Introduction
Inflammation is a fundamental defense mechanism that protects the host from tissue injury and microbial invasion. However, when excessive or persistent, inflammation can damage host tissues and ultimately lead to progressive inflammatory disorders [1]. Periodontitis is one of the most common inflammatory conditions originating from the oral cavity. It is characterized by progressive destruction of periodontal tissues and alveolar bone, and its prevalence increases with age [2]. In modern societies with aging populations, the incidence of periodontitis continues to rise, making it the second most prevalent disease after the common cold [3]. Despite its high prevalence, significant impact on systemic health, and associated economic burden, current treatment of periodontal disease still largely depends on mechanical or surgical approaches, which are often insufficient to prevent recurrence [4]. Therefore, novel strategies for effective prevention and modulation of periodontal inflammation are urgently needed.
Cytokines play a pivotal role in periodontal inflammation. Among them, tumor necrosis factor (TNF)-α, interferon-γ, interleukin (IL)-1β, IL-6, and IL-18 have been identified as major mediators [5]. IL-1β, in particular, is secreted predominantly by activated monocytes and macrophages and acts on multiple cell types to amplify inflammatory responses [6]. Elevated IL-1β levels have been strongly correlated with the initiation and progression of periodontal disease [7,8]. Expression of IL-1β in the oral environment is associated with the upregulation of adhesion molecules and can induce IL-8 expression in oral epithelial cells [9]. IL-8 then recruits and activates neutrophils, which are the most abundant immune cells found in the subgingival sulcus [10]. These neutrophils migrate from surrounding capillaries in response to IL-8 and contribute to the amplification of inflammation. The IL-1β–IL-8 axis thus plays an essential role in sustaining neutrophil recruitment and driving the progression of periodontal tissue destruction [11].
Peptide-based therapeutic approaches have recently gained attention because of their high specificity, biocompatibility, and low cytotoxicity, making them promising candidates for the modulation of inflammatory signaling in periodontal disease [12,13]. Stromal cell–derived factor-1 (SDF-1), also known as CXCL12 is a chemokine that binds to the receptor CXCR4 and activates signaling pathways including PI3K, mitogenactivated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK), and JAK/TYK2 [14]. SDF-1 plays critical roles in stem cell migration, proliferation, hematopoietic cell trafficking, and angiogenesis [15]. During embryonic development, SDF- 1 guides hematopoietic cells from the fetal liver to the bone marrow, while in adulthood it recruits endothelial progenitor cells to promote angiogenesis [16]. In addition, SDF-1 has been implicated in lymphocyte chemotaxis, adult stem cell recruitment, and tissue regeneration following injury [17]. These biological properties suggest that SDF-1 not only contributes to immune regulation but may also participate in host repair mechanisms.
In the present study, we focused on the role of IL-1β–induced IL-8 expression in oral epithelial cells, which is closely linked to the initiation and progression of periodontal inflammation. We further sought to investigate the functional effects of novel SDF-1–derived peptides on modulating this pathway. Specifically, we evaluated whether these peptides could suppress IL-1β–induced IL-8 production in oral epithelial cells and thereby provide a potential therapeutic strategy for the prevention and control of periodontitis.
Materials and Methods
1. Cell culture and treatment
KB cells, a human oral epithelial cell line, were maintained in Roswell Park Memorial Institute medium (RPMI-1640; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific). Immortalized human oral keratinocytes (IHOK) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) and Ham’ s F-12 Nutrient Mixture (Thermo Fisher Scientific) mixed at a ratio of 3:1 with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. To stimulate the cells, they were seeded in multi-well plates and cultured overnight. After replacing the medium, the cells were stimulated with fresh medium containing IL-1β (5 pg/mL; PeproTech). To inhibit IL-1β–induced cell activation, cells were pretreated for 30 minutes with MAPK signaling inhibitors—pyrrolidine dithiocarbamate (Sigma- Aldrich), PD98059 (Cell Signaling Technology), SB203580 (Cell Signaling Technology), and SP600125 (Sigma-Aldrich)— or SDF-1–derived peptides synthesized by PeproTech at the indicated concentrations. Supernatants were collected for IL-8 enzyme‐linked immunosorbent assay (ELISA), and cells were harvested for western blot analysis.
2. Synthesis of SDF-1–derived peptide
SDF-1–derived peptide samples and their sequences were kindly provided by Dr. Hyung Keun Kim. The sequences were obtained from the SDF-1 isoform delta precursor [Homo sapiens] (accession: NP_001171605.1). The complete amino acid sequences of the SDF-1δ–derived peptides cannot be disclosed due to ongoing intellectual property and patent application procedures. Each peptide is composed of 15 contiguous amino acids derived from the SDF-1δ precursor using an overlapping design strategy. After initial testing with these peptide samples, recombinant proteins corresponding to the selected sequences were synthesized by PeproTech.
3. Measurement of IL-8
Quantities of IL-8 in IL-8 released to the culture medium after IL-1β stimulation were analyzed using an ELISA kit (Biolegend) according to manufacturer instructions. A standard or sample solution was added to an ELISA well plate. After incubation for 2 hours at room temperature, anti-IL-8 antibody conjugated with biotin was added to the solution and incubated for 2 hours at room temperature. Streptavidin conjugated with horseradish peroxidase (HRP) was added and allowed to react for 30 minutes. Tetramethylbenzidine substrate solution was then added and stopped with stop solution 2M H2SO4 after allowed to react for 15 minutes. Level of cytokine expression was assessed by an ELISA reader (TECAN) at 450 nm/570 nm. Each densitometric value was obtained from three independent experiments and expressed as mean ± standard deviation.
4. Western blot analysis
The cells were lysed in RIPA (radioimmunoprecipitation) buffer (Cell Signaling Technology) supplemented with complete EDTA (ethylene-diamine-tetra-acetic acid)-free protease inhibitor (Sigma Aldrich) and a phosphatase inhibitor cocktail (Genedepot). Cell lysates, containing equal amounts of protein (20 μg), were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto PVDF (polyvinylidene difluoride) membranes (Merck Millipore), which were then immunoblotted using the following specific antibodies: phospho-NF-κB (Santa Cruz Biotechnology), NF-κB (Cell Signaling Technology), phospho–c-Jun Nterminal kinase (phospho-JNK; Cell Signaling Technology), JNK (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), p38 (Cell Signaling Technology), phospho-ERK (Cell Signaling Technology), ERK (Cell Signaling Technology), and β-actin (Santa Cruz Biotechnology). The blots were then incubated with HRP-conjugated anti-mouse, anti-rabbit immunoglobulin G secondary antibodies (1:5,000). Signal detection was performed using enhanced chemiluminescence, and the resulting signals were visualized using the Super Signal West Femto maximum sensitivity substrate (Pierce) and a LAS-4000 Immuno-Image Analyzer (Fuji Film). Band intensities were quantified using Image J software (version 1.53t; National Institutes of Health) with normalized versus β-actin. In this study, n = 3 indicates that each experimental condition was tested three times independently to ensure reliability and reproducibility.
5. Statistics
Data were analyzed using a statistical software package (SPSS v.21.0; IBM Corp.). One-way analysis of variance was used for multiple comparisons and the Pearson correlation coefficient for correlation analysis between IL-1β and IL-8. p ＜ 0.05 was considered to indicate significance. Data are presented as mean ± standard deviation. Each experiment was repeated three times and carried out in triplicate or more.
Results
1. IL-1β induces IL-8 via NF-κB/MAPK signaling in oral epithelial cells
To determine whether IL-1β regulates pro-inflammatory responses in oral epithelial cells, IL-8 secretion was measured in KB and IHOK cells. KB cells treated with IL-1β exhibited significant increase in secretion of IL-8 compared with untreated controls. IL-1β–induced IL-8 production was reduced approximately 2-fold by NF-κB inhibitor, 4-fold by ERK inhibitor, 10- fold by p38 inhibitor, and 3-fold by JNK inhibitor (Fig. 1A). In IHOK cells, IL-1β stimulation also induced significant increase in secretion of IL-8. This response was suppressed by approximately 3-fold by NF-κB inhibitor, 2.5-fold by ERK inhibitor, and 2-fold by p38 inhibitor. In contrast, JNK inhibition did not produce a significant reduction, indicating that JNK signaling does not play a major role in IL-1β–induced IL-8 expression in IHOK cells (Fig. 1B).
To evaluate the time-dependent activation of NF-κB and MAPK signaling pathways, phosphorylation of NF-κB p65, ERK, JNK, and p38 was examined in KB and IHOK cells following IL-1β stimulation (Fig. 2). In KB cells, IL-1β stimulation led to a rapid and transient activation of both NF-κB and MAPKs. Phosphorylation of NF-κB p65 began to increase at 10 minutes, showing approximately a 1.1-fold enhancement, and reached its peak at 20 minutes with about a 1.5-fold elevation compared with the control. ERK phosphorylation displayed a similar pattern, increasing by roughly 1.2-fold at 10 minutes and reaching about 1.5-fold at 20 minutes before returning close to baseline at 30 minutes. Phosphorylation of p38 also peaked around 20 minutes with a comparable 1.5-fold increase, whereas JNK phosphorylation showed only minor fluctuation, remaining near basal levels throughout the 30-minute stimulation period. These results indicate that IL-1β induces a transient and coordinated activation of NF-κB, ERK, and p38 signaling pathways in KB cells, with maximal activity observed at approximately 20 minutes post-stimulation. In IHOK cells, IL-1β–induced a different activation pattern. Phosphorylation of NF-κB p65 rapidly increased at 10 minutes by approximately 1.6-fold and remained elevated up to 30 minutes, indicating sustained activation. In contrast, ERK phosphorylation rose slightly at 10 minutes (about 1.2-fold) but decreased below basal levels by 30 minutes. JNK phosphorylation exhibited a sharp but transient increase, showing about a 1.7-fold rise at 10 minutes followed by a marked reduction thereafter. Phosphorylation of p38 showed only a mild change, with less than a 1.2-fold variation during the entire time course. These results demonstrate that IL-1β activates NF-κB and MAPKs in a timedependent manner in both KB and IHOK cells, but with distinct temporal characteristics. NF-κB, ERK, and p38 are transiently activated in KB cells, whereas IHOK cells display sustained NF-κB activation and transient ERK and JNK phosphorylation, suggesting differential regulatory mechanisms between the two oral epithelial cell types.
Taken together, these findings demonstrate that IL-1β induces phosphorylation of NF-κB and MAPKs in both oral epithelial cell types, but with distinct activation kinetics. In KB cells, phosphorylation of NF-κB, ERK, and p38 occurs rapidly and transiently, corresponding to the peak of IL-8 production. In contrast, IHOK cells exhibit sustained NF-κB activation and relatively weaker, short-lived phosphorylation of ERK and JNK, suggesting a cell type–specific regulatory pattern. These differences imply that IL-1β–mediated IL-8 expression in oral epithelial cells is primarily driven by NF-κB and the ERK/p38 MAPK pathways, whereas JNK contributes minimally, particularly in IHOK cells.
2. S12 attenuates IL-1β–induced IL-8 secretion in oral epithelial cells
A library of 18 small peptides covering all isoforms of human SDF-1δ was synthesized and sequentially named S1 to S18. All peptides consisted of 15 amino acids, with the final five C-terminal residues overlapping with the N-terminus of the following peptide. The peptide concentration used in this study was chosen according to our previous study [18] showing that 1 μg/mL of SDF-1–derived peptides produced effective biological responses in oral epithelial cells. Based on the initial screening of 18 peptides (S1–S18) by ELISA, three candidate peptides (S10, S11, and S12) were selected for further investigation due to their apparent inhibitory effects on IL-1β– induced IL-8 secretion. The complete screening results for all 18 peptides are provided in Supplementary Fig. 1. An initial ELISA-based screening of all 18 peptides (S1–S18) was performed as a preliminary exploratory experiment to identify candidates showing inhibitory tendencies toward IL-1β–induced IL-8 secretion. Based on these preliminary observations, three peptides S10, S11, and S12 were selected for further evaluation. To validate these findings, KB and IHOK cells were pretreated with the selected peptides prior to IL-1β stimulation, and IL-8 production was quantified by ELISA (data not shown). In KB cells, IL-1β elevated IL-8 secretion compared with untreated controls. Pre-treatment with peptides S10, S11, and S12 all reduced IL-8 expression; however, S12 demonstrated the most significant suppression among the three peptides, indicating a stronger inhibitory capacity in KB cells (Fig. 3A). In IHOK cells, IL-1β stimulation also induced increase in IL-8 expression. S10 showed only a slight decreasing tendency that did not reach statistical significance, whereas S11 and S12 both significantly suppressed IL-8 production to a similar extent (Fig. 3B). These results suggest that, while S10, S11, and S12 all exhibited inhibitory effects on IL-1β–induced IL-8 production in KB cells, S12 showed the most inhibitory effect among the tested peptides. In IHOK cells, S11 and S12 significantly reduced IL-8 levels, whereas S10 displayed only a nonsignificant trend. Taken together, these findings indicate that S12 exerts consistent and the most inhibitory effects across both oral epithelial cell types, and therefore was selected as the primary candidate for subsequent experiments.
3. S12 inhibits phosphorylation of NF-κB and MAPKs in oral epithelial cells
To investigate the molecular mechanisms underlying the inhibitory effect of S12 on IL-1β–induced inflammation, phosphorylation of NF-κB and MAPKs was examined by western blotting in KB and IHOK cells (Fig. 4). In both cell types, IL-1β stimulation induced phosphorylation of NF-κB, accompanied by enhanced phosphorylation of ERK, JNK, and p38 MAPKs. Pre-treatment with S12 suppressed IL-1β–induced phosphorylation of NF-κB in both KB and IHOK cells. Similarly, S12 reduced the phosphorylation of ERK, JNK, and p38 MAPKs, indicating a broad inhibitory effect on MAPK signaling pathways. The degree of inhibition was consistent across both cell types, although the reduction appeared particularly prominent for p- ERK and p-p38. This western blot analysis was performed as a single mechanistic experiment to confirm IL-1β–induced phosphorylation and the inhibitory effect of S12; therefore, quantitative densitometry is not presented.
These findings suggest that S12 exerts its anti-inflammatory effects in oral epithelial cells, at least in part, by attenuating IL-1β–induced activation of NF-κB and MAPK signaling pathways. Given that these pathways are critical for IL-8 gene transcription, the inhibition of NF-κB and MAPK phosphorylation by S12 likely contributes to the reduced IL-8 production observed in IL-1β-stimulated oral epithelial cells.
Discussion
In this study, we identified and characterized a novel human SDF-1–derived peptide, S12, that effectively suppresses IL-1β–induced IL-8 secretion in oral epithelial cells. Given that IL-8 plays a pivotal role in neutrophil recruitment and the amplification of inflammatory responses in the oral mucosa, our findings suggest that S12 acts as a potent modulator of epithelial inflammatory signaling. We demonstrated that this peptide exerts its anti-inflammatory effects through the inhibition of NF-κB and MAPK signaling pathways, thereby reducing the transcriptional activation of IL-8 and providing a molecular basis for its potential use as a host-modulation agent in periodontal inflammation.
At first, we examined that IL-1β markedly induces IL-8 secretion in oral epithelial cells, and this response is critically mediated through NF-κB and MAPK signaling pathways (Figs. 1 and 2). These findings are consistent with previous studies showing that IL-1β functions as a key pro-inflammatory cytokine in periodontal tissues and plays a central role in amplifying host immune responses. IL-8 acts as a chemokine that recruits neutrophils from the circulation into periodontal lesions, where they release proteolytic enzymes and reactive oxygen species [19]. Such neutrophil-driven activity contributes to connective-tissue degradation and alveolar-bone resorption, thereby playing an essential role in the initiation and progression of periodontitis [20-22]. These observations provide strong evidence that the IL-1β–IL-8 signaling axis constitutes a fundamental regulatory mechanism in periodontal inflammation.
At the molecular level, IL-1β stimulation activates canonical NF-κB signaling [23]. Upon receptor engagement, IκBα is phosphorylated and degraded, permitting nuclear translocation of the NF-κB p65 subunit to induce transcription of proinflammatory genes including IL-8 [24,25]. In parallel, IL-1β triggers phosphorylation of MAPKs such as ERK, JNK, and p38 (Fig. 2), which further regulate transcription factors such as AP-1 [26,27]. Together, these cascades cooperate to amplify IL-8 gene expression and link extracellular cytokine stimulation to an enhanced inflammatory output. Our inhibitor assays revealed that blocking ERK and p38 significantly attenuated IL-8 production, demonstrating that these kinases play predominant roles in IL-1β-driven responses in oral epithelial cells. In contrast, JNK inhibition had minimal effect in IHOK cells, suggesting that MAPK family members function in a cell-type-specific manner.
SDF-1 is a chemokine that regulates cell migration, proliferation, and tissue repair through its receptor CXCR4. Beyond its chemotactic activity, SDF-1 signaling is involved in modulating inflammatory responses and maintaining tissue homeostasis. Based on the multifunctional properties of native SDF-1, we designed a series of short peptides (S1–S18) that cover overlapping regions of the human SDF-1δ isoform, with the aim of identifying peptide fragments capable of retaining its immunomodulatory potential while minimizing structural complexity. Among the 18 designed SDF-1δ–derived peptides, S12 exhibited the most prominent inhibitory effect on IL-1β– induced IL-8 production (Fig. 3). S12 consistently reduced IL-8 secretion in both KB and IHOK cells, whereas S10 and S11 showed only moderate or cell-specific effects. These results indicate that S12 has a broader inhibitory capacity and may act on multiple intracellular signaling nodes. Western blot analysis confirmed that S12 significantly suppressed phosphorylation of NF-κB p65 as well as ERK, JNK, and p38 MAPKs (Fig. 4). Notably, the inhibition of ERK and p38 phosphorylation was strongly reduced, indicating that these kinases are major molecular targets of S12 activity.
Mechanistically, the reduction in NF-κB phosphorylation by S12 suggests that this peptide may interfere with upstream signaling components that connect IL-1β receptor activation to downstream transcriptional events. The concurrent inhibition of MAPK phosphorylation implies that S12 dampens multiple convergent pathways, providing a broader and more sustained anti-inflammatory effect. Such dual regulation may explain the consistent suppression of IL-8 observed in both KB and IHOK cells (Figs. 3 and 4). Importantly, this indicates that S12 not only reduces IL-8 secretion but also modulates the intracellular signaling network that underlies periodontal inflammation.
Our findings are in agreement with previous reports showing that peptide-based therapeutics can modulate host responses and suppress excessive inflammation in periodontal disease. However, the present study extends this concept by identifying a novel SDF-1–derived peptide that simultaneously inhibits NF-κB and MAPK activation in oral epithelial cells. This dual inhibition may contribute not only to the suppression of proinflammatory cytokine expression but also to the maintenance of periodontal tissue homeostasis.
In conclusion, this study provides compelling evidence that the SDF-1–derived peptide S12 suppresses IL-1β–induced IL-8 expression in oral epithelial cells by attenuating NF-κB and MAPK signaling pathways. These findings highlight the potential of S12 as a new host-modulatory therapeutic candidate for controlling periodontal inflammation. Future studies using in vivo periodontitis models will be necessary to validate its efficacy, elucidate its upstream molecular targets, and evaluate whether S12 could prevent systemic inflammatory complications associated with periodontal disease.
