MZB1 Drives Periodontitis via ER Stress-Mediated Inflammation and Osteogenic Differentiation

Abstract

Introduction and aims

Increasing evidence suggests that marginal zone B and B1 cell-specific protein (MZB1) plays crucial roles in inflammatory responses, endoplasmic reticulum (ER) stress, and cell survival, but its function in periodontitis is still unclear. Periodontitis is a chronic inflammatory disease. This study aimed to investigate the expression profile and functional role of MZB1 in periodontitis.

Methods

Transcriptomic analysis and RT-qPCR were performed to evaluate MZB1 expression in gingival tissues of periodontitis patients. An in vitro inflammatory model of periodontal ligament stem cells (PDLSCs) was established using lipopolysaccharide (LPS), and an in vivo rat periodontitis model was constructed. Loss-of-function assays were conducted via shRNA or adeno-associated virus (AAV)-mediated knockdown of MZB1. The effects of MZB1 knockdown on cell proliferation, apoptosis, ER stress, and osteogenic differentiation were assessed.

Results

Transcriptomic analysis revealed that MZB1 was significantly upregulated in gingival tissues of periodontitis patients and enriched in inflammation-related pathways. Knockdown of MZB1 alleviated LPS-induced damage in PDLSCs, improved cell viability and proliferation, reduced ER stress and apoptosis. Moreover, MZB1 depletion significantly restored the osteogenic differentiation potential of PDLSCs, as evidenced by increased mineralised nodule formation, alkaline phosphatase (ALP) activity, and elevated expression of osteogenesis-related proteins (RUNX2, BMP2, and Collagen I). In vivo, AAV-mediated MZB1 knockdown ameliorated periodontal inflammation, tissue destruction, and functional impairment.

Conclusion

MZB1 downregulation alleviates inflammation, suppresses ER stress and apoptosis, and enhances the osteogenic capacity of PDLSCs, indicating that MZB1 plays a crucial role in the development and progression of periodontitis.

Clinical Relevance

Targeting MZB1 represents a promising therapeutic strategy. Inhibiting MZB1 could alleviate ER stress, enhance bone regeneration, and restore periodontal tissue homeostasis, potentially leading to more effective and regenerative treatments for periodontitis.

Introduction

Periodontitis is a chronic, multifactorial inflammatory oral disease that primarily affects the supporting structures of the teeth, including the gingiva, periodontal ligament, alveolar bone, and cementum.^1,2 It is one of the most prevalent oral diseases worldwide and the leading cause of tooth loss in adults.³ The progression of periodontitis results from a complex interplay between a dysbiotic microbial community within the dental plaque biofilm and the host immune-inflammatory response, ultimately leading to the destruction of periodontal connective tissue and alveolar bone resorption.^4,5 Clinical diagnosis typically relies on multiple parameters, including clinical attachment loss (CAL), probing depth (PD), bleeding on probing (BOP), and radiographic evidence of alveolar bone loss.^6,7 However, due to the lack of prominent symptoms in the early stages, periodontitis is often diagnosed only after significant disease progression. In advanced stages, persistent inflammation and limited regenerative capacity pose major challenges to achieving long-term disease control.⁸

Porphyromonas gingivalis, a key periodontal pathogen, exerts much of its pathogenicity through its lipopolysaccharide (LPS), which has been shown to induce endoplasmic reticulum (ER) stress in host cells.^9,10 This stress response contributes to reduced cell viability, increased apoptosis, and impair osteogenic differentiation.^11,12 In the context of periodontitis, prolonged ER stress can activates the unfolded protein response (UPR), stimulates pro-inflammatory transcriptional programs, and trigger apoptotic pathways, thereby exacerbating alveolar bone loss.¹³ Periodontal ligament stem cells (PDLSCs), which are essential for maintaining periodontal tissue homeostasis and mediating regeneration, are especially susceptible to ER stress under inflammatory conditions. Studies have shown that ER stress is a key factor in suppressing the osteogenic of PDLSCs in such environments.^14,15 Given the pivotal role of PDLSCs in maintaining periodontal homeostasis and promoting tissue regeneration, elucidating the mechanisms regulating their inflammatory response and differentiation is of great significance for developing effective therapeutic strategies for periodontitis.

Marginal zone B and B1 cell-specific protein (MZB1) is an ER-resident co-chaperone protein predominantly expressed in immune cells, especially B cells and plasma cells, which functions in immunoglobulin folding and secretion.^16,17 Increasing evidence indicates that MZB1 plays important roles in modulating inflammatory responses, ER stress, and cell survival.¹⁸ MZB1 overexpression can improve mitochondrial function, increase ATP production, reduce inflammation, and regulate apoptosis.¹⁹ Notably, MZB1 is among the most upregulated genes in periodontitis,²⁰ and it can modulate the migration of human periodontal ligament cells (hPDLCs) via the NF-κB signaling pathway, thereby contributing to alveolar bone loss.²¹ Considering the chronic inflammatory nature of periodontitis and the close association between ER stress and PDLSC dysfunction, MZB1 may play a crucial role in periodontal tissue destruction. Moreover, modulation of MZB1 expression may influence the inflammatory response and regenerative capacity of PDLSCs, offering a potential therapeutic target for periodontitis.

This study aims to investigate the expression profile of MZB1 in periodontitis and elucidate its functional role in inflammatory responses and the osteogenic differentiation of periodontal stem cells. Transcriptomic analysis of gingival tissues from periodontitis patients was conducted to assess MZB1 expression levels and associated signaling pathways. An in vitro inflammatory model of PDLSCs was established using LPS stimulation, alongside an in vivo rat model of periodontitis. Functional loss-of-function experiments were carried out using short hairpin RNA (shRNA) or adeno-associated virus (AAV)-mediated MZB1 knockdown to evaluate its effects on PDLSC proliferation, apoptosis, ER stress response, and osteogenic differentiation potential.

Materials and methods

Data source

Transcriptomic data of gingival tissue samples were obtained from 2 publicly available datasets in the Gene Expression Omnibus (GEO) database: GSE16134 (69 healthy samples and 241 periodontitis samples) and GSE10334 (64 healthy samples and 183 periodontitis samples).

Identification of differentially expressed genes (DEGs) and enrichment analysis

Based on the GPL570 platform, Affymetrix probe IDs from the microarray data were annotated to gene symbols. The expression matrices were normalised using the normalizeBetweenArrays method from the limma R package.²² Batch effects were removed using the “Combat” function from the sva R package. The merged dataset included 424 periodontitis samples and 133 healthy controls. DEGs were defined as those with |log₂FoldChange| > 1 and P < .05. Biological pathway enrichment analysis, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, was conducted using the Metascape platform (http://metascape.org) based on DEGs.

Clinical sample collection and preprocessing

This study recruited 60 participants (30 with periodontitis and 30 healthy controls) from our Hospital. All participants signed informed consent, and the study protocols were approved by the institutional ethics committee. Periodontitis patients met the specific clinical criteria: probing depth (PD) ≥ 5 mm, positive bleeding on probing (BoP), clinical attachment loss (CAL) ≥ 3 mm, alveolar bone resorption (ABL) ≥ 15%) and required surgery after initial treatment. The control group consisted of surgical patients who showed no signs of gingival inflammation. Gingival tissues were divided into 2 portions: one was fixed with 4% paraformaldehyde, dehydrated, and paraffin-embedded for histological analysis; the other was snap-frozen in liquid nitrogen and stored at −80 °C for subsequent molecular assays.

RT-qPCR

Total RNA was extracted using TRIzol reagent. RNA concentration and integrity were assessed by spectrophotometry and agarose gel electrophoresis. First-strand cDNA was synthesised from 1 μg of RNA using 5 × All-In-One RT MasterMix (GeneCopoeia) and incubated at 37 °C for 15 minutes and 60 °C for 10 minutes. The resulting cDNA was diluted 1:1 with RNase-free water and stored at −80 °C. RT-qPCR was performed using BlasTaq 2 × qPCR Master Mix (Applied Biological Materials). Specific primers for MZB1 and β-actin (internal control) were listed in Table S1. Cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative expression levels were calculated using the 2^−ΔΔCt method.

Cell culture and LPS treatment

Rat PDLSCs were purchased from Fenghui Biotechnology. Cells were cultured in mesenchymal stem cell growth medium (MSCGM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO₂ incubator. To induce inflammation, cells were seeded and allowed to adhere for 48 hours, after which the medium was replaced with fresh MSCGM containing 1 μg/mL LPS and the cells were incubated for an additional 72 hours.

Transfection and grouping

PDLSCs were seeded at 1 × 10⁵ cells/mL (2 mL per well in 6-well plates) and incubated overnight, followed by transfected with either control lentivirus or LV-MZB1-RNAi lentivirus targeting 3 specific sites at a multiplicity of infection (MOI) of 100. Transfection efficiency of MZB1 knockdown was validated by RT-qPCR and Western blot. The specific MZB1 siRNA sequences were as follows: shRNA-1: 5’-GATGAAGAGAAGTACGCATCC-3’; shRNA-2: 5’-GCGAAGAGCAGAGGCTAA TCT-3’; shRNA-3: 5’-GCAGTCCTATGGAGTCCAAGA-3’ (Shanghai Genechem Co., Ltd., China). Following transfection, PDLSCs were assigned to 4 experimental groups: (1) Control (untreated), (2) LPS-treated, (3) LPS+shRNA-NC (cells transduced with non-targeting shRNA), and (4) LPS+shRNA-MZB1 (cells transduced with MZB1-specific shRNA).

CCK-8 assay

Cells (5 × 10⁴ cells/mL) in the logarithmic growth phase were seeded in 96-well plates. Then 100 μL of CCK-8 solution (10%) was added to each well. After incubation at 37 °C for 1 hours, absorbance was measured at 450 nm using a microplate reader.

Wound healing assay

PDLSCs (5 × 10⁵ cells/mL) were seeded in 24-well plates. Upon reaching of confluence of 100%, a straight scratch was made using a sterile pipette tip. Serum-free medium was added, and images were captured at 0 hours and 24 hours. Scratch area was quantified using ImageJ to evaluate cell migration.

Crystal violet staining

PDLSCs in 24-well plates were passaged every 3 days. At passage 6, cells were fixed with 10% neutral formalin for 30 minutes, washed with PBS, and stained with 0.1% crystal violet for 3 minutes. Morphology was observed under a microscope. For quantification, 1 mL of 33% glacial acetic acid was added to dissolve the dye, and absorbance at 570 nm was measured.

Alizarin red staining

PDLSCs were treated as described above and cultured in osteogenic induction medium supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, 50 ng/mL ascorbic acid, 10% FBS in DMEM-F12). On day 21, cells were fixed and stained with Alizarin Red S for 1 to 5 minutes. Calcium deposition (orange-red) was observed under a microscope. For quantification, 2% cetylpyridinium chloride was added, and absorbance was measured at 560 nm.

Alkaline phosphatase (ALP) activity assay

Cells were cultured in osteogenic induction medium (DMEM-F12) supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, 50 ng/mL ascorbic acid, and 10% FBS. On day 21, cells from each group were harvested, and total protein was extracted. After determining protein concentrations, ALP activity was measured using an ALP activity assay kit (Nanjing Jiancheng Bioengineering Institute, China).

Flow cytometry

PDLSCs were digested with trypsin, washed twice with cold PBS, and resuspended in 500 μL 1 × binding buffer. After filtration through a 200-mesh strainer, 5 μL Annexin V-PE and 10 μL 7-AAD were added. Cells were incubated at 4 °C for 10 minutes in the dark and analyzed by flow cytometry.

Rat periodontitis model and grouping

Thirty-eight male Wistar rats (SPF grade) were obtained from SBF Biotechnology (Suzhou, China). Animals were housed under standard conditions (22 ± 2 °C, 60–80% humidity, 12 hours light/dark cycle) with ad libitum food and water. Periodontitis was induced by ligating the bilateral maxillary first and second molars using 3-0 sutures and 0.2 mm orthodontic wire. Rats were fasted 8 hours before surgery. Ligatures were placed around the cervical region and embedded into the gingival sulcus. LPS (1 mg/mL, 60 μL/site) was injected between the molars 3 times per week for 3 weeks to exacerbate inflammation.

Rats were randomly assigned to 4 groups: (1) Sham (n = 8): subjected to saline injection without ligation; (2) periodontitis (n = 10): ligation combined with LPS injection via tail vein; (3) periodontitis + shRNA-NC (n = 10): tail vein injection of negative control AAV 1 week prior to periodontitis induction; (4) periodontitis + shRNA-MZB1 (n = 10): AAV-MZB1 knockdown virus injection 1 week prior to periodontitis induction.

After anesthesia, blood was collected from the abdominal aorta, and serum was stored at low temperature. Rats were euthanised, and bilateral maxillary bones were harvested. The right maxilla was fixed, decalcified, and embedded for histological sectioning. Gingival tissues from the left side were collected, washed, and frozen for molecular analyses.

H&E staining

Rat maxillary tissue sections embedded in paraffin were subjected to deparaffinisation, rehydration, hematoxylin and eosin staining, and dehydration. Tissue morphology was observed and imaged under a light microscope.

TUNEL assay

After deparaffinisation and antigen retrieval, sections were digested and blocked. TUNEL staining was performed to detect DNA fragmentation. DAB or fluorescence was used for visualisation. Slides were counterstained, dehydrated, and sealed for microscopic analysis.

Cytokine detection by ELISA

Serum cytokine levels, including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), IL-17 and transforming growth factor β1 (TGF-β1), were quantified by ELISA following standard protocols. Absorbance was measured, and cytokine concentrations were calculated using standard curves.

Western blot

Total protein was extracted from PDLSCs and ground maxillary bone tissues. For bone tissues, samples were ground in liquid nitrogen, and 100 mg of tissue powder was lysed in 400 μL RIPA buffer. All lysates were incubated on ice for 60 minutes, followed by centrifugation at 12,000 rpm for 15 minutes at 4 °C to obtain the supernatant. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 hours at room temperature and incubated overnight at 4 °C with the following primary antibodies (all from Abcam): β-actin (1:1000), CHOP (1:800), MZB1 (1:1000), PERK (1:500), p-PERK (1:400), ATF4 (1:500), GRP78 (1:500), Bcl-2 (1:500), BAX (1:500), RUNX2 (1:500), BMP2 (1:500), α-SMA (1:500), and Collagen I (1:400). After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:5,000, Abcam) for 1 hours at room temperature. Protein bands were visualised using an ECL detection system and imaged with ChemiScope mini. Densitometric analysis was performed using ImageJ software.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Inter-group comparisons were analysed using Student’s t test; multiple groups were compared using 1-way ANOVA. Statistical analysis was performed using SPSS 19.0. A P value of less than .05 was considered to be significant.

Results

MZB1 is highly expressed in gingival tissues of periodontitis patients

Transcriptomic analysis of gingival tissues from periodontitis patients revealed 139 upregulated and 81 downregulated DEGs compared to healthy controls (Figure 1A), indicating widespread gene dysregulation during the onset and progression of periodontitis. Notably, MZB1 mRNA exhibited the highest fold increase among all upregulated genes (Figure 1B), suggesting that MZB1 is strongly transcriptionally activated in periodontitis.

Fig. 1.

MZB1 is significantly upregulated in gingival tissues of periodontitis patients. (A) Histogram of the number of transcriptomics differential genes in gingival tissues of patients with periodontitis. (B) Volcano plot of differentially expressed genes, highlighting MZB1 as the gene with the most significant upregulation. (C) GO biological process and KEGG pathway enrichment analysis of differentially expressed genes; upper right quadrant shows enrichment of upregulated genes, while lower left indicates downregulated genes. (D) Quantitative RT-PCR analysis of MZB1 mRNA expression in gingival tissues from periodontitis patients and healthy controls (N = 20).

Enriched signaling pathways of DEGs

Functional enrichment analysis of the upregulated DEGs (Figure 1D) revealed significant associations with inflammatory and immune-related pathways, including response to bacterium, regulation of immune responses, cell surface receptor signaling pathways, malaria, leukocyte transendothelial migration, PID integrin 2 pathway, regulation of leukocyte differentiation, cellular response to cytokine stimulus, and mature B cell differentiation. MZB1 was notably enriched in the malaria-associated inflammatory response pathway, implying a retained immune function within gingival tissues. Conversely, downregulated genes were enriched in pathways related to epidermal development and keratinocyte development.

Expression of MZB1 in periodontal tissues

We also determined the expression of MZB mRNA in periodontal tissues. RT-qPCR analysis (Figure 1C) confirmed a significant upregulation of MZB1 in gingival tissues from periodontitis patients compared to controls, consistent with the transcriptomic data.

MZB1 knockdown alleviates LPS-induced inflammatory injury and promotes PDLSC proliferation

RT-qPCR analysis demonstrated that 3 different shRNAs significantly reduced MZB1 mRNA expression in PDLSCs (Figure 2A). Among them, shRNA-3 knockdown was the most efficient and was selected for the subsequent experiments. Western blot analysis further confirmed that MZB1 protein was downregulated (Figures 2B and C), validating successful knockdown.

Fig. 2.

Lentiviral knockdown of MZB1 attenuates LPS-induced cytotoxicity and enhances PDLSC migration and viability. (A) Knockdown efficiency of MZB1 in PDLSCs transduced with lentivirus-mediated shRNA. (B) Western blot analysis of MZB1 protein expression. (C) Quantification of MZB1 protein levels normalised to β-Actin based on grayscale intensity (N = 3). (D) Cell viability of PDLSCs following lentiviral transduction and LPS stimulation, as measured by CCK-8 assay (N = 5). (E) Quantification of wound closure rates in the scratch assay (N = 3). (F) Quantitative analysis of cell viability by absorbance at 570 nm following crystal violet staining. (G) Representative images of wound healing assay at 24 hours post-injury. (H) Representative microscopic images of PDLSCs stained with crystal violet (comparisons in panel B are relative to Control shRNA).

To simulate periodontitis in vitro, PDLSCs were treated with LPS, a well-established pro-inflammatory agent that activates TLR4 signaling. LPS exposure significantly reduced PDLSC viability (Figure 2D), confirming effective induction of inflammatory damage. Notably, PDLSCs with MZB1 knockdown exhibited significantly higher viability following LPS treatment compared to control shRNA-transfected cells, indicating that MZB1 suppression alleviated LPS-induced cytotoxicity.

Wound healing (Figures 2E and G) revealed that LPS markedly inhibited PDLSC proliferation, whereas MZB1 knockdown partially restored proliferation rates to near-normal levels. Similarly, crystal violet staining (Figures 2F and H) showed that LPS reduced staining intensity, indicating impaired adhesion and proliferation. This effect was reversed in the presence of MZB1-knockdown, where staining intensity increased, supporting the protective role of MZB1 knockdown in preserving cell proliferation under inflammatory conditions.

MZB1 knockdown reduces ER stress induced by inflammation

To investigate the role of MZB1 in LPS-induced ER stress, we assessed the expression of ER stress markers. As shown in Figures 3A and B, LPS treatment significantly upregulated the expression of ER stress-related proteins, including p-PERK, ATF4, CHOP, and GRP78, indicating a robust ER stress response. However, in MZB1-knockdown cells, the expression of these markers was markedly reduced. These results suggest that MZB1 knockdown attenuates ER stress activation, which may partially explain the improved cell viability observed in previous experiments.

Fig. 3.

Knockdown of MZB1 alleviates LPS-induced ER stress and apoptosis in PDLSCs. (A) Western blot chemiluminescence imaging of endoplasmic reticulum stress-related proteins, including p-PERK, ATF4, CHOP, and GPR78, in PDLSCs cells under LPS treatment. (B) Quantitative analysis of protein band intensities for p-PERK, ATF4, CHOP, and GRP78, normalised to β-Actin (N=3). (C) Flow cytometric analysis of apoptosis using Annexin V-PE and 7-AAD staining. (D) Quantification of apoptosis rates based on flow cytometry results (N = 3). (E) Western blot chemiluminescence imaging of apoptosis-related proteins BCL-2 and BAX in PDLSCs cells under LPS treatment. (F) Quantitative analysis of BCL-2 and BAX protein expression normalised to β-Actin (N=3).

MZB1 knockdown enhances PDLSC survival in inflammatory microenvironments by inhibiting apoptosis

Flow cytometry analysis (Figures 3C and D) showed that LPS significantly increased apoptosis in PDLSCs. In contrast, MZB1 knockdown significantly reduced the apoptotic rate, suggesting a protective effect against LPS-induced cell death. Further analysis of apoptosis-related proteins (Figures 3E and F) revealed that LPS decreased the expression of the anti-apoptotic protein BCL-2 and increased the pro-apoptotic protein BAX, indicating activation of apoptotic signaling pathways. MZB1 knockdown reversed these changes, restoring BCL-2 levels and reducing BAX expression.

MZB1 knockdown restores osteogenic differentiation capacity of PDLSCs under inflammatory conditions

Periodontitis-associated inflammation is known to inhibit the osteogenic differentiation and mineralisation potential of PDLSCs. To assess the effect of MZB1 on PDLSC differentiation, Alizarin Red S staining was performed under osteogenic induction conditions. The results (Figures 4A and B) showed that LPS treatment significantly reduced mineralised nodule formation, while MZB1 knockdown markedly restored both the number and distribution of nodules, suggesting improved osteogenic differentiation under inflammatory suppression.

Fig. 4.

Knockdown of MZB1 restores osteogenic differentiation capacity of PDLSCs under inflammatory conditions (A) Microscopic images of alizarin red S staining showing calcium nodule formation after osteogenic induction of PDLSCs under different conditions. (B) Quantification of alizarin red S staining by absorbance at 560 nm (N = 3). (C) Quantitative analysis of alkaline phosphatase (ALP) activity following osteogenic induction of PDLSCs (N = 3). (D) Western blot images of osteogenic differentiation-related proteins (RUNX2, BMP2, α-SMA, and Collagen I) in PDLSCs. (E) Quantification of Western blot bands normalised to β-Actin, representing relative expression levels of osteogenic markers (N = 3).

ALP activity, a marker of early osteogenesis, was examined next. ALP staining (Figure 4C) revealed that LPS significantly suppressed ALP activity in PDLSCs. However, this inhibition was reversed following MZB1 knockdown, consistent with the trend observed in calcium deposition, further supporting the positive effect of MZB1 silencing on osteogenesis.

Western blot analysis (Figures 4D and E) showed that LPS reduced the expression of osteogenic markers including RUNX2, BMP2, α-SMA, and Collagen I, indicating impaired differentiation. In contrast, MZB1 knockdown significantly upregulated the expression of these proteins, reinforcing its role in enhancing PDLSC osteogenic differentiation.

AAV-mediated knockdown of MZB1 alleviates periodontal tissue damage

To further investigate the functional role of MZB1 in periodontitis, an in vivo rat model was established, and AAV-mediated shRNA delivery was used to silence MZB1 expression in periodontal tissues. Western blot analysis confirmed that MZB1 protein levels were significantly elevated in periodontitis rats, but were restored to near-baseline levels following AAV-MZB1 shRNA treatment (Figures 5A and B), confirming effective knockdown.

Fig. 5.

AAV-mediated knockdown of MZB1 alleviates periodontal inflammation and tissue damage in a rat model of periodontitis. (A) Western blot analysis of MZB1 protein expression in rat periodontal tissues. (B) Quantitative densitometry of MZB1 protein expression normalised to β-Actin (N = 3). (C) Body weight changes in rats after AAV-mediated knockdown of MZB1 in the periodontitis model (N = 6). (D) Representative H&E-stained images of rat periodontal tissue sections. (E) Quantitative analysis of histopathological parameters, including inflammatory cell infiltration, alveolar bone resorption, and loss of periodontal attachment (N = 6). (F) ELISA-based quantification of inflammatory cytokines TNF-α, IL-6, IL-17A, and anti-inflammatory factor TGF-β1 in rat periodontal tissues (N = 6).

H&E staining (Figures 5D and E) revealed that the periodontitis model and control shRNA groups exhibited pronounced alveolar bone loss, periodontal ligament disorganisation, and inflammatory cell infiltration. In contrast, the MZB1 knockdown group showed attenuated tissue damage, with better-preserved periodontal ligament architecture and reduced bone resorption and inflammation.

Body weight monitoring also suggested systemic involvement: rats in the periodontitis group showed weight loss starting from week 4, while the MZB1 knockdown group exhibited a recovery trend, albeit without statistical significance (Figure 5C), suggesting a potential role in modulating systemic inflammatory responses.

MZB1 knockdown reduces inflammation and modulates immune cytokine expression

To further explore immune response modulation, inflammatory cytokine levels in periodontal tissues were measured (Figure 5F). ELISA results showed that pro-inflammatory cytokines TNF-α, IL-6, and IL-17A were significantly elevated in the periodontitis and control shRNA groups, while the anti-inflammatory cytokine TGF-β1 was decreased. In the MZB1 knockdown group, pro-inflammatory cytokine levels were reduced and TGF-β1 expression was significantly increased, indicating that MZB1 silencing may contribute to immune rebalancing and suppression of inflammatory activation.

MZB1 knockdown alleviates ER stress in periodontal tissues

To assess the regulatory effect of MZB1 on ER stress in vivo, the expression of ER stress-related proteins was evaluated by Western blot (Fig. S1A-B). Compared to controls, the periodontitis and control shRNA groups showed significantly increased expression of p-PERK, ATF4, CHOP, and GRP78. Notably, these markers were markedly reduced following MZB1 knockdown, indicating that MZB1 silencing effectively attenuates ER stress in periodontal tissues.

MZB1 knockdown restores osteogenic capacity

Lastly, we examined the expression of osteogenic markers (RUNX2, BMP2, α-SMA, and Collagen I) in periodontal tissues via Western blot (Figure S1C and D). In the periodontitis and control shRNA groups, all osteogenesis-related proteins were markedly downregulated, indicating impaired osteogenesis. However, in the MZB1 knockdown group, expression levels of these proteins were restored, suggesting that MZB1 inhibition supports the maintenance or regeneration of periodontal osteogenic capacity.

MZB1 knockdown inhibits apoptosis

TUNEL staining (Figures 6A and B) revealed a significant increase in apoptotic cells in the periodontal tissues of both the periodontitis and control shRNA groups. In contrast, the MZB1 knockdown group exhibited a marked reduction in apoptosis signals. Western blot analysis further confirmed this observation: BAX, a pro-apoptotic protein, was elevated and BCL-2, an anti-apoptotic protein, was suppressed in the disease model, whereas in the MZB1-silenced group, BAX expression was reduced and BCL-2 levels were restored (Figures 6C and D). These findings suggest that MZB1 knockdown protects periodontal tissues by inhibiting apoptosis signaling pathways and mitigating tissue destruction.

Fig. 6.

Knockdown of MZB1 reduces apoptosis in periodontal tissues of rats with periodontitis. (A) Representative TUNEL-stained images (80 ×) of rat periodontal tissue sections. Apoptotic cells are labeled with green fluorescence, and nuclei are counterstained with DAPI (blue). (B) Quantitative analysis of apoptotic cell ratio in TUNEL-stained sections (N = 5). (C) Western blot analysis of apoptosis-related proteins (BAX and BCL-2) in rat periodontal tissues. (D) Quantification of BAX and BCL-2 protein expression normalised to β-Actin (N = 3). (*P < .05, **P < .01, ***P < .001, ****P < .0001).

Discussion

This study demonstrates that MZB1 is significantly upregulated in the gingival tissues of patients with periodontitis and plays a role in the inflammatory microenvironment by modulating ER stress, apoptosis, and osteogenic differentiation of PDLSCs. Using both in vitro and in vivo models, we confirmed that MZB1 knockdown alleviates LPS-induced cellular damage, restores regenerative capacity, and attenuates periodontal tissue inflammation and destruction. These findings reveal a previously unrecognised role of MZB1 in the pathogenesis of periodontitis and suggest it as a promising therapeutic target for modulating periodontal inflammation and promoting tissue repair.

Periodontitis is characterised by chronic inflammation resulting from a dysregulated immune response to bacterial biofilms.²³ Transcriptomic analysis of gingival tissues from periodontitis patients revealed significant MZB1 overexpression, and its enrichment in inflammation-related pathways suggests a potential immunomodulatory role. While previous transcriptomic and proteomic studies have identified MZB1 as a candidate gene in chronic periodontitis. Li et al.²¹ found that MZB1 was one of the most significantly upregulated genes in periodontitis tissues compared to healthy tissues from transcriptomic analysis. Guzeldemir-akcakanat et al.²⁴ integrated transcriptomic and proteomic data revealed MZB1 as a potent candidate for chronic periodontitis. However, its functional investigations have been lacking. MZB1 was originally described as an ER-resident protein involved in immunoglobulin folding and secretion in B cells and plasma cells.²⁵ Emerging evidence suggests its involvement in cellular stress responses and inflammation in non-lymphoid tissues.²⁶ Several studies have also implicated MZB1 in inflammatory regulation in other disease models. Yue et al.²⁷ reported that MZB1 deficiency led to reduced intestinal IgA production and aggravated inflammation, accelerating azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colorectal cancer. Xiong et al.²⁸ found that MZB1 promotes J-chain-containing dimeric IgA secretion, which is essential for suppressing intestinal inflammation. Xu et al.²⁶ demonstrated that MZB1 modulates cell proliferation, mitochondrial dysfunction, and inflammation in acute pancreatitis via the PI3K-Akt signaling pathway. Consistent with these findings, our LPS-induced periodontitis rat model also showed significant upregulation of MZB1, supporting its role in responding to inflammatory stimuli. Importantly, MZB1 knockdown reduced the expression of pro-inflammatory cytokines and mitigated LPS-induced cellular injury, indicating a pro-inflammatory function in the periodontium. These results suggest that MZB1 may amplify inflammation through modulation of ER stress-related signaling pathways, which are closely linked to both inflammation and apoptosis.

ER stress has been identified as a key pathological mechanism in periodontitis, particularly under persistent inflammatory and oxidative stress conditions.²⁹ LPS stimulation is known to trigger ER stress in PDLSCs, leading to activation of the UPR and subsequent apoptosis if unresolved.^30,31 In our study, MZB1 knockdown significantly reduced the expression of ER stress markers such as GRP78 and CHOP, and attenuated apoptosis in PDLSCs. These findings suggest a protective role of MZB1 inhibition against LPS-induced ER dysfunction. Previous research has shown that MZB1 is a cochaperone of Grp94 and that MZB1 associates with Grp94 in an ATP-dependent manner following exposure to ER stress.³² Notably, Kapoor et al.³³ reported that MZB1 enhances plasmacytoid dendritic cells to secrete high amounts of IFNα by mitigating ER stress via the ATF6-mediated UPR. Xue et al.³⁴ found that puerarin protects the heart from oxidative and ER stress-induced injury by upregulating the KLF4/MZB1 pathway. Furthermore, MZB1 has been shown to regulate mitochondrial function and apoptosis.³⁵ In an atherosclerosis model, MZB1 enhanced mitochondrial integrity by restoring ATP production, reducing membrane potential loss, and lowering mitochondrial ROS, ultimately attenuating cell death.³⁶ Collectively, these findings suggest that MZB1 may serve as a regulator of ER homeostasis in PDLSCs. However, under inflammatory conditions, its overexpression could exacerbate ER dysfunction and promote apoptosis. Thus, targeting MZB1 may represent a novel strategy for reducing ER stress–related cell death and preserving periodontal tissue integrity.

One of the hallmark pathological features of periodontitis is alveolar bone resorption, primarily driven by inflammation-induced impairment of PDLSC osteogenesis.^37,38 In this study, LPS stimulation significantly reduced ALP activity, calcium deposition, and the expression of osteogenic genes such as RUNX2, BMP2, and COL1A1. Notably, MZB1 knockdown reversed these effects, suggesting its negative regulatory role in PDLSC osteogenic potential under inflammatory conditions. This finding is supported by prior research showing that MZB1 overexpression suppresses ALP and RUNX2 expression.²¹ RUNX2 is a master transcription factor essential for osteogenic differentiation, directly regulating the expression of bone-associated proteins.³⁹ Studies have shown that RUNX2 plays a key role in the osteogenic differentiation of hPDLSCs.^40,41 Suppression of ER stress has been shown to downregulate RUNX2 expression, thereby reducing intracellular calcium deposition and impairing matrix mineralisation.⁴² BMP2, one of the most potent osteoinductive factors, can promote osteogenesis via signaling pathways activated by mild ER stress.⁴³ The ER stress pathway, inositol-requiring enzyme-1 alpha-X-box binding protein-1 (IRE1α-XBP1) promotes the proliferation via regulating the autophagy and apoptosis, and enhances expression of osteogenic genes in hPDLCs. XBP1s may activate the PERK-ATF4 pathway through interaction with BMP2, thereby enhancing the expression of osteogenic-related genes and promoting the formation of mineralised nodules.⁴⁴ However, under conditions of persistent inflammatory stimulation, this stress response may become excessively activated, leading to the suppression of osteogenic differentiation and potentially triggering cell death. Our data suggest that MZB1 may modulate the intensity of ER stress in PDLSCs, and its overexpression could amplify inflammation-induced stress, impairing osteogenic capacity. Therefore, MZB1 may act as a key modulator in PDLSC dysfunction, and its knockdown could relieve ER stress, reactivating BMP2 and RUNX2-mediated osteogenesis and enhancing bone regeneration. This provides a rationale for targeting MZB1 as a strategy to promote periodontal tissue regeneration.

This study has several limitations. Although we demonstrated the effects of MZB1 knockdown in PDLSCs and animal models, the specific downstream signaling pathways of MZB1 remain to be elucidated. Future transcriptomic and proteomic profiling following MZB1 silencing may help clarify its regulatory network. Additionally, while PDLSCs are relevant models for periodontal regeneration, the role of MZB1 in resident immune cell populations (eg, macrophages, B cells) within the periodontium warrants further investigation, given its origin in immunobiology. Finally, our Western blot analysis and previous studies support the expression of MZB1 in PDLSCs, but we did not perform immunofluorescence co-localisation experiments to verify its primary cellular origin in the periodontal microenvironment. This lack of spatial validation limits our ability to ultimately determine whether PDLSCs are the primary source of MZB1 in vivo. Therefore, future studies combining immunofluorescence or single-cell analysis are needed to address this deficiency.

Conclusion

In summary, this study provides compelling evidence that MZB1 is a critical mediator of periodontitis progression by regulating PDLSC inflammation, ER stress, apoptosis, and osteogenic differentiation. Targeting MZB1 may offer a promising dual-function therapeutic strategy for modulating the periodontal immune microenvironment and enhancing regenerative outcomes. Thus, MZB1 represents a novel and potentially valuable therapeutic target in the treatment of chronic periodontitis.

Ethics approval and consent to participate

The study was approved by the Animal Ethics Committee of Newloong Youshu life Technology (Hangzhou) Co., Ltd. (No: NLYS-r202401002). The study protocol involving human participants was consistent with the ethical principles of the Helsinki and was approved by the Ethics Committee of the South China Hospital of Shenzhen University (No. HNLS20241223003-A). All participants signed informed consent.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

Fei Ge: Formal Analysis, Resources, Investigation, Validation, Visualisation, Writing – original draft. Yang Zhao: Data curation, Investigation, Validation, Visualisation, Writing – original draft. Qun Xiang: Data curation, Methodology, Software, Writing – original draft. Manjuan Chen: Resources, Software, Visualisation, Writing – original draft. Xianmei Hu: Methodology, Software, Writing – original draft. Lei Wang: Software, Visualisation, Writing – original draft. Qipeng Jiang: Software, Visualisation, Writing – original draft. All authors read and approved the final manuscript.

Conflict of interest

None disclosed.