Abstract
Periodontitis is an oral immunoinflammatory disease, and macrophages play a crucial role in its pathophysiology. However, macrophage death during antibacterial activities will exacerbate inflammation and tissue damage. Porphyromonas gingivalis is a major constituent of subgingival biofilm plaques in periodontitis, but the effects and precise molecular mechanisms by which it triggers macrophage death remain unknown. Here we found that P. gingivalis infection notably activated multiple death pathways in bone-marrow-derived macrophages, including pyroptosis, apoptosis and necrosis. Furthermore, using RNA sequencing, we identified that P. gingivalis infection markedly increased the expression of Z-DNA binding protein 1 (Zbp1) in bone-marrow-derived macrophages. Initially identified as an interferon-induced tumor-associated protein, Zbp1 serves as an upstream sensor that regulates cell death by activating PANoptosis. Mechanistically, P. gingivalis induced a mitochondrial stress response, prompting the release of mitochondrial DNA. This mitochondrial DNA then interacted with Zbp1, consequently augmenting its downstream PANoptosis signals. In addition, P. gingivalis stimulated macrophage Zbp1 expression through the Tlr2/4–JNK–Stat3/5 pathway, exacerbating macrophage death. Importantly, blocking the biosynthesis of endogenous Zbp1 by pharmacological delivery with microneedles improved the survival of P. gingivalis-infected macrophages and inhibited periodontal tissue destruction. These findings highlight Zbp1 as a potential therapeutic target for P. gingivalis-induced periodontitis.
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Introduction
Periodontitis is an oral immunoinflammatory disease characterized by pathological loss of the periodontal ligament and alveolar bone, driven by intricate interactions between specific pathogens and destructive immune responses1. Porphyromonas gingivalis, an anaerobic Gram-negative rod, is a key constituent of subgingival biofilm plaques and is recognized as a primary predisposing factor in the development of chronic periodontitis (CP)2. In periodonitis pathophysiology, P. gingivalis disrupts host–microbe homeostasis, evades immune surveillance and manipulates immune signaling pathways to establish a state of chronic inflammation. Its array of virulence factors, including lipopolysaccharides (LPS), gingipains and fimbriae, directly interact with immune cells, subverting their functions and contributing to periodontal tissue destruction3.
Macrophages are essential components of the innate immune system and are actively recruited to infection sites during inflammatory responses. They play a pivotal role in clearing microorganisms and cellular debris through phagocytosis and cytokine release4. Paradoxically, macrophage death during the antibacterial response may amplify proinflammatory signaling5. This phenomenon can not only exacerbate immune-mediated tissue damage but also contribute importantly to periodontal tissue destruction and alveolar bone resorption. Therefore, exploring the mechanisms underlying macrophage death and its impact on periodontal pathogenesis is critical to elucidating the disease’s progression and developing targeted therapeutic strategies.
Recent studies indicate that multiple cell death modes, including pyroptosis, apoptosis and necroptosis, have been detected simultaneously in rodent periodontitis models and cellular experiments6,7. He et al.8 reported that glycolysis mediates macrophage pyroptosis in periodontal diseases, resulting in increased inflammation and bone resorption. Moreover, periodontal pathogens can induce macrophage apoptosis, enabling deeper tissue invasion and exacerbating pathology9. MLKL-mediated necroptosis in macrophages promotes the upregulation of TNF-α, IL-1β, IL-6, COX-2 and MMP9, contributing to alveolar bone resorption in mouse periodontitis models10. These findings highlighted the extensive involvement of various cell death processes in periodontitis pathophysiology.
In 2019, Malireddi et al.11 introduced the concept of PANoptosis, where multiple cell death pathways occur simultaneously, enormously amplifying inflammatory signals. Jiang et al. further explored the association between P. gingivalis-induced cell death and periodontitis progression, proposing that PANoptosis contributes to the immune evasion of P. gingivalis and represents a potential pathogenic mechanism in periodontal disease12. Z-DNA binding protein 1 (Zbp1), identified as an interferon (IFN)-induced tumor-associated protein, contains two left-handed helical nucleic acid-binding domains (Zα) and two RIP homotypic interaction motif (RHIM) domain that facilitates protein–protein interactions13. Zbp1 is initially acknowledged for its involvement in antiviral responses, aiming to characterize the upstream receptors that mediate the activation of PANoptosis pathways during influenza A virus infection14. Recent studies have expanded their role in inducing cell death during bacterial infections15. Although moderate Zbp1 activation contributes to the host defense against certain pathogens, evidence suggests that excessive Zbp1 induction may paradoxically exacerbate chronic inflammation15. For example, key necrosis-related regulatory pathways predicted by the ceRNA network indicated that the RP11-138A9.1–hsa-miR-98-5p–Zbp1 axis markedly amplified periodontal inflammation16. In addition, metformin was used to alleviate apical periodontitis by inhibiting Zbp1-mediated necrosis17. Thus, Zbp1 may contribute to tissue damage through the activation of the cell death pathway in immune regulatory processes. However, its role in P. gingivalis-induced periodontal inflammation is not fully understood.
In this study, we characterized the abundance of macrophages within periodontitis tissues using single-cell RNA sequencing analysis. Subsequently, we presented evidence that P. gingivalis infection substantial intensified Zbp1-mediated macrophage death and periodontal inflammation through interactions with mitochondrial DNA (mtDNA) and activation of the Tlr2/Tlr4–JNK–Stat3/Stat5 signaling pathways. The potential of Zbp1 as a therapeutic target for alleviating P. gingivalis-induced macrophage death and periodontal resorption has also been explored, suggesting a theoretical basis for novel targeted tissue regeneration strategies in periodontitis.
Materials and methods
Specimen collection and preparation
Subgingival plaque samples were obtained from six patients with CP and five healthy individuals during health examinations. Sterile curette scalers were used to collect subgingival plaques from the areas between the gingival margin and 3 mm below it, and the samples were promptly stored at −80 °C. The inclusion criteria for CP in this study followed the 2018 updated classification of the periodontal workshop18, encompassing patients who (1) had not received treatment for periodontitis in the preceding 6 months, (2) exhibited normal blood glucose levels without diabetes mellitus, (3) had a minimum of four teeth with a probing depth ≥6 mm, (4) had clinical attachment loss ≥3 mm, (5) had a bleeding on probing index ≥2 and (6) had evident bone loss observed through radiographic examination. The clinical characteristics of the patients are summarized in Supplementary Table 1.
Gingival specimens were obtained during crown lengthening or oral implant surgery from five patients with CP and five healthy individuals (Supplementary Table 2). The collected gingival tissues were fixed in 4% paraformaldehyde for 24 h, followed by dehydration, embedding and sectioning into 4-μm frozen slices. The protocols for collecting subgingival plaque samples and gingival specimens were approved by the Research Ethics Committee of Nanjing Medical University (PJ2023-089-001), and informed consent was obtained from all participants.
16S rRNA sequencing
16S rRNA gene amplicon sequencing and analysis were conducted by OE Biotech. Bacterial DNA was extracted from subgingival plaque samples using the DNeasy PowerSoil kit (Qiagen) following the manufacturer’s instructions. DNA concentration and integrity were assessed using NanoDrop 2000 (Thermo Fisher Scientific). Polymerase chain reaction (PCR) amplification of the V3–V4 hypervariable regions of the bacterial 16S rRNA gene was performed using universal primer pairs (343 F: 5′-TACGGRAGGCAGCAG-3′; 798 R: 5′-AGGGTATCTAATCCT-3′). Subsequent sequencing was conducted using the Illumina NovaSeq6000 platform (Illumina). Relative abundance of each operational taxonomic unit was calculated. The microbial diversity of the samples was assessed using alpha diversity indices, including abundance-based coverage estimator, observed operational taxonomic units, whole trees and Chao1 indexes.
Mouse periodontitis model
Male wild-type (WT) C57BL/6N mice (8 weeks old) were housed under specific-pathogen-free conditions with a standard 12-h/12-h light/dark cycle. Mice were randomly divided into control and P. gingivalis-infected groups with five mice in each group. Antibiotics (amoxicillin and metronidazole) were administered in drinking water for 7 days to mitigate the impact of the resident oral microbiota19,20. After a 3-day antibiotic recovery period, silk ligatures (5–0) were placed around the maxillary second molars. Subsequently, P. gingivalis (ATCC 33277, 1 × 109) suspended in phosphate-buffered saline (PBS, 20 μl) were administered onto the alveolar crest every other day, while PBS alone were injected in the control group. The maxillary bones were excised 14 days after the first injection and immediately fixed in 4% paraformaldehyde solution for 48 h. The maxillary jaws were subsequently decalcified with 20% ethylene diamine tetraacetic acid at 4 °C for 30 days. After decalcification, standard procedures for dehydration and paraffin embedding were used for histological analyses.
Zbp1−/− mice were established by Cyagen Biosciences using the CRISPR–Cas9 technology. Animal experimental procedures were approved by the Laboratory Animal Care and Use Committee of Nanjing Medical University (approval no. IACUC - 1905049).
Cell isolation and culture
The culture and differentiation of bone-marrow-derived macrophages (BMDMs) adhered to procedures detailed in a previous study21. In brief, femurs and tibias of 8-week-old C57BL/6N mice were aseptically extracted. Subsequently, the cells were flushed from the bone marrow cavities, followed by blood cell lysis, and cultured in DMEM supplemented with 2% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) for 4 h at 37 °C in a 5% CO2 atmosphere. All nonadherent cells were collected and reseeded in DMEM containing 10% fetal bovine serum, 2% penicillin–streptomycin and murine M-CSF (10 ng/ml, PeproTech), with medium renewal every 3 days. BMDMs adhering to the culture flasks were collected after 7 days and used for subsequent experiments.
Co-culture of BMDMs and P. gingivalis
P. gingivalis (ATCC 33277) was inoculated onto anaerobic blood agar medium (10% sheep blood, 10 µg/ml hemin chloride and 1.0 µg/ml vitamin K1) and incubated under anaerobic conditions (85% nitrogen, 10% hydrogen and 5% carbon dioxide) at 37 °C for 48 h. After incubation, bacterial colonies were collected and washed with PBS. P. gingivalis was inoculated into BMDMs at various multiplicities of infection (MOI) and cultured for 24 h. The co-culture environment was maintained under the same conditions as those used for BMDM cultivation.
Fluorescence in situ hybridization
A fluorescence in situ hybridization (FISH) assay was performed to identify the localization of P. gingivalis following the manufacturer’s instructions for the Ribo Fluorescent In Situ Hybridization Kit (RiboBio). Cell or periodontal tissue samples were treated with 0.1% Triton X-100 (Beyotime), followed by prehybridization solution incubation at 37 °C for 30 min. Subsequently, they were incubated overnight in darkness at 37 °C with hybridization solution containing Cy3-labeled FISH probe (POGI: 5′-CAATACTCGTATCGCCCGTTATTC-3′, Generay Biotech). Before sealing, the nuclei were stained with DAPI (Beyotime), and images were captured using confocal laser scanning microscopy (Zeiss).
RNA sequencing
The differentially expressed genes (DEGs) between PBS-treated BMDMs and P. gingivalis-infected BMDMs (MOI of 100 for 24 h) were identified through RNA sequencing, with each group comprising four cell samples (n = 4). Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) was used for cDNA synthesis and library construction. Subsequently, the constructed libraries underwent quality assessment using an Agilent 2100 Bioanalyzer (Agilent Technologies) and were sequenced using an Illumina HiSeq6000 sequencer after passing the quality check. Gene expression was quantified in fragments per kilobase per million reads using the limma package in R software. The threshold for identifying differentially expressed genes (DEGs) was set at log2 fold change (FC) ≥1.5 with P < 0.05.
DNA extraction
Subcellular fractionation was performed according to the previously described method22. In brief, BMDMs (1 × 106) were collected and centrifuged (400g, 3 min, 4 °C), and the pellet was subsequently resuspended in hypotonic buffer and incubated on ice for 30 min. Swollen cells were homogenized 50 times with a 26-gauge needle. Low-speed centrifugation (2,500g, 5 min, 4 °C) was used to pellet nuclei and cellular debris. The supernatant was transferred to a new centrifuge tube, and after high-speed centrifugation (17,000g, 30 min, 4 °C), the supernatant was the cytoplasmic fraction. Mitochondria were enriched in the pellet, which was then washed three times with hypotonic buffer. DNA extraction was carried out separately from the cytoplasmic and mitochondrial fractions using the Quick-gDNA MiniPrep Kit (Thermo Fisher Scientific).
Genomic DNA was extracted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific). BMDMs (1 × 106) were collected and centrifuged (250g, 5 min, 4 °C). Lysis buffer and proteinase K solution were added, and samples were incubated at 56 °C for 10 min until cells were completely lysed. Subsequently, the lysate was transferred to DNA purification columns and purified using wash buffer. Genomic DNA was obtained by eluting with elution buffer and centrifugation (8,000g, 1 min).
Chromatin immunoprecipitation assay
The SimpleChIP Plus Sonication ChIP kit (CST) was used for chromatin immunoprecipitation (ChIP) assays. BMDMs (1 × 106) were fixed using 1% formaldehyde and subsequently collected by scraping and lysis. Chromatin was collected and fragmented into 300-bp segments by sonication. The fragments were then subjected to immunoprecipitation using anti-Zbp1 antibody (sc-271483, Santa Cruz, 5 μg) and Protein A/G. Subsequently, the protein–DNA cross-links were reversed, and the DNA was purified. The expression of mitochondrial genes Cyt B and Nd2 were assessed by PCR to validate the binding of mtDNA to Zbp1. The fragments were also subjected to immunoprecipitation using antibodies against Stat3 (#12640, CST, 1:50) and Stat5 (#94205, CST, 1:50). Subsequently, the enrichment of putative binding sites for Stat3/Stat5 in the Zbp1 promoter was evaluated via quantitative real-time PCR (qRT–PCR). The sequences of ChIP primer are listed in Supplementary Table 3.
Microneedle patch construction and characterization
Microneedle (MN) patch was used for the delivery of salvianolic acid B (Sal B), a compound extracted from Salvia miltiorrhiza, into periodontal tissues. In brief, Sal B (0.5 or 1 mg, Aladdin) was dissolved in ddH2O (50 μl), followed by addition of polyethyleneimine (PEI, Aladdin) solution (50 μg/ml, 50 μl) and hyaluronic acid (Aladdin) solution (50 μg/ml, 50 μl). The mixture was vortexed for 30 s, and the resulting solution was allowed to stand for 1 h. Subsequently, the complex solution was added to the polydimethylsiloxane mold with a distribution area of needles measuring 4.8 mm × 4.8 mm, and arranged in an array of 10 × 10. After this, the mold was subjected to vacuum treatment for 30 min to fill the cavities. The mold was dried in a vacuum desiccator at 25 °C for 30 min. Next, an 18% aqueous polyvinyl alcohol (PVA) solution was added to the mold, which underwent vacuum treatment for an additional 30 min. The mold was stored in a vacuum desiccator at 25 °C overnight to obtain MN patches.
A texture analyzer (Brookfield CT3) was used to assess the mechanical strength of the MN patches. The MN patches were positioned with the needle tips facing upward, and the force magnitude and deformation of the MNs were recorded under a vertical compression force applied above the MNs. To further validate the effective delivery of Sal B loaded into the MNs, patches were immersed in 1 ml of PBS for 10, 20 and 30 s. The solution was then collected, and the concentration of Sal B was measured using Agilent 1260 Infinity II LC System (Agilent) to assess the release efficiency from MN loading.
Statistical analysis
Data are presented as mean ± standard deviation, and statistical analyses were carried out using GraphPad Prism 9 software. Statistical significance of two-group comparisons was assessed using a two-tailed unpaired Student’s t-test. The statistical significance of the differences between more than two groups was calculated using one-way analysis of variance with Tukey’s post-hoc test. Significance was set at P < 0.05.
The sequences of small interfering RNAs (siRNAs) used in this study are listed in Supplementary Table 4. For complete materials and methods, please refer to the Supplementary Information.
Results
P. gingivalis infection triggered macrophage infiltration and induced apoptosis and necrosis in periodontal tissues
To investigate the cell population composition and regulatory pathways in periodontitis, we analyzed periodontal samples from four healthy individuals and five patients with severe periodontitis using the single-cell sequencing dataset GSE171213 (ref. 23) (Fig. 1a, b). Given the pivotal role of monocyte clusters in the innate immune response, we focused on their involvement in periodontitis pathophysiology (Fig. 1c and Supplementary Fig. 1a). The analysis revealed a statistically significant increase in the abundance of macrophage clusters, particularly proinflammatory M1 macrophages, in periodontitis compared with healthy controls (Fig. 1d–f and Supplementary Fig. 1b).
a t-Distributed Stochastic Neighbor Embedding (t-SNE) plot and stacked bar plots showing cell subtypes of periodontal samples from the single-cell sequencing dataset GSE171213. b Dot plots showing average expression levels (Z-scaled) and percentage of expressed cells (dot size) of marker genes in major cell types. c Cell subtypes of monocyte clusters. d t-SNE plot representing the macrophage clusters of cells identified across healthy and periodontitis. e Increased abundance of macrophage clusters in periodontitis samples. f Elevated proportion of proinflammatory M1 macrophages in periodontitis. g Top 10 bacterial genera in subgingival plaque. h FISH and IF staining showing increased P. gingivalis accumulation and macrophage infiltration in periodontitis tissues. n = 5. Scale bar, 50 μm. i KEGG enrichment analysis revealing abnormal increase in apoptosis and necrosis pathways in periodontitis macrophages. j, k Elevated TUNEL-positive and p-Mlkl+ cells in periodontitis tissues. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.
Oral microbiota is vital in the development of periodontitis. Therefore, subgingival plaque samples were collected from five healthy individuals and six patients with periodontitis, and subsequently subjected to 16S rRNA sequencing. Microbiota analysis revealed increased alpha diversity in patients with periodontitis, reflecting a richer microbial community compared with healthy controls, and beta diversity analysis revealed notable differences in microbial composition between samples (Supplementary Fig. 1c, d). Figure 1g delineates the top 10 bacterial genera in abundance, with a notable enrichment of P. gingivalis in patients with periodontitis compared with healthy individuals. FISH and immunofluorescence (IF) staining confirmed a substantial increase in P. gingivalis accumulation and macrophage infiltration in gingival samples from patients with periodontitis (Fig. 1h).
Studies have shown that, during immune response, macrophage death can accelerate the release of inflammatory factors, promoting gingival destruction and periodontal bone loss24,25. Notably, we analyzed upregulated DEGs in macrophages from periodontitis patients compared with healthy individuals, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed a marked increase in apoptosis and necrosis pathways in periodontitis samples (Fig. 1i). IF staining confirmed the enhanced expression of proinflammatory cytokines IL-1β and TNF-α in periodontitis tissues (Supplementary Fig. 1e). Furthermore, sagittal cross-sections of gingival tissues revealed a statistically significant enrichment of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive and phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)+ cells in the periodontitis group compared with healthy individuals (Fig. 1j, k), which highlighted the substantial activation of cell death pathways in the context of periodontal inflammation.
Increased cell apoptosis and necrosis in P. gingivalis-infected mouse periodontal tissues
To further explore the potential impact of sustained P. gingivalis infection on periodontal inflammation, we established a mouse model of periodontitis involving the maxillary second molar (Fig. 2a). Antibiotics were administered to the mice for 7 days, resulting in a statistically significant reduction in bacterial colonization in the oral cavity (Fig. 2b). Subsequently, the mice with silk ligatures were randomly divided into two groups, receiving either PBS or P. gingivalis suspension treatment. Micro-computed tomography (micro-CT) imaging (Fig. 2c) and hematoxylin and eosin (HE) staining (Fig. 2d) indicated a statistically significant increase in both gingival recession and alveolar bone resorption in P. gingivalis-infected mice compared with control mice. Tartrate resistant acid phosphatase (TRAP)-positive osteoclasts were markedly increased in the infected mice (Fig. 2e). In addition, we noted significant macrophage infiltration within the P. gingivalis-infected mice (Fig. 2f). Moreover, upregulated expression of proinflammatory markers Il-1β and Tnf-α was observed in P. gingivalis-infected periodontal tissues (Supplementary Fig. 2). Importantly, a significant increase in cell apoptosis and necrosis was detected in periodontitis tissues, along with higher relative proportions of TUNEL+F4/80+ and p-Mlkl+F4/80+ macrophages within the apoptotic and necrotic cell populations (Fig. 2g, h). These findings further indicated that P. gingivalis infection can exacerbate periodontitis and contribute to increased periodontal cell death.
a Establishment of a mouse periodontitis model involving the maxillary second molar. n = 5. b Reduced bacterial colonization after antibiotics treatment. n = 10. Representative micro-CT (c) and HE (d) staining images illustrating gingival recession and alveolar bone resorption in P. gingivalis-infected mice. n = 5. Scale bar, 50 μm. e Enhanced TRAP-positive osteoclasts in P. gingivalis-infected periodontitis tissues. n = 5. Scale bar, 50 μm. f–h Increased macrophage infiltration (f), cell apoptosis (g) and necrosis (h) in periodontal tissues from mice infected with P. gingivalis. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.
P. gingivalis infection activated Zbp1 in BMDMs
To further explore the impact of P. gingivalis infection on macrophages phenotype, we co-cultured mouse BMDMs with P. gingivalis (MOI of 100) for 24 h. The results revealed significantly higher expression of IL-1β and TNF-α, along with an increased M1 polarization in P. gingivalis-infected BMDMs, compared with BMDMs treated with PBS (Supplementary Fig. 3a–c). Moreover, YO-PRO-1/propidium iodide (PI) staining revealed that P. gingivalis stimulation increased apoptosis and necrosis in BMDMs (Supplementary Fig. 3d).
Currently, there is limited information on the mechanism by which oral microbiota induce macrophage death in periodontitis. Therefore, we subjected total RNA isolated from BMDMs treated with PBS or P. gingivalis to RNA sequencing (n = 4). A volcano plot displayed a total of 1068 identified DEGs (log2FC ≥1.5 and P < 0.05), including 500 upregulated genes and 568 downregulated genes (Fig. 3a). Immune-response-related markers were dysregulated in P. gingivalis-infected BMDMs (Supplementary Fig. 3e). Subsequently, we sought to uncover the intersection between upregulated DEGs and genes associated with cell death, and 18 genes were screened common to both datasets (Fig. 3b and Supplementary Fig. 3f). Importantly, qRT–PCR assays revealed the significant upregulation of Zbp1 in P. gingivalis-infected BMDMs (Fig. 3c).
a Volcano plot of 1,068 DEGs (500 upregulated, 568 downregulated) in BMDMs treated with PBS or P. gingivalis (n = 4, log2FC ≥1.5 and P < 0.05). b Eighteen upregulated DEGs associated with cell death. c qRT–PCR showing significant upregulation of Zbp1 expression in P. gingivalis-infected BMDMs. n = 3. GO (d) and KEGG (e) enrichment analyses revealing cell death-related pathways abnormalities. f FISH and IF staining showing increased Zbp1 expression in P. gingivalis-infected BMDMs. n = 3. Scale bar, 10 μm. g Higher Zbp1 signals in P. gingivalis-induced BMDMs, displaying both dose- and time-dependent regulation. n = 3. h–j qRT–PCR analysis revealing elevated IL-1β, TNF-α and ZBP1 expression in periodontitis tissues. n = 5. *P < 0.05. k Western blotting analyses of Zbp1 expression in gingival tissues from healthy and periodontitis individuals. n = 5. l, m IHC and IF assays confirming upregulated Zbp1 expression in CD68+ and F4/80+ macrophages. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.
As a crucial innate immune sensor, Zbp1 mediates a wide range of cell death processes, leading to PANoptosome assembly26. Gene Ontology (GO) and KEGG enrichment analyses further indicated upregulation of pathways related to PANoptosis (including pyroptosis, apoptosis and necrosis) in P. gingivalis-infected macrophages compared with the control group (Fig. 3d, e). FISH and IF staining demonstrated that, after infection with P. gingivalis, the bacteria were engulfed by BMDMs, accompanied by increased Zbp1 expression in the infected macrophages (Fig. 3f). Moreover, western blotting assays revealed significantly higher Zbp1 signals in P. gingivalis-infected BMDMs than in PBS-treated cells, displaying both dose- and time-dependent regulation (Fig. 3g). Analyses of gingival tissues from healthy and periodontitis individuals demonstrated a statistically significant increase in the expressions of IL-1β, TNF-α and ZBP1 in infected tissues (Fig. 3h–k). Immunohistochemistry (IHC) and IF assays of human and mouse periodontal tissues further confirmed the upregulation of Zbp1 expression, particularly in CD68+ and F4/80+ macrophages (Fig. 3l, m). In summary, these data support the finding that P. gingivalis infection triggers Zbp1 expression in macrophages.
P. gingivalis infection amplified Zbp1-mediated PANoptosis signaling
To elucidate the regulatory role of Zbp1 in P. gingivalis-mediated macrophage PANoptosis, Zbp1-specific siRNAs were used (Fig. 4a). The enzyme-linked immunosorbent assay (ELISA) revealed a significant reduction in the expression levels of IL-1β and TNF-α in the supernatants of P. gingivalis-infected macrophages upon Zbp1 inhibition (Fig. 4b), accompanied with a decrease in lactate dehydrogenase (LDH) release (Fig. 4c). Moreover, YO-PRO-1/PI staining confirmed a significant increase in apoptosis and necrosis upon P. gingivalis infection, which was partially mitigated by Zbp1 siRNAs (Fig. 4d).
a Zbp1 knockdown using Zbp1-specific siRNAs. n = 3. ELISA assay showing suppressed IL-1β, TNF-α (b) and LDH (c) release upon inhibition. n = 3. d YO-PRO-1/PI staining showing decreased apoptosis and necrosis with Zbp1 siRNA treatment. n = 3. Scale bar, 50 μm. Western blotting (e) and quantitative analysis (f) demonstrated decreased p-Ripk3, cleaved Caspase8, cleaved Caspase3, Nlrp3, cleaved Gsdmd and p-Mlkl levels after Zbp1 knockdown. n = 3. g Increased ROS levels in BMDMs upon P. gingivalis infection. n = 3. Scale bar, 50 μm. h JC-1 staining showing reduced ΔψM after P. gingivalis infection. n = 3. Scale bar, 50 μm. i Swollen mitochondrial morphology in P. gingivalis-stimulated BMDMs. Scale bar, 500 nm. j, k Elevated mtROS levels and impaired ATP generation in P. gingivalis-infected BMDMs. n = 3. Scale bar, 50 μm. l Fluorescence of MitoTracker revealing increased mitochondrial fragmentation upon P. gingivalis stimulation. n = 3. Scale bar, 10 μm. m PCR assays showing significant cytoplasmic expression of mitochondrial genes Cyt B and Nd2 after P. gingivalis infection. n = 3. n Interactions between mtDNA and Zbp1 identified using a ChIP experiment. n = 3. o Undetectable Cyt B and Nd2 in Zbp1-bound immunoprecipitates after EtBr treatment (10 μg/ml). n = 3. All data were derived from independent experiments. ns, no significance, *P < 0.05.
Previous studies have suggested the role of the ZBP1–receptor-interacting protein kinase 3 (RIPK3) complex in triggering pyroptosis and apoptosis through the recruitment and activation of cysteine–aspartic acid protease 8 (Caspase8)15,27. In addition, RIPK3 is associated with the phosphorylation and subsequent activation of mixed-lineage kinase domain-like protein (MLKL), leading to necrosis28. In this study, the siRNA targeting Zbp1 significantly reduced the P. gingivalis-induced upregulation of Zbp1 expression (Supplementary Fig. 4a). Importantly, Zbp1 knockdown resulted in a reduction in the levels of p-Ripk3, cleaved Caspase8, cleaved cysteine–aspartic acid protease 3 (Caspase3, a crucial enzyme in the apoptosis pathway), NOD-like receptor pyrin domain-containing 3 (Nlrp3) and cleaved Gasdermin D (Gsdmd, a key protein in the pyroptosis pathway), and p-Mlkl (a crucial protein in the necroptosis pathway) in P. gingivalis-infected BMDMs (Fig. 4e, f), indicating a comprehensive inhibition of PANoptosis pathways. Consistently, IF assays showed decreased expression of cleaved Gsdmd and cleaved Caspase3 in the Zbp1 siRNA groups infected with P. gingivalis (Supplementary Fig. 4b). Furthermore, the detection of p-Mlkl with the antibody revealed distinct localization of p-Mlkl fluorescence on the plasma membrane upon P. gingivalis stimulation, consistent with the translocation observed during necrotic activation. By contrast, co-transfection with Zbp1 siRNAs reduced the plasma membrane localization of p-Mlkl (Supplementary Fig. 4c). Together, these results suggested that Zbp1 knockdown inhibited the activation of P. gingivalis-mediated macrophage pyroptosis, apoptosis and necrosis, further confirming the crucial role of Zbp1 in P. gingivalis-induced PANoptosis.
During bacterial infections, host immune cells are activated, leading to elevated oxidative stress levels. Excessive reactive oxygen species (ROS) can compromise mitochondrial function by increasing the permeability of the mitochondrial outer membrane, facilitating the release of mtDNA into the cytoplasm. Given the DNA recognition properties of ZBP1, the released mtDNA may be recognized by ZBP1, triggering the activation of downstream signaling pathways. Therefore, investigating the changes in mitochondrial function is essential for elucidating the mechanism of ZBP1-mediated cell death signal amplification during bacterial infections29. Figure 4g demonstrated a significant increase of ROS levels in BMDMs after P. gingivalis infection. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining indicated a decreased red/green fluorescence ratio after P. gingivalis infection, suggesting a notable decline in mitochondrial membrane potential (ΔψM) (Fig. 4h). In addition, transmission electron microscopy revealed swelling mitochondrial morphology in infected BMDMs (Fig. 4i). MitoSOX staining and adenosine triphosphate (ATP) detection suggested elevated mitochondrial ROS (mtROS) levels and impaired ATP production in the infected BMDMs (Fig. 4j, k). IF staining of MitoTracker showed increased fragmentation of mitochondrial morphology upon P. gingivalis stimulation (Fig. 4l). PCR assays revealed statistically significant cytoplasmic expression of mitochondrial genes Cyt B and Nd2 after P. gingivalis infection, confirming the release of mtDNA into the cytoplasm (Fig. 4m).
To determine whether mtDNA can bind to the Zα domains of Zbp1 and facilitate downstream signal amplification, a ChIP experiment using Zbp1 antibody was conducted. As depicted in Fig. 4n, the detection of Cyt B and Nd2 in the Zbp1 immunoprecipitates of the P. gingivalis-stimulated group indicated interactions between mtDNA and Zbp1 (Fig. 4n). Furthermore, depleting mtDNA with ethidium bromide (EtBr) led to undetectable levels of Cyt B and Nd2 in Zbp1-bound immunoprecipitates (Fig. 4o) and reduced expression of PANoptosis-related indicators (Supplementary Fig. 4d,e). In summary, P. gingivalis infection induced mitochondrial oxidative stress and promoted mtDNA release, thereby amplifying Zbp1-mediated downstream PANoptosis signaling.
Deletion of Zbp1 protected against PANoptosis activation and periodontal tissue destruction in P. gingivalis-induced mouse periodontitis
To further elucidate the regulatory role of Zbp1-mediated PANoptosis in P. gingivalis-induced periodontitis, Zbp1-knockout mice (mZbp1KO, Zbp1−/−) mice were generated and IHC staining confirmed the deficiency of Zbp1 in the periodontal tissues (Fig. 5a). The levels of IL-1β and TNF-α after P. gingivalis infection decreased in the BMDMs derived from Zbp1−/− mice compared with those from WT mice (Supplementary Fig. 5a, b). The YO-PRO-1/PI staining assay further verified that BMDMs derived from Zbp1−/− mice showed marked inhibition of macrophage apoptosis and death induced by P. gingivalis infection (Fig. 5b), and a similar expression pattern of LDH was observed (Fig. 5c).
a IHC staining confirming Zbp1 deficiency in periodontal tissues of Zbp1−/− mice. Scale bar, 50 μm. Decreased apoptosis, necrosis (b) and LDH release (c) in BMDMs from Zbp1−/− mice. n = 3. Scale bar, 50 μm. d Establishment of a mouse periodontitis model in WT and Zbp1−/− mice. n = 5. e Representative micro-CT images revealing diminished periodontal tissue destruction in Zbp1−/− mice. n = 5. Diminished periodontal tissue destruction (f) and reduced osteoclastogenesis (g) in Zbp1−/− mice after P. gingivalis challenge. n = 5. Scale bar, 50 μm. h Reduced cleaved Gsdmd, cleaved Caspase3 and p-Mlkl expression in periodontitis lesions of Zbp1−/− mice. Scale bar, 50 μm. i, j Protein analysis from periodontal tissues showing Zbp1 deficiency alleviating upregulation of p-Ripk3, cleaved Caspase8, cleaved Caspase3, Nlrp3, cleaved Gsdmd and p-Mlkl induced by P. gingivalis infection. n = 3. All data were derived from independent experiments. *P < 0.05.
Based on a ligature-induced mouse model of periodontitis, P. gingivalis was injected vertically into the alveolar crest of WT and Zbp1−/− mice (Fig. 5d). Zbp1−/− mice exhibited diminished periodontal destruction, reduced osteoclastogenesis and reduced proinflammatory cytokine expression (Fig. 5e–g and Supplementary Fig. 5c,d). IF staining showed reduced expression of cleaved Gsdmd, cleaved Caspase3 and p-Mlkl in periodontitis tissues from Zbp1−/− mice (Fig. 5h). Furthermore, protein analysis from periodontal tissues revealed that Zbp1 deficiency mitigated the upregulation of PANoptosis signaling stimulated by P. gingivalis (Fig. 5i, j). These findings suggest that the specific deletion of Zbp1 protects against PANoptosis activation and periodontal tissue destruction in P. gingivalis-induced periodontitis.
P. gingivalis activated Zbp1 in BMDMs via the Tlr2/4–JNK pathways
Previous studies have highlighted the pivotal role of Toll-like receptor (TLR) family in mediating interactions between microbial flora and immune cells30. Our KEGG enrichment analysis verified substantial activation of the TLR pathway in P. gingivalis-stimulated BMDMs (Fig. 3e). To further investigate the pattern recognition receptors responsible for macrophage recognition by P. gingivalis, we filtered the relative expression of Tlr genes (Fig. 6a) and identified pronounced upregulation of Tlr2 and Tlr4 by qRT–PCR (Supplementary Fig. 6a). Myeloid differentiation primary response 88 (Myd88) is an essential bridging protein for downstream signaling by all TLRs except Tlr3 (ref. 31). In this study, IHC assays further demonstrated that P. gingivalis increased Tlr2, Tlr4 and Myd88 expression in P. gingivalis-induced mouse periodontitis compared with that in the controls (Fig. 6b and Supplementary Fig. 6b). Furthermore, specific inhibitors targeting the Tlr2 (C29), Tlr4 (TAK-242) and Myd88 (T6167923) pathways effectively counteracted the induction of Zbp1 mRNA and protein expression in BMDMs stimulated with P. gingivalis (Fig. 6c, d and Supplementary Fig. 6c). YO-PRO-1/PI staining confirmed that treatment with C29 and TAK-242 significantly suppressed apoptosis and necrosis induction in P. gingivalis-stimulated BMDMs (Supplementary Fig. 6d). Similar observations were made by IF staining of cleaved Gsdmd, cleaved Caspase3 and p-MLKL (Supplementary Fig. 6e,f). Importantly, daily systemic administration via intraperitoneal injection of C29 (5 mg/kg) and TAK-242 (3 mg/kg) protected against increased alveolar bone resorption and Zbp1 activation in mice infected with P. gingivalis (Fig. 6e–h).
a A heat map showing Tlr gene expression profiles in BMDMs stimulated with P. gingivalis. b IHC assays revealing increased levels of Tlr2 and Tlr4 in P. gingivalis-induced periodontal tissues. n = 5. Scale bar, 50 μm. Tlr2 and Tlr4 inhibitors effectively counteracted Zbp1 gene (c) and protein expression (d) in the infected BMDMs. n = 3. e P. gingivalis-infected mouse periodontitis model with daily intraperitoneal administration of C29 (5 mg/kg) and TAK-242 (3 mg/kg). n = 5. Micro-CT (f), IHC (g) and IF staining (h) showing reduced alveolar bone resorption and Zbp1 activation with C29 and TAK-242 treatment. n = 5. Scale bar, 50 μm. i Western blotting showing increased phosphorylation levels of p65, ERK, JNK and p38 in BMDMs after P. gingivalis stimulation. n = 3. j The JNK inhibitor dose-dependently inhibited Zbp1 expression in the infected BMDMs. n = 3. All data were derived from independent experiments. ns, no significance, *P < 0.05.
Tlr–Myd88 pathway activation can signal downstream to NF-κB (p65) and MAPK (including ERK, JNK and p38) pathways, leading to the production of proinflammatory cytokines32. In this study, KEGG enrichment analysis indicated that p65 and MAPK pathways were enriched after P. gingivalis infection (Fig. 3e). Western blotting assay further demonstrated that P. gingivalis stimulation increased the phosphorylation of p65, ERK, JNK and p38 in BMDMs (Fig. 6i). Notably, noncytotoxic doses of a JNK inhibitor (SP600125) significantly inhibited Zbp1 expression in P. gingivalis-induced BMDMs in a dose-dependent manner, indicating a predominant role for the JNK signaling pathway downstream of Tlr2/4 in controlling Zbp1 transcription (Fig. 6j). These findings demonstrate that Tlr2/4 is involved in the recognition of P. gingivalis by macrophages, which subsequently activates the JNK pathway, leading to the activation of Zbp1 and driving periodontal inflammation and bone resorption.
P. gingivalis activated transcription factors (TFs) Stat3 and Stat5 to promote Zbp1 transcription
To delve into the intricate mechanism by which Tlr2/4–JNK promotes Zbp1 activation after P. gingivalis stimulation, we conducted a detailed bioinformatics analysis to identify potential TFs involved in Zbp1 transcription using the Jasper database (https://jaspar.genereg.net/). The predicted scores indicated that Zbp1 transcription may be governed by the binding of multiple TFs, including Myc, SP1, Stat3, Sox2 and Stat5 (Supplementary Fig. 7a). We subsequently performed qRT–PCR and found that the increased Zbp1 expression was blunted by Stat3 inhibitor (Stattic) and Stat5 inhibitor (AC-4-130), implying that Stat3 and Stat5 are crucial TFs for Zbp1 induction (Fig. 7a). Western blotting assays demonstrated that P. gingivalis infection significantly enhanced the phosphorylation levels of Stat3 and Stat5 (Fig. 7b). In addition, IF staining revealed the nuclear translocation of Stat3 and Stat5 in BMDMs after P. gingivalis infection (Supplementary Fig. 7b,c). More importantly, pharmacological blocking and genetic depletion of the Tlr2/Tlr4–JNK pathway were shown to block the phosphorylation of Stat3 and Stat5, confirming their activation by the Tlr2/Tlr4–JNK pathway in P. gingivalis-stimulated BMDMs (Fig. 7c–e).
a P. gingivalis-induced BMDMs showing decreased Zbp1 expression with Stattic and AC-4-130 treatment. n = 3. b Western blotting showing increased phosphorylation of Stat3 and Stat5 after P. gingivalis infection. n = 3. Pharmacological inhibition (c, d) and genetic depletion (e) of the Tlr2/Tlr4–JNK pathway reduced the phosphorylation of Stat3 and Stat5 in P. gingivalis-stimulated BMDMs. n = 3. f Putative binding sites of Stat3 and Stat5 in the Zbp1 promoter regions. g ChIP assays revealing significant Stat3 and Stat5 enrichment in the predicted binding regions of Zbp1 promoter. n = 3. h Luciferase reporter assays validating the regulatory roles of Stat3 and Stat5 in Zbp1 transcription. n = 3. i P. gingivalis-infected mouse periodontitis model with daily intraperitoneal administration of Stattic (5 mg/kg) and AC-4-130 (5 mg/kg). n = 5. Micro-CT images (j), IHC (k) and IF staining (l) showing decreased alveolar bone resorption and Zbp1 activation with Stattic and AC-4-130 treatment. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. ns, no significance, *P < 0.05.
Further analysis of the putative binding sites of Stat3 and Stat5 in the Zbp1 promoter revealed predicted binding regions with high affinity score (Fig. 7f). Primers were designed accordingly, and a ChIP assay was performed using specific Stat3 and Stat5 antibodies. The qRT–PCR showed statistically significant difference in enrichment of Zbp1 in this binding region compared with the IgG control (Fig. 7g). Furthermore, luciferase reporters were constructed to insert the Zbp1 promoter DNA sequence into the reporter gene vector (pGL3-Basic). Results showed that co-transfection with Stat3 and Stat5 plasmids enhanced luciferase activity, validating their regulatory roles in Zbp1 transcription (Fig. 7h). In a mouse model of periodontitis, daily intraperitoneal administration of Stat3 (5 mg/kg) and Stat5 (5 mg/kg) inhibitors significantly reduced P. gingivalis-induced periodontal resorption and Zbp1 induction (Fig. 7i-l). Collectively, these findings suggested that Stat3 and Stat5 activation mediated by the Tlr2/Tlr4–JNK pathway regulated Zbp1 induction in P. gingivalis-stimulated BMDMs.
MNs delivery of Sal B alleviated P. gingivalis-induced periodontal resorption and promoted mucosal wound healing
To expand our search for potential bioactive compounds targeting Zbp1, we utilized the Comparative Toxicogenomics Database (https://pwaqa40.ctdbase.org/) and predicted the interactions between Danshen extract and the target protein Zbp1 (Supplementary Fig. 8a). Danshen is a traditional Chinese medicine widely used to prevent and treat vascular diseases33. Subsequently, a water-soluble polyphenolic antioxidant extracted from Danshen—Sal B (Fig. 8a)—was chosen for subsequent investigation owing to its reported inhibition of proinflammatory pathways34,35. Molecular docking simulations were performed to explore the interaction between Sal B and Zbp1 (Fig. 8b), revealing binding energies lower than −5 kcal/mol (−7.2 kcal/mol), indicative of a favorable binding activity. In addition, in vitro experiments showed that 50 μM Sal B was nontoxic and notably suppressed the upregulation of inflammatory markers induced by P. gingivalis (Supplementary Fig. 8b,c). Further characterization of YO-PRO-1/PI staining revealed that Sal B intervention inhibited apoptosis and death associated with P. gingivalis stimulation (Fig. 8c). Western blotting assays indicated that Sal B treatment significantly reduced Zbp1 and p-Ripk3 levels (Fig. 8d), leading to inhibited downstream PANoptosis signal transduction (Supplementary Fig. 8d,e).
a The chemical structure of Sal B. b Molecular docking simulations revealing Sal B binds favorably to Zbp1. c YO-PRO-1/PI staining showing inhibited apoptosis and necrosis with Sal B intervention. n = 3. Scale bar, 50 μm. d Sal B treatment significantly reduced Zbp1 and p-Ripk3 levels. n = 3. e Encapsulation of Sal B (0.5 mg or 1 mg) into PVA MN patches. f Sal B@MNs demonstrating mechanical strength exceeding 1 N per needle. g Dissolution of the modified MN tips in PBS. h Delivery efficiency of Sal B-1@MN and Sal B-2@MN. n = 3. i P. gingivalis-infected mouse periodontitis and hard palate wound healing models with daily administration of Sal B@MNs. n = 5. Representative images and HE staining illustrating reduced periodontal tissue resorption (j, k) and and near-complete wound healing (l, m) in mice treated with Sal B@MNs. n = 5. Scale bar, 50 μm. IHC images showing lower Zbp1 expression in the periodontitis (n) and hard palate wound healing (o) tissues of Sal B@MNs-treated mice. n = 5. Scale bar, 50 μm. All data were derived from independent experiments. *P < 0.05.
MNs have been reported as a novel physical transdermal technology, effectively facilitating drug delivery36. In this study, we encapsulated either 0.5 mg or 1 mg Sal B into a 10 × 10 array of the PVA MN patches (labeled as Sal B-1@MN and Sal B-2@MN), respectively (Fig. 8e). The MNs exhibited a mechanical strength exceeding 1 N per needle, indicating their ability to penetrate the oral mucosal tissue (Fig. 8f). In vitro experiments confirmed the dissolution of the modified MN tips in PBS within 30 s, whereas the backing layer remained intact (Fig. 8g). The delivery efficiency was further validated before and after MN immersion in PBS for 30 s, reaching up to 83.88% and 80.85% for Sal B-1@MN and Sal B-2@MN, respectively (Fig. 8h). We subsequently established a ligature-induced periodontitis model and a hard palate wound healing model in mice, in which a P. gingivalis suspension was injected vertically onto the alveolar crest or wound edge every other day (Fig. 8i). Sal B@MNs were administered daily to the local mucosa surrounding periodontitis or palatal wounds. By day 14 after model establishment, the mice treated with P. gingivalis and Sal B @MN showed reduced periodontal tissue resorption (Fig. 8j, k) and nearly complete wound healing (Fig. 8l, m). No notable difference was observed in the therapeutic effects of the two concentrations of Sal B. IHC images revealed a lower expression of Zbp1 in the periodontal and palatal mucosa tissues of Sal B @MN treatment mice than in the P. gingivalis-stimulated groups (Fig. 8n, o). In summary, these findings suggest that the local delivery of Sal B by MNs might prevent P. gingivalis-mediated Zbp1 activation, thereby suppressing periodontal inflammation and tissue resorption.
Discussion
Macrophages are crucial for combating persistent infections and providing protection to infected tissues. However, pathogen-induced cell death during this process can lead to damage and severe disease37. Accumulating evidence indicates that different death processes are not mutually exclusive; instead, they interact as a collective series of events7. Although apoptosis has traditionally been considered immunologically silent, studies on the crosstalk between apoptosis, pyroptosis and necroptosis suggest that apoptotic cell death can also cause inflammation in certain cell types38. In this study, we propose a novel pathogenic model of P. gingivalis-induced periodontitis, suggesting that P. gingivalis activates Zbp1-mediated PANoptosis in macrophages, thereby contributing to periodontal tissues damage. More importantly, Zbp1 deletion in mice alleviated periodontal cell death and tissue resorption. Therefore, Zbp1 may be a crucial therapeutic target for blocking proinflammatory cell death and preventing P. gingivalis-mediated periodontal damage.
Under conditions of periodontitis, sustained inflammation and tissue damage contribute to elevated oxidative stress39. The interaction of P. gingivalis lipopolysaccharides and ATP has been shown to elevate intracellular ROS levels, activating NF-κB–NLRP3 signaling and inducing gingival fibroblast death40. In this study, we validated the involvement of mtROS disruption and mtDNA, into the cytoplasm. Cytoplasmic mtDNA acts as an intracellular damage signal recognized by DNA sensors, triggering inflammatory pathways41. Our investigation revealed the interaction of Zbp1 with mtDNA, eliciting downstream PANoptosis signal amplification. In addition, studies have reported that intracellular receptor cGAS can bind to sting, further catalyzing cGAMP production, thereby activating the STING pathway and ultimately facilitating intracellular inflammatory responses and programmed immune cell death42. Furthermore, cytoplasmic mtDNA release can directly activate the NLRP3 complex, prompting the maturation and release of proinflammatory cytokine IL-1β and IL-18, further propagating cellular apoptosis43. Therefore, a future research direction may involve a more comprehensive elucidation of the complex mechanisms controlling oxidative stress and cell death in periodontal pathology.
Tlr2 and Tlr4 are well established as central pathogens recognition receptors in immune cells30. Bacterial pathogen-associated molecular patterns activate the Tlr2/Tlr4–Myd88 pathway in pathological environments, converting infection signals into bone resorption signals, activating RANKL expression and inducing bone destruction44. However, the precise role of these receptors in periodontitis-related cell death remains unclear. Our data suggested that the activation of Tlr2/4 was essential for the recognition of P. gingivalis by macrophages, mediating Zbp1-activated downstream cell death pathways. Mechanistically, activation of the Tlr2/Tlr4–JNK pathway increased the phosphorylation of Stat3 and Stat5, and blocking these TFs prevented Zbp1 induction in macrophages. Studies have reported that Stat3 and Stat5 facilitate the transcription of various inflammatory mediators, including IL-6 and IL-17, by directly targeting their promoter regions, thereby amplifying the inflammatory response45. However, their involvement in cell-death-related pathways is multifaceted. Under normal conditions, Stat3 and Stat5 serve as antiapoptotic factors, shielding cells from damage46. Conversely, in certain circumstances, the activation of Stat3 and Stat5 may also contribute to the modulation of inflammation-associated cell death processes, promoting apoptosis and necrosis47. This duality could be influenced by factors such as cell type, environmental cues and upstream signaling pathway regulation, warranting further investigation in specific contexts.
Sal B is a bioactive component isolated from Danshen, involving diverse anti-inflammatory mechanisms33. Functioning as a natural antioxidant, Sal B demonstrates substantial antioxidative activity, mitigating the adverse effects of oxidative stress during inflammation48. Furthermore, Sal B can modulate various inflammation-related signals, including NF-κB, MAPK and STAT pathways35,49. Studies have also indicated that Sal B administration reduces the prevalence of M1-polarized macrophages and alleviates the release of inflammatory factors50. In this study, MNs loaded with Sal B have been developed to facilitate drug penetration. Upon penetration of the tissues by the crosslinked MNs, the interstitial fluid swelled, leading to the release of Sal B loaded inside through free diffusion. The treatment inhibited local Zbp1 expression, reduced periodontal tissue damage and enhanced healing of the palatal mucosa. Studies have demonstrated that Sal B can influence protein expression through several pathways, including: (1) promoting protein degradation, such as via the ubiquitin-proteasome pathway51; (2) inhibiting transcription or translation, thus reducing protein synthesis52; and (3) altering protein stability or intracellular localization53. Based on the finding that Sal B reduces Zbp1 protein expression, we hypothesize that Sal B may directly bind to Zbp1, inducing conformational changes that lead to its degradation or disrupt its interactions with other molecules; however, further investigations are required to elucidate the precise mechanism. Overall, our findings suggest that Sal B may offer novel therapeutic potential for periodontitis and other inflammatory diseases of the oral cavity, expanding its current applications.
Collectively, our results highlight the crucial role of Zbp1-mediated PANoptosis in periodontitis. As a crucial oral pathogen, P. gingivalis triggers a mitochondrial stress response, leading to the release of mtDNA. This mtDNA then interacte with Zbp1, consequently augmenting its downstream PANoptosis signals. In addition, P. gingivalis stimulates macrophage Zbp1 expression through the Tlr2/4–JNK–Stat3/5 pathway, exacerbating periodontal inflammation and tissue destruction. The pharmacological delivery of Sal B through MNs effectively inhibited Zbp1 levels, prevented severe periodontal damage. In conclusion, Sal B@MNs and other drugs targeting Zbp1 and the downstream regulatory mechanism of PANoptosis may represent new strategies for alleviating periodontal tissue destruction and treating periodontitis. These approaches hold promise for advancing therapeutic interventions for periodontal health.
Data availability
The single-cell sequencing dataset GSE171213 is publicly available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/). All data are available from the corresponding author on reasonable request.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China under grant no. 82170993; National Natural Science Foundation of China under grant no. 82201097; National Natural Science Foundation of China under grant no. 82303457; Natural Science Foundation of Jiangsu Higher Education Institutions of China under grant no. 23KJB320003; Open Project Fund of Jiangsu Provincial Key Laboratory of Oral Disease Research under grant no. JSKLOD-KF-2304; China Postdoctoral Science Foundation under grant no. 2023M741789; and China Postdoctoral Science Foundation under grant no. 2023M741788 to X.H.
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J.W., X.L. and C.T. developed the study concept and design. J.W., Z.G. and Y.S. performed experiments. Z.Z., X.H. and J.Z. provided acquisition, analysis and interpretation of data, and statistical analysis. L.W. and K.C. were responsible for the production of MNs. J.W. and Z.G. performed writing, review and revision of the paper. All authors proofread and approved the final paper.
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Wu, J., Guo, Z., Wang, L. et al. Porphyromonas gingivalis induces Zbp1-mediated macrophages PANoptosis in periodonitis pathophysiology. Exp Mol Med 57, 964–978 (2025). https://doi.org/10.1038/s12276-025-01443-y
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DOI: https://doi.org/10.1038/s12276-025-01443-y
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