Application of ganoderic acid A in preparation of drugs and oral care products for preventing and treating periodontitis diseases

Abstract

The invention discloses application of ganoderic acid A in preparation of drugs and oral care products for preventing and treating periodontitis diseases. Research finds that GAA (ganoderic acid A) can improve pyroptosis caused by periodontal pathogenic bacteria and regulate and control expression of pyroptosis-related proteins, and animal experiments prove that GAA can improve mouse periodontitis diseases. GAA has a potential drug effect of regulating and controlling periodontitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically discloses the application of Ganoderic A (GAA) in the preparation of medicines for preventing and treating periodontal inflammatory diseases and oral care products. Background Art

[0002] Periodontitis is a chronic inflammatory disease initiated by a microbial plaque film. It is primarily influenced by the interplay of multiple factors, including plaque microbes, the host immune response, and environmental factors. The excessive host immune inflammatory response to the invasion of pathogenic microorganisms leads to degradation and destruction of periodontal tissues, ultimately leading to tooth loss.

[0003] Cell pyroptosis is a highly inflammatory form of programmed cell death that is closely related to local inflammation and is usually accompanied by a strong inflammatory response. Pyroptosis is closely related to the progression of periodontitis. Increased levels of pyroptosis in periodontal tissues can lead to overactivation of the host immune response and promote the massive secretion of inflammatory factors IL-1β and IL-18, resulting in an overactive immune response and degradation of collagen in periodontal tissues, causing damage to periodontal soft tissues. At the same time, pyroptosis also weakens the activity of osteoblasts, increases the proliferation rate and activity of osteoclasts, aggravates the bone resorption process, and ultimately leads to significant destruction of alveolar bone. Clinical studies have shown that the levels of pyroptosis-related markers NLRP3 and GSDMD in the gingival tissue of patients with periodontitis are significantly higher than those in healthy subjects, and are correlated with the degree of periodontal inflammation.

[0004] Traditional Chinese medicines are primarily derived from natural plants, are widely available, and are inexpensive. They are generally considered to possess excellent immunomodulatory properties. Ganoderma lucidum, a precious medicinal fungus first mentioned in Shennong's Classic of Materia Medica, is known as the "immortal herb" of traditional Chinese medicine. Ganoderic acid A (GAA), the primary active ingredient in Ganoderma lucidum, possesses multiple pharmacological effects. However, there are currently no reports demonstrating the therapeutic effects of GAA or other Ganoderma lucidum extracts on periodontitis. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of GAA in the preparation of drugs and oral care products for the prevention and treatment of periodontal inflammatory diseases. By exploring the regulatory mechanism of GAA in cell pyroptosis, the present invention confirms that GAA can regulate the classic cell pyroptosis pathway NLRP3-caspase1-GSDMD, reduce the expression of pyroptosis-related proteins NLRP3, caspase1, N-GSDMD, IL-1β, and IL-18, improve cell pyroptosis, and improve periodontal inflammation in periodontitis mice. The purpose of the present invention is achieved by the following technical solutions:

[0006] The application of ganoderic acid A in the preparation of drugs for preventing and treating periodontal inflammatory diseases and oral care products is characterized in that: the molecular formula of ganoderic acid A is C 30 H 44 O7, the structural formula is as follows:.

[0007]

[0008] As a solution, the ganoderic acid A is used as the sole active ingredient in the preparation of drugs for preventing and treating periodontal inflammatory diseases and oral care products.

[0009] In a further optimization, the drug further comprises a pharmaceutically acceptable carrier.

[0010] Furthermore, the carrier is a polar solvent and a non-polar solvent; the polar solvent is selected from one or more of water, ethanol, glycerol, propylene glycol and DMSO, and the non-polar solvent is selected from one or two of fatty oil and liquid paraffin.

[0011] Furthermore, the drug also includes supplementary additives, which are selected from one or more of diluents, buffers, encapsulating agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents and adsorption carriers.

[0012] Furthermore, the dosage form of the drug is a suspension, emulsion, granule, spray, injection, transdermal absorbent, dosage form suitable for transfection, tablet, powder, granule or capsule.

[0013] Furthermore, the oral care products include toothpaste, mouthwash, oral spray, oral gel, film and chewable colloid.

[0014] The advantages and beneficial effects of the present invention are:

[0015] The present invention discovered that GAA can ameliorate pyroptosis caused by periodontal pathogens and regulate the expression of pyroptosis-related proteins. Animal experiments have also demonstrated that GAA can improve periodontitis in mice. Therefore, GAA has the potential to be a drug for regulating periodontal inflammation. This discovery of a novel function provides new insights into the treatment of periodontal inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be further described below with reference to the accompanying drawings and examples.

[0017] Figure 1 Effects of GAA on THP-1 cell viability. (a) Live-dead staining shows the effects of different concentrations of GAA on Porphyromonas gingivalis (Pg)-induced pyroptosis in THP-1 cells. Black scale bar 4×: 200 μm. (b) CCK8 assay assesses the effects of Pg and GAA on THP-1 cell viability. **: P < 0.01, ****: P < 0.0001.

[0018] Figure 2 Effect of GAA on Pg-induced pyroptosis in THP-1 cells. Figure 2 (a)-(e) Changes in the mRNA levels of key molecules of the classical pyroptosis pathway, NLRP3, caspase1, GSDMD, IL-1β, and IL-18, in THP-1 cells of each group; Figure 2 (f) Changes in the expression levels of key proteins of the classical pyroptosis pathway in THP-1 cells in each group; Figure 2 (g1-g5) Quantitative analysis of key proteins of the classical pyroptosis pathway in THP-1 cells in each group; ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0019] Figure 3 NLRP3 staining results in mouse periodontitis. Figure 3 (a) Expression of NLRP3 in mouse maxillary tissue, scale bar 4×: 200 μm; Figure 3 (b) and (c) show the expression of NLRP3 in the furcation and interproximal regions of the second molar of mice, scale bar 10×: 100 μm; Figure 3 (d) Percentage of NLRP3-positive staining area; *: P < 0.05, ***: P < 0.001.

[0020] Figure 4 GSDMD staining results of periodontitis in mice. Figure 4 (a) Expression of GSDMD in mouse maxillary tissue, scale bar 4×: 200 μm; Figure 4(b) and (c) show the expression of GSDMD in the furcation and interproximal regions of the second molar of mice, scale bar 10×: 100 μm; Figure 4 (d) is the percentage of GSDMD-positive areas; **: P < 0.01.

[0021] Figure 5 Analysis of Micro-CT results of periodontitis in mice. Figure 5 (a) Schematic diagram of Micro-CT 3D reconstruction of mouse maxilla, palatal side; Figure 5 (b) Micro-CT two-dimensional reconstruction of the cross-section of the second molar of the mouse alveolar bone; Figure 5 (c) is the distance from the cementoenamel junction to the crest of the bone (CEJ-ABC); Figure 5 (d) is the percentage of bone volume within the ROI (BV / TV); Figure 5 (e) Bone mineral density (BMD) within ROI; ns: P>0.05, *: P<0.05, ****: P<0.0001.

[0022] Figure 6 HE staining results of mouse periodontitis. Figure 6 (a) Schematic diagram of HE staining of mouse maxillary bone, black scale bar 4×: 200 μm; Figure 6 (b) Schematic diagram of HE staining of the furcation area of the second molar of mice, black scale bar 10×: 100 μm; Figure 6 (c) Schematic diagram of HE staining of the interproximal area of the second molar of mice, black scale bar 10×: 100 μm.

[0023] Figure 7 TRAP staining results of mouse periodontitis. Figure 7 (a) Schematic diagram of TRAP staining of mouse maxillary bone, scale bar 10×: 100 μm; Figure 7 (b) TRAP-positive cell counts; ns: P>0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. DETAILED DESCRIPTION

[0024] Source of Ganoderic acid A: Ganoderic acid A was purchased from MedChemExpress, with a molecular weight of 516.67 and a molecular formula of C 30 H 44 O7, CAS number 81907-62-2, catalog number HY-N1447, purity ≥99%.

[0025] GAA can regulate the classical pyroptosis pathway NLRP3-caspase1-GSDMD, reduce the expression of pyroptosis-related proteins NLRP3, caspase1, N-GSDMD, IL-1β, and IL-18, improve cell pyroptosis, and improve periodontal inflammation in periodontitis mice, reducing periodontal tissue inflammation and alveolar bone resorption. First aspect: Ganoderma lucidum A can inhibit cell pyroptosis induced by periodontal pathogen Pg, and inhibit cell pyroptosis in the periodontal tissues of periodontitis mice (Examples 1 and 2). Second aspect: Ganoderma lucidum A can inhibit periodontal inflammation in periodontitis mice (Example 3)

[0026] Example 1

[0027] To study the effect and mechanism of GAA on pyroptosis of human mononuclear macrophage THP-1 cells induced by periodontal pathogen Pg.

[0028] Experimental methods:

[0029] Recovery and Culture of Pg bacteria: Frozen Pg W83 lyophilized powder (provided by Professor Lai Chen-hsiung of Kaohsiung Medical University, Taiwan, and previously cultured and sequenced by our research group before freeze-drying) was thawed in a 37°C water bath and then dissolved and mixed in 1 mL of BHI liquid medium. The bacterial suspension was then plated onto BHI solid blood plates and cultured in anaerobic bags for 5-7 days. Once monoclonal colonies formed, they were picked and inoculated into BHI liquid medium and cultured anaerobically for another 1-2 days. The culture was then subcultured and expanded for subsequent experiments.

[0030] Recovery, culture, passage and macrophage induction of monocyte THP-1: Thaw the frozen THP-1 cells in a 37°C water bath until the cell suspension is completely melted. The cell suspension is then inoculated into a T75 cell culture flask, 15mL RPMI 1640 complete medium is added, pipetted to mix, and cultured in a 37°C, 5% CO2 cell culture incubator. The culture medium is changed every 2-3 days. When passaged, aspirate part of the culture medium (including suspended THP-1 cells) and centrifuge at 1000rpm for 5 minutes at room temperature. Discard the supernatant, add 2mL RPMI 1640 complete medium and resuspend. After resuspension, count the cells and continue to culture them in a T75 cell culture flask or a 10cm cell culture dish according to the number of cells. When it is necessary to induce macrophages, observe the cell morphology under an optical microscope. After confirming that the cell morphology is normal and there is no growth retardation, every 1×10 6 The cells were added with 1 μL of 100 ng / mL phorbol methyl paraformaldehyde (PMA) induction solution, placed in a cell culture incubator, and stimulated for 48 hours to induce adherent macrophage-like cells.

[0031] Construction of Pg invasion THP-1 cell model:

[0032] (1) Cell culture and counting: THP-1 cell culture medium was collected, centrifuged at 1000 rpm for 5 min, resuspended in 1640 ml complete culture medium, and counted. The cells were then inoculated into cell culture dishes and cell culture plates at an appropriate density, PMA was added, and the cells were placed in a cell culture incubator and cultured for 48 hours to induce adherence to macrophages.

[0033] (2) Bacterial count: Take the bacterial suspension in the logarithmic growth phase, centrifuge it at 6000 rpm for 10 min at 4°C, remove the supernatant, and resuspend it in sterile PBS. Adjust the concentration of the resuspended bacterial solution so that its OD value at 600 nm is 0.5, which corresponds to a bacterial concentration of about 10 8 pieces / mL.

[0034] (3) Stimulation of THP-1 cells with different infection concentrations of Pg: Remove the cells from (1), discard the old culture medium, add new culture medium without double antibodies (90% RPMI 1640 culture medium + 10% FBS), and then aspirate the corresponding resuspended bacterial solution and add it to the new culture medium according to the multiplicity of infection (MOI, the average number of pathogens infected per cell) of 1:1, 10:1 and 100:1. After culturing for 8 hours, collect the cells and supernatant for subsequent experiments.

[0035] Preparation of GAA and pretreatment of THP-1 cells: Dissolve ganoderic acid A powder in DMSO in a biosafety cabinet to prepare a GAA solution of appropriate concentration. Centrifuge and shake until the GAA powder is completely dissolved. Store at 4°C until ready for use. If GAA is needed to pretreat THP-1 cells, add GAA 30 minutes before Pg stimulation. Collect cells and supernatant for subsequent experiments.

[0036] Live and dead cell staining:

[0037] (1) Cell inoculation and stimulation: THP-1 cells were plated at 2×10 5 Cells were seeded at a density of 1 / well in a 12-well plate. PMA was added to induce adherence and the cells were cultured in a cell culture incubator for 48 hours. The old medium was aspirated and replaced with fresh medium. The cells were then cultured for an additional 8 hours according to the following groups: a. Control group (no treatment); b. Pg group (cells stimulated with Pg at an MOI of 100:1); c. Treatment group 1 (cells stimulated with Pg and pretreated with 8 μM GAA); d. Treatment group 2 (cells stimulated with Pg and pretreated with 16 μM GAA).

[0038] (2) Prepare the live-dead staining working solution: Prepare the working solution according to the table below, store in a dark place, and use immediately after preparation.

[0039] Table 1 Preparation of live-dead staining working solution

[0040]

[0041]

[0042] (3) Staining: Aspirate the culture medium, wash three times with PBS, aspirate the PBS, add 400 μL of live-dead assay working solution to each well, and incubate at 37°C in the dark for 30 minutes. After incubation, aspirate the working solution, gently wash three times with PBS, aspirate the PBS, and photograph using an inverted fluorescence microscope.

[0043] CCK8 detection

[0044] (1) Cell inoculation and stimulation: Collect THP-1 culture medium, centrifuge and resuspend the cells, count them, add appropriate amount of PMA induction solution, and then inoculate them into 96-well plates, with 1×10 cells per well. 4 Cells were cultured in a 100 μL cell suspension, with a blank group containing culture medium without cells. The cells were placed in a cell culture incubator and incubated for 48 hours. The old culture medium was discarded, replaced with fresh medium, and cultured for another 8 hours according to the grouping procedure (same live-dead cell staining for each group).

[0045] (2) Add CCK8 working solution: Dilute the CCK8 solution with serum-free 1640 medium at a ratio of 10:1 to make CCK8 working solution. Protect from light. Aspirate the old medium in the well plate and add 100 μL of CCK8 working solution to each well. Incubate in the incubator in the dark for 1 hour.

[0046] (3) Determine absorbance: Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0047] (4) Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0048] A (drug addition): absorbance of the wells containing cells, CCK8 working solution, and drug stimulation;

[0049] A(0 drug addition): absorbance of the wells with cells, CCK8 working solution, and no drug stimulation; (control group)

[0050] A (blank): absorbance of the well without cells and with CCK8 working solution;

[0051] Extraction and detection of cellular RNA

[0052] (1) Extraction of total cell RNA: Collect cells from different treatment groups, rinse gently with PBS three times, aspirate PBS, add 500 μL Trizol to each well plate, shake rapidly on a horizontal shaker for 5 minutes, collect cell lysate in a 1.5 mL centrifuge tube. Add 200 μL chloroform to each tube, cover the tube tightly and shake to mix, let it stand at room temperature for 3 minutes until obvious stratification, centrifuge at 4°C, 13000 rpm for 10 minutes, and the mixture will be divided into three layers after centrifugation: the lower layer is a red organic phenol chloroform layer, the middle layer is a white thin layer, and the upper layer is a colorless water layer. Pipette the upper layer into a new centrifuge tube. Add an equal volume of isopropanol, shake and mix after inversion, let it stand at room temperature for 10 minutes, and then centrifuge at 4°C, 13000 rpm for 15 minutes, discard the supernatant, and retain the white precipitate at the bottom. Add 75% alcohol (diluted with DEPC water) and gently invert the tube to resuspend the precipitate. Centrifuge at 13,000 rpm at 4°C for 5 minutes, discard the supernatant, and be sure to retain the RNA precipitate at the bottom. Repeat twice. Allow the precipitate to dry at room temperature until translucent. Depending on the amount of precipitate, add 20-40 μL of sterile, enzyme-free water to fully dissolve the RNA. Then, use a NanoDrop to determine the RNA concentration and purity. Samples are considered qualified if the absorbance ratio of 260 / 280 is between 1.8 and 2.0 and the ratio of 260 / 230 is greater than 1.8.

[0053] (2) Reverse transcription to obtain cDNA: Reverse transcription was performed using the ABScript II cDNA First Strand Synthesis Kit. The RNA volume required for 1 μg was calculated based on the previously measured RNA concentration. The components were added to a 200 μL sterile PCR tube as shown in Table 2. The volume was made up to 20 μL with Nuclease-free H2O, and the tube was shaken and mixed. The tube was centrifuged at 400 rpm for 1 min at room temperature. Reverse transcription was performed using a SimpliAmp PCR thermal cycler. The reaction procedure was as follows: incubate at 37°C for 2 min, incubate at 55°C for 15 min, heat at 85°C for 5 min to inactivate the enzyme, and keep warm at 4°C.

[0054] Table 2 Volume of each component of the reverse transcription system

[0055]

[0056] (3) Real-time qPCR: The following components were added according to the SYBR Green PCR Master Mix reagent instructions to prepare the PCR reaction system. The group ratios are shown in Table 3. The primer sequences for each gene are shown in Table 4. A 96-well plate was used, with three replicate wells designed for each gene in each sample. The plates were centrifuged at 2500 rpm for 1 min at room temperature. The 96-well plate was placed in a QuantStudio 3 Real-time PCR instrument, and the reaction program was set as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, and 40 cycles.

[0057] Table 3 Volume of each component of the reverse transcription system

[0058]

[0059]

[0060] (4) Result calculation and analysis: The relative expression level of the target gene in each sample was calculated using the relative quantification method (ΔΔCT method).

[0061] Table 4 Human gene primer sequences

[0062]

[0063] Western-blot:

[0064] (1) Extraction of total cell protein: Collect cells from different treatment groups, rinse gently with PBS three times, aspirate the PBS, add 80 μL of cell protein lysate to each well plate, place on a horizontal shaker and shake quickly for 10 minutes, use a cell spatula to collect the protein lysate in a 1.5 mL sterile centrifuge tube. Then use an ultrasonic disruptor to disrupt the protein on ice, with intermittent disruption, each lasting 3 seconds, interrupted for 3 seconds, and disrupted for 2 minutes, at a power of 35%. After the ultrasonication is completed, centrifuge at 4°C, 13,000 rpm for 10 minutes, collect the supernatant into a new centrifuge tube, and store in a -80°C refrigerator.

[0065] (2) Protein concentration determination: Dilute the BCA protein standard using the gradient dilution method according to the instructions. Then dilute the sample to be tested with 2 μL protein sample + 18 μL PBS. Add 20 μL of PBS, BCA standard diluent, and sample to be tested to the 96-well plate in sequence. Then prepare the BCA working solution at a ratio of 50:1 of reaction solution A: reaction solution B, add 200 μL of working solution to each well and mix well. Gently shake to mix, incubate at 37°C for 30 minutes, and measure the absorbance (OD value) of each well at a wavelength of 570 nm using a microplate reader. Calculate the sample protein concentration based on the OD value and the standard curve, average the values of the duplicate wells to obtain the final concentration, add 5× Loading Buffer at a volume ratio of 1:4 of Loading Buffer: protein sample, mix well, place in a metal bath, heat at 100°C for 10 minutes, rotate at 300 rpm, and finally store in a -80°C refrigerator.

[0066] (3) Western-blot experiment

[0067] Prepare gel according to the molecular weight of the target protein. Then, add 500 mL of running buffer (100 mL of 5× running buffer + 400 mL of distilled water). Add 40 μg of the prepared protein sample to each well. Add 5 μL of prestained marker to the front and back wells. Run at a constant voltage of 120-125 V until the sample reaches the bottom of the separating gel. After electrophoresis, transfer the protein from the gel to a PVDF membrane using transfer buffer at a constant current of 300 A for 90 minutes in a cold room at 4°C. The membrane is then blocked in TBST solution on a shaker for 1.5 hours. Trim the PVDF membrane, place it in a hybridization bag, add the primary antibody (diluted in TBST, 1:1000), and incubate on a shaker at 4°C overnight. Wash the PVDF membrane three times with TBST for 15 minutes each. Add a goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody (diluted in TBST, 1:10,000) and incubate on a shaker at room temperature for 1 hour. Wash with TBST solution three times for 15 minutes each. Prepare ECL supersensitive chemiluminescent solution and drop it onto the PVDF membrane. Use a gel imaging system to expose and develop the color. Image J software is used to quantitatively analyze the grayscale value of the bands.

[0068] Experimental results: The results of live-dead cell staining showed that GAA could protect THP-1 cells. After Pg infection, some THP-1 cells died, but pretreatment with 8μM and 16μM GAA could significantly reduce the number of dead cells ( Figure 1a). CCK8 assay for cell viability revealed that THP-1 cell viability decreased significantly after Pg infection, and decreased with increasing bacterial infection multiplicity, with statistically significant differences. Pre-treatment with GAA significantly improved THP-1 cell viability compared to simple Pg stimulation, with 16μM GAA providing a greater improvement. Figure 1 b). Therefore, GAA can improve Pg-induced cell death. RT-qPCR and WB were further used to detect the changes in the mRNA and protein levels of key molecules in the classical pyroptosis pathway, NLRP3, Caspase1, GSDMD, N-GSDMD (which plays a role in cell membrane perforation), IL-1β, and IL-18, after Pg stimulation and pretreatment with 8μM and 16μM GAA. Figure 2 As can be seen, after Pg stimulation, the mRNA levels of NLRP3, Caspase1, GSDMD, IL-1β, and IL-18 were significantly increased, and the protein levels of NLRP3, Caspase1, N-GSDMD, IL-1β, and IL-18 were also significantly increased. The differences were all statistically significant, verifying that Pg stimulation of THP-1 cells can indeed activate the NLRP3 / Caspase1 / GSDMD classical pathway to induce cell pyroptosis. After GAA pretreatment, the mRNA and protein levels of key proteins in the pathway decreased, indicating that GAA can ameliorate Pg-induced cell pyroptosis by inhibiting the NLRP3 / Caspase1 / GSDMD classical cell pyroptosis pathway.

[0069] Example 2

[0070] To investigate the effect of GAA on the expression of NLRP3 and GSDMD in the periodontal tissues of mice with periodontitis.

[0071] Experimental methods:

[0072] Periodontitis modeling: After the mice were adaptively raised for 3-5 days, a silk thread ligature periodontitis mouse model was established. 1% sodium pentobarbital was used as an anesthetic, and 100-120 μL was injected intraperitoneally per mouse. After the mouse was fully anesthetized, it was fixed on the experimental table. An opener was used to keep the mouse in an open state to expose the maxillary molar area. First, a probe was used to gently separate the gap between the second molar of the mouse and the surrounding molars. Then, a micro needle holder was used to clamp the two ends of the ligature. The 5-0 ligature was gently inserted into the mesial and distal spaces of the second molar, respectively, so that the ligature remained on the neck of the maxillary second molar. The ligature was gently moved to ensure that the ligature was close to the tooth surface, and then it was fixed on the palatal side of the second molar.

[0073] Drug preparation and administration: Dissolve ganoderic acid A powder in DMSO and add GAA solution at an appropriate concentration. Centrifuge and shake until the GAA powder is completely dissolved. Store at 4°C until use. For administration, anesthetize and secure the mouse. Expose the maxillary second molar, open the mouth angle, and use a microsyringe to gently insert the needle tip into the periodontal pocket of the second molar. Inject slowly, ensuring that the drug reaches the periodontal pocket on the mesiobuccal, distal buccal, mesiopalatal, and distal palatal sides of the second molar. Dosing should be continued once daily, starting on the day after ligation, until sacrifice.

[0074] Immunohistochemistry (IHC) staining

[0075] The maxillary bone tissue of mice was fixed in 4% tissue cell fixative for 1-2 days. Decalcification was then carried out using 10% EDTA decalcification solution for 14 days, with the decalcification solution replaced every 2-3 days. After decalcification, a syringe needle was used. If it could be inserted without resistance, decalcification was successful. If there was resistance, decalcification was continued. Use tissue scissors to trim the mandibular tissue, rinse it with running water, place it in an automatic dehydrator, and complete dehydration according to the hard tissue dehydration program. After dehydration, place it in a tissue embedding instrument and embed the buccal side of the tissue facing down. After embedding, use a Yamato microtome or a wheel microtome to cut the wax block into slices with a thickness of 5μm. During the slicing process, adjust the slice angle at all times to ensure the slice direction. Place the cut wax slices in a 37°C water bath on a slide table to heat the slides. After picking them up, bake them on a baking table for 2 hours, dry them in a 60°C oven overnight, and store them at room temperature for later use.

[0076] 1) Dewaxing of sections. Place the slides in a constant temperature incubator for 2 hours for gradient dehydration. Soak in xylene I, II, and III for 30 minutes, 20 minutes, and 10 minutes, respectively. Soak in anhydrous ethanol I, II, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 minutes each.

[0077] 2) Remove peroxide. After removing the slides from the 70% ethanol, immerse them in PBS buffer and wash them three times for 5 minutes each on a horizontal shaker. Immerse the slides in 30% hydrogen peroxide and incubate them at room temperature for 20 minutes.

[0078] 3) Antigen repair. After discarding the hydrogen peroxide, immerse the slides in 1×PBS buffer and wash them 3 times on a low-speed decolorization shaker for 5 minutes each time. A. High-pressure repair method: Preheat the appropriate antigen repair solution (sodium citrate or EDTA) in a pressure cooker water bath, immerse the slides in the antigen repair solution, cover the pressure cooker and heat it. When the jet valve starts to spray, start timing for 2 minutes. Rinse the pressure cooker with running water to cool and depressurize it, then take out the box containing the antigen repair solution and cool it in a ventilated place. Once it cools to room temperature, proceed to the next step.

[0079] 4) Blocking. After discarding the antigen retrieval solution, immerse the slide in 1× PBS buffer and wash three times for 3 minutes each on a low-speed decolorization shaker. Wipe away any moisture surrounding the tissue on the slide. Add a drop of goat serum blocking solution to completely cover the tissue. Place the slide in a humidified chamber and incubate at room temperature for 30–60 minutes.

[0080] 5) Incubate with primary antibody. After discarding the antigen retrieval solution, wipe away any moisture surrounding the tissue on the slide. Dispense 50 μL of the diluted primary antibody to completely cover the tissue. Place the slide in a humidified chamber and incubate overnight at 4°C.

[0081] 6) Incubate the secondary antibody. The next day, after discarding the primary antibody, immerse the slide in 1×PBS buffer and wash it three times on a low-speed decolorization shaker for 3 minutes each time. Wipe off the moisture around the tissue on the slide, add the antibody booster solution in the secondary antibody kit to the covered tissue, and incubate it at room temperature in a wet box for 20 minutes. After discarding the antibody booster solution, immerse the slide in 1×PBS buffer and wash it three times on a low-speed decolorization shaker for 3 minutes each time. Wipe off the moisture around the tissue on the slide, add the streptavidin affinity secondary antibody in the secondary antibody kit to the covered tissue, and incubate it at room temperature in a wet box for 20 minutes.

[0082] 7) DAB color development. After discarding the secondary antibody, immerse the slide in 1× PBS buffer and wash three times for 3 minutes each on a low-speed decolorizing shaker. Prepare DAB color development solution by diluting it proportionally and prepare it immediately before use. Wipe away any moisture around the tissue on the slide. Add approximately 50 μL of color development solution and observe under a microscope until a yellow-brown positive result appears. Then, immerse the slide in tap water to terminate the reaction.

[0083] 8) Hematoxylin counterstaining: Incubate with hematoxylin solution for 2 minutes, observe the staining effect under a microscope, and rinse with running water for 10 minutes to return to blue.

[0084] 9) Dehydrate, seal with gum, and air dry in a fume hood before taking photos and observing under a microscope.

[0085] Experimental results: This study used immunohistochemical staining to detect the expression and distribution of pyroptosis-related proteins NLRP3 and GSDMD in the periodontal tissues of the second molar area in the control group, periodontitis group and drug-treated group in the silk thread ligature model of mice. Figure 3 As shown in the results, compared with the control group, the expression of NLRP3 in the periodontal tissues around the second molars in the periodontitis group was higher, especially in the root bifurcation area of the second molars, as well as in the epithelium and connective tissue. The expression of NLRP3 in the drug-treated group was reduced compared with the periodontitis group, especially in the root bifurcation area, while the expression of NLRP3 in the epithelium was higher. The expression of GSDMD was similar to that of NLRP3. It was significantly more expressed in the periodontal tissues of the second molars in the periodontitis group, while the expression in the drug-treated group was reduced compared with the periodontitis group ( Figure 4). ImageJ was further used to quantitatively analyze the expression areas of NLRP3 and GSDMD and compare the percentages of protein staining-positive areas. It was found that the percentages of NLRP3 and GSDMD-positive areas in the periodontitis group were significantly higher than those in the control group, while those in the GAA-administered group were significantly lower than those in the periodontitis group. The differences were statistically significant.

[0086] Based on Examples 1 and 2, Pg stimulation can induce pyroptosis in THP-1 cells, and GAA can inhibit the classical pyroptosis pathway, reduce the expression of pyroptosis-related proteins, improve Pg-induced pyroptosis in THP-1 cells, and exert an anti-pyroptosis protective effect. In a periodontitis mouse model, silk ligation can induce pyroptosis in the periodontal tissues around the second molars of mice, including an increase in the number of pyroptotic cells and increased expression of pyroptosis-related proteins NLRP3 and GSDMD; local administration of GAA can improve pyroptosis in the periodontal tissues of mice.

[0087] Example 3

[0088] To investigate the effect of ganoderic acid A on periodontitis in mice

[0089] Periodontitis modeling, drug preparation and administration methods were the same as in Example 2.

[0090] Experimental Grouping: A total of 24 mice were enrolled, with 6 mice per group. They were divided into four groups: the control group received no treatment; the periodontitis group received silk ligature alone; the blank drug group (DMSO) received ligature and daily local injections of the same dose of DMSO solvent; and the GAA group (GAA) received ligature and daily local injections of 2 μL of 1 mg / kg GAA. The experiment lasted for 7 days. On the 7th day after ligature, the mice were sacrificed by cervical dislocation, and maxillary bone tissue was obtained for subsequent analysis.

[0091] Detection method:

[0092] (1) Micro-CT scanning and reconstruction were performed using the Inveon MM system. The maxilla of mice was obtained for bone mass and microstructure scanning. Three-dimensional reconstruction was performed on the maxilla of each group. The region of interest (alveolar bone around the second molar of the maxillary mouse, the mesiodistal range was from the distal side of the distal root of the first molar to the mesial side of the mesial root of the third molar, and the coronal-root range was from the bifurcation to the root apex of the second molar) was selected for data analysis. The vertical distance from the alveolar bone crest to the cementoenamel junction, the bone mass percentage, and the periodontal bone density were obtained, and the average value was taken for each group of mice.

[0093] (2) The scanned samples were fixed with a fixative for 36 h, and then placed in a 10% EDTA decalcification solution. The samples were decalcified at room temperature for 2 weeks, with the decalcification solution changed every 2 days. The samples were then dehydrated, embedded, sectioned, and stained with HE and TRAP to evaluate alveolar bone absorption and local inflammation.

[0094] Experimental results: The data obtained by Micro-CT scanning are as follows Figure 5 As shown in Figure 2, the distance from the bone crest to the cementoenamel junction in the drug-treated group was significantly less than that in the periodontitis group, and the bone volume percentage and bone density in the region of interest in the drug-treated group were significantly higher than those in the periodontitis group. Figure 6 As shown in Figure 2, the attachment loss and alveolar bone resorption in the treatment group were less than those in the periodontitis group, and the inflammatory cells infiltrating the gingival lesion area in the treatment group were less than those in the periodontitis group. Figure 7 As shown, the number of osteoclasts increased significantly in the periodontitis group, while it decreased significantly in the treatment group. In summary, local administration of GAA can improve alveolar bone resorption, ameliorate attachment loss, alleviate inflammatory cell infiltration, reduce osteoclast numbers, and lower the levels of inflammatory factors in periodontal tissues. This suggests that GAA can improve alveolar bone resorption and periodontal inflammation in mice with silk ligature-induced periodontitis.

[0095] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of ganoderic acid A in the preparation of drugs for preventing and treating periodontal inflammatory diseases and oral care products, characterized in that: The structural formula of Ganoderic acid A is as follows:

2. The use according to claim 1, characterized in that: The ganoderic acid A is used as the sole active ingredient in the preparation of medicines for preventing and treating periodontal inflammatory diseases and oral care products.

3. The use according to claim 1 or 2, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The carrier is a polar solvent and a non-polar solvent; the polar solvent is selected from one or more of water, ethanol, glycerol, propylene glycol and DMSO, and the non-polar solvent is selected from one or two of fatty oil and liquid paraffin.

5. The use according to claim 1 or 2, characterized in that: The medicine further comprises supplementary additives, which are selected from one or more of diluents, buffers, encapsulating agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents and adsorption carriers.

6. The use according to claim 5, characterized in that The dosage form of the drug is a suspension, emulsion, granule, spray, injection, transdermal absorbent, dosage form suitable for transfection, tablet, powder, granule or capsule.

7. The use according to claim 1 or 2, characterized in that: The oral care products include toothpaste, mouthwash, oral spray, oral gel, film and chewable colloid.

8. The use according to claim 1 or 2, characterized in that: Ganoderic acid A regulates the cell pyroptosis pathway NLRP3-caspase1-GSDMD, reduces the expression of pyroptosis-related proteins NLRP3, caspase1, N-GSDMD, IL-1β and IL-18, improves cell pyroptosis, and thus improves periodontal inflammation.

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