Application of FTO inhibitor in preparation of periapical periodontitis alveolar bone absorption treatment medicine

Abstract

The invention provides application of an FTO inhibitor in preparation of a periapical periodontitis alveolar bone absorption treatment medicine. The application proves that Dac51 can reduce the FTO level in periapical periodontitis alveolar bone tissue, down-regulate the expression of osteoclast specific factors and reverse the periapical periodontitis alveolar bone absorption process, and a new medicine is provided for treating periapical periodontitis alveolar bone absorption.

Description

Field of the Invention

[0001] The present application belongs to the field of dentistry and bone disease treatment. Specifically, the present application provides the use of FTO inhibitors in the preparation of drugs for treating alveolar bone resorption in apical periodontitis. Background Art

[0002] Apical periodontitis is a non-specific chronic inflammatory reaction of the periapical tissue caused by microbial infection in the root canal. It is a common infectious disease in stomatology with a prevalence of 52%. Clinical manifestations include pain, recurrent swelling, and sinus tracts. Apical alveolar bone resorption is an important clinical feature of apical periodontitis. As the degree of apical alveolar bone resorption increases, it can lead to alveolar bone defects. In severe cases, it can cause mandibular osteomyelitis and eventually lead to tooth loss. Although clinical root canal treatment technology has intervened in the cause of the disease and improved the survival rate of teeth with apical periodontitis through major technical means such as clearing infection, the persistent problem of periapical lesions and alveolar bone resorption seriously affects the quality of life of patients. There is currently a lack of effective treatment methods, which is the difficulty in the clinical treatment of endodontic diseases. Therefore, it is urgent to further clarify the occurrence and development mechanism of alveolar bone resorption in apical periodontitis and seek effective treatment targets.

[0003] Epigenetic modifications regulate gene expression and translation, affecting cell development and differentiation. 6 A) is the most common RNA modification on eukaryotic mRNA, affecting RNA transcription, splicing, nuclear transport, degradation and translation to regulate gene expression, thereby participating in the regulation of various biological processes. 6 A occurs at the adenosine base at the nitrogen 6 position of mRNAs and is regulated by methyltransferases, demethylases, and methylation reader proteins. Fatmass and obesity-associated protein (FTO) is the first mRNA to be discovered. 6 A demethylase. Studies have shown that m 6 A promotes or inhibits bone formation in different ways, among which the demethylase FTO may be involved in the occurrence and development of bone-related diseases.

[0004] Alveolar bone resorption caused by apical periodontitis is a difficult point to treat in stomatology. 6 The role of A methylation modification, especially the demethylase FTO, in alveolar bone resorption in apical periodontitis remains unknown. To date, the use of FTO inhibitor Dac51 in the treatment of alveolar bone resorption in apical periodontitis has not been disclosed or used. Summary of the invention

[0005] The present invention found that in the alveolar bone tissue of rat periapical periodontitis model, m 6A methylation level was downregulated, and the expression of demethylase FTO increased significantly. Dac51 reduced FTO expression, downregulated the expression of osteoclast-specific factors, reversed the alveolar bone resorption process of apical periodontitis, and alleviated the pathological changes of the animal model of apical periodontitis. It shows that the FTO inhibitor Dac51 can be used to prepare drugs for the treatment of alveolar bone resorption in apical periodontitis. A new use of the FTO inhibitor Dac51 for the treatment of alveolar bone resorption in apical periodontitis is provided.

[0006] On the one hand, the present application provides the use of FTO inhibitors in the preparation of drugs for treating alveolar bone resorption caused by apical periodontitis.

[0007] Furthermore, the FTO inhibitor is Dac51.

[0008] Furthermore, the apical periodontitis is apical periodontitis caused by microbial infection.

[0009] Furthermore, the drug reduces the number of osteoclasts in alveolar bone tissue around periapical periodontitis.

[0010] Furthermore, the drug reduces the level of FTO in alveolar bone tissue of apical periodontitis.

[0011] Furthermore, the drug reduces the level of osteoclast-specific factors in alveolar bone tissue of apical periodontitis.

[0012] Furthermore, the osteoclast-specific factor is selected from one or more of NFATc1, MMP9, and CTSK.

[0013] Furthermore, the drug is an injection, a local administration preparation or an oral preparation.

[0014] Furthermore, the drug is an injection or a local administration preparation.

[0015] The topical administration preparations include, but are not limited to, subcutaneous implants.

[0016] In another aspect, the present application provides a non-therapeutic method for inhibiting alveolar bone resorption in a subject, comprising administering the FTO inhibitor Dac51 to the subject.

[0017] Such non-therapeutic methods include, but are not limited to, procedures involving animal models used in medical research.

[0018] The CAS number of Dac51 described in this application is: 2243944-92-3, and the chemical formula is as follows. This compound is a known FTO inhibitor and can be obtained through commercial routes:

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 In the alveolar bone tissue of rat periapical periodontitis model, m 6 A methylation level was downregulated, and the expression of demethylase FTO increased significantly;

[0021] Figure 2 It showed that after treatment with FTO inhibitor Dac51, the range of periapical alveolar bone resorption in animal periapical periodontitis models was significantly reduced;

[0022] Figure 3 It showed that after treatment with FTO inhibitor Dac51, the number of osteoclasts in the periapical alveolar bone tissue of the animal periapical periodontitis model was significantly reduced;

[0023] Figure 4 It showed that after treatment with the FTO inhibitor Dac51, the expression of FTO and osteoclast markers NFATc1, MMP9, and CTSK in the periapical alveolar bone tissue of the animal periapical periodontitis model was significantly reduced. DETAILED DESCRIPTION

[0024] Example 1 Rat periapical periodontitis model

[0025] Healthy Wistar male rats aged 8 weeks and weighing 180-220 g were selected. Rats in each group were housed in separate cages at an ambient temperature of 21-25°C with a light / dark cycle of 12 h and fed with standard rat chow.

[0026] The rat periapical periodontitis model was established by deep anesthesia with sodium pentobarbital. After anesthesia, a 1 / 4 ball drill was used to expose the coronal pulp of the right mandibular first molar, and a No. 10 K file was used to clear the root canal. A small cotton ball was placed at the bottom of the cavity to open the pulp cavity. Routine feeding was performed and the oral cavity was observed regularly.

[0027] Animal grouping: Ten Wistar rats were randomly selected and euthanized by carbon dioxide at the 1st, 3rd, and 6th week after modeling, and used as the experimental group for 1 week (1week), 3 weeks (3weeks), and 6 weeks (6weeks). The left mandibular first molar was used as the control group.

[0028] Example 2 Dotblot detection m 6 AOverall Level

[0029] The mandible was separated, and the soft tissue was removed using hemostats and scissors. The alveolar bone of the first molar was cut along the lower edge of the junction between the first molar and the anterior and posterior teeth, and 1 mm away from the first molar. The first molar was removed with hemostats, the periodontal membrane was scraped off with a small spoon, and the alveolar bone below the mandibular nerve canal was cut off. TRIzol was used to extract RNA from the alveolar bone.

[0030] Dotblot detection 6 AOverall level:

[0031] (1) Take out the RNA sample to be tested, measure the RNA concentration after thawing, and operate on ice throughout the process.

[0032] (2) The sample concentration was unified and serially diluted in new enzyme-free tubes and mixed to a final concentration of 200 μg / μL.

[0033] (3) After centrifugation, the RNA sample was heated at 95°C for 3 min to destroy the secondary structure and then immediately cooled on ice to prevent the re-formation of the secondary structure.

[0034] (4) Cut the NC membrane into appropriate sizes and transfer it to a clean plastic box.

[0035] (5) Drop 2 μL of RNA sample onto the NC membrane in sequence. Be careful not to let the pipette tip touch the membrane. The sample should diffuse naturally on the membrane. Replace the pipette tip each time you add the sample.

[0036] (6) After the NC membrane is slightly dried at room temperature, RNA is cross-linked to the membrane and placed in a 37°C oven for 30 min.

[0037] (7) Wash the membrane with 10 mL TBST for 5 min to remove unbound RNA.

[0038] (8) Discard TBST, add 10 mL of 5% BSA blocking solution, and incubate with shaking at room temperature for 1 h.

[0039] (9) Discard the blocking solution and add m 6 A primary antibody (1:1,000 dilution) was incubated overnight at 4°C.

[0040] (10) Wash the membrane three times with 10 mL of TBST, each time for 10 min.

[0041] (11) Incubation with secondary antibody: IRDye 680RD anti-rabbit IgG, incubate at 37°C for 1 h.

[0042] (12) Wash the membrane three times with 10 mL of TBST, each time for 10 min.

[0043] (13) Color development: Images were acquired using LI-COR's Odyssey imaging system.

[0044] The results are as follows Figure 1 As shown in part a of the figure, in the alveolar bone tissue of the rat periapical periodontitis model, m 6 A methylation level was downregulated.

[0045] Example 3 Immunoblotting WB detection of FTO, NFATc1, MMP9, and CTSK expression

[0046] Lysis solution was added at a ratio of 60 μL RIPA lysis solution per 20 mg alveolar bone, and the tissue was ground in a pre-cooled high-speed tissue grinder until fully lysed. After standing for 30 minutes, centrifuged at 4°C, 12,000g for 10 minutes, and the supernatant was taken as the extracted protein. The protein concentration was determined using the BCA protein content detection kit. Then, the expression of FTO, NFATc1, MMP9, and CTSK was detected by immunoblotting WB.

[0047] Western Blot:

[0048] (1) Protein denaturation: Add 5× loading buffer at a ratio of 1:4 between sample volume and 5× loading buffer. Cover the EP tube with a lid, vortex, and place in a 95°C water bath for 5 min.

[0049] (2) Glue preparation: Wash the glass plate used for glue pouring, blow dry, and place it in a sandwich. After the glue solidifies (about 15 minutes at room temperature), remove the comb teeth and use it for electrophoresis.

[0050] Preparation of the lower layer of gel: Take equal volumes of Resolver A and Resolver B, 2.7 mL each, mix well, add 60 μL of APS, mix thoroughly immediately, and inject into the gel-making glass plate so that the liquid level is about 1.5 cm from the upper edge of the short glass plate.

[0051] Preparation of the upper layer gel: Take equal volumes of Stacker A and Stacker B, 0.75 mL each, mix well, add 15 μL of APS, and mix thoroughly immediately. Without waiting for the lower layer gel to solidify, the mixed solution can be gently injected into the glass plate for gel making, and the comb teeth can be gently inserted.

[0052] (3) Sample loading: Add electrophoresis buffer to the electrophoresis tank. The liquid level must be higher than the top of the gel. Pull out the comb teeth vertically and gently. Take 40 μg of denatured protein and load it into the loading well. Add a prestained protein marker on one side to facilitate the determination of the molecular weight of the protein band.

[0053] (4) Electrophoresis: Constant voltage 80 V, 30 min. After running through the stacking gel, adjust the voltage to 120 V. Stop electrophoresis when bromophenol blue reaches the bottom of the gel.

[0054] (5) Transfer: According to the size of the gel to be transferred, cut the filter paper and PVDF membrane. Soak the PVDF membrane in methanol solution for 60 seconds before use to activate it. Spread out the transfer clip in the pre-cooled 1× Transfer buffer, with the black side of the transfer clip facing up, and place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in order. Be careful not to have bubbles between the layers. After the clip is placed in the Bio-rad transfer tank, the transfer device is placed in an ice bath, and the power is turned on. Be careful not to connect the electrodes in the wrong direction. Constant voltage 100V, transfer for 52 minutes.

[0055] (6) Blocking: Block with 5% BSA for 1 h at room temperature.

[0056] (7) Incubation with primary antibody: dilute the primary antibody with primary antibody diluent. Incubate at 4°C overnight. Wash with PBST for 5 min. Repeat the wash three times.

[0057] (9) Incubate with secondary antibodies: IRDye 680RD anti-rabbit IgG and IRDye 800CW anti-mouse IgG (1:10,000 dilution), incubate at 37°C for 1 h, wash with PBST for 5 min, and repeat washing three times.

[0058] (10) Color development: Images were acquired using LI-COR's Odyssey imaging system.

[0059] The results are as follows Figure 1 As shown in part b, the expression of demethylase FTO was significantly increased in the alveolar bone tissue of the rat periapical periodontitis model.

[0060] In the model of Example 4, the same method was used to detect the results. Figure 4 As described above, after treatment with the FTO inhibitor Dac51, the expression of FTO and osteoclast markers NFATc1, MMP9, and CTSK in the periapical alveolar bone tissue of the animal periapical periodontitis model was significantly reduced.

[0061] Example 4: Use of Dac51 in the treatment of an animal model of periapical periodontitis

[0062] One week after the root canal was opened, the pulp chamber roof was removed using a #1 / 2 round drill, and the root canal was unblocked using a 25#04ProtperNext nickel-titanium file. Using the standard clinical irrigation procedure, 1% NaClO and 17% EDTA solutions were alternately rinsed, and finally rinsed with normal saline. The sterilized paper tip was dried. Subsequently, Dac51 was injected into the apical area of ​​the mandibular first molar at a volume of 2 mg / kg using a microsyringe, and a #15K file was used to assist in the delivery of the root canal drug to the outside of the root apex. Finally, glass ionomer was used for sealing. Three weeks later, the rats were euthanized with an overdose of isoflurane. The mandibular alveolar bone tissue of the mandibular first molar was collected.

[0063] Example 5 Micro-CT detection of alveolar bone resorption range

[0064] The samples were scanned using a SkyScan 1176 Micro-CT imaging system with the following image acquisition parameters: 385 μA, 65 kV, 1 mm thick aluminum filter, and 340 ms exposure time. NRecon software (version: 1.6.10, Bruker) was used to reconstruct the mandibular 3D image and calculate the area and volume of alveolar bone resorption around the apex of the mandibular first molar.

[0065] The results are as follows Figure 2 As shown in the figure, after treatment with FTO inhibitor Dac51, the scope of periapical alveolar bone resorption in the animal periapical periodontitis model was significantly reduced.

[0066] Example 6 TRAP staining to detect osteoclasts

[0067] (1) The mandibular tissue was fixed and decalcified, embedded in paraffin, and sliced ​​and baked at 50°C for 20 min.

[0068] (2) Gradient dewaxing of anhydrous ethanol, 95% ethanol, and 80% ethanol.

[0069] (3) Rinse the dewaxed sections in a sink for 2 minutes.

[0070] (4) Add 0.5 mL of Fast garnet GBC Base solution and 0.5 mL of Sodium Nitrite Solution to a 1.5 mL EP tube, mix gently for 30 seconds, and let stand at room temperature for 2 minutes.

[0071] (5) In a 50 mL centrifuge tube, prepare the mixture according to the following ratio:

[0072]

[0073] (6) After mixing the above reagents, add 70 μL per slice onto the slice.

[0074] (7) Place in an incubator and incubate at 37°C in the dark for 2 h.

[0075] (8) After incubation for 2 h, wash the sections thoroughly with deionized water until blue nuclei appear.

[0076] (9) Allow to dry naturally, seal with glycerol gelatin, and observe under a microscope.

[0077] The results are as follows Figure 3 As shown, after treatment with FTO inhibitor Dac51, the number of osteoclasts in the apical alveolar bone tissue of the animal periapical periodontitis model was significantly reduced.

Claims

1. Application of FTO inhibitors in the preparation of drugs for the treatment of alveolar bone resorption caused by apical periodontitis.

2. The use according to claim 1, wherein the FTO inhibitor is Dac51.

3. The use according to claim 1 or 2, wherein the apical periodontitis is apical periodontitis caused by microbial infection.

4. The use according to claim 1, wherein the drug reduces the number of osteoclasts in the alveolar bone tissue around periapical periodontitis.

5. The use according to claim 1, wherein the drug reduces the level of FTO in alveolar bone tissue of apical periodontitis.

6. The use according to claim 1, wherein the drug reduces the level of osteoclast-specific factor in alveolar bone tissue of apical periodontitis.

7. The use according to claim 6, wherein the osteoclast-specific factor is selected from one or more of NFATc1, MMP9, and CTSK.

8. The use according to claim 1 or 2, wherein the drug is an injection, a topical preparation or an oral preparation.

9. The use according to claim 8, wherein the drug is an injection or a local administration preparation, preferably a subcutaneous implant.

10. A non-therapeutic method for inhibiting alveolar bone resorption in a subject, characterized in that: The method comprises administering to the subject an FTO inhibitor, Dac51.

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