endothelial cells ( ecs ) are key cellular components of blood vessels , functioning as selectively permeable barriers between blood and tissues . under pathological conditions , endothelial cell ( ec ) apoptosis leads to excess neointima formation [ 1 , 2 ] , a lipid transport disorder [ 3 , 4 ] , and plaque rupture . thus , maintaining endothelial cell viability by inhibiting the induction of apoptosis could be used in the prevention and/or treatment of atherosclerosis [ 6 , 7 ] . oxidatively modiflied ldl ( oxldl ) has been implicated in the development of atherosclerosis and plaque rupture by promoting lipid accumulation , proinflammatory responses , release of metalloproteinases , and apoptotic cell death of ecs [ 8 , 9 ] . oxldl also increases endothelial expression of adhesion molecules , which recruit inflammatory cells that adhere to and migrate through the endothelial barrier . these processes are followed by endothelial dysfunction and loss of expression of antiapoptotic proteins , which in turn causes ecs to become apoptotic [ 1012 ] . lectin - like oxldl receptor-1 ( lox-1 ) is considered the major receptor for oxldl in human and various animal vascular endothelial cells ( ecs ) . 6-shogaol is the major bioactive compound present in zingiber officinale which possesses antitumor , antioxidant , anti - inflammatory [ 1618 ] , antiplatelet aggregation , antihypertensive [ 20 , 21 ] , and antiatherosclerosis [ 22 , 23 ] effects . the mechanism of anti - as action of ginger extract is associated with a significant reduction in plasma and ldl cholesterol levels and a significant reduction in the ldl basal oxidative state , as well as their susceptibility to oxidation and aggregation [ 22 , 23 ] . the present study evaluated effects of 6-shogaol on oxldl - induced insults to huvecs and its possible molecular mechanisms . lucigenin , dimethylsulfoxide ( dmso ) , 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide ( mtt ) , diphenyleneiodonium ( dpi ) , was obtained from sigma ( st . louis , mo ) . 6-shogaol was purchased from national institute for the control of pharmaceutical and biological products ( beijing , china ) . the identity and purity of the compound were determined using hplc ( high - performance liquid chromatography ) and 2d nmr and were > 99% . blood was processed for ldl separation within 1 day by sequential flotation in nabr solution containing 1  mg / ml edta . cu - modified ldl ( 1.0  mg protein / ml ) was prepared by exposure of ldl to 5  mm cuso4 for 18  h at 37c . the extent of ldl oxidation was determined by thiobarbituric acid - reactive substances ( tbars ) . this experiment was approved by the research ethics committee of shanghai east hospital , tongji university school of medicine . after receiving written consent from the parents , we obtained fresh human umbilical cords from normal full - term neonates shortly after birth and suspended them in hanks ' balanced salt solution ( hbss ; gibco ) at 4c . briefly , huvecs were removed from human umbilical veins after collagenase type i digestion and cultured in medium 199 containing 20% fetal calf serum , penicillin ( 100  u / ml ) , streptomycin ( 100  u / ml ) , and heparin ( 50  u / ml ) , supplemented with l - glutamine ( 2  mm ) , sodium pyruvate ( 1  mm ) , and endothelial cell growth factor ( b - ecgf , 5  ng / ml ) , at 37c in 5% co2 on 0.1% gelatin - coated culture flasks . 6-shogaol was dissolved in dimethyl sulfoxide ( dmso ) and stored at 20c until use . final concentration of dmso in culture media was 0.1% huvecs were randomly divided into six groups : a normal control group , a oxldl group , four 6-shogaol groups . the huvecs in the oxldl group were incubated for 24 hours with medium containing 200  g / ml oxldl . in the 6-shogaol groups , the cells were preincubated for 2  h with different final concentrations of 6-shogaol : 1  m , 5  m , 10  m , and 30  m , followed by a 24-hour incubation with 200  g / ml ox - ldl . thp-1 , a human monocytic leukemia cell line , was obtained from atcc ( rockville , md ) and cultured in rpmi with 10% fbs at a density of 25  10 cells / ml as suggested in the product specification sheet provided by the vendor . cells were seeded at density of 5  10 cells / ml in 96-well plates and the cell viability was measured using the mtt assay . briefly , at the indicated time points after the treatment as before , the culture supernatant was removed , and the cells were washed with pbs , incubated with mtt ( 5  mg / ml ) in culture medium at 37c for another 3  h. after mtt removal , the colored formosan was dissolved in 100  l of dmso . the absorption values were measured at 490  nm using a sunrise remote microplate reader ( grodlg , austria ) . the viability of huvecs in each well was presented as percentage of control cells . briefly , 5  10 huvecs were cultured in 12-well tissue culture plates overnight and then cotreated with drugs and 2,7-dichlorofluorescin diacetate . relative fluorescence intensities of cells were quantified using a flow cytometer ( facs calibur , becton dickinson , san jose , ca , usa ) . to evaluate the role of nadph oxidase and lox-1 in oxldl - induced ros generation , we preincubated cells with the flavoprotein inhibitor dpi ( 5  m ) and anti - lox-1 monoclonal antibody ( mab ; 40  g / ml ) for 2  h before exposure to oxldl . sod activity in the homogenate was determined by an enzymatic assay method using a commercial kit ( calbiochem ) according to the manufacturer 's instructions . specific nadph - dependent o2 production was measured by lucigenin ( 5  mm ) chemiluminescence as previously described . the cells were pretreated with various concentrations of 6-shogaol or vehicle for 60  min , after which oxldl ( 200  g / ml ) was added for additional 60  min . then cells were scraped into ice - cold hbss supplemented with 0.8  mm mgcl2 and 1.8  mm cacl2 , disrupted by rapid freezing in liquid nitrogen followed by sonication . oxygen radical production was measured in the presence of 5  mm lucigenin , with or without nadph ( 100  mm ) for 10  min . the relative light units ( rlu ) of chemiluminescence were read in turner td 20/20 luminometer . apoptosis was also examined by analysis of dna fragmentation using flow cytometry [ 27 , 28 ] . huvecs were washed and double - stained by using an annexin v - fitc apoptosis detection kit . annexin v has a strong ca - dependent affinity for phosphatidylserine ( ps ) , which translocates from the internal to the external surface of the plasma membrane as a probe for detecting apoptosis . cells that have the loss of membrane integrity will show red staining ( propidium iodide , pi ) throughout the nucleus and therefore will be easily distinguished between the early apoptotic cells and the late apoptotic cells or necrotic cells . samples were incubated at room temperature for 15  min in the dark with annexin v and pi and quantitatively analyzed by a facs vantage se flow cytometer . the activities of caspase-3 , caspase-8 , and caspase-9 were measured according to the kit manufacturers ' instructions . in brief , after 24  h treatment by different medium conditions , huvecs of each group were lysed and removed from culture dishes , washed twice with pbs , and pelleted by centrifugation . cell pellets were then treated for 10 minutes with iced lysis buffer supplied by the manufacturers : caspase-3 , caspase-8 assay cellular activity kit , and caspase-9 assay kit ( calbiochem ) . then the suspensions were centrifuged at 10000  g for 10 minutes , and the supernatants were transferred to a clear tube . to each tube , specific substrate conjugate [ acetyl - asp - glu - val - asp - p - nitroaniline ( ac - devd- p - na ) for caspase-3 , acetyl - ile - glu - thr - aspaminotrifluoromethyl coumarin ( ac - ietd - afc ) for caspase-8 and acetyl - leu - glu - his - asp - p - nitroaniline ( ac - lehd - p - na ) for caspase-9 ] was added and the tubes were incubated at 37c for 2 hours . during incubation , the caspases cleaved the substrates to form p - na or afc . caspase-3 and -9 activities were read in a microtiter plate reader at 405  nm . caspase-8 activity was read in a fluorescent plate reader at 400  nm for excitation and at 505  nm for emission . assays were performed in triplicate and three independent experiments were performed in this study . mrna from huvecs exposed to 6-shogaol , oxldl , or a combination of 6-shogaol and oxldl were prepared for real - time polymerase chain reaction ( pcr ) analyses of lox-1 and gapdh mrna . the oligonucleotides for the pcr analyses of lox-1 and gapdh mrna were designed and synthesized by invitrogen laboratories ( palo alto , ca , usa ) . the oligonucleotide sequences for these mrna analyses were 5-tcttagcatgaatttggaaat-3 and 5-cccagctaaagggcccatgg-3 for lox-1 , and gapdh ( forward : 5-cca - ccc atg gca aat tcc atg gca-3 and reverse : 5-tct aga cgg cag gtc agg tcc acc-3. a real - time pcr analysis was performed using iqsybr green supermix ( bio - rad , hercules , ca , usa ) and the myiq single - color real - time pcr detection system ( bio - rad ) . cells were lysed in a modified ripa buffer ( 150  mm nacl , 10  mm tris , ph 7.4 , 1  mm edta , 1% triton x-100 , 1% deoxycholic acid , 1  mm pmsf , with addition of complete tm protease inhibitor cocktail ) . protein concentrations were determined by a bca assay and separated by an 8% sdspage , and then transferred to a pvdf membrane ( millipore , usa ) . the membrane was blocked at rt for 2  h in 5% nonfat dry milk diluted with tbst ( in mm : tris - hcl 20 , nacl 150 , ph 7.5 , 0.1% tween 20 ) . the membrane was incubated overnight at 4c with a polyclonal rabbit anti - human lox-1 and bcl-2 ( 1  :  500 dilution ; santa cruz biotechnology inc . the membrane was incubated for 1  h with a goat anti - rabbit igg conjugated to horseradish peroxidase ( 1  :  10000 dilution ; santa cruz biotechnology inc . at last , the levels of lox-1 and bcl-2 protein were determined using amersham ecltm western blotting detection reagents ( ge healthcare , uk ) . incubation with polyclonal rabbit -actin antibody ( 1  :  1000 dilution ; santa cruz biotechnology inc . relative intensities of protein bands were analyzed by scan - gel - it software . pcmv6-xl5-lox-1 plasmids , which were constructed with full - length human lox-1 cdna , were purchased from origene technologies ( rockville , md , usa ) . pcmv6-xl5-lox-1 plasmids were transfected into huvecs usinga fugene 6 transfection reagent ( roche diagnostics , mannheim , germany ) as previously described . briefly , huvecs were cultured in antibiotic - free dulbecco 's modified eagle 's medium at 37c for 24 hours , then the sirna duplex solution was added . activation of nf-b was assessed by measuring the p65 protein - dna binding activity in nuclear extracts of huvecs . when the cells were 7090% confluent , the medium was changed to 15  ml of complete m199 ( 20% fbs ) and incubated for 2  h in the absence or the presence of 6-shogaol or anti - lox-1 monoclonal antibody ( mab ; 40  g / ml ) , after which huvecs were challenged with 200  g / ml ox - ldl . for comparison purposes , huvecs were also incubated for 1  h with pyrrolidine dithiocarbamate ( pdtc ) , a known inhibitor of nf-b activation [ 30 , 31 ] . after 1  h of incubation with ox - ldl , the reaction was stopped by washing the cells with cold pbs . nuclear extracts were prepared according to the manufacturer 's instructions , and the dna binding activity of p65 was measured by elisa ( active motif , carlsbad , ca ) . briefly , nuclear extracts were added to wells previously coated with dna containing specific sequences for the binding of p65 . after incubation at room temperature for 1  h , wells were washed and sequentially incubated for 1  h with a primary antibody raised against p65 and a secondary enzyme - linked antibody . the plate was developed by addition of chromogen , and the absorbance at 450  nm was recorded in a plate - reader spectrophotometer ( spectromax 190 ) . huvecs were grown to confluence , then pretreated with 6-shogaol for 2  h , and stimulated cells with oxldl ( 200  g / ml ) for 24  h. at the end of stimulation , huvecs were harvested and incubated with fitc conjugated anti - icam-1 , anti - e - selectin , and anti - mcp-1 ( r&d systems ) for 45  min at room temperature . after the huvecs had been washed three times , their immunofluorescence intensity was analyzed by flow cytometry using a becton dickinson facscan flow cytometer ( mountain view , ca ) huvecs at 1  10 cells / ml were cultured in 96-well flat - bottom plates ( 0.1  ml / well ) for 1 - 2 days . cells were then pretreated with the indicated concentrations of 6-shogaol for 2  h and incubated with oxldl ( 200  g / ml ) for 24  h. the medium was then removed , and 0.1  ml / well of thp-1 cells ( prelabeled with 4  m bcecf - am for 30  min in rpmi at a 1  10  cell / ml density ) were added in rpmi . the cells were allowed to adhere at 37c for 1  h in a 5% co2 incubator . the number of adherent cells was estimated by microscopic examination ; the cells were then lysed with 0.1  ml of 0.25% triton x-100 . the fluorescence intensity was measured at 485-nm excitation and 538-nm emission using a labsystems fluorescence microplate reader . data analysis values were expressed as mean  sd , statistical significance was determined by student 's two - tailed t - test or one - way anova followed by bonferroni posttests when more than two treatments were compared . concentration - response curves were analysed by two - way anova followed by bonferroni posttests . treatment of mouse huvecs with 1 , 5 , 10 , and 30  m 6-shogaol for 24 , 48 , and 72 hours did not affect cell viability ( data not shown ) . meanwhile , after exposure for 72 hours , 6-shogaol at 60  m caused a significant 27% decrease in cell viability . when the treated concentration reached 100  mol / l , administration of  6-shogaol  for 48 and 72 hours , respectively , decreased the viability of huvecs by 41% and 61% ( data not shown ) . next , we observed the  6-shogaol  antioxidation activity by mda measurement and used fluorescence microscopy to analyze the effect of  6-shogaol  on the lox-1 mediated redox - sensitive signaling pathway in endothelial cells . as shown in figure 1(a ) , when ldl was reacted with copper sulfate for 1 , 6 , 12 , 18 , and 24 hours , amounts of malondialdehyde significantly increased by 3.62- , 8.18 , 12.74- , 16.47- , and 19.44-fold , respectively . 6-shogaol at 0.5  m did not affect the oxidation of ldl by copper sulfate ( figure 1(b ) ) . meanwhile , when the concentrations reached 1 , 5 , 10 , 30  m , 6-shogaol decreased copper sulfate - caused oxidation of ldl by 10.3% , 29.1% , 39.1% , and 59.8% , respectively . the intracellular ros concentration in huvecs was determined by measuring the intensity of dcfh fluorescence . when dcfh - da - labeled cells were incubated in the medium for 2  h , a sudden increment in fluorescence intensity indicated the oxidation of dcfh - da by intracellular radicals ( figure 1(c ) ) . the production of dcfh fluorescence in huvecs with oxldl increased significantly to 364% of the vehicle - treated control group , whereas preincubation with 6-shogaol ( 130  m ) significantly reduced the increased fluorescence induced by oxldl in a concentration - dependent manner . in addition , oxldl - induced ros was abrogated by pretreatment with monoclonal antibody of lox-1 ( anti - lox-1 mab ) or dpi ( figure 1(c ) ) . in addition , the involved ros is able to inactivate antioxidative enzymes that additionally increase the imbalance in favor of oxidative stress . we next turned our attention to the total activity of sod in endothelial cells in response to oxldl . as shown in ( figure 2(a ) ) , 6-shogaol at 5 , 10 , and 30  m significantly decreased the suppression of sod activity caused by oxldl . endothelial nadph oxidase is a major source of ros in vascular endothelial cells , and atherogenic levels of ldl have been shown to induce a marked increase in nadph oxidase - generated ros by the endothelium . as shown in figure 1(d ) , incubation of huvecs with oxldl ( 200  g / ml ) for 1  h increased the nadph oxidase activity by 132% ( p 6-shogaol at 10 and 30  m significantly reduced nadph oxidase activity of huvecs from 87.18  5.06 rlu / s / mg protein to 63.92  6.82  rlu / s / mg protein and 55.53  3.95 rlu / s / mg protein , respectively ( p < 0.05 ) . as shown in figure 2(b ) , the survival rate of huvecs was about 52.37  6.59% after exposure to 200  g / ml oxldl . however , preincubation of huvecs with different concentrations of 6-shogaol ( 1 , 5 , 10 , 30  m ) markedly increased the viability of oxldl - treated huvecs in a concentration - dependent manner . the treatment with 1 , 5 , 10 , and 30  m concentrations of 6-shogaol increased the viability of huvecs in a statistically significant fashion to 69.68    4.40% , 76.91    3.06% , and 83.15    3.07% , respectively . in addition , no difference was seen in cell viability between cells treated with 6-shogaol ( 130  m ) alone and controls ( data not shown ) . these results suggest that 6-shogaol protected huvecs from oxidative stress - related cellular injuries . as shown in figure 2(c ) , in the vehicle - treated control group , the percentage of apoptotic cells was 4.78%    1.40% . after exposure to 200  g / ml oxldl for 24  h , the percentage of apoptosis increased to 32.43%    2.75% . nonetheless , preincubation with 6-shogaol ( 130  m ) for 2  h prior to ox - ldl exposure concenteration - dependently arrested the apoptosis , and the values of apoptosis were decreased to 27.93%    2.85% , 21.58%    2.27% , and 16.53%    2.37%% , respectively ( p < 0.05 ) . moreover , the induction of apoptosis in huvecs treated with 6-shogaol ( 130  m ) alone was not observed ( data not shown ) . application of lox-1 sirna into huvecs for 24 and 48 hours decreased the levels of lox-1 receptor ( figure 3(a ) ) . exposure to lox-1 sirna for 24 and 48 hours caused significant 32% and 79% decreases in the levels of lox-1 . exposure of huvecs to 6-shogaol or lox-1 sirna alone did not induce cell apoptosis ( figure 3(c ) ) . treatment with 6-shogaol and lox-1 sirna , respectively , caused significant 58% and 65% decreases in oxldl - induced huvecs apoptosis . cotreatment with 6-shogaol and lox-1 sirna synergistically reduced oxldl - caused cell apoptosis by 88% ( figure 3(c ) ) . by comparison , overexpression of lox-1 alone in huvecs did not affect cell apoptosis but completely attenuated  6-shogaol  involved protection against oxldl - induced apoptotic insults ( figure 3(b ) ) . consistent with previous study , incubation of huvecs with oxldl ( 200  g / ml ) enhanced lox-1 expression at both the gene ( figure 4(a ) ) and protein levels ( figure 4(b ) ) . pretreatment of huvecs with 6-shogaol for 2  h before exposure to oxldl for 24  h resulted in suppression of lox-1 expression in a concentration - dependent manner . notably , pretreatment with dpi , an inhibitor of ros production , markedly inhibited oxldl - induced lox-1 upregulation ( figures 4(a ) and 4(b ) ) , strongly suggesting that ros plays a critical role in the increased expression of lox-1 . oxldl binding to lox-1 decreased the expression of antiapoptotic proteins such as bcl-2 and c - iap-1 , subsequently activated apoptotic signaling pathway caspase-9 and caspase-3 , and finally resulted in apoptosis . consistent with these studies , oxldl treatment decreased the expression of antiapoptotic protein bcl-2 ( figure 5(a ) ) , while 6-shogaol significantly blocked the decreasing bcl-2 expression induced by oxldl on huvecs . caspase-3 is one of the downstream effectors of the caspase family and is involved in both the mitochondrial apoptotic pathway and the death receptor pathway . the activity of caspase-3 and caspase-9 were not affected by 6-shogaol ( figure 5 ) . treatment of huvecs with 200  g / ml oxldl led to a significant increase in activity of caspase-9 ( figure 5(b ) ) and caspase-3 , not for caspase-8 ( figures 5(b ) , 5(c ) , and 5(d ) ) as compared to control ; however , 6-shogaol administration significantly decreased oxldl - induced caspase-3 and caspase-9 activation ( figures 5(b ) and 5(c ) , p < 0.05 ) . devd - cho ( 25  mol / l ) , an inhibitor of caspase-3 , was applied to huvecs to further evaluate the roles of this protease in 6-shogaol - caused protection ( figure 6(a ) ) . treatment with devd - cho significantly decreased oxldl - induced augmentation of caspase-3 activity by 68.5% ( figure 6(a ) ) . cotreatment with 6-shogaol and z - veid - fmk completely lowered oxldl - caused enhancement of caspase-3 activity . the oxldl - caused huvecs apoptosis was significantly ameliorated by 65.45% following administration of devd - cho ( figure 6(b ) ) . simultaneous exposure to 6-shogaol and devd - cho completely lowered oxldl - induced cell apoptosis . oxldl - induced ros can activate nf-b activation , which facilitates nuclear translocation and subsequent regulation of proinflammatory gene expression [ 32 , 33 ] . a shown in figure 7(a ) , activation of nf-b , as indicated by nuclear translocation and dna binding of its p65 subunit , was decreased by 6-shogaol in a concentration dependent manner , meanwhile 10  m pyrrolidine dithiocarbamate and 40  g / ml anti - lox-1 monoclonal antibody also exerted strong inhibition ( figure 7(a ) ) . the effect of 6-shogaol on the surface expression of adhesion molecules on huvecs exposed to oxldl was subsequently examined . as shown in figure 7(b ) , the expression levels of icam-1 , mcp-1 , and e - selectin were significantly higher in huvecs that had been treated with oxldl ( 200  g / ml ) for 24  h than in the control cells ( 229 , 304 , and 460% , resp . flow cytometry revealed that the induction of adhesion molecule expression was significantly ameliorated by the presence of 130  m 6-shogaol . in addition , oxldl - induced  expression of adhesion molecules  was abrogated by pretreatment with monoclonal antibody of lox-1 ( anti - lox-1 mab ) or sirna ( figure 7(b ) ) . oxldl - enhanced recruitment , retention , and adhesiveness of human monocytes and monocytic cell lines to endothelium have been implicated in the initial stage of atherogenesis . to test the effect of 6-shogaol on monocyte adhesion to huvecs , confluent monolayers of huvecs were pretreated with various concentrations of 6-shogaol or anti - lox-1 monoclonal antibody ( mab ; 40  g / ml ) for 2  h and then stimulated with oxldl ( 200  g / ml ) for 24  h , followed by incubation with thp-1 cells for 1  h at 37c . as shown in figure 7(c ) , oxldl stimulated an increase in adherence of thp-1 cells to huvecs ( 472  17% , p < 0.05 ) ; however , the effect was significantly inhibited by 6-shogaol treatment in a concentration - dependent manner ( all p < 0.05 ) . oxldl is an important initiating factor for endothelial activation and injury contributing to endothelial dysfunction , one of the earliest hallmarks of atherosclerosis [ 8 , 34 , 35 ] ; lox-1 , as the primary oxldl receptor on endothelial cells , plays an important role in the pathogenesis of atherosclerosis [ 3638 ] . the binding of oxldl to lox-1 initiates ros formation , which in turn upregulates lox-1 expression , thereby contributing to further ros generation . the present study shows the effectiveness of 6-shogaol , the major bioactive compound present in zingiber officinale , in suppressing endothelial lox-1 expression and lox-1-mediated proatherogenic effects . this effect of 6-shogaol on endothelial lox-1 expression appears to be exerted at the transcriptional level , as reflected by the parallel decrease in lox-1 mrna and protein levels in 6-shogaol -treated cells ( figure 3 ) . furthermore , pretreatment with dpi or blockade of lox-1 with anti - lox-1 mab or sirna - lox-1 prevented oxldl - induced ros generation and cell apoptosis , which suggests that the binding of oxldl to lox-1 and the consequent formation of ros may be the first event in lox-1-mediated endothelial dysfunction ( figures 1 and 4 ) . because regulation of lox-1 gene expression is redox sensitive , suppression of oxldl - induced ros production by 6-shogaol may contribute to the reduction of lox-1-mediated expression of a number of proinflammatory molecules and cell apoptosis . nadph oxidase is recognized as the major source of ros in endothelial cells and the increased nadph activity has been detected in atherosclerotic arterie . it has been shown that oxldl - induced endothelial dysfunction is caused by an increase in nadph oxidase - generated superoxide concentrations and a decrease in antioxidative enzyme activity , resulting in the activation of multiple ros - sensitive signaling pathways . consistent with the literature , our data show that 6-shogaol treatment significantly reduced the level of oxldl - induced ros generation ( figures 1(c ) and 1(d ) ) and increased the level of sod activity ( figure 2(a ) ) . ros can activate nf-b and enable nuclear translocation and subsequent regulation of proinflammatory molecules , including cytokines , chemokines , enzymes , and adhesion molecules . in the present study , ros production in huvecs occurred within 5  min ( data not shown ) , and nf-b was activated within 1.5  h of the addition of oxldl . however , pretreatment with anti - lox-1 mab , ros production , and nf-b activation , and icam-1 , mcp-1 , and e - selectin expression were decreased markedly , which suggests that the binding of oxldl to lox-1 and the consequent nf-b activation . furthermore , our stuy showed that 6-shogaol inhibited nf-b activation ( figure 7(b ) ) and repressed the oxldl - induced icam-1 , mcp-1 , and e - selectin expression ( figure 7(c ) ) . from these , we speculated that 6-shogaol protection against oxldl - induced endothelial dysfunction may be by blockading the binding of oxldl to lox-1 , and subsequently decrease intracellular ros generation and the proinflammatory molecules expression . all of these findings strongly indicate that 6-shogaol elicits antioxidative and anti - inflammatory effects . apoptosis , also called programmed cell death , is an important process of many pathological conditions including atherosclerosis . lox-1 activation by oxldl stimulates endothelial proinflammatory gene expression and production of superoxide radicals   and leads to activation of apoptotic signaling pathway [ 13 , 45 ] . their findings suggested that oxldl binding to lox-1 subsequently decreased the expression of antiapoptotic proteins , such as bcl-2 and c - iap-1 , then activated apoptotic signaling pathway caspase-9 and caspase-3 , and finally resulted in apoptosis . consistent with previous reports , the results presented here indicated that oxldl induced decrease in bcl-2 expression , but 6-shogaol completely normalized this oxldl - induced alterations . application of lox-1 small interference ( si)rna into huvecs simultaneously increased 6-shogaol protection from oxldl - induced cell apoptosis . by comparison , overexpression of lox-1 attenuates 6-shogaol protection . both 6-shogaol and therefore , this study shows that 6-shogaol may protect huvecs from oxldl - induced apoptotic insults via downregulating lox-1-mediated activation caspase protease pathway .   in summary , the results from our experiments indicate that 6-shogaol prevents the oxldl - induced lox-1-mediated biological events in huvecs , probably via its antioxidative and anti - inflammatory functions . our work adds 6-shogaol to the growing list of herbal remedies whose mode of action has been at least partially revealed on a molecular level .
endothelial dysfunction and oxldl are believed to be early and critical events in atherogenesis . 
 6-shogaol is the major bioactive compound present in zingiber officinale and possesses the anti - atherosclerotic effect . 
 however , the mechanisms remain poorly understood . 
 the goal of this study was to investigate the effects of 6-shogaol on oxldl - induced human umbilical vein endothelial cells ( huvecs ) injuries and its possible molecular mechanisms . 
 hence , we studied the effects of 6-shogaol on cell apoptosis , cellular reactive oxygen species ( ros ) , nf-b activation , bcl-2 expression , and caspase -3 , -8 , -9 activities . 
 in addition , e - selectin , mcp-1 , and icam-1 were determined by elisa . 
 our study show that oxldl increased lox-1 expression , ros levels , nf-b , caspases-9 and -3 activation and decreased bcl-2 expression in huvecs . 
 these alterations were attenuated by 6-shogaol . 
 cotreatment with 6-shogaol and sirna of lox-1 synergistically reduced oxldl - induced caspases -9 , -3 activities and cell apoptosis . 
 overexpression of lox-1 attenuated the protection by 6-shogaol and suppressed the effects of 6-shogaol on oxldl - induced oxidative stress . 
 in addition , oxldl enhanced the activation of nf-b and expression of adhesion molecules . 
 pretreatment with 6-shogaol , however , exerted significant cytoprotective effects in all events . 
 our data indicate that 6-shogaol might be a potential natural antiapoptotic agent for the treatment of atherosclerosis .