival crevicular fluid ( gcf ) is increased in periodontal disease   and is decreased by oral administration or mouthwash with nsaids [ 27 , 28 ] . considering that both nsaids and kakkonto suppress pge2 production
, it is possible that administration of kakkonto also decreases the pge2 concentration in gcf and results in the improvement of gingival inflammation . therefore , kakkonto may be useful for the improvement of gingival inflammation in periodontal disease .
importantly , kakkonto did not affect the basal level of pge2 , although kakkonto decreased cox-1 activity to approximately 70% . because pge2 produced by cox-1 protects gastric mucosa , these results suggest that kakkonto may cause minimal gastrointestinal dysfunction .
we demonstrated that kakkonto suppresses lps - induced erk phosphorylation , resulting in the suppression cpla2 activation and further pge2 production by hgfs .
these results suggest that kakkonto is clinically useful for the improvement of inflammatory responses in periodontal disease .
 periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues , leading to alveolar bone loss in severe clinical cases . 
 the chemical mediator prostaglandin e2 ( pge2 ) and cytokines such as interleukin- ( il-)6 and il-8 have been known to play important roles in inflammatory responses and tissue degradation . in the present study , we investigated the effects of a kampo medicine , kakkonto ( tj-1 ) , on the production of prostaglandin e2 ( pge2 ) , il-6 , and il-8 by human gingival fibroblasts ( hgfs ) treated with lipopolysaccharide ( lps ) from porphyromonas gingivalis . 
 kakkonto concentration dependently suppressed lps - induced pge2 production but did not alter basal pge2 levels . 
 in contrast , kakkonto significantly increased lps - induced il-6 and il-8 production . 
 kakkonto decreased cyclooxygenase- ( cox-)1 activity to approximately 70% at 1  mg / ml but did not affect cox-2 activity . 
 kakkonto did not affect cytoplasmic phospholipase a2 ( cpla2 ) , annexin1 , or lps - induced cox-2 expression . 
 kakkonto suppressed lps - induced extracellular signal - regulated kinase ( erk ) phosphorylation , which is known to lead to erk activation and cpla2 phosphorylation . 
 these results suggest that kakkonto decreased pge2 production by inhibition of erk phosphorylation which leads to inhibition of cpla2 phosphorylation and its activation 
 . therefore , kakkonto may be useful to improve gingival inflammation in periodontal disease . 