dentin pulp complex injuries are often induced by invasion of microorganisms and their components via dentinal tubules towards the pulp . caries , cracks , fractures , and leakage from restorations provide pathways for microorganisms and their toxins to enter the pulp . the inflammation can spread to the surrounding alveolar bone and cause periapical pathosis . in this process , bacterial lipopolysaccharides ( lpss ) play a potential role in several responses to pulpal infection . lipopolysaccharide ( lps ) can induce the expression of proinflammatory cytokines and chemokines such as tnf- and il-6 and elicit the innate immune response in dental pulp cells ( dpcs ) . signaling pathways initiated by engagement of toll - like receptors ( tlrs ) , such as tlr2 and tlr4 , by bacterial products lead to enhanced transcription of genes responsible for the expression of cytokines , chemokines , adhesion molecules , and other mediators of the inflammatory response associated with bacterial infection . of note , the activation of mitogen - activated protein kinases ( mapks ) is important in the production of inflammatory cytokines by lps stimulation . the mapk family includes extracellular - signal - related protein kinase ( erk ) , c - jun n - terminal kinase / stress - activated protein kinases ( jnk / sap ) and p38mapk . the mapk signaling pathway is involved in various kinds of cellular processes including differentiation , development , proliferation , and survival , as well as cell death , depending on cell type and stimulus [ 5 , 6 ] . pulpal p38mapk signaling is activated by lps stimulation during the induction of local proinflammatory response [ 79 ] . telomeres are specialized structures at the ends of chromosomes that have a role in protecting the chromosome ends from dna repair and degradation . telomerase is a cellular reverse transcriptase ( tert , telomerase reverse transcriptase ) which prevents premature telomere attrition and maintains normal length and function . human reverse transcriptase subunit of telomerase ( htert ) has become an attractive target for cancer vaccines due to it being expressed in 8590% of human cancer tissues , whereas it is almost never expressed in normal tissues . gv1001 peptide , which is a peptide corresponding to amino acids 611626 of htert ( earpalltsrlrfipk ) , has been developed as a vaccine against various cancers and has been reported to have the ability to penetrate into various cells , including cancer cell lines and primary blood cells . gv1001 was found to localize predominantly in the cytoplasm and could successfully deliver macromolecules such as proteins , dna , and sirna into cells . because of this novel pharmaceutical potential and cell - penetrating ability , as well as its own anticancer activity , gv1001 peptide is very promising for use in the medical field . here , we observed that this peptide could also penetrate into human dental pulp stem cells and , furthermore , that it had a self - anti - inflammatory effect without affecting cell viability . the purpose of this study was to evaluate the cell - penetrating function of gv1001 peptide in human dental pulp cells ( hdpc ) and to investigate the anti - inflammatory effect of gv1001 and its related mechanism in p. gingivalis lps - induced inflammation through regression of inflammatory cytokine production . all of the peptides used in this study were synthesized by the fmoc- ( 9-fluorenylmethoxycarbonyl- ) based solid - phase method and characterized by peptron inc . the purities of all peptides used in this study were greater than 95% , as determined by high - performance liquid chromatography . the impacted third molars of human adults were collected from 18- to 22-year - old patients after obtaining informed consent . the isolated dental pulp was cut into small pieces and digested in a solution of 3  mg  ml type i collagenase and 4  mg  ml dispase for 3060  min at 37c ( sigma aldrich , st . subsequently , the solution was filtered through a 70  mm cell strainer ( becton / dickinson , franklin lakes , nj , usa ) . the single - cell suspensions were seeded in 35 or 60  mm culture dishes and maintained in a culture media consisting of -minimum essential medium ( -mem ; invitrogen , carlsbad , ca , usa ) supplemented with 15% fetal bovine serum ( fbs ; gibco - brl , life technologies inc . , gaithersburg , md , usa ) , 0.292  mg  ml glutamine ( invitrogen ) , 100 units ml penicillin g , 100  mg  ml streptomycin , and 50  mg  ml ascorbic acid ( sigma ) . the cells were incubated in a humidified atmosphere containing 5% co2 at 37c and cells between 3 and 4 passages were used in the following experiments . porphyromonas gingivalis atcc 33277 was purchased from the american type culture collection ( atcc ; manassas , va ) and cultured with brain heart infusion broth ( bhi ; bd bioscience , sparks , md , usa ) supplemented with hemin ( 1  g  ml ) and vitamin k ( 0.2  g  ml ) in anaerobic conditions at 37c . lipopolysaccharide was extracted from p. gingivalis cultured according to the method described by lee et al . . hdpcs were seeded and cultivated in 2-chamber glass slides ( nunc , roskilde , denmark ) for 12  h. after washing with pbs , cells were incubated in serum - free opti - mem for an hour . fluorescein isothiocyanate- ( fitc- ) labeled 1 , 10 , and 50  m of gv1001 peptides were added to cells and incubated for 2  h. the cells were fixed with 4% paraformaldehyde solution for 20  min at room temperature . cells were stained with to - pro-3 iodide 642/661  nm ( invitrogen , grand island , ny , usa ) to visualize nuclei and were subjected to confocal microscopy . colocalization of the peptides and nuclei was assessed using an fv1000 lager scanning confocal microscope ( olympus ) . after addition of 0.75  m10  m gv1001 to lps - treated cells , the samples were prepared for electrophoresis and were separated using a 10% sodium dodecyl sulfate - polyacrylamide ( sds - page ) gel with a previously established buffer system . after electrophoresis , the proteins were transferred onto a nitrocellulose membrane and blocked by tris - buffered saline ( tbst , 0.05% tween-20 ) containing 5% nonfat dried milk . the membranes were then incubated with anti - p - erk and anti - p - p38 ( cell signaling technology , beverly , ma , usa ) antibodies and subsequently washed with tbst . antigen - antibody complexes were visualized using an enhanced chemiluminescent detection system ( west - zol , intron , seoul , korea ) by incubating membranes with goat anti - rabbit igg or goat anti - mouse igg antibody coupled to horseradish peroxidase ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) , diluted at 1  :  2000 . the blots were stripped and reprobed with an anti--actin polyclonal antibody to ensure that equal amounts of protein were used . after treatment with 0.7520  g  ml lps for 2 , 4 , 6 , and 8  h , total rna was isolated from the cells using trizol reagent ( life technologies inc . , gaithersburg , md , usa ) according to the manufacturer 's suggested protocol and treated with dnase i ( rnase - free , rq1 ; promega , madison , wi , usa ) . one microgram of total rna was used as a template to create first - strand cdna with oligo - dt priming using an omniscript rt kit ( qiagen inc . , the quantitative real - time pcr analyses were performed using an abi prism 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) with a sybr premix ex taq ii kit ( takara , otsu , japan ) and 35 cycles of pcr . the denaturing , annealing , and extension conditions of each pcr cycle were 95c for 10  s , 60c for 15  s , and 72c for 10  s , respectively . the relative amount or fold change of the target gene was normalized relative to the level of the control ( untreated cells ) . the following primer sequences were used in the real - time pcr reactions : 5-cga aag tca act cca tct gcc-3 and 5-ggc aac tgg ctg gaa gtc tct-3 for il-6 gene ; 5-cca gga gaa agt cag cct cct-3 and 5-tca tac cag ggc ttg agc tca-3 for tnf- gene ; 5-gtg gtg gac ctg acc tgc-3 and 5-tga gct tga caa agt ggt cg-3 for gapdh gene . to evaluate the cytotoxicity of gv1001 on hdpcs , cells ( 2  10/well ) were treated with several concentrations of gv1001 ( 0 , 1 , 5 , 10 , and 50  m / well ) for 48 hours . mol  l of mtt solution for 4  h in a tissue - culture incubator . a 200-ll quantity of dimethyl sulfoxide solution was then added to the cell - culture wells , and the plates were shaken for 10  min at room temperature to dissolve the precipitated formazan crystals . the solution was centrifuged for 10  min , and the optical density of the supernatant was measured at wavelength of 540  nm using an elisa plate reader ( powerwave x 340 ; biotek instruments , vt ) . the data are expressed as the mean  standard deviation ( sd ) of at least 3 separate experiments . to confirm the cell - penetrating activity of gv1001 , fitc was conjugated to the c - terminus of 1 , 10 , and 50  m gv1001 peptides ( gv1001-f ) and subjected to an internalization assay using confocal microscopy . as shown in figure 1 , gv1001-f showed mostly cytoplasmic distribution in hdpcs in various levels of peptide concentration . to verify the effects of various doses of lps on inflammatory cytokine expression in hdpcs , the levels of tnf- and il-6 expression were evaluated using real - time pcr after exposing hdpc cells to lps of p. gingivalis or 1  g ml of e. coli at concentrations of 1 , 5 , 10 , or 20  g  ml for 10  hrs . the results showed that il-6 transcript was profoundly induced in the presence of 1 , 5 , 10 , or 20  g ml of p. gingivalis lps or 1  g  ml of e. coli lps ( p < 0.05 ) , and tnf- transcript was profoundly induced in the presence of 10 or 20  g ml of p. gingivalis or 1  g  ml of e. coli lps ( p < 0.05 ) ( figure 2(a ) ) . to evaluate the effects of lps on inflammatory cytokine expression in hdpcs over a period of time , the levels of tnf- and il-6 expression were evaluated using real - time pcr after exposing hdpcs to 20  g  ml of p. gingivalis lps or 1  g  ml of e. coli lps for 2 , 4 , 6 , or 8  hrs . upregulation was found in hdpcs after 4 to 8  hrs of exposure to 20  g  ml of p. gingivalis lps or 1  g  ml of e. coli lps ( p < 0.05 ) ( figure 2(b ) ) . the expression of tnf- and il-6 in hdpcs were upregulated by lps in a time- and dose - dependent manner . we measured tnf- and il-6 expression after lps stimulation to determine the anti - inflammatory activity of gv1001 peptide by real - time pcr . lps - stimulated tnf- and il-6 production was significantly inhibited ( p < 0.05 ) ( figures 3(a ) and 4(a ) ) . to investigate whether the anti - inflammatory effect of gv1001 is mediated by the suppression of erk and p38mapk activation , regulation of phosphorylated erk and phosphorylated p38mapk was evaluated by western blot analysis . as shown in figures 3(b ) and 4(b ) , gv1001 decreased lps - induced phosphorylation of erk and p38mapk . incubating gv1001 peptide with hdpcs for 48  h did not affect cell viability in all concentrations tested ( 050  m ) , which showed a pattern similar to that seen with 0.9% nacl ( p < 0.05 ) ( figure 5 ) . although endodontically treated teeth can maintain their function for a prolonged period of time , there are many advantages to maintaining pulp vitality . in immature permanent teeth with incomplete apical and dentinal wall development , reparative dentin formation is critical for further development of the teeth . maintaining the vital pulp also has the benefit of reducing the occurrence of apical periodontitis by blocking bacterial infections . based on these advantages , it is preferable to maintain or renew pulp vitality rather than use current endodontic therapies . recently , successful pulp regeneration and revascularization techniques have been developed and are gaining popularity . p. gingivalis is an obligately anaerobic , gram - negative bacterium that has been positively associated with destructive periodontal disease [ 17 , 18 ] . it is one of the most pathogenic species among black - pigmented gram - negative anaerobes ; it is frequently present in root canal infections and other odontogenic abscesses of patients not suffering from periodontal disease , which confirms its pathogenic role . p. gingivalis may also play a role in symptomatic infections , such as acute apical abscess [ 21 , 22 ] . furthermore , there is a positive association between porphyromonas spp . and pain , mechanical allodynia , swelling , and purulent exudates in root canals [ 22 , 23 ] . pathogens are generally detected by specific receptors , one of which is the toll - like receptor ( tlr ) family . tlr stimulation initiates host defense through the activation of several intracellular signaling pathways , including activation of the mitogen - activated protein ( map ) kinases . exposure of cells to inflammatory stimuli , including lps and proinflammatory cytokines , results in phosphorylation of p38mapk . once the tlr is stimulated by a pathogen , proinflammatory cytokines and chemokines are produced by the odontoblast , resulting in recruitment and stimulation of immune effector cells and also direct bacterial killing . although these biological responses protect the host against invading pathogens , the inflammatory response also leads to host tissue damage . the extra - telomeric functions of htert are suggested regarding cellular proliferation , stem cell mobilization , and antiapoptotic , antiaging , and antioxidant effects through mitochondrial stabilization , transcriptional regulation [ 26 , 27 ] . if htert peptide treatment that mimics the extra - telomeric functions of htert could be developed , it could be used in the treatment of pulpal diseases . in this study , the potential use of a small peptide of human origin to control pulpal inflammation as a therapeutic agent was investigated . the anti - inflammatory effect of gv1001 is achieved by modulating the suppression of the activation of erk and p38 mapk and the subsequent cytokine production induced by p. gingivalis lps stimulation . p. gingivalis and e. coli lpss induced tnf- and il-6 expression in hdpc in a time- and dose - dependent manner , while gv1001 peptide downregulated inflammatory cytokines , il-6 and tnf-. hdpcs expressed tlr2 ( data are not shown ) and upregulated phosphorylated erk and p38 mapk in response to stimulation by lps . from the similar suppression patterns of the two , it could be assumed that the downregulation of inflammatory il-6 and tnf- by gv1001 was dependent on p38 mapk and erk - signaling , respectively , thus indicating that mapk signaling was required in downregulation of inflammatory cytokines in dental pulp stem cells by gv1001 peptide . as shown in figure 3 , higher dose of gv1001 exhibited a lower effect on tnf induced by p. gingivalis lps . one of the possible reasons is that gv1001 can partially aggregate in the high concentration in cell culture medium because cell culture medium does not include carrier protein . thus , the lower concentration of gv1001 except aggregating gv1001 penetrates into the cells and reacts with signaling molecules related with inflammation . in another previous study , gv1001 peptide was reported to have the ability to penetrate into various cells , including cancer cell lines and primary blood cells , without affecting cell viability . gv1001 was predominantly located in the cytoplasm and was used to successfully deliver macromolecules such as proteins , dna , and sirna into cells . these cell - penetrating peptides ( cpps ) have become one of the most popular and efficient tools for delivering various molecules into cells owing to the fact that they have the ability to enter cells independently of a membrane receptor , and they show no cell - type specificity . this study is significant in that it is the first to demonstrate gv1001 peptide as a cpp of human origin with a self - anti - inflammatory effect and without affecting cell viability . the use of gv1001 peptide can be a potential therapeutic approach for treating pulpal inflammation and the peptide can also be used as an intracellular delivery tool for bioactive molecules . using gv1001 peptide as a pulp - capping agent on reversibly inflamed pulp or an alternative to antibiotics in regeneration therapy may be effective . conjugating growth factors such as tgf-s and bmps with gv1001 can facilitate induction of hdpcs to differentiate effectively into odontoblast - like cells . moreover , gene therapy can be implemented using gv1001 to fuse a growth / differentiation factor for application in vital pulp therapy , regenerative endodontic fields and tissue engineering . the results from this study may support further research on the gv1001 peptide and its various clinical applications . the downregulation of lps - induced tnf- and il-6 expression was mediated through erk and p38 map kinase pathways . these findings provide mechanistic insight into the ability of gv1001 peptide to cause anti - inflammatory actions in lps - stimulated pulpal inflammation without significantly affecting cell viability .
porphyromonas gingivalis is considered with inducing pulpal inflammation and has lipopolysaccharide ( lps ) as an inflammatory stimulator . 
 gv1001 peptide has anticancer and anti - inflammation activity due to inhibiting activation of signaling molecules after penetration into the various types of cells . 
 therefore , this study examined inhibitory effect of gv1001 on dental pulp cells ( hdpcs ) stimulated by p. gingivalis lps . 
 the intracellular distribution of gv1001 was analyzed by confocal microscopy . 
 real - time rt - pcr was performed to determine the expression levels of tnf- and il-6 cytokines . 
 the role of signaling by map kinases ( erk and p38 ) was explored using western blot analysis . 
 the effect of gv1001 peptide on hdpcs viability was measured by mtt assay . 
 gv1001 was predominantly located in hdpc cytoplasm . 
 the peptide inhibited p. gingivalis lps - induced tnf- and il-6 production in hdpcs without significant cytotoxicity . 
 furthermore , gv1001 treatment markedly inhibited the phosphorylation of map kinases ( erk and p38 ) in lps - stimulated hdpcs . 
 gv1001 may prevent p. gingivalis lps - induced inflammation of apical tissue . 
 also , these findings provide mechanistic insight into how gv1001 peptide causes anti - inflammatory actions in lps - stimulated pulpitis without significantly affecting cell viability .