cigarette smoke ( cs ) is associated with the development of inflammation - related diseases such as chronic obstructive pulmonary disease and vascular diseases , including atherosclerosis and stroke .
several studies have revealed that cs is a major contributor to vascular diseases because it accelerates the development of atherosclerotic plaques .
the relationship between cs and the increased incidence of atherosclerosis has been reported [ 57 ] , which may be a consequence of direct endothelial damage , increased proliferation of smooth muscle in atherosclerotic lesions , and/or decreased vasodilation .
endothelial damage has also been suggested as the initial cause of development of vascular diseases . in a previous study , it was shown that inhibition of oxidative stress exerts protection in human endothelial cells , which could be an effective strategy in the treatment of vascular diseases .
a number of studies support that reactive oxygen species ( ros ) causing oxidative stress may play an essential role in mediating endothelial cell death .
oxidative stress is a major factor in vascular diseases such as hypertension , stroke , and atherosclerosis .
korean red ginseng ( krg ) is a popular traditional herbal medicine that has been widely used to treat several diseases such as cancer and vascular diseases .
recent research shows that ginseng may have therapeutic potential in the treatment of alzheimer 's disease , diabetes , cancer , and cardiovascular diseases , through its antioxidant , antithrombotic , antihyperlipidemic , and anticancer effects [ 1215 ] . in endothelial cells
, krg simulates no production in vivo and in vitro , suggesting that krg has antihypertensive effects .
krg also promotes angiogenesis through the activation of the signaling pathway , indicating that krg can be implicated in potential angiogenic therapies for improving tissue repair , wound healing , and cardiovascular diseases .
in addition , our previous study suggested that krg exerts a cytoprotective effect through the induction of heme oxygenase ( ho)-1 expression , suggesting a possible therapeutic mechanism of krg in cardiovascular diseases .
it is well known that chronic inflammation contributes to the pathogenesis of many human diseases such as atherosclerosis .
accumulating evidence suggests that krg is involved in the regulation of inflammatory responses , suggesting an anti - inflammatory effect of krg .
cyclooxygenase ( cox ) catalyzes the conversion of arachidonic acid to prostaglandins that play vital roles in multiple physiological and pathophysiological processes , including inflammation .
in particular , cox-2 is normally undetectable in most tissues and is induced in response to numerous stimuli .
vascular diseases may , in part , be caused by cox-2 upregulation at sites of inflammation and vascular injury .
cox-2 plays an important role in inflammation , therefore , inhibition of cox-2 expression may participate in the treatment of inflammation - related diseases such as vascular diseases .
the objective of our study was to investigate the vascular protective effect of krg in acrolein - stimulated human umbilical vein endothelial cells ( huvecs ) .
therefore , we examined the involvement of cox-2 expression via p38 mitogen - activated protein kinase ( mapk ) , intracellular ros , and apoptosis in acrolein - stimulated huvecs .
krg powder was obtained from the korea ginseng corporation ( daejeon , korea ) . m199 medium and fetal bovine serum
krg powder was soaked in water ( 1:25 , w / w ) for 3  h , and boiled for 40  min .
following centrifugation at 1,900 g for 60  min , supernatants of ginseng extract were further centrifuged at 10,000  g for 30  min and lyophilized .
the general composition of the product offered by the korea ginseng corporation is as follows : moisture 36% , solid volume 64% , ash 2.5% , total fat 0.05% , total crude saponin 70  mg / g , and total ginsenosides 20  mg / g .
huvecs were maintained in m199 medium and supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin , 10  ng / ml human fibroblast growth factor , and 18  mu / ml heparin .
huvecs were grown to 80% confluence , maintained with fresh medium described above , and subcultured every 2 or 3  d. the cells were used within nine passages during these experiments .
we applied 20 or 40  g of the whole cell lysate proteins to each lane and analyzed them with western blotting .
western blotting was performed using primary antibodies as follows : anti - cox-2 , p38 mapk , phopho - p38 , cyclic amp - responsive element - binding protein ( creb ) , phospho - creb ( cell signaling , danvers , ma , usa ) and anti - glyceraldehyde 3-phosphate dehydrogenase ( abfrontier , seoul , korea ) .
horseradish - peroxidase - conjugated anti - igg antibodies were used as the secondary antibody to detect the above - mentioned protein bands by enhanced chemiluminescence westsave - up ( abfrontier ) .
the rna pellets were washed in 70% ethanol , dried completely , and dissolved in diethylpyrocarbonate to inhibit rnase .
total rna was quantified using a nd-100 spectrometer ( nanodrop technologies , wilmington , de , usa ) .
polymerase chain reaction ( pcr ) was performed using the synthesized cdna as a template and using specific primers for cox-2 or -actin as a loading control .
the primer sequence for human cox-2 was 5-gacagtccaccaacttacaat-3 ( forward ) and 5-catctctccatcaattatctgat-3 ( reverse ) .
the amplified products were resolved by 1% agarose gel electrophoresis , stained with ethidium bromide , and photographed under ultraviolet light .
huvecs were cultured in a glass culture chamber slide ( falcon plastics , london ontario , canada ) and processed for immunofluorescence analysis .
the amount of prostaglandin ( pg)e2 in the culture medium was measured using the pge2 eia kit according to the manufacturer 's protocol ( cayman chemical company , ann arbor , mi , usa ) .
samples as well as standards were applied to a 96-well plate , precoated with goat anti - mouse igg , and incubated with pge2 acetylcholinesterase tracer and pge2 antiserum .
all the wells were emptied , rinsed five times , and incubated with ellman 's reagent for 60  min in the dark with gentle rocking to produce 5-thio-2-nitrobenzoic acid , which has a strong absorbance at 405  nm ; the plate was read at 405  nm in an enzyme - linked immunosorbent assay reader ( el 800 ; bio - tek , winooski , vt , usa ) .
we calculated the results using the standard curve , which were expressed as picograms per milliliter .
intracellular ros in acrolein - stimulated huvecs is analyzed using a fluorescent dye , 2,7-dichlorofluorescein diacetate ( dcf / da ) . in the presence of oxidants ,
after 18  h incubation with 25  m acrolein in the presence or absence of krg , cells were stained with 10  m dcf / da , and fluorescence was analyzed by a facs vantage flow cytometer ( becton dickinson , san jose , ca , usa ) and fluorescence microscopy ( eclipse 50i ; nikon , japan ) . to clarify whether krg - mediated inhibition of acrolein - induced cox-2 expression plays a significant role in cytoprotection against oxidative stress , acrolein - stimulated cells were pretreated with krg ( 1  mg / ml ) or untreated , and cell death was measured by in situ terminal transferase dutp nick end labeling ( tunel ) assay . to measure dna fragmentation , the commercially available in situ death detection kit ( roche diagnostics , mannheim , germany )
huvecs were cultured in a glass culture chamber slide and fixed for 30  min in 10% neutral buffered formalin solution at room temperature .
a tunel assay system was used , according to the manufacturer 's instructions , for examination under a fluorescence microscope , with excitation at 488  nm and emission at 525  nm .
fluorescein isothiocyanate ( fitc ) ( bd pharmingen , san diego , ca , usa ) and propidium iodide ( pi ) staining of necrotic and apoptotic cells .
cells were washed in pbs , resuspended in 100  l binding buffer containing 5  l annexin v  fitc and 1  g / ml pi , and incubated for 10  min at room temperature in the dark .
positioning of quadrants on the annexin v / pi dot plots was performed as previously described .
data were expressed as mean    standard deviation . statistical analysis was performed using one - way analysis of variance ( graphpad prism version 4 ; graphpad software , san diego , ca , usa ) followed by bonferroni 's multiple comparison test .
a previous study found that acrolein in cs induces cox-2 expression in human endothelial cells .
krg inhibited acrolein - induced cox-2 protein expression in a concentration - dependent manner ( fig . 
after pretreatment of acrolein - stimulated cells with krg , the cells were fixed , and cox-2 localization in huvecs was observed by immunofluorescence staining with an anti - cox-2 antibody followed by a fluorescence - tagged secondary antibody .
immunofluorescence analysis showed that acrolein - induced cox-2 protein levels were inhibited in huvecs after treatment with krg ( fig . 
the induction of cox-2 expression is known to be responsible for pge2 release in the culture medium of cells stimulated with  acrolein .
acrolein increased pge2 secretion , which was dramatically reduced by krg ( fig .  2 ) .
this result indicates that krg leads to the reduction of cox-2 protein expression and  subsequently pge2 biosynthesis in acrolein - stimulated huvecs .
thus , we examined the effect of krg on ros production in acrolein - stimulated cells .
the shift to the right of the curve due to increased fluorescence indicates an increase in the intracellular levels of ros .
the results indicate that ros generation in cells treated with acrolein increased compared to untreated cells , whereas krg inhibited acrolein - induced ros generation ( fig . 
these results indicate that krg may play a role in the inhibition of cox-2 expression via reduction of acrolein - generated ros in acrolein - stimulated huvecs .
thus , to determine the upstream signaling pathway involved in krg - mediated cox-2 inhibition , we measured the activation of p38 and creb by detecting increased phospho - p38 and phospho - creb levels in acrolein - stimulated cells and found that phosphorylation of p38 and creb was strongly reduced by krg in acrolein - stimulated cells ( fig .  4 ) .
these results demonstrate the role of p38 and creb signaling in the inhibition of acrolein - mediated cox-2 induction .
fluorescence - activated cell sorting showed that while the number of apoptotic cells increased following treatment with acrolein , pretreatment with krg reduced the number of apoptotic cells ( fig . 
to confirm this result , we evaluated the presence of dead cells by tunel staining , which is widely used in detecting dna fragmentations in situ .
the tunel assay indicates cell death , including apoptosis , by detection of the appearance of intensely stained nuclei , which indicates incorporation of labeled dutp into the 3-end of fragmented dna derived from apoptotic nuclei . as illustrated in fig . 
5b , acrolein treatment significantly increased the proportion of tunel - positive cells , which was restored by krg pretreatment .
these results revealed that the vascular protective effect of krg is mediated by the inhibition of cox-2 expression in acrolein - stimulated huvecs .
in this study , we explored the inhibition of an inflammatory mediator , cox-2 , by krg water extract in huvecs .
we found that krg inhibited both mrna and the protein level of cox-2 and its cytoprotective effect in acrolein - stimulated huvecs .
there is increasing evidence that ,-unsaturated aldehydes in cs , including acrolein and crotonaldehyde play an important pathophysiological role in vascular diseases such as atherosclerosis and alzheimer 's disease .
exposure to ,-unsaturated aldehydes is critical to the inflammatory response via activation of the proinflammatory signaling pathway and redox - sensitive transcription factors .
furthermore , ,-unsaturated aldehydes increase oxidative stress , which plays a crucial role in the pathogenesis of vascular diseases via direct injury to the endothelium .
cox-2 , a key enzyme for prostaglandin biosynthesis , is an inducible enzyme that is rapidly induced during inflammatory reactions .
numerous studies have reported the involvement of cs in vascular diseases through cox-2 and endothelial no synthase activity .
chronic inflammation plays an important role in vascular diseases , therefore , cox-2 may participate in the development of inflammation - related diseases , including vascular diseases .
ginseng has been used as a general tonic for > 2000 years in east asia , and it has become a famous herbal medicine for treatment of various diseases , including vascular disorders .
krg has been reported to have effective pharmacological activities , including antioxidant , anticarcinogenic , and ameliorative effects on blood circulation .
recently , the diverse effects of several constituents of krg , including ginsenoside , on endothelial cells have been extensively studied .
hien et  al demonstrated the anti - inflammatory and antiatherosclerotic activities of ginsenoside rg3 in human endothelial cells , with a decrease of cell adhesion molecules and proinflammatory cytokines .
moreover , the cytoprotective effect of ginsenoside rb1 in endothelial cell damage mediated by oxidized low - density lipoprotein has been reported .
several constituents of red ginseng have been reported to regulate proliferation and migration and to protect oxidative stress - mediated damage in human endothelial cells .
there is evidence demonstrating the presence of major ginsenosides including rb1 and rg1 in krg water extract .
thus , these components could also contribute to the diverse retinue of protective actions of krg .
a previous study showed that the induction of ho-1 expression may exert protective effects in krg - treated human endothelial cells .
however , there have been no reports revealing the mechanism underlying krg - inhibited cox-2 expression in acrolein , ,-unsaturated aldehydes in cs , stimulated huvecs .
we have established that the major signaling pathway of cox-2 ( i.e. , p38 mapk  creb ) and intracellular ros generation are involved in this inhibition of cox-2 expression in acrolein - stimulated huvecs by krg . as mentioned above
therefore , the inhibition of cox-2 expression following krg water extract treatment may be associated with its strong protective effect in acrolein - stimulated huvecs . in conclusion ,
we propose that the krg water extract may exert a cytoprotective effect through the inhibition of cox-2 induction and that this reduction of cox-2 in acrolein - stimulated huvecs is mediated by the p38 mapk 
 cigarette smoke is considered a major risk factor for vascular diseases . 
 there are many toxic compounds in cigarette smoke , including acrolein and other ,-unsaturated aldehydes , which are regarded as mediators of inflammation and vascular dysfunction . 
 furthermore , recent studies have revealed that acrolein , an ,-unsaturated aldehyde in cigarette smoke , induces inflammatory mediator expression , which is known to be related to vascular diseases . in this study , we investigated whether korean red ginseng ( krg ) water extract suppressed acrolein - induced cyclooxygenase ( cox)-2 expression in human umbilical vein endothelial cells ( huvecs ) . 
 acrolein - induced cox-2 expression was accompanied by increased levels of phosphorylated p38 in huvecs and krg inhibited cox-2 expression in huvecs . 
 these results suggest that krg suppresses acrolein - induced cox-2 expression via inhibition of the p38 mitogen - activated protein kinase signaling pathway . 
 in addition , krg exhibited an inhibitory effect on acrolein - induced apoptosis , as demonstrated by annexin v  
 propidium iodide staining and terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling assay . 
 consistent with these results , krg may exert a vasculoprotective effect through inhibition of cox-2 expression in acrolein - stimulated human endothelial cells . 
Introduction
Materials and methods
Results
Discussion
Conflicts of interest