innate immune responses provide four anatomical , physiological , phagocytic , and inflammatory barriers . among these barriers ,
macrophages produce proinflammatory cytokines and other signaling molecules such as interleukins ( ils ) , tumor necrosis factor ( tnf)- , and prostaglandins ( pgs ) , all of which are essential for the initial inflammatory response .
thus , the initial activation of macrophages is a significant event in inflammatory processes . to activate the inflammatory response , macrophage surface receptors such as toll - like receptors ( tlrs ) interact with their cognate ligands . activated macrophages consequently upregulate intracellular signaling pathways to activate the nuclear factor ( nf)-b pathway and the activator protein- ( ap- ) 1 pathway , leading to the expression of inflammatory genes [ 46 ] . to cure inflammatory diseases , effective regulation of the inflammatory response
therefore , targeted inhibition of intracellular inflammatory - related signaling pathways has been considered to be a promising therapeutic strategy for treating various immunological diseases such as cancer , septic shock , diabetes , and atherosclerosis [ 711 ]
biological hydrogels consisting of self - assembling peptide nanofibers have been shown to have a broad range of potential applications [ 1215 ] .
self - assembling peptides consist of short peptides with 8 to 16 residues that are 2.5 to 5  nm in length , depending on conditions such as ph and ionic strength .
these structural features have made self - assembling systems an attractive option for tissue culture - based and tissue - based research into the mechanisms that control various cellular processes .
although recent studies have indicated that self - assembling peptides have potential applications in drug delivery as a nanomedicine platform , the molecular mechanisms underlying these applications are not yet fully understood . in our previous study , we determined the immune - regulatory functions of a self - assembling peptide , k5 , radaradaradarada - kkkkk . in the present study
, we investigated another peptide named s5 , radaradaradarada - sssss , with 5 serine resides , which was developed to investigate polarity effects on self - assembling property , plasma protein interactions , and platelet activation .
while the k5 peptide exhibits lysine residue - driven net positive charge at a ph of 7.4 , disadvantageous in terms of membrane permeability , s5 peptide only maintains free hydroxyl group .
considering chemical property of s5 , it is hypothesized that the penetration of s5 into cytoplasmic compartment in a cell might be better than k5 . in this study , we aimed to demonstrate the immunopharmacological improvement of the s5 peptide in cellular and molecular inflammatory responses .
tetrazole ( 3 - 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , prednisolone and indomethacin , and lipopolysaccharide ( lps , e. coli 0111:b4 ) were purchased from sigma chemical co. ( st . louis , mo , usa ) .
enzyme immunoassay ( eia ) kits and enzyme - linked immunosorbent assay ( elisa ) kits for determining the levels of pge2 and tnf- were purchased from thermo scientific ( nyse : tmo ) and amersham ( little chalfont , buckinghamshire , uk ) . fetal bovine serum ( fbs ) , rpmi1640 and dmem media were purchased from thermo scientific ( nyse : tmo ) .
raw264.7 and hek293 cells were obtained from atcc ( rockville , md , usa ) .
phospho- and total protein antibodies against c - jun , extracellular signal - related kinase ( erk ) , c - jun n - terminal kinase ( jnk ) , p38 , map kinase kinase 3/6 ( mkk3/6 ) , tgf- activated kinase 1 ( tak1 ) , lamin a / c , and -actin were purchased from cell signaling ( beverly , ma , usa ) and santa cruz ( santa cruz , ca , usa ) .
male icr mice ( 6 weeks old , 17 to 21  g ) were purchased from daehan biolink ( chungbuk , korea ) and housed in plastic cages under conventional conditions .
water and food pellets ( samyang , daejeon , korea ) were supplied ad libitum .
all studies were carried out in accordance with the guidelines established by the institutional animal care and use committee of sungkyunkwan university ( approval i d : skkubbi 12 - 6 - 2 ) .
bone marrow- ( bm- ) derived macrophages were obtained from icr male mice ( 6 weeks old , 17 to 21  g ) by severing the bone of the femoral region .
bone marrow - derived macrophages were flushed out into petri dishes with pbs . after harvesting bone marrow cells by centrifugation at 1,200  rpm for 5 minutes at 4c
finally , bone marrow - derived macrophages ( 1  10 cells / ml ) were seeded in 100  mm tissue culture dishes and grown for 4  h at 37c in a humidified atmosphere with 5% co2 .
primary cells ( bone marrow - derived macrophages ) and cell lines ( raw264.7 and hek293 cells ) were cultured with rpmi1640 medium supplemented with 10% heat - inactivated fbs , glutamine , penicillin , and streptomycin at 37c under 5% co2 .
raw264.7 cells and bone marrow - derived macrophages ( 1  10 cells / ml ) were preincubated for 18  h , after which cells were treated with the s5 peptide ( 0 to 100  g / ml ) or standard compounds ( prednisolone and indomethacin ) for 30  min and then additionally treated with lps ( 1  g / ml ) for 24  h. production and the release of pge2 and tnf- were determined by eia and elisa kits , as described previously .
bone marrow - derived macrophages or raw264.7 cells ( 1  10 cells / ml ) were treated with s5 peptide ( 0 to 100  g / ml ) for 24  h. cytotoxic effect of s5 peptide was evaluated by conventional mtt assay as described previously [ 21 , 22 ] . to evaluate inflammatory gene mrna expression levels , raw264.7 cells pretreated with s5 peptide ( 0 to 100  g / ml ) for 30  min were incubated with lps ( 1  g / ml ) for 6  h. total rna from the cells
was then isolated with trizol reagent ( gibco brl ) according to the manufacturer 's instructions and stored at 70c until use .
the levels of il-1 , tnf- , and cox-2 mrna expression levels were quantified by real - time reverse transcriptase - polymerase chain reaction ( rt - pcr ) with sybr premix ex taq according to the manufacturer 's instructions ( takara , japan ) .
reactions were run in a real - time thermal cycler ( bio - rad , usa ) , as reported previously [ 23 , 24 ] .
results are expressed as relative ratios of gene expression normalized to the internal control gapdh .
primers were ordered from bioneer ( seoul , korea ) and their sequences are listed in table 1 .
preparation of total lysates and nuclear extracts from lps - treated raw264.7 cells pretreated with s5 peptide was used , a method previously published .
immunoblotting analysis of phosphorylated and total protein [ c - jun , lamin a / c , mapk ( erk , p38 , and jnk ) , mkk3/6 , mek1/2 , mkk4/7 , mkk4 , tak1 , lamin a / c , and -actin ] levels was performed , according to published methods . for immunoprecipitations ,
cell lysates were generated from raw264.7 cells ( 1  10 cells / ml ) that had been treated either with or without lps ( 1  g / ml ) for 90  min .
lysates containing equal amounts of protein ( 500  g ) were precleared with 10  l protein a - coupled sepharose beads ( 50% v / v ) ( amersham , uk ) for 1  h at 4c .
immune complexes were captured with 10  l protein a - coupled sepharose beads ( 50% v / v ) by rotation for 3  h at 4c .
data ( figures 1 and 2 ) are expressed as means  standard deviations ( sds ) and were calculated from one of two independent experiments .
the data shown in this paper are representative of three independent experiments , each of which yielded similar results . for statistical comparisons ,
results were analyzed using analysis of variance / scheffe 's post hoc test and the kruskal - wallis / mann - whitney tests .
we previously reported that the self - assembling k5 peptide , applied as 3d scaffolds for tissue engineering or drug delivery [ 18 , 27 ] , downregulates the macrophage - mediated inflammatory responses .
the s5 peptide is part of a family of self - assembling peptides that includes the k5 peptide .
however , in contrast to the k5 peptide , the s5 peptide lacks positive charge in this backbone .
since these two peptides exhibit reasonably similar structures with only a minor difference , we hypothesized that the s5 peptide would also exert anti - inflammatory effects .
similar to the pharmacological profiles of k5 peptide , the s5 peptide appears to be a promising therapeutic molecule for dampening the inflammatory response .
for instance , the s5 peptide decreased the production of pge2 ( figure 1(a ) ) and tnf- ( figure 1(b ) ) in both malignant raw264.7 cells and in bm - derived lps - activated macrophages in a dose - dependent manner as seen in the cases of standard compounds prednisolone and indomethacin ( figure 1(c ) ) ; importantly , no cytotoxicity was observed in these cells ( figure 1(d ) ) .
these data raised a possibility that s5 peptide can be a peptide drug with anti - inflammatory property as previously suggested with synthetic tip - like peptide ap318 , peptides from bovine -casein , cathelicidin , and n15p polypeptide [ 2931 ] .
since elucidating the molecular mechanisms of these effects is important for initiation of drug discovery process , we next examined the anti - inflammatory mechanism of the s5 peptide . to examine the effects of the s5 peptide on the inflammatory response on the transcriptional level , the mrna levels of cox-2 ( figure 2(a ) ) , tnf- ( figure 2(b ) ) , and il-1 ( figure 2(c ) )
were measured by real - time pcr in cells that had been stimulated with lps .
these experiments showed that the s5 peptide ( 100  g / ml ) downregulated the expression of cox-2 , tnf- , and il-1. to investigate whether the s5 peptide regulates dna promoter activity , a luciferase reporter assay was used that resulted in cell luminescence .
the s5 peptide remarkably decreased ap-1-mediated transcriptional activation in response to pma , myd88 , or trif ; moreover , this effect was dose - dependent ( figures 3(a ) , 3(b ) , and 3(c ) ) .
activation of the inflammatory response is known to result in nuclear translocation of the ap-1 transcription factor , which results in transcription of ap-1-driven target genes . to examine the nuclear translocation of c - jun in the inflammatory response
, we determined the levels of c - jun in the nuclear fractions of lps - treated raw264.7 cells .
interestingly , these experiments showed that the s5 peptide prevented the nuclear translocation of c - jun at 60 and 90  min ( figure 3(d ) ) .
this result implies that the s5 peptide may regulate the ap-1 signaling pathway . to investigate the activation status of upstream signaling molecules in the ap-1 pathway , the phosphorylation of various mapks ( erk , p38 , and jnk ) was analyzed by western blotting .
the phosphorylation of p38 and jnk was decreased at 30 and 15  min , while erk phosphorylation was not decreased by the s5 peptide ( figure 4(a ) ) .
interestingly , the s5 peptide only prevented phosphorylation of mkk3/6 , an upstream kinase of p38 , whereas the s5 peptide did not prevent the phosphorylation of the upstream molecule jnk , which is downstream of mkk4/7 .
phosphorylation of tak1 , a kinase that is upstream of the mkks , was also decreased by the s5 peptide ( figure 4(b ) ) . to determine whether the effects of the s5 peptide were recapitulated in primary cells , peritoneal macrophages were harvested from icr mice and the phosphorylation status of various signaling molecules was investigated by western blotting .
this experiment revealed that the s5 peptide decreased the phosphorylation of c - jun , p38 , and mkk3/6 in lps - driven signaling ( figure 4(c ) ) .
moreover , the s5 peptide also dramatically blocked the interaction of phospho - p38 with c - jun and its phosphoform , as revealed by immunoprecipitation and immunoblotting analyses ( figure 4(d ) ) .
based on these results , we conclude that the s5 peptide is capable of preventing ap-1 signaling events .
some of strong anti - inflammatory remedies such as medicinal plant - derived extracts including dryopteris crassirhizoma and archidendron clypearia and their individual components including andrographolide , ginsenosides , curcumol , and lutein are known to suppress ap-1 function in their pharmacological actions [ 3438 ] , suggesting that ap-1 could play a critical role in inflammation - regulatory functions . due to their size and charge ,
most hydrophobic small chemicals are much assessable to be penetrated into the membrane and display higher possibility to directly interact with cytosolic target enzymes . however , chemical drugs showing effective inhibition property toward important signaling enzymes are faced to have another side effect causing nonspecific toxicity . in this respect ,
peptide drugs are considered to be favorable in terms of drug development , due to their selectivity .
previously , we reported that peptide k5 is able to suppress tnf- production in lps - treated raw264.7 cells up to 48% at 100  g / ml . on the other hand , chemically modified s5 peptide having free oh groups exhibited stronger activity ( 80% inhibition at 100  g / ml ) under the same conditions ( figure 1(b ) ) , implying that membrane permeability of peptide s5 could be more improved than k5 . nonetheless , the fact that k5 was revealed to block the release of tnf- and pge2 between 50 to 100  g / ml   is still raising a possibility that some of peptide receptor such as pept1 , oatp2ba , or oarp1b3 [ 39 , 40 ] could contribute to the uptake of peptides k5 and s5 into cytoplasm .
the point that s5 peptide exhibits higher anti - inflammatory property under improved membrane permeability seems to impose s5 peptide to be developed as anti - inflammatory peptide drug .
indeed , some peptides such as glp and its analogs , extracellular matrix protein - derived peptides , and integrin - binding n - terminal peptides   are closed to be developed as antitumorigenic remedy , although the therapeutic use of these peptides is still limited due to high cost , poor stability , and mediocre pharmacokinetics . in cases of the s5 and k5 peptides ,
these are known to be self - assembling nanopeptides forming a hydrogel applied for drug delivery system and have been shown to be nonimmunogenic and noninvasive in the filling of cavities [ 15 , 18 , 43 ] . according to our results
, these peptides are found to have anti - inflammatory properties which can give additional benefits when they are used as carrier to deliver immunomodulatory drugs .
since the s5 peptide itself suppresses events in the tlr4 signaling pathway , including activation of mapk and ap-1 , it may exert a synergetic effect if carrier molecules are nf-b - targeted anti - inflammatory drugs . to sum up these results
, here we showed that the s5 peptide dampens various components of the tlr4-mediated inflammatory response , such as the production of pge2 and tnf-. in addition , the s5 peptide reduces ap-1 signaling by preventing the phosphorylation of mkk3/6 and tak1 , as summarized in figure 5 . since the s5 peptide is a self - assembling peptide that could be used for drug delivery , it is possible that the s5 peptide could be loaded with therapeutics that target ap-1 or other compounds that synergize with inflammatory molecules .
future studies will test the efficacy of the s5 peptide as a drug carrier and as an anti - inflammatory agent in an animal model .
these experiments have the potential to contribute to the development of an efficacious and novel drug delivery system .
 peptide - based therapeutics have received increasing attention in medical research . 
 however , the local delivery of such therapeutics poses unique challenges . 
 self - assembling peptides that use decorated nanofibers are one approach by which these therapeutics may be delivered . 
 we previously found that the self - assembling k5 peptide affects the anti - inflammatory response . 
 the aim of the present study was to investigate another self - assembling peptide , s5 . 
 unlike the k5 peptide which has a positive charge , the s5 peptide has a free hydroxyl ( -oh ) group . 
 we first examined whether the s5 peptide regulates the inflammatory response in primary cells and found that the s5 peptide reduced the production of prostaglandin e2 ( pge2 ) and tumor necrosis factor ( tnf)- in lipopolysaccharide- ( lps- ) treated bone marrow - derived macrophages . 
 moreover , the s5 peptide significantly downregulated cyclooxygenase- ( cox- ) 2 , tnf- , and interleukin- ( il- ) 1 expression by blocking the nuclear translocation of c - jun . 
 consistent with this finding , the s5 peptide diminished the activation of inflammatory signaling enzymes related to p38 . 
 the s5 peptide also inhibited the formation of the p38/c - jun signaling complex in raw264.7 cells . 
 similarly , p38 and mkk3/6 were inhibited by the s5 peptide in lps - activated peritoneal macrophages . taken together 
 , these results strongly suggest that the s5 peptide could exert anti - inflammatory effects by inhibiting the c - jun / p38 signaling pathway . 
1. Introduction
2. Materials and Methods
3. Results and Discussion