Application of inhibiting histone demethylase KDM4D in prevention and treatment of periodontitis

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of inhibiting histone demethylase KDM4D in prevention and treatment of periodontitis. By constructing a mouse periodontitis and periodontitis animal model accompanied by systemic diseases, the influence of KDM4D intervention on periodontitis and periodontitis accompanied by systemic diseases is studied, and experimental results show that inhibition of KDM4D can down-regulate the expression level of osteoclast related genes, inhibit osteoclast differentiation, relieve alveolar bone resorption and bone loss, inhibit osteoclast differentiation and inhibit osteoclast differentiation. The traditional Chinese medicine composition has an obvious curative effect on periodontitis and periodontitis accompanied by systemic diseases, and a new prevention and treatment strategy is provided for periodontitis and periodontitis accompanied by systemic diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of inhibiting histone demethylase KDM4D in the prevention and treatment of periodontitis. Background Art

[0002] Periodontitis is a common and frequently occurring oral disease and the leading cause of tooth loss in adults. Periodontitis is an inflammatory response of the periodontal tissue caused by dental plaque biofilm. The lesions involve periodontal attachment and supporting tissues, including the periodontal ligament, cementum, alveolar bone, and gums, causing dissolution and destruction of collagen fibers in the gums and periodontal ligament, as well as absorption of alveolar bone, ultimately leading to loose and falling teeth, reduced chewing efficiency, and even harm to overall health in severe cases. Most tissue damage in periodontitis is not only directly caused by infecting microorganisms, but is also caused and aggravated by the host's response to the infection. Systemic diseases are important risk or promoting factors for the occurrence and development of periodontitis, including diabetes, rheumatic autoimmune diseases, chronic kidney disease, etc. Therefore, it is of great significance to conduct research on the prevention and treatment of periodontitis and periodontitis associated with systemic diseases.

[0003] Treatment for periodontitis includes oral hygiene education, supragingival scaling, subgingival scaling, periodontal surgery, and medication. The effectiveness of oral hygiene education depends largely on patient compliance. Supragingival scaling, subgingival scaling, and periodontal surgery are painful and uncomfortable for patients. Currently, commonly used medications primarily involve antibiotics. Oral antibiotics such as metronidazole and tinidazole have side effects such as gastrointestinal reactions, headaches, and dizziness, while topical minocycline hydrochloride has limited short-term anti-inflammatory efficacy. Most importantly, the use of antibiotics can lead to the emergence of drug-resistant and cross-resistant strains. Using supragingival scaling, subgingival scaling, periodontal surgery, and antibiotics in the treatment of periodontitis, it is difficult to simultaneously achieve effective inhibition of osteoclast differentiation and alveolar bone destruction. Furthermore, periodontitis associated with systemic diseases often presents with more severe alveolar bone resorption symptoms and a more complex disease background. Therefore, there is an urgent need to develop safe, more effective, and less side-effect drugs for periodontitis and periodontitis associated with systemic diseases to slow down alveolar bone resorption exacerbated by excessive osteoclast differentiation.

[0004] The pathogenesis of periodontitis is complex. Among them, excessive differentiation of osteoclasts aggravates alveolar bone resorption, which is an important pathological mechanism of periodontitis and the key to the aggravation of periodontitis by some systemic diseases. Osteoclasts are derived from the monocyte / macrophage hematopoietic lineage and differentiate from bone marrow-derived monocytes / macrophages (BMDMs) under the stimulation of monocyte / macrophage colony-stimulating factor M-CSF and RANKL. Drugs that target the inhibition of osteoclast differentiation have become the focus of research on the prevention and treatment of periodontitis. In the Chinese patent application CN202210261643.6 "The use of ceramide binding to the CD300lf receptor on the surface of osteoclasts in the preparation of drugs for the treatment of periodontitis", bone resorption and osteoclast differentiation are inhibited by downregulating CD300lf on the surface of osteoclast precursors to treat periodontitis. Chinese patent application CN202411609140.9, "Application of simeprevir in the preparation of antibacterial agents and drugs for the prevention and treatment of periodontitis," describes the use of simeprevir's antibacterial properties to prepare antibacterial agents or preparations for the prevention and treatment of periodontitis, reducing the destruction of periodontal tissue, alveolar bone resorption, and osteoclast infiltration, for the preparation of preparations that protect periodontal tissue or alveolar bone. None of these targeted drugs address the efficacy of periodontitis associated with systemic diseases.

[0005] Histone lysine demethylase 4D (KDM4D) is a member of the Jumonji C domain (JMJD)-containing histone demethylase family. It catalyzes the removal of methyl groups from protein lysine residues, regulates chromatin structure, participates in the fine-tuning of gene transcription, and maintains the balance between active and inactive chromatin. Consequently, it participates in various biological processes, including cell proliferation, differentiation, ontogeny, energy metabolism, and disease development. Previous studies have found that KDM4D is involved in the regulation of bone metabolism. Under iron deficiency conditions, the H3K9me3 demethylase activity of KDM4D is significantly reduced, inhibiting PIK3R3 expression. Subsequently, it inhibits the activation of quiescent MSCs through the PI3K-Akt-Foxo1 pathway, resulting in impaired activation of bone marrow MSCs and impaired osteogenesis. Furthermore, other JMJD histone demethylase family members, such as KDM5C, KDM4B, JMJD5, and KDM6B, have also been reported to participate in the epigenetic regulation of osteoclastogenesis. However, there have been no reports on whether KDM4D can affect osteoclast differentiation and thus treat and alleviate periodontitis and periodontitis associated with systemic diseases. Summary of the Invention

[0006] The purpose of the present invention is to select KDM4D as the research object, explore its mechanism of action in regulating osteoclastogenesis, and explore the efficacy of intervening KDM4D in treating and alleviating periodontitis and periodontitis associated with systemic diseases, and provide the application of developing new strategies for the treatment of periodontitis by intervening in KDM4D.

[0007] In order to achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0008] In a first aspect, the present invention provides use of a KDM4D inhibitor in the preparation of a medicament for preventing or treating periodontitis.

[0009] Furthermore, the KDM4D inhibitor is selected from the group consisting of the specific inhibitor KDM4D-IN-1, a viral shRNA targeting KDM4D, or a genetic tool that specifically knocks out KDM4D in osteoclasts. The selection or design of a viral shRNA targeting KDM4D or a genetic tool that specifically knocks out KDM4D in osteoclasts to inhibit KDM4D expression is a routine technique in the art and can be accomplished by those skilled in the art without requiring creative effort.

[0010] Furthermore, the periodontitis is simple periodontitis (not associated with systemic diseases) or periodontitis associated with systemic diseases, wherein the periodontitis associated with systemic diseases includes periodontitis associated with chronic kidney disease (CKD).

[0011] Furthermore, KDM4D inhibitors can alleviate alveolar bone resorption and bone loss.

[0012] Furthermore, KDM4D inhibitors significantly inhibited osteoclast differentiation and downregulated the expression of osteoclast-related genes.

[0013] Furthermore, the drug uses a KDM4D inhibitor as a single active ingredient, or a KDM4D inhibitor in combination with existing periodontitis medications. Existing periodontitis medications include systemic and topical medications; systemic medications primarily include antibacterial drugs (metronidazole, minocycline, amoxicillin, etc.) and nonsteroidal anti-inflammatory drugs (ibuprofen, ibuprofen, etc.); topical medications include compound chlorhexidine solution, cetylpyridamole, povidone iodine, minocycline ointment, etc.

[0014] Furthermore, the dosage form of the drug includes but is not limited to paste, gel, spray, granules, capsules, tablets, powders, oral liquids, suspensions or emulsions. The administration mode is local or systemic administration.

[0015] In one embodiment, the drug is in the form of an injection and is administered by local injection.

[0016] In a second aspect, the present invention provides a pharmaceutical composition for preventing or treating periodontitis, comprising an active ingredient, wherein the active ingredient is a KDM4D inhibitor, or a KDM4D inhibitor and an existing periodontitis drug.

[0017] Furthermore, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that when the carrier is properly administered to an animal or human, it does not produce adverse, allergic or other untoward reactions. The pharmaceutically acceptable carrier may be selected from one or more of a solvent, a diluent, a filler, a surfactant, an absorption enhancer, a disintegrant, a wetting agent, a dispersant, and the like.

[0018] In one embodiment, the pharmaceutical composition comprises the active ingredient KDM4D-IN-1 and the solvent components DMSO, PEG300, Tween-80 and normal saline.

[0019] The present invention has the following beneficial effects:

[0020] The present invention explores the effects of intervening KDM4D on periodontitis and periodontitis associated with systemic diseases by constructing an animal model of periodontitis in mice and periodontitis associated with systemic diseases. The results of animal model experiments confirmed that inhibiting KDM4D can inhibit osteoclast differentiation, reduce the area of ​​the cementoenamel junction-alveolar ridge crest (CEJ-ABC), increase the bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) of periodontal bone tissue, and downregulate the trabecular separation (Tb.Sp), thereby alleviating alveolar bone resorption caused by periodontitis; in animal experiments, mice did not experience side effects such as nausea, vomiting, and anorexia. The present invention also confirmed the effect of inhibiting KDM4D on osteoclast differentiation in in vitro cell experiments, which is consistent with the results of animal experiments. TRAP staining results show that inhibiting KDM4D can inhibit the differentiation of BMDMs into osteoclasts, while downregulating the expression levels of osteoclast-related genes.

[0021] Therefore, the present invention confirms that inhibiting KDM4D can effectively inhibit osteoclast differentiation and alleviate alveolar bone resorption, providing a new strategy for the prevention and treatment of periodontitis and periodontitis associated with systemic diseases, and solving the technical problem that drug treatment in the prior art is not effective in preventing or treating periodontitis and periodontitis associated with systemic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :A model diagram of treatment strategies for periodontitis and periodontitis associated with chronic kidney disease based on KDM4D intervention.

[0023] Figure 2Evaluation of the efficacy of a KDM4D inhibitor on alveolar bone loss in mice with periodontitis and periodontitis associated with CKD. (A) Micro-CT analysis shows representative images of the maxilla of the model mice. The red arrow in the two-dimensional cross-sectional image indicates alveolar bone loss along the mesio-distal direction of the maxillary first molar. Scale bar, 500 μm. (B) Quantitative analysis of the area between the CEJ and ABC of the maxillary first molar (n = 5). (C) Quantitative analysis of micro-CT bone morphological parameters, including bone volume percentage (BV / TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) (n = 5). Data are expressed as mean ± SD. ns, not statistically significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

[0024] Figure 3 Effect of KDM4D inhibitors on osteoclast differentiation. (A) Representative images of TRAP staining following in vitro osteoclast differentiation in BMDMs isolated from model mice. Scale bar, 200 μm. (B) Quantitative analysis of the number and area of ​​TRAP-positive cells (n = 5). (C) qPCR analysis of mRNA expression levels of osteoclast-related genes (n = 5). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] (1) Preparation of drugs, reagents and consumables:

[0028] KDM4D inhibitor KDM4D-IN-1 (KI) (MedChemExpress), DMSO (Sigma), Tween-80 (Sigma), PEG300 (Selleck), tartrate-resistant acid phosphatase (TRAP) staining kit (Selleck), Realtime PCR primers (Sangon Biotechnology), conventional PCR kit (Roche), α-MEM medium (Hyclone), fetal bovine serum (Thermo Fisher Scientific), penicillin / streptomycin double antibody solution (Gibico), M-CSF (PeproTech), RANKL (PeproTech), 8-0 silk thread (Gold Ring), etc.

[0029] (2) Animal model construction and grouping:

[0030] 8-week-old C57BL / 6 male mice were selected and Figure 1The process shown was used to construct two types of periodontitis disease models: ① The silk ligation method was used to establish a periodontitis model mouse model (PD group); ② The silk ligation method was combined with the two-stage 5 / 6 nephrectomy method to induce a CKD periodontitis mouse model with systemic disease and periodontitis comorbidity (CKD-PD group) for drug intervention studies.

[0031] The periodontitis mouse model was induced as follows: After anesthesia, male C57BL / 6 mice were anesthetized and an 8-0 silk thread was placed around the cervical area of ​​the maxillary first molars (M1) bilaterally. The thread was completely embedded in the gingiva and then ligated and fixed at the cervical area of ​​the M1 tooth. This was maintained for 10 days to establish a periodontitis mouse model (PD group). The non-ligated group served as the control group for the periodontitis model.

[0032] The CKD-associated periodontitis mouse model was induced as follows: 8-week-old C57BL / 6 male mice were anesthetized with isoflurane gas (induction concentration 3-4%, maintenance concentration 1-2%) and immobilized in the prone position on an operating table. The surgical area was prepared and disinfected locally. An incision approximately 1 cm long, obliquely extending outward and downward, was made at the left lower costal margin near the spine. The soft tissue was separated layer by layer to expose the left kidney. After stripping the renal capsule, one-third of each of the upper and lower poles of the kidney were removed. After compression and hemostasis, the kidney was repositioned and sutured layer by layer. Two weeks later, the mice underwent a second-stage surgery under anesthesia to expose the right kidney. The renal pedicle was ligated, the entire right kidney was removed, and sutured. The sham-operated group underwent only incision, repositioning, and suturing. Twelve weeks after surgery, a 5 / 6 nephrectomy-induced chronic kidney disease mouse model (CKD group) was established. Ten days before the induction of the CKD model, experimental periodontitis (PD group) was induced using the aforementioned silk suture ligation method until chronic kidney disease and periodontitis lesions were established.

[0033] Two weeks before CKD was induced by 5 / 6 nephrectomy, the KDM4D inhibitor KDM4D-IN-1 (KI) was dissolved in a working solution consisting of 10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline (% by volume) to prepare the desired concentration. Mice in the KI-treated group (KI group) received intraperitoneal injections of the KI working solution every two days for two weeks at a dose range of 0.1-1.0 μg / g body weight. Mice in the control group (DMSO group) received an equal volume of DMSO vehicle solution until the end of the experiment. To investigate the effects of KDM4D intervention on periodontitis, four mouse models were included in this study: PD+DMSO group, PD+KI group, CKD-PD+DMSO group, and CKD-PD+KI group, with five mice in each group. At the end of the experiment, mice were euthanized and samples were collected for subsequent experiments.

[0034] (3) Evaluation of the efficacy of KDM4D inhibitors in periodontitis:

[0035] ① Micro-CT imaging and analysis of alveolar bone height and bone morphology parameters: Mouse jaw samples were collected for fixation and Micro-CT scanning, and three-dimensional reconstruction and data analysis were performed to observe alveolar bone resorption, quantitatively detect the area of ​​the cementoenamel junction to the alveolar crest (CEJ-ABC) of the mouse maxillary first molar, and analyze the bone volume fraction (BV / TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). The results are as follows Figure 2 As shown in A, the CKD-PD mouse model showed more severe alveolar bone resorption than the PD mouse model. After treatment with the KDM4D inhibitor KI, alveolar bone resorption in both types of periodontitis mouse models was alleviated. Quantitative analysis showed that the alveolar bone resorption area in the CKD-PD group was larger than that in the PD group ( Figure 2 B), BV / TV and Tb.Th are smaller, Tb.Sp is larger ( Figure 2 C), indicating more obvious bone loss; after KI treatment, alveolar bone resorption was significantly reduced ( Figure 2 B), BV / TV and Tb.Th increased significantly, while Tb.Sp decreased ( Figure 2 C), that is, inhibition of KDM4D can alleviate alveolar bone resorption and bone loss.

[0036] ② Evaluation of osteoclast differentiation: Bone marrow was isolated from the femur and tibia of model mice and cultured in vitro in α-MEM complete medium containing 30ng / ml M-CSF, 10% fetal bovine serum and penicillin-streptomycin double antibody to obtain primary osteoclast precursor cells BMDMs. Osteoclast differentiation was induced in vitro with 50ng / ml RANKL for 8 days and stained with TRAP kit. The number and area of ​​osteoclasts were counted by Image J. The results are shown in the figure. Figure 3 As shown in Figures AB, osteoclast differentiation was more pronounced in the CKD-PD group than in the PD group. Treatment with a KDM4D inhibitor significantly inhibited osteoclast differentiation, with the number and area of ​​osteoclasts significantly reduced, suggesting that KDM4D inhibition negatively regulates osteoclast differentiation.

[0037] ③ Evaluation of osteoclast-related genes: Primary osteoclast precursor cells (BMDMs) were isolated and cultured from the femur and tibia bone marrow of model mice. Osteoclast differentiation was induced by RANKL in vitro, and the expression levels of osteoclast-related genes Acp5 and Ctsk were detected by qPCR. Similar conclusions were found with the aforementioned Micro-CT and osteoclast differentiation assays: the expression of osteoclast-related genes was higher in the CKD-PD group than in the PD group. After treatment with KDM4D inhibitors, the expression of osteoclast-related genes was significantly downregulated ( Figure 3 C).

[0038] The above results suggest that in periodontitis and periodontitis associated with systemic diseases (such as periodontitis associated with CKD), inhibiting KDM4D can effectively inhibit osteoclast differentiation and thereby alleviate alveolar bone resorption, which is a potential new strategy for periodontal treatment.

[0039] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of KDM4D inhibitors in the preparation of drugs for preventing or treating periodontitis.

2. The use according to claim 1, characterized in that The KDM4D inhibitor is selected from a specific inhibitor KDM4D-IN-1, a viral shRNA targeting KDM4D, or a gene tool targeting osteoclasts to specifically knock out KDM4D.

3. The use according to claim 1, characterized in that The periodontitis is simple periodontitis or periodontitis associated with systemic diseases.

4. The use according to claim 3, characterized in that The periodontitis associated with systemic diseases includes periodontitis associated with chronic kidney disease.

5. The use according to claim 1, characterized in that KDM4D inhibitors can alleviate alveolar bone resorption and bone loss.

6. The use according to claim 1, characterized in that KDM4D inhibitors significantly inhibited osteoclast differentiation and downregulated the expression of osteoclast-related genes.

7. The use according to claim 1, characterized in that The drug uses a KDM4D inhibitor alone as an active ingredient, or uses a KDM4D inhibitor in combination with an existing periodontitis drug as an active ingredient.

Images