Xanthine Derivative KMUP‐3 Alleviates Periodontal Bone Resorption by Inhibiting Osteoclastogenesis and Macrophage Pyroptosis

ABSTRACT

Aim

This study investigated the function effects of KMUP‐3, a self‐developed synthetic xanthine‐based derivative, in suppressing Porphyromonas gingivalis (Pg‐LPS)‐aggravated osteoclastogenesis and pyroptosis as a potential treatment for periodontitis.

Methods

In vitro, the effects of Pg‐LPS and KMUP‐3 on osteoclast formation and macrophage pyroptosis were investigated using the receptor activator of nuclear factor‐κB ligand (RANKL)‐primed RAW264.7 macrophages. In vivo, the therapeutic effects of KMUP‐3 were evaluated in a model of experimental periodontitis induced by gingival ligature placement.

Results

We reveal that KMUP‐3 suppressed osteoclastogenesis, inducible nitric oxide synthase activation, and reduced nitric oxide production enhanced by Pg‐LPS in RANKL‐primed RAW264.7 cells while also decreasing TLR4/NF‐κB p65 pathway activation and decreased pro‐inflammatory cytokine production; moreover, Pg‐LPS promoted NLRP3 activation and exacerbated pyroptosis induction effects that were abolished by KMUP‐3. Finally, KMUP‐3 ameliorated alveolar bone loss and IL‐1β levels in the gingival crevicular fluid in the rat ligature periodontitis model.

Conclusions

Our study demonstrated that KMUP‐3 attenuates Pg‐LPS‐enhanced osteoclastogenesis and macrophage pyroptosis. Notably, KMUP‐3 alleviates alveolar bone loss in experimental periodontitis rats and thus suggests its certain role in safeguarding against periodontal bone resorption.

This study reveals that our self‐developed xanthine derivative, KMUP‐3, suppresses osteoclastogenesis, inflammation, and pyroptosis by inhibiting the NLRP3 inflammasome. It also alleviates periodontal bone loss in periodontitis rats, highlighting its potential as a novel therapeutic option for periodontitis.

Summary.

• Background

  • ○
    Lipopolysaccharides from Porphyromonas gingivalis (Pg‐LPS) are the key pathogenic factors in periodontitis, driving inflammation, osteoclastogenesis, and contributing to alveolar bone resorption. KMUP‐3 is a self‐developed synthetic xanthine‐based derivative, demonstrating promising properties, including the inhibition of phosphodiesterase, modulation of nitric oxide (NO), and anti‐inflammatory effects. However, its impact on periodontal bone loss remains unclarified.
• Added value of this study

  • ○
    Initially, KMUP‐3 revealed promise as a novel agent for suppressing osteoclastogenesis.
  • ○
    We also showed that KMUP‐3 inhibited NO and pro‐inflammatory cytokine release.
  • ○
    Next, we demonstrated that KMUP‐3 treatment ameliorates pyroptosis by inactivating NLRP3 inflammasome.
  • ○
    Finally, the anti‐osteoclastogenesis and anti‐pyroptosis effects of KMUP‐3 make it a potential treatment for experimental periodontitis rats.
• Clinical implications

  • ○
    We discovered that the xanthine derivative KMUP‐3 reduces osteoclast formation, inflammation, and pyroptosis caused by bacterial macrophage stimulation. Notably, it also inhibits periodontal bone loss in experimental periodontitis rats, highlighting its ability to mitigate periodontitis‐related bone damage and serve as an adjunctive therapy in oral care for humans.

1. Introduction

Periodontitis, a common inflammatory disease causing soft and hard tissue destruction in the periodontal region, is a leading cause of tooth loss and is linked to systemic diseases [1]. Porphyromonas gingivalis (Pg), a Gram‐negative bacterium, is an important etiologic agent of periodontitis in humans [2], while lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg‐LPS) plays a central role in periodontitis by inducing gingiva inflammation, which disrupts bone homeostasis under inflammatory conditions and subsequent alveolar bone resorption [3, 4]. Pg‐LPS is considered one of the main pathogenic factors eliciting immune‐inflammatory responses leading to elevated osteogenic receptor activator of NF‐κB ligand (RANKL) and osteoclast differentiation in the periodontitis lesions [5]. It can activate multiple cell types in the synthesis of pro‐inflammatory cytokines through toll‐like receptor‐4 (TLR4) [6, 7] where such gene and protein polymorphisms are potentially used as biomarkers in assessing periodontal diseases [8].

Nitric oxide (NO) is a reactive free radical essential for the host immune response to infections [9]. NO release in the periodontium, primarily from inducible nitric oxide synthase (iNOS) in activated macrophages and osteoclasts, is key in periodontitis pathogenesis. An animal study showed that oral aminoguanidine, an iNOS inhibitor, reduces alveolar bone loss in experimental periodontitis rats [10]. Low amounts of NO may be essential for normal osteoclast activity [11]. Alternatively, some early studies have shown that NO inhibits osteoclastic bone loss at high concentrations [12, 13], with one specific study indicating that another iNOS inhibitor, high‐dose oral NG‐nitro‐L‐arginine methyl ester, and aminoguanidine can both reduce bone mass in growing rats [14]. However, another report supports that iNOS‐derived NO mediates bone resorption by modulating the pro‐inflammatory cytokines, such as interleukin (IL)‐1, in an enhanced nuclear activation of NF‐κB [15].

Collectively, the function of iNOS‐derived NO production involved in Pg‐LPS direct stimulation remains obscure. Macrophages are key components of the host immune system, responding to periodontitis‐related irritants (e.g., bacterial factors) by differentiating into pro‐ or anti‐inflammatory subtypes or undergoing cell death, which can affect tissue homeostasis and the severity of periodontitis [16]. Evidence suggests that macrophage pyroptosis aggravates periapical periodontitis and accelerates bone loss by Enterococcus faecalis infections [17, 18] and is driven by the NOD‐like receptor protein 3 (NLRP3) inflammasome, involving NLRP3, apoptosis‐associated speck‐like protein containing a CARD (ASC), and pro‐Caspase‐1 [19]. Upon activation, NLRP3 recruits ASC and pro‐Caspase‐1, leading to Caspase‐1 activation. This process triggers macrophages to release a substantial amount of interleukin‐1β (IL‐1β), causing excessive inflammation [20]; accordingly, targeting macrophage pyroptosis with pharmacological inhibitors offers a promising therapy for periodontitis.

KMUP‐3 is a chemically synthesized xanthine‐based derivative with para‐nitro groups in its structure. It has been shown to exert vasorelaxant effects, inhibit phosphodiesterase (PDE), and activate potassium channels [21] while also inhibiting TNF‐alpha (TNF‐α)‐induced increases of iNOS expression and NO levels as a cyclic guanosine monophosphate (cGMP) enhancer, demonstrating anti‐inflammatory potential in rat tracheal smooth muscle cells [22, 23]. Treatment with KMUP‐3 enhances cardiac output and improves outcomes in the hypoperfused myocardium of rats by inducing calcium sensitization [24]. It offers cardioprotection to attenuate ventricular remodeling and antimycin A‐induced cardiotoxicity via an endothelial NO synthase‐dependent mechanism, reducing acute inflammation and chronic fibrosis [25, 26]; furthermore, KMUP‐3 treatment notably suppresses abdominal aortic aneurysm formation in mice by decreasing pro‐inflammatory cytokines, preventing vascular smooth muscle cell phenotypic switching, and limiting macrophage infiltration [27]; although our aforementioned studies confirm the viability of KMUP‐3 and its anti‐inflammatory properties, it remains unclear as to whether KMUP‐3 affects osteoclastogenesis and pyroptosis.

This study hypothesizes that KMUP‐3 might suppress Pg‐LPS‐aggravated osteoclastogenesis and pyroptosis. We first assessed its anti‐osteoclastogenic, anti‐inflammatory, and anti‐pyroptotic properties in RANKL‐primed macrophages; then, we evaluated its protective effect against alveolar bone loss in ligature‐induced experimental periodontitis rats, providing new insights into its therapeutic potential for periodontitis.

2. Methods

2.1. Reagents and Chemicals

The lipopolysaccharide from Porphyromonas gingivalis (Pg‐LPS) was purchased from InvivoGen (Catalog. tlrl‐pglps, San Diego, CA, USA). Recombinant mouse RANKL (Catalog. 462‐TEC‐010) was purchased from R&D Systems (Minneapolis, MN, USA). KMUP‐3 was synthesized in our laboratory. Thiazolyl blue tetrazolium bromide (MTT) powder (M5655) was purchased from Sigma‐Aldrich (St. Louis, MO, USA), while Dulbecco's modified Eagle's medium/Nutrient Mixture F‐12 (DMEM‐F12), alpha MEM (α‐MEM), fetal bovine serum (FBS), streptomycin, penicillin, and all other cell culture reagents were purchased from GIBCO BRL Life Technologies (Grand Island, NY, USA).

2.2. Measurement of Cell Viability

Murine RAW264.7 macrophages (BCRC6001) were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and were cultured in DMEM‐F12 with 10% FBS at 37°C with 5% CO₂. Cells were cultured with various concentrations of KMUP‐3 for 24 h; then, after washing with phosphate‐buffered saline (PBS), 0.5 mg/mL MTT solution was added and incubated for 4 h at 37°C. Isopropanol was used to dissolve the formazan crystals, and absorbance was measured at 540 and 630 nm using an enzyme‐linked immunosorbent assay (ELISA) reader (DYNEX Technologies, Germany).

2.3. Cell Culture and RANKL‐Primed Osteoclast Formation

RAW264.7 cells were cultured in a differentiation medium (α‐MEM containing 1% FBS) and incubated with RANKL (20 ng/mL) to induce pre‐osteoclast generation. After 24 h, the medium was replaced with fresh α‐MEM‐containing Pg‐LPS (1 μg/mL) with or without pretreatment of KMUP‐3 for the specified time.

2.4. Tartrate‐Resistant Acid Phosphatase (TRAP) Staining and Activity

After Pg‐LPS stimulation, cells were fixed with 4% paraformaldehyde solution for 10 min and replaced with 0.1% Triton X‐100. TRAP expression was examined using a TRAP staining kit from Sigma‐Aldrich (387A, USA); then, TRAP‐positive multinucleated cells (TRAP⁺ MNCs) with three or more nuclei were identified and quantified under a microscope. TRAP activity was measured spectrophotometrically in 50 M citrate buffer with 10 mM sodium tartrate and 3.7 mM 4‐nitrophenyl phosphate at 37°C for 30 min. The reaction was stopped with 0.1 M sodium hydroxide, and absorbance at 405 nm was measured using an ELISA reader [28].

2.5. Fluorescence Staining of Actin Filaments

After treatment, cells were fixed with 4% formaldehyde for 30 min and permeabilized with 0.1% Triton X‐100 for 5 min at 4°C. The nonspecific protein binding sites were blocked with 10% bovine serum albumin for 1 h. Cells were then incubated with phalloidin (Alexa Fluor Plus 647, 1: 500; Invitrogen, CA, USA) at room temperature for 2 h. After washing, coverslips were mounted with the mounting medium (S3023; Dako), and fluorescence was visualized using a confocal laser scanning microscope (Olympus Fluoview FV100; Olympus Optical Co, Tokyo, Japan). Three random fields of view per well were analyzed for mature osteoclasts to quantify the F‐actin ring area [28]. Further, the nucleus number of mature osteoclasts was counterstained with DAPI and then calculated using AxioVision Rel 4.8 software (Carl Zeiss, Oberkochen, Germany) [29].

2.6. Measurement of Nitric Oxide (NO)

Nitrite accumulation as an indicator of NO production was measured using the Griess reagent (1% sulfanilamide and 0.1% N‐1‐naphthylethylenediamine dihydrochloride in 5% phosphoric acid); then, the culture medium was mixed with an equal volume of Griess reagent and incubated for 10 min at room temperature. Absorbance was measured at 540 nm with an ELISA reader [30].

2.7. Enzyme‐Linked Immunosorbent Assay (ELISA)

Conditioned medium was collected to measure mouse iNOS (E‐EL‐M0696; Elabscience, USA), TNF‐α (DY410, R&D Systems), IL‐6 (DY406; R&D Systems), MCP‐1 (DY479; R&D Systems), and IL‐1β (DY401; R&D Systems) levels using commercial ELISA kits. IL‐1β levels in rat gingival crevicular fluid were also analyzed using a precoated ELISA kit (E‐EL‐R0012; Elabscience) as per the manufacturer's instructions.

2.8. Western Blotting

Cells were treated and harvested at the indicated times. Total protein extracts were prepared in the lysis buffer, centrifuged, and concentrations quantified using the Bio‐Rad Protein Assay (#5000002, California, USA). Proteins were separated by SDS‐PAGE (10%–12%) and transferred to polyvinylidene difluoride membranes, blocked, and incubated with primary antibodies. The primary antibodies we used in the study were anti‐iNOS (ab15323; Abcam), rabbit TLR4 (GTX13867; GeneTex), anti‐TRAF6 (#67591; Cell signaling), anti‐phospho‐IκBα (Ser32/36, #9246; Cell signaling), anti‐IκBα (ab7217; Abcam), anti‐NFκB p65 (sc‐8008; Santa Cruz), anti‐NFκB p65 (Ser276, GTX54672; GeneTex), anti‐NLRP3 (tcba2712; Taiclone), anti‐PYCARD (tcba1690; Taiclone), anti‐Caspase 1 (#22915‐1‐AP; proteintech), anti‐IL1 beta (GTX74034; GeneTex), and anti‐beta‐actin (A5441; Sigma‐Aldrich). The membrane was then treated with the secondary antibodies attached to horseradish peroxidase (Merck Millipore). All immunoreactive bands were detected using enhanced chemiluminescence reagents (Merck Millipore, USA) and then captured with a charged‐couple device camera, and the values of each band intensity were quantified with ImageJ software (National Institutes of Health, Bethesda, USA).

2.9. Experimental Periodontitis

Thirty male Wistar rats (4 weeks old, 180–200 g) were purchased from BioLASCO Taiwan Co. Ltd. This study was approved by the Animal Care and Use Committee of the Kaohsiung Medical University with an IACUC number of 108118, while the animals were cared for according to the guiding principles of the National Institutes of Health of the United States.

The ligature model was conducted as previously described with some modifications [31, 32]. Briefly, the experimental rats were randomly divided into five groups: (1) Sham group received glycerol (G2025, Sigma‐Aldrich, USA), (2) KMUP‐3 group received 1 mg/kg of KMUP‐3, (3) ligature group received glycerol, (4) ligature + KMUP‐3 group received KMUP‐3, and (5)l igature + doxycycline (DOX) group received 20 mg/kg of DOX (D3072; Sigma‐Aldrich, USA). In this model, ligatures were placed around the second maxillary molar, whereas all drugs and glycerol were applied through intraperitoneal injection daily for 30 days. Before sacrifice, the nylon thread ligates were removed, sample sites were isolated, and the tooth surfaces were air‐dried. The gingival crevicular fluid (GCF) was collected using paper strips (Periopaper, ProFlow Inc., Amityville, NewYork, USA) [33]; then GCF sampling with paper strips was stored in tubes containing the ELISA assay diluent at −20°C until the day of the ELISA analysis.

2.10. Micro‐Computed Tomography and TRAP Histochemical Staining

A calibrated examiner used the high‐resolution micro‐CT system (SkyScan 1076; Bruker, Kontich, Belgium) to scan the fixed maxilla samples, incorporating density phantoms for calibration. Three‐dimensional (3D) Visualization Software and CT Analysis Software (Bruker, Kontich, Belgium) were used to analyze the three‐dimensional structure and bone parameters. The 3D images measured the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) of the second molar, while the 2D images analyzed bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) [32].

The teeth and maxillae of rats were fixed in 10% paraformaldehyde with 0.5 M ethylenediaminetetraacetic acid. The paraffin‐embedded tissue section with 5 μm thickness was used for TRAP staining (387A; Sigma‐Aldrich, USA) where the number of TRAP⁺ osteoclasts in the alveolar bone sections was quantified and normalized to a consistent area size by AxioVision Rel 4.8 software (Carl Zeiss, Oberkochen, Germany).

2.11. Statistical Analysis

Data were expressed as mean ± standard error of the mean (SEM) for continuous variables. Continuous variables were compared with one‐way ANOVA, followed by Tukey's post hoc analysis where significant differences were found. A value of p < 0.05 was defined as being statistically significant. All data were statistically analyzed using Prism 5 (GraphPad Software, San Diego, CA).

3. Results

3.1. Xanthine Derivative KMUP‐3 Suppresses Osteoclast Differentiation Augmented by Pg‐LPS in RANKL‐Primed Macrophages

The chemical structure of xanthine derivative KMUP‐3 is depicted in Figure 1A. Initially, we revealed that KMUP‐3 exhibited no cytotoxic effects on the RAW264.7 macrophage at concentrations below 20 μM, as assessed by cell viability using the MTT assay (Figure 1B). To assess KMUP‐3 impacts on Pg‐LPS‐enhanced osteoclastogenesis, nitric oxide, pro‐inflammation cytokines, and pyroptosis, cells were pre‐incubated with RANKL (20 ng/mL) for 24 h and then stimulated with Pg‐LPS (1 μg/mL) and KMUP‐3 at various concentrations, added 1 h before Pg‐LPS (Figure 1C). Osteoclast formation was visible under an optical microscope after 4 days of RANKL and Pg‐LPS stimulation (Figure 1D, upper panel). Moreover, co‐stimulation of RANKL and Pg‐LPS, but not RANKL alone, increased the number of TRAP‐positive multinucleated cells (TRAP⁺ MNC), suggesting that Pg‐LPS treatment significantly amplified RANKL‐induced osteoclastogenesis (Figure 1D,E). KMUP‐3 treatment at several concentrations (1, 5, and 10 μM) decreased the TRAP⁺ MNC enhanced by Pg‐LPS stimulation; consistently, these treatments suppressed TRAP activity in the Pg‐LPS‐stimulated osteoclasts (Figure 1F), with the data suggesting that KMUP‐3 attenuates Pg‐LPS‐augmented osteoclastogenesis in a dose‐dependent manner.

FIGURE 1.

Xanthine derivative KMUP‐3 suppresses osteoclast differentiation augmented by Pg‐LPS in RANKL‐primed macrophages. (A) Chemical structure of KMUP‐3. (B) Cell viability was measured by MTT assay (n = 6). (C) Outline of the experimental protocol of the in vitro study. (D) Representative images of TRAP staining were observed under the optical microscope. (E) Quantification of TRAP‐positive multinucleated cell (TRAP⁺ MNC) number from the images in (D) performed using Carl Zeiss AxioVision software (n = 6). (F) Quantitation of TRAP activity assay (n = 6). Yellow arrows indicate TRAP⁺ MNCs. All scale bars represent 100 μm. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 and ^$ p < 0.05 compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. **p < 0.01 and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

3.2. KMUP‐3 Reduces Pg‐LPS‐Enhanced Osteoclast Multinucleation by Inhibiting iNOS‐Derived Nitric Oxide

Next, we evaluated the effects of inducible nitric oxide synthase (iNOS)‐derived nitric oxide (NO) in contributing to osteoclastogenesis in the Pg‐LPS‐treated cells. The induction of iNOS expression and subsequent NO production are key contributors to inflammation and periodontitis [10]. Fluorescent staining showed that Pg‐LPS‐enhanced osteoclast maturation and multinucleation were dose‐dependently reversed by KMUP‐3 treatment (Figure 2A). The quantitative analysis of the F‐actin ring area per field (Figure 2B) and the measurement of nucleus number per MNC (Figure 2C) aligned with the fluorescent staining results. Additionally, the Griess reagent assay result showed that treatment of KMUP‐3 also significantly decreased NO production enhanced by Pg‐LPS‐treated osteoclasts (Figure 2D). High concentrations of NO are produced by iNOS, a soluble enzyme, which is activated by bacterial pathogens or immune‐stimulating cytokines [34], and in line with this, Pg‐LPS significantly elevated the expression level of soluble iNOS while being suppressed by KMUP‐3 (Figure 2E). Consistently, the intracellular iNOS activation by Pg‐LPS in the osteoclast was dose‐dependently reversed by KMUP‐3 (Figure 2F,G). These results revealed that KMUP‐3 reduced the NO level, a pro‐osteoclastogenesis factor in the Pg‐LPS‐enhanced osteoclast multinucleation via iNOS inactivation.

FIGURE 2.

KMUP‐3 reduces Pg‐LPS‐enhanced osteoclast multinucleation by inhibiting iNOS‐derived nitric oxide. (A) Representative images of F‐Actin rings in osteoclasts by immunofluorescence staining. (B) Quantification of the area of F‐Actin ring per field (n = 6). (C) Measurement of the nucleus number per multinucleated cell (MNC) using Carl Zeiss AxioVision software (n = 6). (D) Griess assay was used to identify nitrite products in the culture medium (n = 6). (E) Soluble enzyme, Inos, in the culture medium was evaluated by ELISA assay (n = 6). (F) Protein expression of iNOS was detected by western blotting and (G) quantitatively analyzed (n = 5). All scale bars represent 100 μm. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. ***p < 0.001 compared with the RANKL + Pg‐LPS group.

3.3. KMUP‐3 Inhibits Inflammatory Cytokine Secretion via Suppressing TLR4/NF‐κB p65 Pathway Aggravated by Pg‐LPS

Toll‐like receptor 4 (TLR4) is crucial for activating innate immunity via recognizing various pathogen‐associated molecular patterns such as LPS and bacteria. Activated TLR4 induces its downstream TRAF6, which triggers IκBα/NF‐κB p65 signaling, leading to an inflammatory response [35]. To assess the KMUP‐3‐interference action on Pg‐LPS involving the TLR4/NF‐κB p65 pathway, we determined that KMUP‐3 treatment suppressed the increased TLR4 protein levels (Figure 3A) and TLR4/β‐actin ratios (Figure 3B) by Pg‐LPS in RANKL‐primed cells. Consistently, the downstream adapter protein tumor necrosis factor receptor‐associated factor 6 (TRAF6) and TRAF6/β‐actin ratios were elevated following Pg‐LPS incubation, and these effects were reversed by KMUP‐3 (Figure 3A,B), while KMUP‐3 treatment also suppressed IκB kinase activation of NF‐κB enhanced by Pg‐LPS (Figure 3C,D). We then verified whether KMUP‐3 exerted an anti‐inflammatory effect with Pg‐LPS exposure, with the secretion of various pro‐inflammatory cytokines, including TNF‐α, IL‐6, monocyte chemoattractant protein‐1 (MCP‐1), and IL‐1β being evaluated by ELISA assay. We revealed that these pro‐inflammatory cytokines exhibited augmented enhancement by Pg‐LPS (Figure 3E–H). KMUP‐3 inhibited TNF‐α, IL‐6, and MCP‐1 releases in the conditioned medium (Figure 3E–G). IL‐1β secretion is a major mediator downstream of inflammasome signaling and induction of cell pyroptosis [36]; similarly, KMUP‐3 suppressed IL‐1β secretion induced by Pg‐LPS (Figure 3H). These results confirm that KMUP‐3 acts as an anti‐inflammatory agent by attenuating the exacerbated activation of TLR4/NF‐κB p65 signaling by Pg‐LPS exposure.

FIGURE 3.

KMUP‐3 inhibits inflammatory cytokines secretion via suppressing TLR4/NF‐κB p65 pathway aggravated by Pg‐LPS. (A) Protein expression of TLR4 and TRAF6 were detected by western blotting and (B) quantitatively analyzed (n = 5). (C) Protein expression of phospho‐IκBα/IκBα and phospho‐p65/p65 were detected by western blotting and (D) quantitatively analyzed (n = 5). (E) TNF‐α, (F) IL‐6, (G) MCP‐1, and (H) IL‐1β in the culture medium were evaluated by ELISA assay (n = 6). Data are expressed as mean ± SEM. ^$ p < 0.05, ^$$ p < 0.01, and ^$$$ p < 0.001, compared with the control group. ^## p < 0.01 and ^### p < 0.001 compared with the RANKL‐only group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

3.4. KMUP‐3 Treatment Mitigates Macrophage Pyroptosis via Suppressing NLRP3 Inflammasome Activation

We then investigated whether KMUP‐3 could reduce Pg‐LPS‐augmented trigger pyroptosis by inactivating the inflammasome signaling. Recent studies have proposed pyroptosis as a key pathway contributing to exacerbated inflammation in periodontal tissues [37, 38], with these western blotting results illustrating that Pg‐LPS significantly enhanced the NLRP3 inflammasome multiprotein complex for 24 h (Figure 4A). Additionally, KMUP‐3 suppressed the ratios of NLRP3 (Figure 4B), ASC (Figure 4C), and Caspase‐1 (Figure 4D) activation elicited by Pg‐LPS stimulation (Figure 4A–D). Subsequently, we examined the downstream pyroptotic cytokine production of intracellular IL‐1β. Pg‐LPS induced the high expression level of IL‐1β, though this effect was reversed by KMUP‐3 treatment (Figure 4E,F). Collectively, these results indicate that KMUP‐3 affords anti‐inflammatory effects to myeloid lineage cells by reducing Pg‐LPS‐triggered osteoclastogenesis and pyroptosis.

FIGURE 4.

KMUP‐3 treatment mitigates macrophage pyroptosis via suppressing NLRP3 inflammasome activation. (A) Protein expression of NLRP3 (B), ASC (C), and Caspase‐1 (D) were detected by western blotting and quantitatively analyzed (n = 5). (E and F) Protein expression of IL‐1β was detected by western blotting and quantitatively analyzed (n = 5). Data are expressed as mean ± SEM. ^$ p < 0.05 and ^$$$ p < 0.001, compared with the control group. ^### p < 0.001 compared with the RANKL‐only group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the RANKL + Pg‐LPS group.

3.5. KMUP‐3 Alleviates Periodontal Bone Loss and Reduces Gingival IL‐1β in Ligature‐Induced Periodontitis Rats

Finally, the beneficial effects of KMUP‐3 treatment on ligature‐induced experimental periodontitis were evaluated in the murine model [31, 32], with these results revealing that the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) distance at the second maxillary molar (M2) in both mesiolingual (ML) and distolingual (DL) roots was increased (Figure 5A–C). KMUP‐3 administration and the comparative drug (doxycycline; Dox) restored the CEJ–ABC distance in the affected area. Ligature placement reduced the bone volume/tissue volume, trabecular thickness, and increased trabecular separation, but KMUP‐3 and Dox effectively ameliorated the abovementioned indicators of pathological bone loss (Figure 5D–G). As excessive osteoclast activity results in pathological bone resorption, as seen in conditions like periodontitis [39], we further investigated KMUP‐3 administration to inhibit osteoclastogenesis and pyroptosis in vivo, with the results of the histological TRAP stain revealing that TRAP‐positive osteoclast numbers were significantly increased in the maxillary alveolar bone by ligature placement (Figure 5H,I). KMUP‐3 and Dox decreased osteoclast numbers distributed in the alveolar bones. Moreover, we assessed the pyroptotic cytokine production of IL‐1β in the gingival crevicular fluid (GCF) by ELISA assay, and in line with this, KMUP‐3 and Dox treatment reduced the IL‐1β level of GCF while being elicited by ligature placement (Figure 5J). In summary, our findings reveal that KMUP‐3 mitigates periodontitis‐aggravated alveolar bone resorption, aligning with in vitro results.

FIGURE 5.

KMUP‐3 alleviates periodontal bone loss and reduces gingival IL‐1β in ligature‐induced periodontitis rats. (A) Representative 3D micro‐CT images of rat palatal maxilla. (B and C) Distance (as indicated by red lines) between the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) at the second maxillary molar (M2) of mesiolingual (ML) and M2 of distolingual (DL) was measured (n = 6). (D) Representative micro‐CT images of the second molar ligature placement in the maxilla. The red circles indicate the area quantitatively analyzed. (E) Bone volume/tissue volume (BV/TV), (F) trabecular thickness (Tb.Th), and (G) trabecular separation (Tb.Sp) were measured (n = 6). (H) Representative images of TRAP staining in the rat maxillary alveolar bone sections were observed under the optical microscope. (I) Statistical analysis of TRAP‐positive (TRAP⁺) osteoclast numbers distributed in the alveolar bones (n = 6). (J) IL‐1β in the gingival crevicular fluid (GCF) was evaluated by ELISA assay (n = 6). The red squares indicate the area quantitatively analyzed. Yellow arrows indicate TRAP⁺ osteoclast. All scale bars represent 50 μm. Dox means doxycycline. Data are expressed as mean ± SEM. Nonsignificant (n.s.) p > 0.05 and ^### p < 0.001 compared with the sham group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the ligature group. Nonsignificant (N.S.) p > 0.05 compared with the ligature + KMUP‐3 group.

4. Discussion

In this study, we demonstrated whether xanthine‐based KMUP‐3 has anti‐inflammation potential to inhibit osteoclastogenesis and pyroptosis in vitro and to reduce bone loss in experimental periodontitis in vivo. Osteoclasts, which are multinucleated cells responsible for bone resorption, originate from the myeloid cells of monocyte/macrophage lineage [40]. The ligand RANKL binds to its receptor RANK, expressed on osteoclasts, and plays a key role of regulator in both osteoclastogenesis and immune system regulation [41]. Pg‐LPS is regarded as a key pathogenic factor that triggers immune‐inflammatory responses, resulting in an increased production of inflammatory cytokines and osteogenic RANKL interaction, perpetuating periodontitis lesions [4, 42]. Our findings showed that, consistent with our previous studies, the xanthine‐based compound KMUP‐3 is also noncytotoxic to cultured RAW264.7 macrophages [30, 43], while KMUP‐3 treatment significantly diminishes osteoclastogenesis aggravated by Pg‐LPS stimulation. Inducible nitric oxide synthase (iNOS) catalyzes nitric oxide (NO) production from L‐arginine and is activated in macrophages by LPS and TNF‐α or interferon‐γ stimulation [44]. It is a key upstream enzyme in NO production, strongly induced by proinflammatory stimuli during the inflammation of macrophages [45]. Furthermore, scientific study has demonstrated that bone marrow‐derived macrophages from iNOS knockout mice exhibit reduced osteoclastogenesis compared to wild‐type mice cells [10]. An early study found that sodium nitroprusside treatment inhibited the formation of multinucleated osteoclasts via the cyclic guanosine monophosphate (cGMP)‐dependent pathway; similarly, the specific phosphodiesterase (PDE) inhibitor, zaprinast, also suppressed osteoclast formation by elevated cGMP level [36]. Our data depicted that similar to treatment with the PDE inhibitor [21, 22, 23], KMUP‐3 represses osteoclast multinucleation by strongly reducing NO levels and downregulating iNOS expression elicited by Pg‐LPS (Figures 1 and 2).

Toll‐like receptors (TLRs) are pattern recognition receptors found in immune cells such as macrophages and dendritic cells [7]. Pg‐LPS is a key pathogenic factor in periodontal bone resorption, utilizing microbe‐associated molecular patterns that are recognized by TLRs in inflammatory immune responses [46]. Our results showed that KMUP‐3 inhibits exacerbated activation of TLR4 by Pg‐LPS incubation; furthermore, downstream blockage of the pro‐inflammatory pathway involved TRAF6, IκBα, and NF‐κB phosphorylation. Studies have shown that pro‐inflammatory cytokines such as IL‐1β and TNF‐α are elevated in the saliva of diseased groups and decreased following periodontal therapy, as observed in both clinical trials and animal experimental periodontal diseases [47]. Accordingly, we also demonstrated that KMUP‐3 attenuates advancement of various pro‐inflammatory cytokines in Pg‐LPS‐induced cells. Pyroptosis is a form of programmed cell death that is characterized by inflammation triggered by inflammasome and Caspase‐1 activation, which results in the intracellular contents of pro‐inflammatory cytokine release [19, 20]. Also, the correlation between pyroptosis and periodontitis has been stressed previously using human THP‐1 macrophages and gingival fibroblasts [17, 48]. Our data showed that Pg‐LPS stimulation of NLRP3 inflammasome and downstream pyroptotic cytokine IL‐1β activation were reversed by KMUP‐3 treatment (Figures 3 and 4). Collectively, KMUP‐3 depicts strong anti‐inflammatory effects that suppress the TLR4/NF‐κB p65 axis and NLRP3 inflammasome‐enhanced pyroptosis.

Our self‐developed xanthine‐based compounds, KMUP‐1 and KMUP‐3, are structurally similar to sildenafil and exert inhibitory activity on phosphodiesterases 3, 4, and 5 and were initially designed for their arterial and airway relaxant effects in rats [21, 22, 23]. Our previous in vitro evidence demonstrates that KMUP‐3 is a nontoxic and safe compound, as verified in rat tracheal smooth muscle cells, myocardial cells, and aortic vascular smooth muscle cells [23, 24, 25, 26, 27]. Importantly, this xanthine‐based compound has shown the suppression effect of osteoclastogenesis in ovariectomized mice and anti‐osteoarthritis in monosodium iodoacetate‐induced rats [30, 43]. Our previous findings indicate that KMUP‐3 exhibits strong anti‐inflammatory effects and has been shown to reduce myocardial infarction area in rats by enhancing eNOS activity and restoring the MMP‐9/tissue inhibitor of metalloproteinase‐1 balance [25]. More recently, it has been found to inhibit the formation of abdominal aortic aneurysms in angiotensin II‐infused mice by reducing pro‐inflammatory cytokine production and macrophage infiltration [27].

Consistent with previous works, we determined that treatment with KMUP‐3 led to a favorable outcome, reducing the ligature‐enhanced CEJ‐ABC distances at the affected maxillary molar roots in rats. Given that KMUP‐3 treatment has been effective in preserving alveolar bone mass in ligature‐induced rats, such treatment has also been found effective in the suppression of osteoclast hyperactivation in periodontic environments, where higher levels of IL‐1β in gingival crevicular fluid (GCF) from the progressing sites were reversed by KMUP‐3 (Figure 5). It is well established that scaling and root planing for dental cleaning can reduce inflammatory cytokines including IL‐1β in GCF from chronic periodontitis patients [49], although several minor studies have indicated conventional scaling or debridement is insufficient to reduce the IL‐1 levels [50]. Accordingly, local and systemic administration of antibiotics (e.g., minocycline and doxycycline) as an adjunctive treatment combined with periodontal scaling effectively reduces elevated IL‐1β levels in GCF associated with periodontitis progression in patients [51]. Notably, KMUP‐3 shows therapeutic effects comparable to doxycycline in experimental periodontitis rats, although limitations remain: Lipophilic drugs exhibit high permeability as they readily diffuse across cell membranes [52], so future efforts should focus on improving KMUP‐3 bioavailability using novel liposoluble substances like liposomes or gelling agents while studies in canine models are needed to better represent human periodontal diseases before clinical trials [53].

5. Conclusions

Our in vitro findings demonstrate that Pg‐LPS aggravates NO and pro‐inflammatory cytokine release within RANKL‐enhanced osteoclastogenesis and pyroptosis. This study is the first to show that KMUP‐3 suppresses iNOS‐derived NO against osteoclastogenesis. We illustrated that KMUP‐3 alleviates experimental periodontitis bone loss in rats by inhibiting NLRP3 inflammasome‐mediated pyroptosis (Figure 6), highlighting its potential as a therapeutic strategy for periodontitis.

FIGURE 6.

Proposed mechanisms of KMUP‐3 treatment alleviate periodontal bone loss. KMUP‐3 treatment ameliorates Pg‐LPS‐enhanced osteoclastogenesis, NO production, pro‐inflammatory cytokine secretion, and pyroptosis in RANLK‐primed macrophages. These effects lead to the inhibition of periodontitis‐induced alveolar bone loss. The image was created using BioRender.com (Nov. 2024).

Author Contributions

J.‐H.H. and J.‐L.Y. contributed to the study conception and study design. S.‐E.H., K.‐F.H., and M.‐X.L. performed the experiments. C.‐J.T., B.‐N.W., and Z.‐K.D. performed data acquisition and analysis. S.‐E.H., K.‐F.H., J.‐H.H., and J.‐L.Y. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Ethics Statement

This study was approved by the Animal Care and Use Committee of the Kaohsiung Medical University. The Institutional Animal Care and Use Committee (IACUC) number is 108118. The animals were cared for according to the guiding principles of the National Institutes of Health of the United States. All rats were allowed free access to water and standard laboratory food during their acclimatization and experiments. The rats were housed in a well‐ventilated room under standard conditions (22°C ± 2°C, 60% ± 10% humidity) with a 12 h light/dark cycle.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data are available upon request to the corresponding authors.