The following strains were used: P. gingivalis (ATCC 33277), Fusobacterium nucleatum (F. nucleatum; ATCC 10953), and Streptococcus sanguinis (S. sanguinis; ATCC 10556). All strains were obtained from the Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling and were grown in brain–heart infusion (BHI) broth with hemin (5 mg/L) and vitamin K (0.5 mg/L) at 37°C in anaerobic conditions, passaged every 2 days. P. gingivalis in the logarithmic growth phase was inoculated into six-well plates (WHB Scientific, Shanghai, China) at a density of 1.0 × 10⁸ CFU/mL and incubated for 2 days in BHI broth with 1% sucrose to establish plaque biofilms. Eighteen 8-week-old healthy SD male rats were purchased from Liaoning Changsheng Biotechnology Co., Ltd., randomly selected, and housed in specific-pathogen-free (SPF) conditions. The room temperature was maintained at 22–24°C, and a 12-h light–dark cycle was maintained. They were allowed free access to tap water and standard chow pellets. Animals were allowed to acclimatize to the environment for 1 week before the experiment. The animal production license number was SCXK (Liao) 2022–0001. The animal experiments were approved by the Ethics Committee on Animal Experimentation of Jilin University, with the approval number SYXK (Ji) 2018–0001.

MSZ (Aladdin Chemistry, Shanghai, China) was dissolved in BHI broth at a concentration of 2 mg/mL as a stock solution. The concentrations of MSZ (0.5 and 1 mg/mL) were diluted with BHI broth from this stock solution. P. gingivalis was grown in BHI broth with different concentrations of MSZ to report the growth of the bacteria (Bao et al., 2023). The control group was grown in BHI broth without MSZ. The blank well, which contained only BHI medium without inoculation of bacteria, was incubated under the same culture conditions as the experimental group. OD values at 600 nm were recorded from 0 to 72 h using Synergy HT (Agilent Technologies, Beijing, China) to describe the growth curves. To eliminate the influence of the culture medium on the absorbance value reading, the blank wells were used as the reference for zeroing. The minimum inhibitory concentration (MIC) values were the lowest concentrations of MSZ at which no visible growth was observed in the 15 mL sterile centrifuge tubes (BKMAM Biotechnology, Changshu, China) after incubation for 48 h.

The percentage of live cells was assessed by flow cytometry assay (He et al., 2017). After 48 h of MSZ treatment, the P. gingivalis suspensions were added to 1 mL tubes and stained with SYTO (KeyGEN Biotech, Jiangsu, China) and PI (Beyotime, Shanghai, China) under light avoidance conditions for 15 min. Live/dead staining was used to differentiate between live and dead cells according to the kit protocols. A 0.9% sodium chloride solution was used as the flow cytometer sheath solution. Results were collected for 20,000 hits using FL1 through FL3 channels for each measurement point using the Accuri C6 Plus flow cytometer (BD Biosciences, USA). The results were assessed using FlowJo software (Tree Star, X.10.0.7, United States).

A total of 48 h after treatment, P. gingivalis suspensions were fixed in 2.5% glutaraldehyde and underwent dehydration through a graded ethanol series from 30 to 100%. Finally, the samples were gold-coated and observed using scanning electron microscopy (SEM) MAGNA (TeScan, Brno, Czech Republic). Atomic force microscopy (AFM) JPK Nano Wizard 3 (JPK Instruments, Berlin, Germany) in combination with an inverted Olympus IX81 light microscope, was used to examine the bacterial surface morphology. Roughness and Young’s modulus were analyzed using JPK Data Processing 7.0.97. P. gingivalis suspensions were placed on poly-L-lysine-coated (Sigma-Aldrich, Shanghai, China) culture plates for 20 min to fix (Benn et al., 2019). Cells were imaged in qualitative imaging mode using MLCT-A cantilevers (Bruker, MA, USA) with a spring constant of 0.07 N/m. The resonant frequency of the cantilever was 22 kHz in liquid.

The effect of MSZ on P. gingivalis biofilm formation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and crystal violet staining. Disruption of pre-formed P. gingivalis biofilms was also assessed by crystal violet staining. P. gingivalis biofilms were fixed with 2.5% glutaraldehyde and stained with 0.1% crystal violet reagent for 20 min, followed by 95% ethanol. OD values at 550 nm were measured to assess the effect of MSZ on P. gingivalis biofilm biomass. To evaluate P. gingivalis biofilm’s activity, 50 μL 0.5% MTT was added to P. gingivalis biofilms, anaerobically incubated in a dark place for 4 h, and then 100 μL of DMSO was added to dissolve the formazan crystals. The OD values at 490 nm were measured.

Confocal laser scanning microscopy (CLSM) was used to observe the effect of MSZ on both plaque biofilm formation and the disruption of established plaque biofilm. P. gingivalis, F. nucleatum, and S. sanguinis at a density of 1.0 × 10⁸ CFU/mL were inoculated to form a multispecies biofilm (Blanc et al., 2014). F. nucleatum, as an important bridging bacterium, can co-aggregate with early colonizing bacteria such as S. sanguinis, along with late colonizing bacteria like P. gingivalis, to form the plaque biofilm (Brennan and Garrett, 2019). P. gingivalis biofilms and multispecies biofilms were stained with SYTO/PI for 15 min in a dark place following the manufacturer’s instructions. Images were observed (490/635 nm for PI, 480/500 nm for SYTO) using FV3000 CLSM (Olympus, Tokyo, Japan). The percentage of live cells was analyzed using ImageJ2 2.3.0 software.

Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to investigate the expression of the P. gingivalis virulence gene using Mx3000P (Agilent Technologies, CA, USA). P. gingivalis plaque biofilms were scraped using a cell scraper (Thermo Fisher Scientific, Shanghai, China) and suspended in sterile PBS. After collection, lysozyme (Solarbio, Beijing, China) and RNAiso Plus (Takara Biomedical Technology, Beijing, China) were added to extract RNA. The lysozyme (12650-88-3, Solarbio, Beijing, China) was used at a concentration of 0.5 mg/mL. Each sample was added with 1 mL of RNAiso Plus (Trizol). cDNA synthesis was performed with Hifair® III 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China), and qRT–PCR was performed with Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) following the manufacturer’s protocol. The total reaction volume was 20 μL and was completed on ice. The reaction program was 10 s at 95°C and 30 s at 60°C for 40 cycles. The 16S rRNA gene was used as the housekeeping amplicon. The levels of mRNA expression were calculated using the 2-ΔΔCt method. Primers (He et al., 2022) were obtained from Bioengineering (Shanghai) Co., Ltd., with the sequences detailed in Table 1.

The chromogenic substrate N-Benzoyl-L-arginine p-nitroanilide (BAPNA) kit (Solarbio, Beijing) was used to assess Arg-gingipain activity (Zhou et al., 2016; Shenbakam et al., 2021). P. gingivalis suspensions (10 μL each) were cultured in a buffer containing 200 mM Tris–HCl, 100 mM NaCl, and 5 mM CaCl₂ at pH 7 to an OD₆₆₀ of 0.5. The activity of the pre-incubated samples was then determined using 0.5 mM chromogenic substrate. The OD values were measured at 409 nm. The degradation observed after a 1-h treatment without drugs was set as the 100% reference value.

Alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) activities were measured to evaluate the impact of MSZ on cell walls and membrane integrity (Li et al., 2024). P. gingivalis cultures in the logarithmic growth phase were centrifuged at 8,500 r/min for 5 min. After centrifugation, the samples were washed and resuspended in PBS. The bacteria were disrupted in an ice bath using the ultrasonic homogenizer JY92-IIDN (Scientz Biotechnology, Ningbo, China), and then the supernatant was collected by centrifugation at 15,000 r/min. A bicinchoninic protein assay kit (NCM Biotech, Suzhou, China) was used to determine the concentration of proteins. The AKP and LDH activities in the supernatant were determined according to the kit scheme (Jiancheng Bioengineering Institute, Nanjing, China). The OD values at 520 nm were measured for AKP activity, and the OD values at 440 nm were measured for LDH activity.

A total of 18 8-week-old male SD rats were randomly assigned to three groups (for each group, n = 6), i.e., the control group, the model group, and the MSZ group (treated with 1 mg/mL MSZ). Periodontitis models were established in the model group and MSZ group. Rats in the control group were anesthetized but not operated to exclude the effect of anesthesia. Rats were anesthetized by isoflurane (RWD Life Science, Shenzhen, China) inhalation (5% for induction and 2% for maintenance) using an anesthesia machine (Yuyan Instruments, Shanghai). An oral health exam was performed before the establishment of the rat periodontitis model. After anesthesia, a steel orthodontic wire with a diameter of 0.2 mm was inserted into the gap between the first molar and second molar, and the ligature was completely inserted into the gingival groove (Xu et al., 2019). The rats were checked every 72 h, and if the wires became loose, they were promptly re-tied. The ligature wire was set for 6 weeks. The success of the periodontitis model was judged by the depth of periodontal probing. After successful modeling, the ligature wires were removed. The rats in the MSZ group were injected with 20 μL of 1 mg/mL MSZ into the submucoperiosteal tissues at the midpoints of the buccal and palatal aspects of the maxillary first molars with an insulin syringe three times weekly for 2 weeks. Rats in both the control and model groups were injected with equal amounts of normal saline.

After 2 weeks of treatment, the rats were euthanized with carbon dioxide. The maxillae were collected and fixed in a 4% paraformaldehyde solution. The samples were scanned using micro-CT (μCT50, Scanco Medical AG, Switzerland). Scanning parameters were 114 mA, 70 kVp, and exposure time 0.3 s. The layer thickness was 18 μm. The focal area was the alveolar bone around the maxillary first molar. Using Image J2 2.3.0 software, we measured the distance of alveolar bone loss from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) at three anatomical sites (proximal, middle, and distal) on the palatal side of the maxillary first molar. In addition, the alveolar bone between the maxillary first and second molars was measured. After that, we used μCT Ray V4.2 software parameters to assess the amount of bone and mineralization, including bone volume fraction (bone volume/tissue volume, BV/TV, %), trabecular thickness (Tb. Th, mm), and trabecular separation (Tb. Sp, mm).

Rat maxillary tissue blocks were fixed in 10% paraformaldehyde for 48 h and then decalcified in 10% EDTA for 3 months, with the solution changed three times weekly. After decalcification, the samples were washed under running water for 20 min, trimmed, and sectioned into appropriate sizes. They were dehydrated using graded ethanol solutions, embedded in paraffin wax and sliced uniformly along the long axis of the teeth on the proximal-medial and distal-medial sides at a thickness of approximately 5 mm. The sections were deparaffinized in xylene, sealed in neutral gum, and stained with hematoxylin–eosin (H&E).

The gingival tissues were rapidly and aseptically isolated and then frozen in liquid nitrogen for transport. Community genomic DNA was extracted using the E.Z.N.A™ Mag-Bind Soil DNA Kit (Omega, M5635-02, United States) according to the manufacturer’s instructions. We focused on the V3–V4 hypervariable region of the bacterial 16S rRNA gene. The sequencing was conducted at Sangon Biotech (Shanghai) Co., Ltd.

Data analysis was conducted using GraphPad Prism 9.3.1, with the results presented as mean ± standard deviation (SD). Each experiment was conducted for a minimum of three times. A t-test was used for comparisons between two groups, while analysis of variance (ANOVA) was employed for comparisons between multiple groups. Significance was expressed as p < 0.05 for statistical differences.

The growth curve showed that various concentrations of MSZ (0.5, 1, and 2 mg/mL) inhibited the growth of P. gingivalis compared to the control group, with a MIC value of 2 mg/mL (Figure 1A). To investigate MSZ’s effect on biofilm formation and to minimize the impact of its antibacterial effect, concentrations were kept below the MIC. Flow cytometry was used to assess P. gingivalis activity in both the experimental and control groups (Figures 1B,C). The percentage of dead cells significantly increased following treatment with MSZ 0.5 mg/mL and 1 mg/mL, compared to the control group.

Using SEM, the morphology of P. gingivalis cells was observed (Figure 1D). After 48 h of co-culture with MSZ at concentrations of 0.5 mg/mL and 1 mg/mL, P. gingivalis exhibited irreversible morphological changes, including invagination, wrinkling, breakage, atrophy, and deformation. In contrast, the untreated P. gingivalis cells maintained a rounded surface, intact structure, neat edges without wrinkles, and clearly visible bacterial pili. These observations suggest that MSZ concentrations of 0.5 mg/mL and 1 mg/mL may have damaged the surface structure and morphology of P. gingivalis. Treatment with 1 mg/mL MSZ transformed the initially smooth outer membrane into a broken and rough surface. The increased permeability of the cell membrane led to extracellular leakage of cellular contents, ultimately resulting in cell rupture and death. These findings indicate that MSZ may inhibit bacterial growth by compromising cell structure.

MSZ was added both before and after biofilm formation to simulate the prevention and treatment of biofilm. Crystal violet staining results showed that the OD_{550 nm} values decreased following treatment with 0.5 mg/mL and 1 mg/mL MSZ, indicating reduced biomass of P. gingivalis (Figures 2A–C). MTT analysis revealed that the activity of P. gingivalis biofilms was inhibited by MSZ at these concentrations (Figure 2D). The P. gingivalis and polymicrobial biofilms were also observed by CLSM (Figures 2E,H). In the control group, the biofilms were thick and densely structured. However, after 2 days of 0.5 mg/mL and 1 mg/mL MSZ treatment, the percentage of live cells was significantly decreased in both newly formed and established plaque biofilms (Figures 2F,G,I,J).

The structure of P. gingivalis was observed by AFM. After 48 h of treatment with 1 mg/mL MSZ, the diameter of P. gingivalis decreased, and the surface became rough and irregular (Figure 3A). The average roughness significantly increased compared to untreated cells (Figure 3B). Young’s modulus was increased after 48 h of treatment, indicating that MSZ reduced the elasticity of the cell membrane (Figure 3C). In this study, we also determined the integrity of the cell wall using AKP analysis and the integrity of the cell membrane by assessing the LDH activity. Treatment with 0.5 mg/mL and 1 mg/mL MSZ led to a significant reduction of intracellular AKP and LDH activity in P. gingivalis compared to the control group (Figures 3D,E).

The effect of MSZ on the expression of virulence genes was assessed by qRT-PCR. The selected genes include a hemagglutinin B (HagB), a lysine-specific cysteine proteinase (Kgp), an arginine-specific cysteine proteinase A (RgpA), an arginine-specific cysteine proteinase B (RgpB), a minor fimbrial antigen (Mfa1), and virulence-modulating gene A (VimA). Treatment with MSZ at concentrations of 0.5 mg/mL and 1 mg/mL significantly reduced the expression of these P. gingivalis virulence genes in both biofilm-forming and pre-formed plaque biofilms (Figure 3G). The activity of gingipains was further evaluated using a BAPNA assay, which showed that 1 mg/mL MSZ could decrease P. gingivalis gingipain activity (Figure 3F).

Subsequently, we assessed the potential of MSZ to induce a comparable effect in vivo, which is crucial for periodontitis treatment. We developed a rat model to study experimental periodontitis (Figure 4A). Given the challenge of rapidly achieving an effective dose at the periodontal infection site with systemic medication, this study utilized local injections of MSZ. After euthanasia, maxillary samples from the rats were collected and analyzed using micro-CT scanning. Bone loss was assessed in three-dimensional palatal images by measuring the CEJ-ABC distance of the first molar’s distal buccal root (Figure 4B). Alveolar bone resorption was observed between the first and second maxillary molars following the installation of ligature wires. The model group showed increased bone loss on the palatal side compared to the controls (Figure 4C), confirming successful model establishment via the elevated CEJ-ABC distance. In the MSZ-treated group, the CEJ-ABC distance was significantly reduced compared to the model group, indicating less alveolar bone resorption. The model group exhibited decreased bone volume fraction and trabecular thickness and increased trabecular separation compared to the control group, confirming successful periodontitis modeling. In contrast, the MSZ group showed a significantly higher bone volume fraction and trabecular thickness, along with significantly lower trabecular separation, compared to the model group. In the model group, MSZ contributed to the partial restoration of bone volume (Figure 4D). Bone volume increased primarily due to reduced trabecular spacing and enhanced trabecular thickness.

To validate the results, the periodontal tissues from each rat group were assessed using H&E staining (Figure 4E). The H&E staining results indicated that the gingival papillae in the control group were tightly integrated with the epithelium and enamel surfaces. In contrast, the model group exhibited eroded gingival papillae, accompanied by epithelial proliferation toward the root and evident alveolar bone resorption, confirming the successful establishment of the periodontitis model. Compared to the model group, the MSZ-treated group showed reduced alveolar bone destruction and less damage to periodontal tissues, indicating that MSZ effectively controlled periodontitis in rats.

We examined the impact of MSZ treatment on the periodontal flora composition, which changes in the presence of periodontitis. P. gingivalis and F. nucleatum are the primary pathogens responsible for this condition. Using a ligature-induced rat model of periodontitis, we identified microorganisms from gingival tissues after 2 weeks of MSZ treatment by 16S rRNA sequencing. Our results showed that the control, model, and MSZ groups shared 57 common genera, with unique species totaling 239, 13, and 14 genera, respectively (Figure 5A). Principal component analysis indicated the differences in the species composition of the samples among the three groups (Figure 5B). In total, 10 bacterial species with significant differences and their proportion in the periodontal microbiota after MSZ treatment were showed in Figures 5C,E. The 16S rRNA identification results showed that the proportion of Porphyromonas, Fusobacterium, and Leptotrichia decreased after MSZ treatment when compared to the model group (Figure 5D). KEGG functional prediction analysis revealed that the most abundant KEGG categories were associated with the iron complex transport system, ABC-2 type transport system, sucrose-6-phosphatase, ATP-binding cassette, peptide/nickel transport system, RNA polymerase, and coproporphyrinogen oxidase (Figure 5F). In the complex oral environment, MSZ can reduce periodontal pathogens and disrupt plaque biofilm formation, leading to decreased local inflammation and prevention of further alveolar bone resorption.