Application of D-mannose in preparation of product for treating periodontitis

Abstract

The invention relates to application of D-mannose in preparation of products for treating periodontitis. The products comprise medicines, daily necessities and foods. The D-mannose provided by the invention can effectively inhibit the alveolar bone resorption of rats induced by LPS, down-regulate the expression of IL-1beta, IL-6, IL-17, TNF-alpha, G-CSF, GM-CSF, MPO, Ly6G, CD11b, TET2, HK1, HK2 and PFPKB3 in rat gingival tissues treated by the LPS, and up-regulate the expression of IL-10. In addition, the D-mannose can also inhibit the activation of neutrophil stimulated by LPS, down-regulate the expression of IL-1beta, IL-6, IL-17 and TET2, and up-regulate the expression of IL-10.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of D-mannose in preparing products for treating periodontitis. Background Art

[0002] D-mannose is a natural, safe and multifunctional small molecule, and its average concentration in mammalian serum is less than one-fiftieth of glucose (DOI:10.1155 / 2018 / 7168231). D-mannose is naturally present in some cells of the human body and participates in cellular immune regulation as a prebiotic (DOI:10.1038 / s41586-018-0350-5). For example, D-mannose can inhibit LPS-induced macrophage activation by inhibiting the production of IL-1β (DOI:10.1038 / s41586-018-0350-5). Another study showed that D-mannose can promote the differentiation of naive T cells into Tregs by reducing the level of IL-6 secreted by human periodontal ligament stem cells (hPDLSCs) (DOI:10.1155 / 2018 / 7168231). Current studies have found that D-mannose has good physiological benefits in anti-inflammation (DOI: 10.3389 / fimmu.2021.756920), immunomodulation, antibacterial and anti-tumor aspects: it has anti-cancer activity, can inhibit cell proliferation, and improve the effect of chemotherapy (DOI: 10.1038 / s41586-018-0729-3; DOI: 10.7554 / eLife.83870); it has good antibacterial effects and can effectively assist in the treatment of urinary tract infections (DOI: 10.1016 / j.euf.2020.09.004). Therefore, the future application prospects of D-mannose in dietary additives and the pharmaceutical industry have attracted widespread attention.

[0003] Periodontitis has become a global public health burden. Current treatments for periodontitis (DOI:10.3760 / cma.j.cn112144-20220719-00394) mainly include basic periodontal treatment, periodontal surgical treatment, restorative treatment, and periodontal supportive treatment. Mechanical treatments such as subgingival scaling and supragingival scraping are difficult to reach in some areas, and microorganisms can also enter the periodontal tissue, making it impossible to completely remove dental plaque. Although traditional periodontal treatment can control periodontal inflammation and delay disease progression, it cannot regenerate periodontal tissue; and new regenerative treatment strategies can restore vertical periodontal tissue loss to a certain extent, but the problems of limited regeneration capacity and poor predictability remain to be solved. Summary of the invention

[0004] The present invention aims at the problem that D-mannose is only used for the prevention and treatment of urinary tract infection but has not been effectively utilized, and provides a new application of D-mannose in the preparation of a product for treating periodontitis.

[0005] The structural formula of D-mannose is as follows:

[0006]

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The technical solution of the present invention is to provide the use of D-mannose in the preparation of products for treating periodontitis, wherein the products include medicines, daily necessities and foods.

[0009] In some specific embodiments, the product for treating periodontitis is a product for inhibiting alveolar bone resorption.

[0010] In some specific embodiments, the product for treating periodontitis is a product that inhibits the recruitment and activation of neutrophils.

[0011] In some specific embodiments, the product for treating periodontitis is a product that downregulates the expression of pro-inflammatory factors and / or upregulates anti-inflammatory factors and / or downregulates glycolytic enzymes and / or downregulates neutrophil markers and / or downregulates TET2 protein.

[0012] In some specific embodiments, the pro-inflammatory factors include IL-1β, IL-6, IL-17, TNF-α, G-CSF, GM-CSF; and the anti-inflammatory factors include IL-10.

[0013] In some embodiments, the glycolytic enzymes include HK1, HK2, and PFPKB3.

[0014] In some specific embodiments, the neutrophil markers include MPO, Ly6G, and CD11b.

[0015] In some embodiments, the drug for treating periodontitis comprises D-mannose and a pharmaceutically acceptable carrier.

[0016] In some specific embodiments, the drug for treating periodontitis is an oral tablet, capsule, granule, paste, oral solution or injection.

[0017] In some specific embodiments, the drug for treating periodontitis is a controlled-release dosage form for oral administration, injection or implantation, or a corresponding dosage form for administration via an interventional catheter.

[0018] In some specific embodiments, the daily necessities for treating periodontitis include toothpaste and mouthwash.

[0019] In some specific embodiments, the food for treating periodontitis includes chewing gum and soft candy.

[0020] Compared with the prior art, the D-mannose provided by the present invention can effectively inhibit LPS-induced alveolar bone resorption in rats, downregulate the expression of IL-1β, IL-6, IL-17, TNF-α, G-CSF, GM-CSF, MPO, Ly6G, CD11b, TET2, HK1, HK2 and PFPKB3 in the gingival tissue of LPS-treated rats, and upregulate the expression of IL-10. In addition, D-mannose can also inhibit the activation of neutrophils stimulated by LPS, downregulate the expression of IL-1β, IL-6, IL-17 and TET2, and upregulate the expression of IL-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of D-mannose on LPS-induced alveolar bone resorption in rats. Data are expressed as mean ± SD (methylene staining n = 6; H&E staining n = 1; micro-CT n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group.

[0022] Figure 2 The effect of D-mannose on LPS-induced inflammation of rat gingival tissue. Data are expressed as mean ± SD (n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group. Figures AD are 200×, and the scale bar represents 100 μm.

[0023] Figure 3 The effect of D-mannose on neutrophils in the gingival tissue of periodontal rats induced by LPS. Data are expressed as mean ± SD (n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group. Figures AB and D are 200×, and the scale bar represents 100 μm.

[0024] Figure 4 D-mannose inhibits LPS-induced neutrophil activation in vitro. Data are presented as mean ± SD (n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group. Figures B and D are 200×, and the scale bar represents 100 μm.

[0025] Figure 5 D-mannose inhibits LPS-induced pro-inflammatory phenotype and TET2 expression of neutrophils in vitro. Data are presented as mean ± SD (n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group. Figures AE are 400×, and the scale bar represents 100 μm.

[0026] Figure 6D-mannose inhibits the expression of key glycolytic enzymes in gingival tissue of rats with periodontitis. Data are presented as mean ± SD (n = 3). *p < 0.05 vs. LPS group, **p < 0.1 vs. LPS group.

[0027] Figure 7 This is an illustration of the working principle of D-mannose of the present invention. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0029] In order to verify the new use of D-mannose proposed in the present invention in the preparation of drugs for treating periodontitis, the following experiments were conducted.

[0030] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0031] (1) Sprague Dawley rats were obtained from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0032] (2) Lipopolysaccharide (LPS) used in rat treatment was purchased from Sigma, USA.

[0033] (3) D-Mannose, purchased from Sigma-Aldrich, USA.

[0034] (4) Antibodies used in IF staining:

[0035] Purchased from ABclonal, China: anti-IL-1β, catalog number A22207; anti-IL-6, catalog number A22222; anti-IL-10, catalog number A12255; anti-IL-17, catalog number A10587; anti-TET2, catalog number A5682; anti-Ly6G, catalog number A22270.

[0036] Purchased from Abmart, China: Anti-MPO, catalog number T62224.

[0037] (5) Hydrogen peroxide blocking reagent, purchased from Thermo Fisher Scientific, USA.

[0038] (6) RIPA lysis buffer, purchased from BioSharp, China.

[0039] (7) BCA protein quantification kit, purchased from Res Biotech, Shanghai, China.

[0040] (8) 5× sample loading buffer, purchased from BioSharp, China.

[0041] (9) Polyvinylidene fluoride (PVDF) membrane, purchased from Merck Millipore, USA.

[0042] (10) Antibodies used in Western Blot: GAPDH, purchased from Proteintech, USA, model number 60004-1-Ig; anti-IL-1β, purchased from Cell Signal, USA, model number 12242S; anti-IL-6, purchased from Abclonal, China, model number A2222); anti-IL-10, purchased from Abclonal, China, model number A2171; anti-IL-17, purchased from Abclonal, China, model number A10587; anti-MPO, purchased from Abcam, UK, model number ab65871; anti-CD11b, purchased from Abcam, UK, model number ab133357; anti-TET2, purchased from Abclonal, China, model number A5682.

[0043] (11) TRIzol, purchased from Thermo Fisher Scientific, USA.

[0044] (12) PrimeScript RT Master Mix, purchased from Takara, Japan, model number: Cat#RR036A.

[0045] (13) Rat neutrophil isolation kit, purchased from Solarbio, P9200, China.

[0046] (14) BV786 mouse anti-rat RP-1 antigen, purchased from BD Biosciences, USA.

[0047] (15) Lipopolysaccharide (LPS) used to treat neutrophils was purchased from Sigma-Aldrich, USA, model number SMB00610, and is a characteristic component of the cell wall of Porphyromonas gingivalis.

[0048] All experimental equipment and procedures were reviewed and approved by the Experimental Animal Ethics Committee of Shanghai Tenth People's Hospital (No. SHDSYY-2022-3063).

[0049] Example 1: Effect of D-mannose on LPS-induced alveolar bone resorption in rats

[0050] 1. Construction of LPS-induced periodontitis model in rats

[0051] Eighteen eight-week-old Sprague Dawley rats (half male and half female) were purchased as experimental animals, and the experimental animals were adaptively fed for 1 week. The rats were anesthetized with 2% sodium pentobarbital (0.2 mL / 100 g), and 2 μL of lipopolysaccharide (LPS) with a concentration of 10 μg / μL was injected once a day into the middle side of the lingual interdental papilla of the first and second molars on both sides of the maxilla to induce periodontitis for the first two days. During the injection, a microsyringe (Hamilton, Switzerland) was required to inject slowly, and the needle was kept in place for several seconds after the injection to ensure that LPS would not be lost after extraction. For D-mannose treatment, rats were orally gavaged with 2 mL of 20% (w / v) D-mannose twice a day starting from the first day of LPS stimulation. During the experiment, the changes in the state of the rats, diet, feces, and periodontal conditions (whether plaque was accumulated, whether the gums were red, swollen, or even bleeding) were observed and recorded every day, and the amount of water the rats drank was recorded. To evaluate periodontitis lesions, the rats were sacrificed after 14 days. The 18 rats were divided into the following experimental groups (n = 6 per group) ( Figure 1 A).

[0052] Control group: Rats received the same procedure as above but were injected with saline instead of LPS and D-mannose.

[0053] LPS group: rats received the above-mentioned LPS-induced periodontitis treatment.

[0054] LPS+Man group: rats received the above-mentioned LPS-induced periodontitis treatment and the above-mentioned D-mannose treatment at the same time.

[0055] 2. Collection and processing of maxillary bone and soft tissue samples

[0056] (2-1) The right maxillary bones and surrounding soft tissues of all specimens were collected as tissue samples for Micro-CT scanning and analysis.

[0057] After fixation in 4% paraformaldehyde for 24 hours, the specimens were placed on the scanning bed parallel to the occlusal surface of the molar crown. Then, Micro-CT (Skyscan1176, Bruker, USA) scanning was performed (pixel resolution = 9 μm, 50 kV, 500 A). The original data were reconstructed by NRecon software (Skyscan, Kontich, Belgium) and transferred to CTAnalyser and CTVOX software (Skyscan, Kontich, Belgium) for further analysis. The triangular area below the furcation of the second molar (with a width of 2 mm and a depth from the furcation point to the point where either root disappears) was selected as the region of interest (ROI) ( Figure 1H, red triangle area). The cancellous bone in the ROI was selected and measured three times. The outcome indicators included: bone volume percentage (bone volume / tissue volume, BV / TV) and trabecular separation (Tb.Sp).

[0058] To quantify bone loss, six points (3 points on the first molar, 2 points on the second molar, and 1 point on the third molar) were selected to measure the distance between the cementum-alveolar junction and the alveolar crest (CEJ-ABC) on the palatal side of each rat. The CEJ-ABC distance data of all selected points were analyzed by three blinded investigators. The mean of three measurements was calculated for further analysis.

[0059] (2-2) The right maxillary bones and surrounding soft tissues of all samples were collected and fixed in 4% paraformaldehyde. Six samples in each group were decalcified in 10% EDTA solution (EDTA 100g, NaOH 11g, Na2HPO4 6g, NaH2PO49g, ddH2O, mixed in 900mL magnetic stirrer, ddH2O volume adjusted to 1000mL, pH adjusted to 7.2-7.4, 4°C). After dehydration, the right maxillary bones and soft tissues were embedded in paraffin to prepare paraffin sections, and the sections were 5mm thick. The paraffin sections were detected by conventional hematoxylin-eosin (H&E) staining and immunofluorescence (IF) staining.

[0060] (2-2-1) IF staining and quantification:

[0061] After the paraffin sections were blocked with hydrogen peroxide blocking reagent, they were incubated overnight at 4°C with the following antibodies: anti-IL-1β (PBS configuration, v:v=1:200), anti-IL-6 (PBS configuration, v:v=1:200), anti-IL-10 (PBS configuration, v:v=1:200), anti-IL-17 (PBS configuration, v:v=1:1000). Then, after washing three times with PBS, they were incubated at room temperature for 2 hours in the presence of the corresponding secondary antibody labeled with Alexa Fluor 594 (red). Finally, DAPI staining (blue) was used to show the cell nucleus, and images were taken with a CarlZeiss LSM710 fluorescence microscope. The magnification of the objective lens was 10x, and the acquisition software was LAS V4.12 software. The images of each group were taken at a resolution of 1920x1440 pixels using uniform parameters (brightness, saturation, gamma adjustment, etc.). The brightness was adjusted consistently when processing the photos. For quantification, the relative fluorescence intensity was measured using ImageJ software. A square ROI of a fixed area was drawn around the tissue, and the mean fluorescence intensity within the ROI was measured. Three independent fields of view were measured for each tissue. The results are shown as mean ± standard deviation (SD). The proportion of positive cells was measured using ImageJ software. The ratio of the number of cells positively stained with the marker to the total number of cells was quantified and expressed as a percentage.

[0062] (2-3) The left maxillary bones of all samples were collected and stained with 1% methylene blue and then examined using a vernier caliper.

[0063] (2-4) The left maxillary perigingival soft tissue of all samples was collected for Western Blot analysis and RT-PCR analysis.

[0064] (2-4-1) Western Blot Analysis

[0065] Gingival tissues from all left maxilla were collected and stored at -80°C for protein extraction. Protein samples from gingival tissues were extracted with 300 μL RIPA lysis buffer. Protein concentrations in the collected lysates were quantified using a BCA protein quantification kit with BSA as the standard. Equal amounts of protein from each group were mixed with 5× sample loading buffer, heated at 95°C for 5 min, and proteins were separated on SDS-PAGE gels according to molecular weight and transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, nonspecific sites on the PVDF membrane were blocked with 5% milk in PBS at room temperature to prevent nonspecific binding of antibodies to target proteins. After blocking for 1 hour, the membrane was incubated with the following primary antibodies at 4°C overnight: GAPDH (PBS, v:v=1:10,000), anti-IL-1β (PBS, v:v=1:1000), anti-IL-6 (PBS, v:v=1:1000), anti-IL-10 (PBS, v:v=1:1000), anti-IL-17 (PBS, v:v=1:1000). The membrane was washed three times with PBST (PBS containing 0.1% Tween-20) for 10 minutes each, and then incubated with the corresponding secondary antibody at room temperature for 1 hour. The specific bands were visualized using the Tanon-5200 Multi chemiluminescence system (Tanon). Gray values ​​were measured and calculated using ImageJ software.

[0066] (2-4-2) RT-PCR analysis

[0067] Total RNA was extracted from the gingival tissue around the left maxillary bone of each group using TRIzol. cDNA was reverse transcribed using PrimeScript RTMaster Mix, and RT-PCR was performed using specific primers as shown in Table 1.

[0068] Table 1. Primer sequences used for RT-qPCR analysis.

[0069]

[0070]

[0071] 3. Statistical analysis

[0072] All data were analyzed using GraphPad Prism version 9.5.1 (733) software. Data from experiments repeated at least three times are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Dunnett's post hoc test, and p < 0.05 was considered statistically significant.

[0073] The results are as follows:

[0074] To investigate the potential of D-mannose in alleviating alveolar bone resorption in rats, an LPS-induced periodontitis animal model was established ( Figure 1 A), and the maxillary bones were subjected to methylene blue staining, H&E staining, and Micro-CT scanning.

[0075] like Figure 1 BC shows the methylene blue staining of left maxillary bone resorption and the quantitative analysis of methylene blue staining data. It can be seen that compared with the LPS group, D-mannose in the LPS+Man group can effectively reduce left maxillary bone resorption.

[0076] like Figure 1 DE shows the H&E staining of bone resorption in right maxillary sections and quantitative analysis of H&E staining data. It can be seen that compared with the LPS group, D-mannose in the LPS+Man group can effectively reduce bone resorption in the right maxilla.

[0077] like Figure 1 FG show the sagittal three-dimensional image of the right maxillary bone and the relative distance of CEJ-ABC evaluated by Micro-CT, respectively. It can be seen that the CEJ-ABC distance in the LPS group increased significantly compared with the control group, indicating that the establishment of the rat periodontitis model was successful. Compared with the LPS group, the CEJ-ABC distance in the LPS+Man group was significantly reduced.

[0078] like Figure 1 IJ show the trabecular separation degree (Tb.Sp) and bone volume percentage (bone volume / tissue volume, BV / TV) evaluated by Micro-CT, respectively. Quantitative analysis of ROI confirmed that compared with the LPS group, the Tb.Sp in the LPS+Man group was significantly reduced, and the BV / TV was significantly increased.

[0079] These results collectively indicate that D-mannose can effectively inhibit maxillary bone resorption in rats with LPS-induced periodontitis.

[0080] In order to explore the effect of D-mannose on gingival inflammation in rats with periodontitis, the researchers determined the expression levels of inflammatory factors in gingival tissue based on the above experiments.

[0081] like Figure 2 The IF results shown in AE showed that compared with the control group, the expression levels of proinflammatory factors (IL-1β, IL-6 and IL-17) in the LPS group were increased, while the expression level of IL-10 was significantly decreased; and compared with the LPS group, the LPS+Man group reduced the expression levels of proinflammatory factors (IL-1β, IL-6 and IL-17) and increased the expression level of anti-inflammatory factor IL-10 due to D-mannose.

[0082] like Figure 2 WB analysis shown in FG showed that the expression levels of IL-1β, IL-6, IL-17, and IL-10 in the LPS+Man group showed a similar trend to the IF results compared with the LPS group.

[0083] like Figure 2 The RT-PCR results shown in H showed that the mRNA levels of IL-1β, IL-6, IL-17, TNF-α, G-CSF and GM-CSF in the gingival tissue after D-mannose treatment in the LPS+Man group were significantly decreased compared with those in the LPS group.

[0084] These findings on the expression levels of inflammatory factors in gingival tissues indicated that D-mannose reduced gingival inflammation in rats with periodontitis.

[0085] Example 2: D-mannose inhibits the recruitment and activation of neutrophils and the expression of TET2 in the gingival tissue of rats with periodontitis

[0086] The following experiment was conducted using the paraffin sections and gingival tissues in Example 1.

[0087] 1. Immunofluorescence (IF) staining and quantification

[0088] Referring to Example 1, the primary antibodies used here are anti-MPO (PBS configuration, v:v=1:200), anti-TET2 (PBS configuration, v:v=1:200), and anti-Ly6G (PBS configuration, v:v=1:200). Then, after washing three times with PBS, incubation with different secondary antibodies labeled with Alexa Fluor 594 (red) or Alexa Fluor 488 (green) for two hours at room temperature for co-localization staining.

[0089] 2. Western Blot Analysis

[0090] Referring to Example 1, the antibodies used here are GAPDH (PBS configuration, v:v=1:10,000), anti-MPO (PBS configuration, v:v=1:1000), anti-CD11b (PBS configuration, v:v=1:1000) and anti-TET2 (PBS configuration, v:v=1:1000)

[0091] 3. RT-PCR analysis

[0092] Referring to Example 1, the specific primers used here are shown in Table 2.

[0093] Table 2. Primer sequences used for RT-qPCR analysis.

[0094]

[0095] 4. Statistical analysis

[0096] All data were analyzed using GraphPad Prism version 9.5.1 (733) software. Data from experiments repeated at least three times are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Dunnett's post hoc test, and p < 0.05 was considered statistically significant.

[0097] The results are as follows:

[0098] Given the importance of neutrophils in periodontitis, the study evaluated the effect of D-mannose on the abundance and functional status of neutrophils in gingival tissue. CD11b and Ly6G are markers of mature neutrophils, while MPO is a marker of neutrophil activation.

[0099] like Figure 3 IF analysis of Ly6G and MPO shown in AC showed that the expression levels of Ly6G and MPO in the LPS group were significantly increased compared with the control group. Compared with the LPS group, the LPS+Man group significantly decreased the expression levels of these markers in gingival tissue in vivo.

[0100] like Figure 3 WB of MPO and CD11b shown in FG showed that the expression of MPO and CD11b in the LPS+Man group was similar to the expression trend shown by IF.

[0101] These results suggest that D-mannose inhibits the recruitment and activation of neutrophils in gingival tissues of patients with periodontitis.

[0102] In addition, since TET2 plays a key role in the innate immune balance, the expression level of TET2 in gingival tissue was also analyzed.

[0103] like Figure 3 IF co-localization staining of TET2 and MPO shown in DE shows that Figure 3 Western blot analysis of TET2 shown in FG and Figure 3H shows the RT-PCR analysis of TET2. The expression levels of TET2 in gingival tissues of the three groups showed the same trend as the expression levels of MPO.

[0104] Taken together, these results suggest that one of the potential mechanisms of D-mannose in treating periodontitis may be to inhibit the recruitment and activation of neutrophils and downregulate the expression of TET2.

[0105] Example 3: D-mannose inhibits LPS-induced neutrophil activation in vitro

[0106] 1. Blood sample collection and neutrophil separation

[0107] Twelve eight-week-old Sprague Dawley rats were purchased, and blood samples were collected from the abdominal aorta of all rats after anesthesia with 2% sodium pentobarbital (0.2 mL / 100 g). Neutrophils were isolated from the blood samples using a rat neutrophil isolation kit.

[0108] 2. Flow cytometric analysis of peripheral blood neutrophils

[0109] The collected neutrophils (2×10 6 Cells) were stained with a specific antibody BV786 mouse anti-rat RP-1 antigen to analyze the frequency of neutrophils. The affected cells were analyzed using a BD FACSCalibur and BD FACSAria III flow cytometer (BD Biosciences, USA). Gating controls were performed using positive gates and appropriate negative controls, where the positive gate was gated after excluding non-target cells, and the negative control was a sample that was not stained with the antibody and only showed background fluorescence signals.

[0110] 3. In vitro neutrophil treatment

[0111] The isolated neutrophils were randomly divided into three groups. All groups were placed in an incubator set at 37°C and 5% CO2. After modeling, the neutrophils were centrifuged at 12000rpm for 2 minutes to remove the supernatant, resuspended and washed with 20μl PBS, centrifuged again, and the supernatant was removed. The cells were then fixed with 4% paraformaldehyde for 30 minutes, centrifuged, and the supernatant was removed. After washing twice with PBS, the cells were spread on a labeled culture plate and dried in an oven at 60°C for about 5 minutes. The subsequent procedures followed the same protocol for tissue IF in Example 1.

[0112] The modeling method is as follows:

[0113] Control group: Neutrophils received no special treatment.

[0114] LPS group: Neutrophils were exposed to LPS for 4 hours (100 ng / mL)

[0115] LPS+Man group: Before being exposed to LPS simultaneously with the LPS group, neutrophils were pre-treated with 10mM, 25mM, 50mM, and 100mM D-mannose for 4h.

[0116] Alternatively, centriole cells were treated with 50 mM mannose for 15 min, 30 min, 1 h, 2 h, and 4 h.

[0117] 4. IF staining and quantification

[0118] Referring to Example 1, the antibody used here is anti-MPO (PBS configuration, v:v=1:200).

[0119] 5. Statistical analysis

[0120] All data were analyzed using GraphPad Prism version 9.5.1 (733) software. Data from experiments repeated at least three times are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Dunnett's post hoc test, and p < 0.05 was considered statistically significant.

[0121] To verify the direct effect of D-mannose on neutrophils, neutrophils were stimulated in vitro with LPS. Neutrophils were isolated from rat peripheral blood and their purity was assessed by flow cytometry, and the results showed that the purity of neutrophils was high, exceeding 80%, and they were used for further cell experiments, such as Figure 4 As shown in A, the blue one is the negative control sample, and the red one is the sample to be tested with the addition of antibodies.

[0122] like Figure 4 BE, IF showed that the effective concentration and duration of D-mannose treatment on neutrophils in vitro were 50 mM and 4 h, respectively, and it was shown that D-mannose inhibited the activation of neutrophils stimulated by LPS in vitro.

[0123] Example 4: D-mannose inhibits LPS-induced pro-inflammatory phenotype of neutrophils and TET2 expression in vitro

[0124] 1. In order to further study the effect of D-mannose on neutrophil activation, the following neutrophil treatments were performed:

[0125] Control group: Neutrophils received no special treatment.

[0126] LPS group: Neutrophils were exposed to LPS (100 ng / mL) for 4 hours

[0127] LPS+Man group: Before being exposed to LPS simultaneously with the LPS group, neutrophils were pre-treated with 50 mM D-mannose for 4 h.

[0128] 2. IF staining and quantification

[0129] With reference to Example 1, the primary antibodies herein are anti-MPO (PBS configuration, v:v=1:200), anti-IL-1β (PBS configuration, v:v=1:200), anti-IL-6 (PBS configuration, v:v=1:200), anti-IL-10 (PBS configuration, v:v=1:200), anti-IL-17 (PBS configuration, v:v=1:1000), anti-TET2 (PBS configuration, v:v=1:200). The secondary antibodies are the corresponding antibodies labeled with Alexa Fluor 594 (red) or Alexa Fluor 488 (green).

[0130] 3. Statistical analysis

[0131] All data were analyzed using GraphPad Prism version 9.5.1 (733) software. Data from experiments repeated at least three times are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Dunnett's post hoc test, and p < 0.05 was considered statistically significant.

[0132] The results are as follows:

[0133] like Figure 5 IF shown in AF shows that LPS in the LPS group can significantly increase the expression levels of MPO, IL-1β, IL-6, IL-17 and TET2, and significantly reduce the expression level of IL-10. However, after treatment with D-mannose in the LPS+Man group, the expression levels of MPO, IL-1β, IL-6, IL-17 and TET2 were reduced, and the expression level of IL-10 was increased. Figure 5 A, Colocalization of TET2 and MPO can also be observed.

[0134] These in vitro results further confirmed the inhibitory effect of D-mannose on neutrophil activation and further demonstrated its ability to inhibit the neutrophil proinflammatory phenotype and TET2 expression in neutrophils.

[0135] Example 5: D-mannose inhibits glycolysis in gingival tissue of rats with periodontitis

[0136] 1. Considering the role of D-mannose in sugar metabolism and the positive correlation between glycolysis and inflammatory response, D-mannose may inhibit glycolysis in the gingival tissue of rats with periodontitis. RT-PCR analysis was performed on key glycolytic enzymes (HK1, HK2, PFKFB3) in gingival tissue. The RT-PCR analysis was referred to Example 1. The specific primers used here are shown in Table 3.

[0137] The same grouping as in Example 1 was used: control group, LPS group, and LPS+Man group.

[0138] Table 3. Primer sequences used for RT-qPCR analysis.

[0139]

[0140] 2. Statistical analysis

[0141] All data were analyzed using GraphPad Prism version 9.5.1 (733) software. Data from experiments repeated at least three times are presented as mean ± standard deviation (SD). Statistical comparisons were performed using one-way ANOVA followed by Dunnett's post hoc test, and p < 0.05 was considered statistically significant.

[0142] The results are as follows:

[0143] like Figure 6 LPS in the LPS group significantly upregulated the mRNA level of HK1, while D-mannose in the LPS+Man group significantly downregulated the mRNA levels of these three enzymes (HK1, HK2, PFKFB3).

[0144] These results suggest that D-mannose may alleviate periodontitis by inhibiting glycolysis.

[0145] Based on Examples 1-5, the present invention combines in vivo and in vitro experiments to verify the effect of D-mannose on periodontitis, which is summarized as follows:

[0146] In order to determine the protective effect of D-mannose on alveolar bone loss, the present invention established an LPS-induced periodontitis rat model, treated it with D-mannose by oral gavage, and analyzed it using methylene blue staining, H&E staining and Micro-CT scanning technology.

[0147] In order to explore the effect of D-mannose on gingival tissue inflammation, neutrophils, and glycolysis, the present invention uses IF, Western Blot and RT-qPCR to determine the expression levels of interleukins (IL-1β, IL-6, IL-17, IL-10), tumor necrosis factor-α (TNF-α), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), myeloperoxidase (MPO), lymphocyte antigen 6G (Ly6G), integrin αM (CD11b), DNA methyltransferase 2 (TET2) and key glycolytic enzymes (HK1, HK2, PFPKB3) in gingival tissue. At the same time, neutrophils were isolated from the peripheral blood of healthy rats, treated with LPS and D-mannose, and the changes in the expression levels of MPO, IL-1β, IL-6, IL-17, IL-10 and TET2 were observed by IF.

[0148] The results showed that D-mannose could inhibit LPS-induced alveolar bone resorption in rats, down-regulate the expression of IL-1β, IL-6, IL-17, TNF-α, G-CSF, GM-CSF, MPO, Ly6G, CD11b, TET2, HK1, HK2 and PFPKB3 in the gingival tissue of LPS-treated rats, and up-regulate the expression of IL-10. In vitro experiments found that D-mannose inhibited the activation of neutrophils stimulated by LPS, down-regulated the expression of IL-1β, IL-6, IL-17 and TET2, and up-regulated the expression of IL-10. Therefore, D-mannose alleviates chronic periodontitis in rats by regulating the function of neutrophils, and its mechanism may be related to down-regulating the expression of TET2 and inhibiting glycolysis. D-mannose can be made into therapeutic drugs for the treatment of periodontitis, such as Figure 7 shown.

[0149] Embodiment 6:

[0150] This example illustrates the method of using D-mannose to prepare a drug for treating periodontitis.

[0151] Generally speaking, D-mannose can be combined with pharmaceutically acceptable carriers to prepare different dosage forms.

[0152] Exemplarily, a specific method for preparing a solid preparation is: mixing D-mannose with pharmaceutical excipients, such as starch and sugar, and then making it into solid dosage forms such as tablets, capsules, granules, etc. for oral use.

[0153] Tablets: D-mannose is combined with different proportions (1-99%) and appropriate amounts of fillers (such as glucose), binders (such as hydroxypropyl methylcellulose), and lubricants (such as magnesium stearate). One of the combinations is: D-mannose, glucose, hydroxypropyl methylcellulose and magnesium stearate. After mixing, granulation, drying, sieving and tableting, chewable tablets are made. Each tablet contains 500 mg of D-mannose for clinical oral administration.

[0154] Capsules: D-mannose is mixed with appropriate amounts of the above-mentioned fillers and lubricants in different proportions (1-99%) and loaded into capsule shells. D-mannose can also be mixed with different solvents to make soft capsules. One combination is: D-mannose, glucose, hydroxypropyl methylcellulose and magnesium stearate. Each capsule contains 500 mg of D-mannose for clinical oral administration.

[0155] Other dosage forms: D-mannose can also be made into other dosage forms according to clinical treatment needs: such as ointment, oral solution, injection, etc.

[0156] D-mannose can also be used to prepare daily necessities for the treatment of periodontitis, such as toothpaste and mouthwash.

[0157] D-mannose can also be used to prepare foods for treating periodontitis, such as chewing gum and soft candy.

[0158] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. The use of D-mannose in preparing a product for treating periodontitis, characterized in that: The products include medicines, daily necessities, and food.

2. The use of D-mannose according to claim 1 in preparing a product for treating periodontitis, characterized in that: The product for treating periodontitis is a product for inhibiting alveolar bone absorption.

3. The use of D-mannose according to claim 1 in preparing a product for treating periodontitis, characterized in that: The product for treating periodontitis is a product for inhibiting the recruitment and activation of neutrophils.

4. The use of D-mannose according to claim 1 in preparing a product for treating periodontitis, characterized in that: The product for treating periodontitis is a product that downregulates the expression of pro-inflammatory factors and / or upregulates the expression of anti-inflammatory factors and / or downregulates the expression of glycolytic enzymes and / or downregulates the expression of neutrophil markers and / or downregulates the expression of TET2 protein.

5. The use of D-mannose in the preparation of a product for treating periodontitis according to claim 4, characterized in that: The pro-inflammatory factors include IL-1β, IL-6, IL-17, TNF-α, G-CSF, and GM-CSF; the anti-inflammatory factors include IL-10.

6. The use of D-mannose in the preparation of a product for treating periodontitis according to claim 4, characterized in that: The glycolytic enzymes include HK1, HK2, and PFPKB3.

7. The use of D-mannose in the preparation of a product for treating periodontitis according to claim 4, characterized in that: The neutrophil markers include MPO, Ly6G, and CD11b.

8. The use of D-mannose according to claim 1 in preparing a product for treating periodontitis, characterized in that: The drug for treating periodontitis comprises D-mannose and a pharmaceutically acceptable carrier.

9. The use of D-mannose in the preparation of a product for treating periodontitis according to claim 1, characterized in that: The daily necessities for treating periodontitis include toothpaste and mouthwash.

10. The use of D-mannose in the preparation of a product for treating periodontitis according to claim 1, characterized in that: The food for treating periodontitis includes chewing gum and soft candy.

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