keishibukuryogan ( kbg , gui - zhi - fu - ling - wan in chinese ) is one of the kampo medicines , and has been widely administered to patients with blood stagnation for improving blood circulation .
it was reported that kbg improves conjunctional microcirculation in patients with cerebrospinal vascular diseases , thus suggesting that it may have beneficial effects on hematological parameters such as blood viscosity and red blood cell deformability [ 24 ] .
moreover , matsumoto et al . have explored a proteomic approach for the diagnosis of blood stasis in rheumatoid arthritis patients treated with kbg .
in addition , kbg is used to treat symptoms of peripheral ischemia such as cold extremities .
furthermore , we recently reported that kbg is effective in patients with chronic pigmented purpura , a group of skin vascular disorders of unknown etiology .  
kbg is composed of five medicinal plants , cinnamomum cassia blume ( cinnamomi cortex ) , paeonia lactiflora pallas ( paeoniae radix ) , paeonia suffruticosa andrews ( moutan cortex ) , prunus persica batsch ( persicae semen ) and poria cocos wolf ( hoelen ) ( table 1 ) .
paeoniae radix and moutan cortex have many known active components in common , including paeoniflorin , paeonol , oxypaeoniflorin , benzoylpaeoniflorin , and palbinone .
paeoniflorin is a characteristic main principal bioactive component of paeoniae radix , which included approximately 5.57% ( w / w ) paeoniflorin , and moutan cortex , which included approximately 3.96% ( w / w ) paeoniflorin .
paeoniflorin has been reported to have many pharmacological effects , such as anti - inflammatory and antiallergic effects .
recently , zheng and wei reported that the total glucosides present in the moutan cortex , which contain paeoniflorin as the principle bioactive component , inhibited primary and secondary inflammation in both collagen - induced arthritis and adjuvant - induced arthritis .
up until now , topical and oral corticosteroids , oral bioflavonoids , ascorbic acid , griseofulvin , and cyclosporine have been suggested as treatments for chronic pigmented purpura [ 13 , 14 ] . however , none of these treatments have proven to be satisfactory .
human dermal microvessel endothelial cells ( hdmecs ) are the prominent cells in dermal skin .
hdmecs produce inflammatory cytokines , such as interleukin ( il)-6 and il-8 when they are exposed to lipopolysaccharide ( lps ) .
we therefore consider that examinations of the effects of kbg and paeoniflorin on hdmecs are important for the therapeutic studies of chronic pigmented purpura in vitro . in this study
, we aimed to evaluate the role of kbg and paeoniflorin , ininhibiting the production of inflammatory cytokines using hdmecs .
kbg ( tj-25 ) was obtained from tsumura & co. ( tokyo , japan ) .
kbg was suspended in cs - c complete medium containing 10% fetal calf serum and 1% penicillin and csc growth factor ( cell systems inc , wa ) and was rotated at 4c overnight .
then , the suspension was centrifuged , and the supernatant was filtered through a 0.45  m - pore membrane .
the following materials were obtained from commercial sources : the isogen rna extraction kit was obtained from nippon gene ( tokyo , japan ) ; m - mlv reverse transcriptase was from gibco ( grand island , ny ) ; taq dna polymerase was from perkin - elmer ( norwalk , co ) ; lps was purchased from sigma ( st . louis ) ; nylon membranes were from schleicher & schuell ( keene , nh ) ; the anticyclooxygenase-2 ( cox-2 ) polyclonal antibody ( pab ) was purchased from cell signaling technology , inc .
( boston ) ; antiinducible nos ( inos ) pab was purchased from enzo life sciences international inc .
; mif , il-6 , and il-8 elisa kits were obtained from r&d systems ( minneapolis ) ; and the western blot detection system was obtained from cell signaling technology ( beverly , ma ) .
primary human dermal endothelial cells ( hdmecs ) were obtained from cell systems inc ( wa ) .
hdmecs were cultivated in conditioned endothelial culture medium ( cs - c complete medium ) containing 10% fetal calf serum and 1% penicillin and csc growth factor at 37c in a 5% co2 atmosphere .
cell viability before treatment was always over 95% as evaluated by trypan blue dye exclusion test . on the day of the experiment ,
cells were collected and suspended in fresh culture medium at a concentration of 1    10  cells / ml . the cells were divided into 4 groups : a control group , a group receiving 10  mg / ml of kbg or 100  g / ml of paeoniflorin as previously described [ 9 , 15 ] , a group treated with 1  g / ml of lps , and another group treated with the combination .
the cells were treated with the various agents , and analyses were performed as described for each procedure .
the number of cells was measured by an mtt assay . in brief , the media were removed by aspiration , and then the cells were treated with 0.5  mg / ml dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide ( mtt , sigma ) in culture medium for 4  hours at 37c . after being washed with pbs once , isopropanol/0.04  m hcl was added , and od570 was measured , and the value of blank was subtracted .
rna reverse transcription was performed with m - mlv reverse transcriptase using random hexamer primers , and subsequent amplification was done using taq dna polymerase .
pcr was carried out for 30 cycles with denaturation at 94c for 30  seconds , annealing from 52 to 64c for 1  minute and extension at 72c for 30  seconds using a thermal cycler ( pe applied biosystems gene amp pcr system 9700 ) .
the human mif primers used were 5-atgccgatgttcatcgtaaac-3   ( forward ) and 5-ggcgaaggtggagttgttcca-3 ( reverse ) .
supernatants were collected after 24  h of incubation with lps ( 1  g / ml ) and/or kbg ( 10  mg / ml ) and paeoniflorin ( 100  g / ml ) .
mif , il-6 , and il-8 were assayed by elisa , according to the manufacturer 's instructions .
the cells were lysed at a density of   110  cells/50  l of ripa buffer ( 1  m tris - hca , 5  m nacl , 1% nonidet p-40 ( v / v ) , 1% sodium deoxycholate , 0.05% sds , 1  mm phenylmethyl sulfonyl fluoride ) for 20  minutes . after brief sonication
, the lysates were centrifuged at 12,000  rpm for 10  minutes at 4c , and the protein content in the supernatants was measured using a bio - rad protein assay kit ( bio - rad , hercles , ca ) .
the protein lysates were denatured at 96c for 5  min after mixing with 5  l of sodium dodecylsulfate ( sds ) loading buffer , applied on an sds polyacrylamide gel for electrophoresis , and transferred to nitrocellulose membranes .
western blot analysis was carried out to detect the expression levels of cox-2 and inos using specific antibodies .
band signals were visualized on x - ray film using chemiluminescence ecl detection reagents ( amersham biosciences , buckinghamshire , uk ) .
the relative amounts of proteins associated with specific antibodies were normalized according to the intensities of -actin .
the values are expressed as the means  sd of the respective test or control group .
statistically significant differences in the stimulation with lps in the groups treated with kbg or paeoniflorin were evaluated by the nonparametric mann - whitney u test .
lps plays a fundamental role in the pathogenesis of a number of inflammatory diseases by inducing a distinctive pattern of inflammatory cytokine release .
the cells were incubated with or without 1  g / ml of lps or 10  mg / ml of kbg for 6 or 24  h , and the cell viability was assessed .
none of the treatments elicited cytoxicity in the cells at the tested concentrations and incubation times ( figure 1(a ) ) .
we examined the anti - inflammatory effects of kbg on the induction of inflammatory cytokines by lps stimulation .
lps - induced protein levels of mif , il-6 , il-8 , and tnf- were significantly inhibited by kbg ( figure 1(b ) ) .
treatment of the cells with 1  g / ml of lps increased in mrna levels of these cytokines from 6  h after stimulation , but these mrna levels were decreased by kbg ( figure 1(c ) ) .
the cells were incubated with or without 1  g / ml of lps or 100  g / ml of paeoniflorin for 6 or 24  h , and the cell viability was assessed .  
figure 2(a ) reveals that the incubation with paeoniflorin did not elicit toxicity in cells at the tested concentration at either incubation time .
lps - induced protein levels of mif , il-6 , il-8 , and tnf- were significantly inhibited by paeoniflorin , and the inhibitory effect was stronger than that of kbg ( figure 2(b ) ) . moreover , to examine the effects of paeoniflorin on the mrna levels of inflammatory cytokines , cells were treated with lps for 6  h in the presence of 100 g / ml of paeoniflorin .
the lps - induced mif , il-6 , il-8 , and tnf- mrna levels were decreased by paeoniflorin ( figure 2(c ) ) .
western blot analyses revealed that the expression of cox-2 and inos was increased by lps stimulation in hdmecs whereas the treatment with paeoniflorin resulted in a decrease in cox-2 and inos expression ( figure 3 ) .
among traditional japanese medicinal compounds , kampo formulas originated from ancient chinese medicine , and are currently recognized and reimbursed by the health ministry in japan for the treatment of a wide variety of conditions .
the kampo formula kbg is frequently used in traditional japanese and chinese medicine to treat several symptoms , including skin diseases .
the preparation has demonstrated anti - inflammatory and free - radical scavenging effects .   in the present study , we have demonstrated that kbg treatment significantly suppressed the protein and mrna levels of mif , il-6 , il-8 , and tnf- in lps - stimulated cultured hdmecs .
chronic pigmented purpura is a disease complex characterized by capillaritis and lymphocytic capillary damage in the papillary dermis .
several lines of evidence suggest the involvement of humoral or cellular immunity , as well as hemostasis , in the pathogenesis of this disease .
indeed , our previous data support the effectiveness of kbg , which led to a significant improvement in patients with chronic pigmented purpura .
it is reported that kbg can prevent the progression of atherosclerosis , and preserve vascular endothelial function in cholesterol - fed rabbits   and in hypertensive rats .
furthermore , in diabetic rats , it was demonstrated to have protective effects against vascular injury , and to delay the development of diabetic kidney disease [ 22 , 23 ] .
moreover , kbg has a favorable effect on impaired glucose metabolism in type 2 diabetes by improving glucose intolerance , and it has been suggested that some of this effect is derived from the reduction of tnf- content in skeletal muscle .
accordingly , kbg may exert beneficial effects that result in the inhibition of inflammatory cytokines in hdmecs .
it was recently reported that mif induces the endothelial expression of il-8 , thus resulting in leukocyte recruitment .
since mif is an initiator of other proinflammatory cytokines such as il-6 , il-8 , and tnf- , and regulates the induction of these cytokines [ 26 , 27 ] , we hypothesized that kbg may inhibit mif - regulated inflammatory mediators .
we also demonstrated that paeoniflorin , a bioactive compound contained in kbg , suppressed the mrna levels of these inflammatory cytokines in lps - stimulated cultured hdmecs , similar to the effects of kbg .
it is known that paeoniflorin has anti - inflammatory and antiallergic activities [ 28 , 29 ] .
in addition , paeoniflorin has reportedly exhibited immunoregulatory effects [ 30 , 31 ] , analgesic effects , neuromuscular blocking [ 33 , 34 ] , cognition enhancement , and steroid protein binding inhibition .
many studies have demonstrated that overproduction of nitric oxide and inflammatory prostaglandins such as pge2 , mainly via inos and cox-2 , respectively , plays an important pathophysiological role in the development of inflammation .
kim and ha reported that paeoniflorin treatment significantly attenuated lps - induced no and pge2 production .
moreover , the extract of kbg has been shown to have cox-2 and inducible inos inhibitory activities . in this context
, we suggest that kbg and paeoniflorin induce their anti - inflammatory effects by inhibiting both proinflammatory cytokines and inhibiting the cascade of the overproduction of cox-2 and inos in hdmecs .
taken altogether , these findings suggest that kbg may be useful to improve microvascular inflammation in patients with skin diseases such as chronic pigmented purpura .
 keishibukuryogan ( kbg ) is one of the traditional herbal formulations widely administered to patients with blood stagnation for improving blood circulation ; currently , it is the most frequently prescribed medicine in japan . 
 kbg has been reported to improve conjunctional microcirculation . 
 the aim of this study was to evaluate the role of kbg and paeoniflorin , a bioactive compound of kbg , in inhibiting the production of inflammatory cytokines using human dermal microvessel endothelial cells ( hdmecs ) . 
 the authors observed that lipopolysaccharide ( lps ; 1  g / ml ) stimulated the secretion of proinflammatory cytokines in hdmecs . 
 kbg treatment ( 10  mg / ml ) significantly suppressed the mrna levels of migration inhibitory factor ( mif ) , interleukin ( il)-6 , il-8 , and tumor necrosis factor ( tnf)- in lps - stimulated cultured hdmecs . similarly , paeoniflorin significantly suppressed the mrna levels of these cytokines in lps - stimulated cultured hdmecs . 
 elisa showed that kbg and paeoniflorin suppressed the production of mif , il-6 , il-8 , and tnf- in lps - stimulated hdmecs . 
 moreover , kbg and paeoniflorin decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase ( inos ) in these cells . 
 these results suggest that kbg may be useful for improving microvascular inflammation in patients with skin diseases . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion