inflammation plays a key role in innate immune responses during infections with invading pathogens such as bacteria , fungus , virus , and parasite .
inflammation can be protective by recruiting immune and inflammatory cells and eliminating pathogens ; however , excessive inflammation with overexpression of inflammatory factors and cytokines commonly leads to kinds of autoimmune diseases and host tissue injury , such as inflammatory bowel disease ( ibd ) , psoriasis , and sepsis [ 24 ] .
pattern - recognition receptors ( prrs ) are essential to regulate immune responses by targeting pathogenic microbes and presenting antigens to the adaptive immune system .
prrs can be divided into four categories : toll - like receptors ( tlrs ) , nucleotide - binding domain leucine - rich repeats ( nlrs ) , nucleotide - binding oligomerization domain ( nod ) , and retinoic acid - inducible gene i - like receptors ( rlrs ) . in the presence of microbial stimuli , host prrs such as tlrs
promote nuclear factor - kappa b ( nf-b ) mediated proinflammatory cytokines expression such as interleukin- ( il- ) 1 , il-6 , tumor necrosis factor- ( tnf- )  , and nitric oxide ( no ) .
meanwhile , another group of prrs including nlr family pyrin domain - containing 3 ( nlrp3 ) recruit the adaptor protein apoptosis - associated speck - like protein containing card ( asc ) and procaspase-1 .
formation of this complex leads to the activation of caspase-1 by triggering procaspase-1 self - cleavage and promotes the precursor forms of cytokines such as pro - il-1 and pro - il-18 into active forms that are secreted .
activation of macrophages triggered by prrs further induces the maturation of dendritic cells ( dcs ) by releasing proinflammatory cytokines and promotes the induction of adaptive immune responses .
the nlrp3 inflammasome is mainly expressed in immune and inflammatory cells such as macrophages , monocytes , neutrophils , and dcs when challenged by microbial stimuli .
it has been well - established that multiple pathways participate in inflammasome activation such as cellular and mitochondrial reactive oxygen species ( ros ) , cathepsin b , and cytosolic protein kinase r ( pkr ) [ 1012 ] . among all of these triggers , ros generation
is reported to play an essential role in the activation of nlrp3 inflammasome when challenged by stimuli such as lipopolysaccharides ( lps ) , adenosine triphosphate ( atp ) , and urate crystals .
more fundamentally , a more recent study demonstrated that ros derived by xo , the oxidized form of xanthine dehydrogenase , is the main source of nlrp3 activation in macrophages , leading to excessive il-1 and il-18 secretion .
inhibition of xo by febuxostat , well - documented xo inhibitor , could significantly decrease the ros generation and il-1 secretion in macrophages stimulated by lps , indicating the predominant role of xo in lps - induced il-1 mature and secretion [ 14 , 15 ] .
hsya ( 2d and 3d structure in figure 1 ) is a water soluble monomer extracted from carthamus tinctorius l. ( safflower ) , which has long been used for treatment of cardiovascular diseases in traditional chinese medicine .
recent researches showed that , besides the therapeutic effects upon cardiovascular system , hsya exhibits promising anti - inflammatory properties by suppressing innate immune tlr4-inducing pathway , bettering lps - induced inflammatory injury , scavenging excessive ros , and inhibiting proinflammatory cytokines generation [ 1719 ] .
however , few studies have attempted to uncover the direct target of hsya and interpret the mechanisms of its anti - inflammatory properties . in this study
, we tried to find the potential target of hsya via inverse prediction method and computation docking and further assessed the role of hsya in regulating nlrp3/caspase-1/il-1 pathway in macrophages .
hsya [ > 98% high - performance liquid chromatography ( hplc ) purity ] was purchased from tauto biotech ( shanghai , china ) .
lps ( escherichia coli o55:b5 ) was purchased from sigma - aldrich chemical ( st . louis , mo , usa ) . fetal bovine serum ( fbs ) , dulbecco 's modified eagle medium ( dmem ) , antibiotic - antimycotic , and trizol reagents
bicinchoninic acid ( bca ) protein assay kit was purchased from pierce ( rockford , il , usa ) .
enzyme - linked immunosorbent assay ( elisa ) kits for mouse il-1 and il-18 were purchased from cusabio ( wuhan , china ) .
antibodies for mouse -actin , gapdh , pro - il-1 , il-1 , nlrp3 , asc , procaspase-1 , and caspase-1 were purchased from cell signaling technology ( danvers , ma , usa ) .
antibody for xo was purchased from santa cruz ( dallas , tx , usa ) .
the goat anti - mouse antibody was purchased from li - cor odyssey ( lincoln , ne , usa ) .
xo activity assay kit ( c / f ) was purchased from biovision ( milpitas , ca , usa ) .
raw264.7 macrophage cell line was purchased from the american type culture collection ( rockville , md , usa ) .
cells were cultured in dmem supplemented with 10% fbs and antibiotics ( 100 u / ml penicillin and 100 u / ml streptomycin ) at 37c in a humidified incubator under 5% co2 . to detect the levels of cytokines ( il-1 and il-18 ) released by raw264.7 macrophages in vitro ,
cells were preincubated in 24-well plates ( 4  10 cells / well ) for 12  h and pretreated by hsya ( 25 , 50 , and 100  m ) for 3  h. cells were then washed twice with pbs and challenged by lps ( 1  g / ml ) for another 24  h at 37c under 5% co2 . the supernatants were collected and assayed immediately using sandwich technique .
cells ( 1  10 ) were preincubated at 37c under 5% co2 for 12  h and then treated by appropriate experimental reagents .
cells were then harvested on ice , washed three times with cold pbs , and suspended in 500  l lysis buffer supplemented with protease inhibitors .
after incubation on ice for 30  min , cell extracts were centrifuged at 14,000  rpm for 15  min at 4c to isolate total cell proteins , which were quantified using a bca protein assay kit .
proteins were separated by sds - page and electrotransferred to nitrocellulose membranes ( pierce , il , usa ) before hybridization with the appropriate detection antibodies ( xo , pro - il-1 , il-1 , nlrp3 , asc , procaspase-1 , and caspase-1 ) .
gapdh was used to correct for differences in loading of the proteins and for normalization of the densitometric values of immunoblot signals obtained from three separate experiments using li - cor odyssey detecting system .
macrophages were preincubated in 6-well plates ( 1  10 cells / well ) for 12  h and pretreated by hsya ( 25 , 50 , and 100  m ) 3  h prior to lps ( 1  g / ml ) stimulation .
the concentration and integrity of total rna samples were evaluated by measurement of the a260/280 ratio . quantitative polymerase chain reaction ( pcr )
analysis was performed using the dna engine mx3000p ( agilent , santa clara , ca , usa ) fluorescence detection system according to optimized pcr protocols .
the following primers were used for amplification : -actin : f : 5-cccatctacgagggctatgc-3 , r : 5-ggtgtaaaacgcagctcagta-3 ; xo , f : 5-cacgatgacgaggacaacgg-3 , r : 5-taggctcaggcttgtttcgg-3. ros production was detected by measuring intracellular ros formation using probe dcfh2da . briefly , raw264.7 cells were pretreated with hsya ( 100  m ) or positive control febuxostat ( 30  m ) for 3  h and then stimulated with lps ( 1  g / ml ) for 6  h to promote ros generation .
cells were washed twice with pbs and then incubated with probe dcfh2da ( 20  nm ) for 15  min .
fluorescence staining was visualized using a fluorescence microscopy ( olympus , ix71 ) and fluorescence assays were measured with a fluorescence microplate reader ( tecan , sunrise ) at excitation / emission 525/610  nm .
theoretically , xo oxidizes xanthine to hydrogen peroxide ( h2o2 ) which reacts stoichiometrically with oxired probe to generate color at  = 570  nm and fluorescence at ex / em = 535/587  nm .
since the color or fluorescence intensity is proportional to xo content , the xo activity can be accurately measured . to detect the direct inhibition of hsya on xo ,
we first added hsya at various concentrations ( 0 , 10 , 20 , 40 , 80 , 160 , 320 , 640 , and 1280  m ) into active xo solution and incubated at 37c for 30  min .
after that , this mixed solution was added into a reaction system that consisted of assay buffer , substrate mix , enzyme mix , and oxired probe and incubated for another 30  min at 37c .
the ic50 of hsya on xo was calculated by spss probit regression . to detect the effect of hsya on xo activity in lps - stimulated raw264.7 macrophages , we treated cells with hsya at different concentrations ( 25 , 50 , and 100  m ) for 3  h before lps challenge ( 1  g / ml , 12  h )
clear xo extract was obtained by centrifuge ( 16,000  g , 10  min ) and the activity of xo from cell extract was added into reaction system and measured as described above .
pharmmapper server is a web server for potential drug targets identification using pharmacophore mapping approach at http://59.78.96.61/pharmmapper/. briefly , 2d mol2 file of hsya ( pubchem cid : 6443665 ) was submitted to pharmmapper server . during the procedure , the maximum conformations were set up to 300 , and the number of reserved matched targets was 300 .
other parameters were kept as default . the submission i d can be stored and used to check the prediction results .
further , to characterize the combination between hsya and xo , we used autodock and autogrid to calculate the binding affinity and binding sites .
briefly , the two - dimensional ( 2d ) structure of hsya was drawn and converted to mol2 format by chemdraw .
then the mol2 file of hsya was optimized and saved as final coordinate pdb file for docking study .
the 3d crystal structure of xo ( entry code 1f04 ) was downloaded from the protein data bank ( pdb ) at http://www.rcsb.org/. the grid procedure was handled , by autogrid , in a grid box of 606060   with a 1.0   to enclose all the active sites and to allow for the flexible rotations of hsya . then
the data were presented as means  standard error of the mean ( sem ) and differences between mean values of normally distributed data were assessed by the one - way analysis of variance ( anova ) followed by duncan 's test for multiple comparisons .
via pharmacophore mapping approach , 300 potential candidates out of 7302 were listed and sorted according to the fit score ( submission i d 151015052728 ) . based on the disease information and potential roles in inflammation and redox related signaling pathways , endothelial nitric oxide synthase ( pdb i
d : 1p6 m ) , nadph cytochrome p450 reductase ( pdb i d : 1j9z ) , and xo ( pdb i d : 1f04 ) were selected as potential targets of hsya .
based on the pharmacophore model , endothelial nitric oxide synthase had one hydrophobic , four donors , and three acceptors ; nadph cytochrome p450 reductase had two hydrophobics , one positive , two negatives , one donor , eight acceptors , and one aromatic ; xo had one negative , two donors , and five acceptors . however , since inhibition tests showed that hsya possessed no depression effects on activities of endothelial nitric oxide synthase and nadph cytochrome p450 reductase ( data not shown ) , only the pharmacophore model of xo was exhibited in figure 2 . to confirm
if there is a direct interaction between hsya and xo , we detected the inhibitory effect of hsya on xo activity both out of cells and within cells .
febuxostat was used as positive control . because hsya liquor appears yellow , we employed fluorescence detection , other than od detection , to reduce error .
results showed that hsya exhibited remarkable inhibitory effect on xo activity with ic50 = 40.04  m out of cells ( figure 3(a ) ) .
the ic50 of positive control febuxostat was 3.24  m ( data not shown ) .
in addition , as shown in figure 3(b ) , 50100  m hsya treatment started to exhibit suppression on the activity of xo in lps - stimulated macrophages ( p < 0.05 ) .
however , results demonstrated that xo treatment had no noticeable action on the protein and mrna expression of xo in macrophages ( p > 0.05 ) ( figure 4 ) .
since we confirmed that hsya inhibits the activity of xo , we tried to further understand the interaction between hsya and xo by computation docking . from the molecular docking data , we concluded hsya directly implanted into the activity pocket of xo by hydrophobic characteristic and hydrogen bonds , which are essential to this interacting system with a binding energy of 5.77  kcal / m ( table 1 ) . in this hsya - xo complex , hsya was surrounded by leu 648 , leu 712 , his 875 , leu 873 , ser 876 , glu 879 , phe 649 , and asn 650 in hydrophobic interactions ( figures 5(a ) and 5(b ) ) .
moreover , glu 879 , asn 650 , and his 875 were observed to form hydrogen bonds with the hydroxyl groups of hsya ( figure 5(c ) ) . as a main source of ros , xo in macrophages
if xo inhibition by hsya results in less ros production , we evaluated the ros level via fluorescence detection .
as shown in figure 6 , lps challenge markedly increased the amount of ros in macrophages ( p < 0.01 ) ; however , 100  m hsya pretreatment for 3  h significantly suppressed lps - induced ros generation ( p < 0.05 ) , with no remarkable difference compared with 30  m positive control febuxostat ( p > 0.05 ) . since latest research demonstrated that activation of nlrp3 inflammasome requires the ros generated by xo , we further detected the effect of hsya on activation of nlrp3 inflammasome .
as shown in figure 7 , 1  g / ml lps challenge significantly enhanced the expression of nlrp3 , asc , and procaspase-1 proteins ( p < 0.01 ) , indicating that nlrp3 inflammasome was activated by lps in macrophages .
hsya treatment from 50 to 100  m notably inhibited the expression of nlrp3 ( p < 0.05 ) ; however , no significant regulatory effects were observed on expression of asc and procaspase-1 ( p > 0.05 ) .
result showed that hsya treatment from 50100  m exhibited remarked inhibitory effect on the mature caspase-1 expression in macrophages ( p < 0.05 ) . to further estimate the inhibition of hsya on nlrp3 inflammasome activation
as shown in figure 8(a ) , hsya treatment at concentrations from 50 to 100  m notably decreased the protein expression of il-1 in a dose - dependent manner ( p < 0.05 ) .
results based on western blot also displayed that 100  m hsya possessed an observable negative regulatory effect on pro - il-1 expression , but with no significant difference compared with lps group .
moreover , data in figure 8(b ) showed that , as expected , hsya treatment at concentrations from 50 to 100  m markedly decreased the secretion level of il-1 and il-18 in supernatant ( p < 0.05 ) .
xo is one of the major enzymatic sources of ros by utilizing molecular oxygen as a substrate to break down xanthine and hypoxanthine into o2 and hydrogen peroxide .
it has been well - documented that the activity of xo can be enhanced by kinds of stimuli including lps and cytokines in macrophages , resulting in many pathologic conditions characterized by oxidative stress and inflammation [ 21 , 22 ] .
inhibition of xo by pharmacological inhibitors such as febuxostat and allopurinol markedly decreases xo - induced excessive ros generation and betters the pathologic conditions , suggesting the essential role of xo in inflammation and providing potential strategy of therapies to cure inflammation diseases [ 14 , 23 , 24 ] . by employing pharmmapper , a server computationally predicting candidates of submitted small molecule , drug , and compounds using inverse docking technique
, we found that endothelial nitric oxide synthase , nadph cytochrome p450 reductase , and xo may be potential disease - related targets of hsya .
however , data from subsequent inhibitory tests showed that hsya only possessed promising regulatory effect on xo instead of endothelial nitric oxide synthase and nadph cytochrome p450 reductase .
the pharmacophore outcome disposed to the molecule characteristics of hsya , which convincingly supports the inverse docking result provided by pharmmapper . to better understand the chemical - protein combination information of hsya and xo , we employed autodock and autogrid to investigate the interaction of hsya - xo complex .
results demonstrated that hsya embedded into the active region of xo via hydrophobic interaction , a critical binding force for complex stabilization , with many amino acids including leu 648 , leu 712 , his 875 , leu 873 , ser 876 , glu 879 , phe 649 , and asn 650 .
besides , the hydrogen bond interactions between hsya and xo at residues such as glu 879 , asn 650 , and his 875 also contribute to the combination and endow the inhibitory effect of hsya on xo activity .
since we proved that hsya directly inhibits the activity of xo , we tried to further investigate whether hsya affects the expression of xo and leads to changes in downstream signaling pathways .
however , results based on rt - pcr and western blot demonstrated that hsya possesses limited regulatory effect on lps - induced overexpression of xo at mrna and protein level , suggesting that hsya exhibits subsequent regulating properties through inhibiting the activity but not amount of xo in lps - challenged macrophages .
ros overproduction triggered by phagocytes is critical for the cellular signaling cascades in macrophages during immune activation [ 2729 ] .
since it has been well - documented that xo plays an important role in ros generation , we then detected the effect of hsya on lps - induced ros production in raw264.7 macrophages .
results indicated that 100  m hsya pretreatment notably suppressed lps - induced excessive ros generation , with no significant difference compared with 30  m xo inhibitor febuxostat . knowing that hsya owns considerable antioxidant property [ 30 , 31 ] , our findings may partially uncover the mechanism of which hsya decreases ros generation . since it has been proved that xo - induced ros generation is the dominant trigger of nlrp3 inflammasome , we further investigated the effect of hsya on nlrp3 inflammasome activation .
nlr family , well - established prr , is critical for promoting inflammation process in host innate immune response when challenged by infectious stimuli . among these inflammasomes , nlrp3 has been characterized in most mammalian cells .
studies in macrophages and various animal models demonstrated that the activation of nlrp3 inflammasome can be observed in kinds of autoimmune and inflammatory diseases such as experimental autoimmune encephalomyelitis ( eae ) , multiple sclerosis , inflammatory bowel disease including ulcerative colitis , and crohn 's disease [ 35 , 36 ] .
moreover , nlrp3 inflammasome activation may contribute to insulin resistance and type ii diabetes , uric acid accumulation , and gout .
mechanically , when stimulated by prrs , nlrp3 inflammasome activates and then promotes the recruitment of asc and the procaspase-1 , resulting in the formation of caspase-1 p10 fragment which contributes to active caspase-1 enzyme that cleaves pro - il-1 into mature il-1 form .
results based on western blot illustrated that hsya treatment notably decreased the cleaved caspase-1 expression , indicating that hsya could suppress lps - induced activation of nlrp3 inflammasome . to better understand the regulatory effect of hsya on nlrp3 inflammasome , we detected the protein expression of nlrp3 , asc , and procaspase-1 complex .
results revealed that hsya notably inhibited lps - induced nlrp3 expression ; however , no noticeable suppressions were observed on the upregulated expression of asc and procaspase-1 .
a more recent study in human and mice macrophages demonstrated that ros generation caused by xo , instead of nox , is the major source that promotes nlrp3 inflammasome activation . taken together , we conferred that hsya treatment restrained the activation of nlrp3 inflammasome via directly binding to xo and inhibiting the ros overproduction . il-1 and il-18 are two well - documented proinflammatory cytokines that play central role in inflammation and autoinflammatory disorders .
it has been established that secretion of il-1 and il-18 needs the participation of nlrp3 inflammasome [ 39 , 40 ] .
hence , we measured the protein expression of pro - il-1 and il-1 in macrophages and the secretion of il-1 and il-18 in the supernatant .
as expected , hsya pretreatment notably decreased lps - induced cleaved il-1 expression , indicating that hsya inhibited the cleaving process of il-1 mainly by suppressing the nlrp3 inflammasome activation .
meanwhile , mild abatement of pro - il-1 expression was observed under hsya pretreatment . considering the important role of ros generation in the activation of nf-b , a classical nuclear transcription factor that encodes kinds of proinflammatory cytokines including il-1
, we speculated that ros scavenging by hsya partially depressed the activation of nf-b and led to lower pro - il-1 protein expression .
however , the inhibition of nlrp3 eventually blocked the transformation process from pro - il-1 to cleaved il-1. detection on secretion of il-1 and il-18 by elisa further proved that hsya inhibited nlrp3 inflammasome activation and finally suppressed its downstream cytokines secretion .
but , since we did not find the suitable antibodies for pro - il-18 and il-18 detection , we could not prove whether the downregulation of il-18 was caused by blocking mature process .
taken together , we found that xo is a potential target of hsya using pharmmapper inverse docking and computer simulation .
the inhibitory effect of hsya on xo activity contributes to the ros scavenging and nlrp3 inflammation suppression triggered by lps , leading to decreased il-1 and il-18 secretion in raw264.7 macrophages ( figure 9 ) .
 hydroxysafflor yellow a ( hsya ) is an effective therapeutic agent for inflammatory diseases and autoimmune disorders ; however , its regulatory effect on nlrp3 inflammasome activation in macrophages has not been investigated . in this study , we predicted the potential interaction between hsya and xanthine oxidase ( xo ) via pharmmapper inverse docking and confirmed the binding inhibition via inhibitory test ( ic50 = 40.04  m ) . 
 computation docking illustrated that , in this hsya - xo complex , hsya was surrounded by leu 648 , leu 712 , his 875 , leu 873 , ser 876 , glu 879 , phe 649 , and asn 650 with a binding energy of 5.77  kcal / m and formed hydrogen bonds with the hydroxyl groups of hsya at glu 879 , asn 650 , and his 875 . 
 we then found that hsya significantly decreased the activity of xo in raw264.7 macrophages and suppressed lps - induced ros generation . 
 moreover , we proved that hsya markedly inhibited lps - induced cleaved caspase-1 activation via suppressing the sensitization of nlrp3 inflammasome and prevented the mature il-1 formation from pro - il-1 form . 
 these findings suggest that xo may be a potential target of hsya via direct binding inhibition and the combination of hsya - xo suppresses lps - induced ros generation , contributing to the depression of nlrp3 inflammasome and inhibition of il-1 secretion in macrophages . 