Application of GDF6 in preparation of medicine for detecting periodontitis or preventing and treating periodontitis

Abstract

The invention discloses application of GDF6 in preparation of a medicine for detecting periodontitis or preventing and treating periodontitis, and belongs to the technical field of biological medicine. The invention finds that GDF6 is obviously reduced in periodontitis gingival tissues; the GDF6 can improve the expression of ALP, cell mineralization and the expression of osteogenic transformation related genes ALP, RUNX2 and OCN by reducing the expression of inflammatory factors of the inflammatory PDLSCs, so that the GDF6 can be used for osteogenic differentiation of the PDLSCs in an inflammatory state. In addition, the GDF6 can significantly improve the level of periodontitis of mice and alveolar bone resorption when applied in vivo, so that the GDF6 has a good treatment effect on periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of GDF6 in the preparation of drugs for the detection or prevention of periodontitis. Background Technology

[0002] Periodontitis is a chronic infectious and destructive disease of the periodontal supporting tissues caused by microorganisms in dental plaque biofilm, leading to gingivitis, periodontal pocket formation, progressive attachment loss, and alveolar bone resorption. Periodontitis is the sixth most common human disease and a leading cause of tooth loss in adults. The results of my country's Fourth National Oral Health Epidemiological Survey showed that only 9.1% of residents aged 35-44 had a periodontal health rate, and only 5.0% of those aged 55-64. Furthermore, various epidemiological and experimental studies have demonstrated that periodontitis can affect overall health and is closely related to the incidence of various chronic non-communicable diseases such as diabetes, pregnancy complications, and cardiovascular diseases. Therefore, periodontitis treatment is of significant clinical importance for maintaining oral and systemic health.

[0003] The development of periodontitis is the result of the interaction between oral pathogenic microorganisms and the host immune microenvironment. Currently, the most common treatment for periodontitis is mechanical removal of pathogenic microorganisms, supplemented by laser and drug therapies. Drug therapy is an important adjunct, especially for areas difficult to reach with mechanical debridement; however, it carries the risk of periodontal microbial dysbiosis and the development of drug resistance in some microorganisms. Therefore, controlling the level of inflammation in the gingival immune microenvironment is another potential strategy for periodontitis treatment. However, controlling inflammation alone can only maintain the existing level of bone resorption. Therefore, to improve periodontitis treatment, combining current clinical methods with factors that assist in controlling local gingival inflammation while promoting bone regeneration has significant research and application value.

[0004] Growth differentiation factor 6 (GDF6) plays an important regulatory role in tumorigenesis and organ regeneration. In osteoarthritis, GDF6 can inhibit arthritis by controlling chondrocyte senescence. Using a rat model, GDF6 can improve the degenerative phenotype of intervertebral disc herniation and inhibit the expression of inflammatory factors. The GDF6-FTO axis enhances the cellular response to human respiratory syncytial virus by inhibiting the inflammatory response. Research on the osteogenic differentiation of mesenchymal stem cells (MSCs) using GDF6 is limited. Human induced pluripotent stem cells (iPSCs) can be induced into mesenchymal stem cells. GDF6-deficient MSCs differentiate into mesenchymal stem cells, and GDF6 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Shen et al. showed that GDF6 inhibits the osteogenic differentiation ability of bone marrow mesenchymal stem cells, but the induction concentrations were 100, 200, and 300 ng / mL; the induction effect at lower concentrations was not investigated. Furthermore, GDF6 expression is reduced in senescent bone marrow mesenchymal stem cells. GDF6 can promote osteogenic differentiation of senescent bone marrow mesenchymal stem cells in vitro. Lipopolysaccharides (LPS) derived from *Porphyromonas gingivalis* are an important inducing factor for gingival tissue inflammation in periodontitis. Whether GDF6 can regulate osteogenic differentiation of periodontal ligament stem cells (PDLSCs) under inflammatory conditions and thus improve periodontitis has not been reported. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of GDF6 in the preparation of drugs for the detection or prevention of periodontitis.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of GDF6 as a biomarker for the detection of periodontitis.

[0007] This invention utilizes RT-qPCR to demonstrate the downregulation of GDF6 gene expression in periodontitis tissues; and uses immunofluorescence to confirm the downregulation of GDF6 protein expression in periodontitis tissues. Therefore, the detection of GDF6 gene and protein expression in gingival tissues, as well as expression in saliva and gingival crevicular fluid, can be used as a biomarker for periodontitis.

[0008] This invention provides the application of GDF6 in the preparation of medicaments for the detection and / or prevention of periodontitis.

[0009] This invention provides the application of GDF6 in the preparation of drugs that promote osteogenic differentiation of PDLSCs under inflammatory conditions.

[0010] Furthermore, the dosage form of the drug includes paste, gel, spray, granules, capsules, tablets, powder, oral liquid, suspension or emulsion.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the drug also includes other substances for the prevention and treatment of periodontitis.

[0013] It contains at least the following beneficial technical effects: This invention reveals that GDF6 is significantly reduced in gingival tissue of patients with periodontitis. GDF6 can reduce the expression of inflammatory factors in inflammatory PDLSCs, increase ALP expression, cell mineralization, and the expression of osteogenic transformation-related genes ALP, RUNX2, and OCN, thus it can be used to promote osteogenic differentiation of PDLSCs under inflammatory conditions. Furthermore, in vivo application of GDF6 can significantly improve the level of periodontitis and alveolar bone resorption in mice, thereby demonstrating a good therapeutic effect on periodontitis. Attached Figure Description

[0014] Figure 1 This image shows the expression of the GDF6 gene in gingival tissue derived from periodontitis, along with immunofluorescence staining and quantification of GDF6 in normal and periodontitis gingival tissues.

[0015] Figure 2 This diagram illustrates how GDF6 inhibits the expression of inflammatory factors in inflammatory PDLSCs.

[0016] Figure 3 The image shows the in vitro osteogenic differentiation of inflammatory PDLSCs promoted by GDF6; where A is ALP staining; B is quantitative ALP staining; C is Alizarin Red staining; D is quantitative ARS; and E is the expression detection of ALP, RUNX2, and OCN.

[0017] Figure 4 Figures showing the effect of GDF6 on alveolar bone resorption in mice; where A is the periodontitis model and injection model; B is the silk suture ligation pattern; C is the reconstruction diagram and mesiodistal section of the palatal second maxillary molar; D is the distance between the enamel / dentin junction (CEJ) and alveolar ridge (ABC) on the buccal and palatal sides. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0024] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0025] The human GDF6 used in this embodiment of the invention has the product code HY-P700022AF and the amino acid sequence is as follows: TAFASRHGKRHGKKSRLRCSKKPLHVNFKELGWDDWIIAPLEYEAYHCEGVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCCVPTKLTPISILYIDAGNNVVYKQYEDMVVESCGCR; The mouse-derived GDF6, catalog number HY-P79333, has the following amino acid sequence: TAFASRHGKRHGKKSRLRCSRKPLHVNFKELGWDDWIIAPLEYEAYHCEGVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCCVPTKLTPISILYIDAGNNVVYKQYEDMVVESCGCR.

[0026] All the human and mouse-derived GDF6 samples were purchased from MCE.

[0027] GDF6 in humans and mice is highly conserved, with only a single amino acid difference in the mature sequence. Unless otherwise specified, all reagents used in the embodiments of this invention are available from the commercially available companies.

[0028] Example 1: Detection of GDF6 RNA Expression Level 1. Gingival sample collection Gingival tissue was collected from 15 healthy individuals and 15 patients with periodontitis. The samples were collected from individuals older than 18 years, with at least 20 teeth, and in good overall health.

[0029] Exclusion criteria: 1) Tooth misalignment due to trauma; 2) Immune system diseases; 3) Blood diseases; 4) Hormone therapy within the past three months; 5) Neurological and psychological diseases; 6) Metabolic and endocrine disorders; 7) Tumors; 8) Antibiotics, cell therapy, etc. within the past three months; 2. Real-time quantitative PCR (RT-qPCR) detection Collected gingival tissue was subjected to low-temperature grinding with TRIZOL and magnetic beads. RNA was extracted from the ground magnetic beads using the TRIZOL method and then reverse transcribed. A 10 μL reverse transcription system contained 500 ng RNA and 2 μL reverse transcriptase. The reverse-transcribed cDNA was detected by quantitative real-time PCR using the SYBR method. The 20 μL reaction system consisted of: 2 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, 10 μL SYBR reagent, and 7.2 μL enzyme-free water (H2O). GADPH was used as an internal control, and relative gene expression was measured using a 2:1 ratio. ΔΔCt Methods and primer sequences are shown in Table 1.

[0030] Table 1 Primer Sequences Test results as follows Figure 1 As shown in A, GDF6 expression is significantly downregulated in periodontitis tissues.

[0031] Example 2: GDF6 Immunofluorescence Detection Gingival tissues were collected from three healthy individuals and three patients with periodontitis. The tissues were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned at a thickness of 4 μm. The sections were then dewaxed in xylene and dewaxed to water. After antigen retrieval, serum was added for blocking. GDF6 antibody (1:500) was incubated overnight. After antibody washing, CY3-labeled goat anti-rabbit IgG (1:300) was added and reacted for 1 h. After antibody washing, DAPI containing an anti-quencher was added for nuclear staining for 10 min. Images were taken using a fluorescence pathology scanner. Fluorescence intensity was quantified using ImageJ software.

[0032] Test results as follows Figure 1 As shown in B and C, GDF6 protein levels are significantly downregulated in periodontitis tissues. The reduced mRNA and protein levels of GDF6 in periodontitis tissues may be a potential risk factor for periodontitis and could serve as a biomarker for periodontitis detection.

[0033] Example 3: Effect of GDF6 on the expression of inflammatory genes in periodontal ligament stem cells.

[0034] 1. Cell treatment Periodontal ligament stem cells were divided into four groups: The control group was prepared with normal culture medium (α-MEM + 10% FBS + 1% P / S). The LPS group was treated with 1 μg / mL of Porphyromonas gingivalis LPS for 48 h and then cultured in bone induction medium (α-MEM complete medium containing 10% FBS with 50 ng / mL vitamin C, 10 mM β-glycerophosphate sodium, and 10 nM dexamethasone). The LPS+50 ng / mL GDF6 group was treated with 1 μg / mL of Porphyromonas gingivalis LPS for 48 h, and then added to bone induction medium (α-MEM complete medium containing 10% FBS with 50 ng / mL vitamin C, 10 mM β-glycerophosphate sodium, and 10 nM dexamethasone), and then 50 ng / mL LDF6 was added for culture. The LPS+100 ng / mL GDF6 group was treated with 1 μg / mL of Porphyromonas gingivalis LPS for 48 h, and then added to bone induction medium (α-MEM complete medium containing 10% FBS with 50 ng / mL vitamin C, 10 mM β-glycerophosphate sodium, and 10 nM dexamethasone), and then 100 ng / mL LDF6 was added for culture.

[0035] 2. RT-qPCR Seven days after osteogenic induction in PDLSCs, RNA was extracted using the TRIzol method. RNA was reverse transcribed using random primers and reverse transcriptase. A 10 μL reverse transcription system contained 500 ng RNA and 2 μL reverse transcriptase.

[0036] The reverse-transcribed cDNA was detected by real-time quantitative PCR using the SYBR method.

[0037] The 20 μL reaction system consisted of the following reaction procedure: 2 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, 10 μL SYBR reagent, and 7.2 μL enzyme-free water (H2O). GADPH was used as an internal control, and relative gene expression was measured using a 2:1 ratio. ΔΔCt Methods and primer sequences are shown in Table 2.

[0038] Table 2 Primer sequences like Figure 2 As shown, the addition of GDF6 can reduce the expression of inflammatory factors, and the inhibitory effect is significant with increasing dosage.

[0039] Example 4: Effect of GDF6 on alkaline phosphatase (ALP) in periodontal ligament stem cells.

[0040] 1. Cell treatment The cell treatment method is the same as that in Example 3.

[0041] 2. ALP staining After 7 days of osteogenic induction in PDLSCs, the culture medium was aspirated, and the cells were washed once with PBS. 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 25 min. The paraformaldehyde was aspirated, and the cells were washed twice with PBS. ALP staining reagent was added, and the cells were stained at room temperature for 10 min.

[0042] Figure 3 Image A shows the staining results of alkaline phosphatase (ALP) in periodontal ligament stem cells in Example 4, and Image B shows the semi-quantitative results of ALP staining.

[0043] ALP plays a crucial role in osteogenic differentiation and is an important marker of osteoblast differentiation. ALP provides high local concentrations of inorganic phosphates by hydrolyzing phosphate esters and phosphoric anhydrides, providing the necessary phosphate for hydroxyapatite formation. Figure 3 As shown in A and B, the addition of LPS can significantly reduce the ALP staining intensity. After the addition of GDF6, the ALP expression of inflammatory PDLSCs increased significantly compared with the LPS group, proving that GDF6 can effectively promote osteogenic differentiation of PDLSCs in an inflammatory environment.

[0044] Example 5: Experiment on the effect of GDF6 on the mineralization of periodontal ligament stem cells.

[0045] 1. Cell treatment The cell treatment method is the same as that in Example 3.

[0046] 2. Alizarin Red staining After 14 days of osteogenic induction, periodontal ligament stem cells were aspirated from the culture medium and washed once with PBS. 4% paraformaldehyde solution was added, and the cells were fixed at room temperature for 25 min. The paraformaldehyde was then aspirated, and the cells were washed twice with PBS. Alizarin Red dye (pH 4.2) was added, and the cells were stained at room temperature for 10 min. Finally, images were taken under a stereomicroscope. Subsequently, the cells were dissolved in 10% sodium 12-alkylsulfonate at room temperature for 1 h, and the OD was measured using a microplate reader. 562 value.

[0047] Alizarin Red S is an anthraquinone derivative that chelates with calcium salts (such as calcium carbonate or calcium phosphate) to form orange-red or deep red complexes. This property makes it a commonly used staining agent for detecting calcium deposition. During osteoblast differentiation, calcium salt deposits in the extracellular matrix bind to Alizarin Red S, appearing red or orange-red under a microscope. Alizarin Red staining is widely used to assess osteoblast differentiation and mineralization capacity. In cell culture, Alizarin Red S can stain calcium nodules, thereby determining whether cells have successfully differentiated into osteoblasts.

[0048] Depend on Figure 3 As shown in C and D, the addition of LPS significantly reduced the intensity of Alizarin Red staining. With the addition of GDF6, inflammatory PDLSCs showed deeper staining and more mineralized calcium nodules. This demonstrates that GDF6 can effectively promote osteogenic differentiation of PDLSCs under inflammatory conditions.

[0049] Example 6: Effects of GDF6 on osteogenic differentiation-related genes of periodontal ligament stem cells.

[0050] 1. Cell treatment The cell treatment method is the same as that in Example 3.

[0051] 2. RT-qPCR Seven days after osteogenic induction, periodontal ligament stem cells underwent RNA extraction using TRIzol, followed by reverse transcription using random primers and reverse transcriptase. A 10 μL reverse transcription system contained 500 ng RNA and 2 μL reverse transcriptase. The transcribed cDNA was detected by quantitative real-time PCR using the SYBR method. The 20 μL reaction system consisted of: 2 μL cDNA, 0.4 μL front primer, 0.4 μL back primer, 10 μL SYBR reagent, and 7.2 μL enzyme-free water (H2O). GADPH was used as an internal control, and relative gene expression was measured using a 2:1 ratio. ΔΔCtMethods and primer sequences are shown in Table 3.

[0052] Table 3 Primer Sequences like Figure 3 As shown in Figure E, GDF6 significantly increased osteogenic markers in LPS-treated PDLSCs. ALP , RUNX2 , OCN The expression.

[0053] Example 7: GDF6 improves periodontitis levels in mice 1. Periodontitis modeling Silk ligation is the most commonly used method for creating models of periodontitis.

[0054] Six-week-old C57BL / 6J mice had their second molars ligated bilaterally using 5-0 silk sutures. Different doses of 75 ng and 150 ng of mouse-derived GDF6 (10 μL system) were injected using a Hamilton microsyringe on day 0 (the day of suture ligation), day 3, and day 5. The control group was injected with 10 μL of sterile ddH2O. Six mice were in each group. Figure 4 A in the diagram represents the animal experiment procedure; Figure 4 Figure B is a schematic diagram of silk thread ligation.

[0055] 2. MicroCT Analysis Nine days later, maxillary bone was collected, and reconstructed and analyzed using microCT to evaluate the potential efficacy of GDF6 in the treatment of periodontitis. Analysis parameters: pixel size 10 μm, aluminum filter 0.25 mm, voltage 70 kV, current density 80 μA. Vertical distances were measured at the following sites: mesiobuccal (BM), midbuccal (B-MD), disbuccal (BD), mesiopalatal (PM), midpalatal (P-MD), and dispalatal (PD).

[0056] Figure 4 The results from the study indicate that GDF6 injection can improve overall alveolar bone resorption.

[0057] Figure 4 The results showed that GDF6 injection improved alveolar bone resorption at different sites: BM, B-MD, BD, PM, P-MD, and PD.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of GDF6 as a biomarker for the detection of periodontitis.

2. Application of GDF6 in the preparation of drugs for the detection and / or prevention of periodontitis.

3. Application of GDF6 in the preparation of drugs that promote osteogenic differentiation of periodontal ligament mesenchymal stem cells under inflammatory conditions.

4. The medicament according to any one of claims 2-3, characterized in that, The dosage forms of the drug include pastes, gels, sprays, granules, capsules, tablets, powders, oral liquids, suspensions, or emulsions.

5. The medicament according to any one of claims 2-3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The medicament according to any one of claims 2-3, characterized in that, The medication also includes other substances for the prevention and treatment of periodontitis.

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