Application of DDO-2728 in preparation of medicine for treating periodontitis

Abstract

The invention belongs to the technical field of periodontal ligament stem cells, and discloses application of DDO-2728 in preparation of a medicine for treating periodontitis. Researches show that DDO-2728 can promote ALP enzyme activity and cell mineralization of periodontal ligament stem cells and expression of osteogenic transformation related genes RUNX2 and OCN, and can be used for inducing and promoting osteogenic differentiation of periodontal ligament stem cells, so that the DDO-2728 has a good treatment effect on periodontitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of periodontal ligament stem cells, and relates to the application of DDO-2728 in the preparation of a medicament for treating periodontitis. Background Art

[0002] Periodontitis is a globally prevalent chronic inflammatory disease, and its clinical manifestations include gingival bleeding, repeated swelling and pain, and alveolar bone resorption. If not treated properly, it may lead to tooth loosening and exfoliation, affecting masticatory function and even having an impact on the digestive system. The most important pathological manifestation of periodontitis is the gradual degradation of the tooth supporting structure. The repair and reconstruction of the periodontal supporting tissue rely on mechanical plaque removal and guided tissue regeneration techniques. Despite various treatment methods from non-surgical interventions to guided tissue regeneration techniques, these methods have shown limited success in achieving meaningful periodontal ligament regeneration. Therefore, reconstructing the periodontal ligament structure and restoring its function is an urgent need currently faced.

[0003] Stem cell therapy is well-known for its regenerative and immunomodulatory potential, providing a promising approach for periodontal tissue repair. The periodontal ligament is a fibrous connective tissue that anchors the tooth to the surrounding alveolar bone. This structure has been reported as a source of adult stem cells capable of regenerating alveolar bone tissue and maintaining the homeostasis of the periodontal tissue. Human periodontal ligament stem cells isolated from extracted third molars have the ability of self-renewal and the potential to differentiate into various specialized cells. Periodontal ligament stem cells have been isolated and used in in vitro and in vivo experiments, showing pluripotent differentiation ability, including differentiation into osteoblasts, nerve cells, and adipocytes. Due to the excellent osteogenic differentiation potential of periodontal ligament stem cells, it brings great hope for periodontal tissue engineering, especially in periodontal tissue regeneration and bone defect repair. Inducing the differentiation of periodontal ligament stem cells into osteoblasts can promote the regeneration of alveolar bone and repair the bone defects caused by periodontitis. By combining with bio-scaffold materials, tissue engineering scaffolds with osteogenic ability can be constructed for the regeneration of periodontal tissue. Inducing the differentiation of periodontal ligament stem cells into osteoblasts can also be used for tooth and periapical tissue regeneration.

[0004] Drugs that induce the differentiation of periodontal ligament stem cells into osteoblasts have broad application prospects in the fields of periodontitis treatment, alveolar bone defect repair, periodontal tissue engineering, and tooth regeneration. DDO-2728 is a novel agonist of N6-methyladenosine (m6A) modification. m6A is the most common dynamic and reversible post-transcriptional modification in eukaryotic mRNA and non-coding RNA, accounting for 80% of RNA methylation modifications. It forms a modification site by adding a methyl group to the 6th nitrogen atom of adenine. AlkB homolog 5 (ALKBH5) is an Fe(II) / 2-oxoglutarate (2-OG)-dependent dioxygenase that can remove monomethylated or dimethylated adenosine. DDO-2728 is not a 2-oxoglutarate analog and can selectively inhibit the demethylation activity of ALKBH5, achieving the effect of increasing the abundance of m6A modification. DDO-2728 can induce apoptosis and cell cycle arrest. It shows the effects of inhibiting tumor cell proliferation and promoting tumor cell apoptosis in acute myeloid leukemia and has good safety. However, there is no research and report on the application of DDO-2728 in the field of periodontal diseases. Summary of the Invention

[0005] The present invention provides an application of DDO-2728 in the preparation of a drug for treating periodontitis. DDO-2728 can promote the ALP enzyme activity, cell mineralization, and the expression of osteogenic transformation-related genes RUNX2 and OCN in periodontal ligament stem cells. Therefore, it can be used for the osteogenic differentiation of periodontal ligament stem cells and thus has a good therapeutic effect on periodontitis.

[0006] In the first aspect of the present invention, there is provided an application of DDO-2728 in the preparation of a drug for treating periodontitis. The molecular formula of DDO-2728 is C 28 H 17 F3N4O7, and its chemical structural formula is shown as follows:

[0007]

[0008] Further, the drug is used for inducing and / or promoting the osteogenic differentiation of periodontal ligament stem cells.

[0009] Further, the drug is used for increasing the expression level and activity of ALP enzyme in periodontal ligament stem cells.

[0010] Further, the drug is used for promoting the mineralization of periodontal ligament stem cells.

[0011] Further, the drug is used for increasing the expression of RUNX2 in periodontal ligament stem cells.

[0012] Further, the drug is used for increasing the expression of OCN in periodontal ligament stem cells.

[0013] Furthermore, the dosage form of the drug is paste, gel, spray, granule, capsule, tablet, powder, oral liquid, suspension or emulsion.

[0014] Furthermore, the DDO-2728 is used alone or in combination with other drugs.

[0015] Furthermore, the dosage form of the drug is paste, gel, spray, granule, capsule, tablet, powder, oral liquid, suspension or emulsion.

[0016] Compared with the prior art, through research, it is found in the present invention that the use of DDO-2728 can increase the expression and activity of ALP in periodontal ligament stem cells, can promote the expression of RUNX2 and OCN, and can make cells more prone to osteogenic mineralization, thus having a good therapeutic effect on periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a statistical chart of the ALP staining result and the ALP enzyme activity detection result of alkaline phosphatase (ALP) of periodontal ligament stem cells in Example 1;

[0018] Figure 2 It is a staining diagram of calcium deposition of periodontal ligament stem cells in Example 2;

[0019] Figure 3 It is a statistical chart of the expression levels of osteogenic differentiation-related genes RUNX2 and OCN of periodontal ligament stem cells in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In order to more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention. For the experimental methods without specific conditions noted in the examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0021] The DDO-2728 used in the present invention has a CAS number of 3029515-97-4, a molecular formula of C 28 H 17 F3N4O7, a molecular weight of 578.45, and is purchased from MCE Company, and the product catalog number is HY-155489.

[0022] Unless otherwise specified, the reagents used in the embodiments of the present invention can all be obtained from sales companies.

[0023] Example 1: Effect of DDO-2728 on alkaline phosphatase (ALP) of periodontal ligament stem cells.

[0024] 1. Cell treatment

[0025] The cells were divided into two groups: the control group was cultured with a conventional osteogenic induction medium (α-MEM complete medium containing 10% FBS supplemented with 100 μM vitamin C, 10 nM β-glycerophosphate, and 10 nM dexamethasone); the experimental group was cultured with 5 μM DDO-2728 combined with the conventional induction medium.

[0026] 2. Real-time PCR

[0027] After 7 days of osteogenic induction of periodontal ligament stem cells, RNA was extracted using TRIzol. RNA was reverse transcribed into cDNA using oligo(dT) primers and reverse transcriptase. The cDNA was placed into the prepared PCR reaction system and the program was initiated on a Real-time PCR instrument. The Real-time PCR instrument automatically recorded the fluorescence intensity of each cycle, and the 2 -ΔΔCt method was used to compare the expression differences of target genes between different samples.

[0028] 3. ALP enzyme activity detection

[0029] After 7 days of osteogenic induction of periodontal ligament stem cells, the cells were rinsed 3 times with PBS, an appropriate amount of cell lysate was added, and the cells were ultrasonically disrupted in an ice bath. The cells were centrifuged at 8000 g for 10 min at 4 °C, and the supernatant was taken and placed on ice for further measurement. The chromogenic reagent was added successively, and after mixing, it was incubated in a water bath at 37 °C for 15 min. Finally, the termination reagent was added, and after mixing, the absorbance was measured at 510 nm. According to the formula, the enzyme activity of ALP was calculated.

[0030] Figure 1 Figure A shows the ALP staining results and Figure B shows the ALP enzyme activity detection results of alkaline phosphatase (ALP) in periodontal ligament stem cells in Example 1.

[0031] ALP is an enzyme widely present in various tissues, especially in high concentrations in bone, liver, kidney, and placenta. ALP plays a key role in the process of osteogenic differentiation and is an important marker in the differentiation and mineralization of osteoblasts. ALP provides a locally high concentration of inorganic phosphate by hydrolyzing phosphate esters and phosphoric anhydrides, providing the necessary phosphate for the formation of hydroxyapatite. From Figure 1 it can be seen that after adding DDO-2728, the ALP expression and activity of periodontal ligament stem cells increased significantly compared with the control group. It was proved that adding DDO-2728 could more efficiently promote the osteogenic differentiation of cells compared with conventional osteogenic inducers.

[0032] Example 2: Experiment on the effect of DDO-2728 on the mineralization of periodontal ligament stem cells.

[0033] 1. Cell treatment

[0034] The cells were divided into two groups: the control group was cultured with a conventional osteogenic induction medium (α-MEM complete medium containing 10% FBS supplemented with 100 μM vitamin C, 10 nM β-glycerophosphate, and 10 nM dexamethasone); the experimental group was cultured with 5 μM DDO-2728 combined with the conventional induction medium.

[0035] 2. Alizarin red staining

[0036] After 7 days of osteogenic induction of periodontal ligament stem cells, the medium was aspirated and the cells were gently washed with PBS.

[0037] 4% paraformaldehyde solution was added and fixed at room temperature for 30 min. The fixing solution was aspirated and the cells were gently washed 3 times with PBS. Alizarin red staining solution was prepared and the cells were stained at room temperature for 10 min. Finally, the staining results were observed under an optical microscope. As Figure 2 shown, among them, the calcium nodules were red or orange-red.

[0038] Alizarin red S is an anthraquinone derivative that can chelate with calcium salts (such as calcium carbonate or calcium phosphate) to form orange-red or dark red complexes. This property makes it a commonly used staining agent for detecting calcium deposition. During the differentiation of osteoblasts, the calcium salt deposition in the extracellular matrix binds to alizarin red S, thus showing red or orange-red under the microscope. Alizarin red staining is widely used to evaluate the differentiation and mineralization ability of osteoblasts. In cell culture, alizarin red S can stain calcium nodules to determine whether the cells have successfully differentiated into osteoblasts. It can be Figure 2 seen that the periodontal ligament stem cells in the group treated with DDO-2728 had darker staining and more calcium nodules, indicating that DDO-2728 was helpful for cell mineralization and could promote its osteogenic differentiation.

[0039] Example 3: Effects of DDO-2728 on osteogenic differentiation-related genes RUNX2 and OCN of periodontal ligament stem cells.

[0040] 1. Cell treatment

[0041] The cells were divided into two groups: the control group was cultured with a conventional osteogenic induction medium (α-MEM complete medium containing 10% FBS supplemented with 100 μM vitamin C, 10 nM β-glycerophosphate, and 10 nM dexamethasone); the experimental group was cultured with 5 μM DDO-2728 combined with the conventional induction medium.

[0042] 2. Real-time PCR

[0043] After 7 days of osteogenic induction of periodontal ligament stem cells, RNA was extracted using TRIzol. RNA was converted to cDNA using oligo(dT) primers and reverse transcriptase. The cDNA was placed into the prepared PCR reaction system and the program was initiated in a Real-time PCR instrument. The Real-time PCR instrument automatically records the fluorescence intensity of each cycle, and the 2 -ΔΔCt -method is used to compare the expression differences of target genes between different samples.

[0044] 3. Western blot

[0045] Cells were collected and lysate and protease inhibitor were added. After ultrasonic fragmentation, the mixture was incubated on ice for 30 min. After high-speed centrifugation to collect the supernatant, the protein concentration was determined using the BCA method. Loading buffer was added in proportion and the proteins were denatured by boiling for 5 min. After cooling to room temperature, the precipitate was removed by centrifugation. An SDS-PAGE gel was prepared and 20 μL of the denatured protein sample and molecular weight standard (Marker) were added to each well. Then electrophoresis, membrane transfer, and blocking were performed. A primary antibody dilution solution was prepared and incubated overnight at 4 °C. It was washed 3 times with TBST. A secondary antibody dilution solution was prepared and incubated at room temperature for 1 h. It was washed 3 times with TBST. The ECL luminescent solution was evenly applied to the membrane. The membrane was placed into a chemiluminescent imaging instrument, exposed, and the signal was recorded. Image J was used to analyze the band intensity. The relative expression level was calculated by comparison with the β-actin internal reference protein.

[0046] Figure 3 It is a statistical chart of the expression levels of the osteogenic differentiation-related genes RUNX2 and OCN in periodontal ligament stem cells in Example 3. Among them, Figure 3 A: Reql-time PCR shows the mRNA expression levels of RUNX2 and OCN; Figure B: Western blot shows the protein expression levels of RUNX2 and OCN.

[0047] RUNX2 is a key transcription factor in osteoblast differentiation and bone development. It plays a crucial role in regulating the formation, differentiation of osteoblasts, and the mineralization process of bone tissue. OCN is a non-collagen protein secreted by osteoblasts. It plays an important role in bone metabolism and osteoblast differentiation and has multiple endocrine functions. OCN is an important component in the bone matrix and participates in the mineralization process of bone tissue. It promotes the mineralization of bone tissue by binding to hydroxyapatite crystals. The expression of OCN usually appears in the late stage of osteoblast differentiation and is an important marker for osteoblast maturation. By Figure 3It can be seen that adding DDO-2728 to the induction solution can significantly increase the mRNA and protein levels of RUNX2 and OCN in periodontal ligament stem cells, indicating that the cells have entered the mineralization stage. This shows that DDO-2728 can play a role in promoting the osteogenic differentiation of periodontal ligament stem cells.

Claims

1. Application of DDO-2728 in the preparation of drugs for treating periodontitis, wherein the molecular formula of DDO-2728 is C 28 H 17 F3N4O7, the chemical structure is shown below:

2. The use according to claim 1, characterized in that: The drug is used for inducing and / or promoting osteogenic differentiation of periodontal ligament stem cells.

3. The use according to claim 1, characterized in that: The medicine is used for increasing the expression level and activity of ALP enzyme of periodontal ligament stem cells.

4. The use according to claim 1, characterized in that: The medicine is used for promoting mineralization of periodontal ligament stem cells.

5. The use according to claim 1, characterized in that: The drug is used for increasing the expression of RUNX2 on periodontal ligament stem cells.

6. The use according to claim 1, characterized in that: The drug is used for increasing the expression of OCN on periodontal ligament stem cells.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug is paste, gel, spray, granule, capsule, tablet, powder, oral solution, suspension or emulsion.

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