Use of prussian blue in the preparation of a medicament for treating chronic periodontitis

Abstract

The application discloses application of Prussian blue in preparation of a medicine for treating chronic periodontitis and belongs to the technical field of medicines. The application solves the problem of treating chronic periodontitis. The application comprises application of Prussian blue in preparation of the medicine for treating chronic periodontitis. The application is mainly used for treating chronic periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of Prussian blue in the preparation of drugs for treating chronic periodontitis. Background Technology

[0002] Chronic periodontitis is a common chronic inflammatory disease characterized by alveolar bone resorption and destruction. This disease can lead to tooth loosening and even eventual tooth loss, severely impacting chewing function and oral aesthetics. There is an urgent need for an effective medication to treat chronic periodontitis. Summary of the Invention

[0003] In view of this, the present invention aims to propose the use of Prussian blue in the preparation of drugs for treating chronic periodontitis.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The present invention provides the application of Prussian blue in the preparation of drugs for treating chronic periodontitis.

[0005] This invention provides the application of Prussian blue in the preparation of drugs for the prevention of chronic periodontitis.

[0006] This invention provides the application of Prussian blue in the preparation of health care products for the adjuvant treatment of chronic periodontitis.

[0007] Further specifying, the concentration of Prussian blue is 5 μg / mL to 10 μg / mL.

[0008] Further specifying the preparation method of Prussian blue: Weigh 3g of polyvinylpyrrolidone and 131.7mg of potassium ferricyanide and add them to a beaker. Pour in 40ml of 0.01M hydrochloric acid solution and stir for 30min at 500rpm. Incubate in an oven at 80℃ for 20h. Cool to room temperature, centrifuge to collect the mixture, and use an ethanol:water (2:1) system to centrifuge and wash the precipitate three times to obtain Prussian blue.

[0009] To further specify, the chemical formula of Prussian blue is: .

[0010] Further specifying, the dosage form of the drug or health product is any one of tablets, capsules, granules, powders, or liquid preparations.

[0011] Compared with the prior art, the beneficial effects of the present invention are: PBNPs have good cell compatibility with MC3T3-E1 cells, and in vitro and in vivo experiments have confirmed that PBNPs have a positive therapeutic effect on promoting bone repair in experimental periodontitis, providing a new approach for the treatment of chronic periodontitis. Attached Figure Description

[0012] Figure 1Example 1 describes the characterization and physicochemical property analysis of PBNPs; A. Morphological characteristics of PBNPs under transmission electron microscopy; B. Analysis of the crystal structure of PBNPs by X-ray diffraction (XRD) pattern; C. Particle diameter of PBNPs by dynamic light scattering (DLS); D. Zeta potential analysis results of PBNPs.

[0013] Figure 2 Example 2: Biosafety experiment of PBNPs; A. CCK8 test results; B. Live and dead cell staining results; C. Phalloidin staining results.

[0014] Figure 3 Effects of PBNPs on osteogenic differentiation of MC3T3-E1 cells in Example 3; A. ALP staining results; B. Alizarin Red staining results; C. RT-qPCR quantitative analysis results; D. GH. Western blot results.

[0015] Figure 4 Figure 4 shows the results of PBNPs promoting osteogenic therapeutic effects in mice with experimental periodontitis; AC.H&E staining results; DG. immunohistochemical staining results.

[0016] Figure 5 Figure 4 shows the results of the osteogenic therapeutic effect of PBNPs on mice with experimental periodontitis. Detailed Implementation

[0017] Example 1. The method for obtaining PBNPs is as follows: Weigh 3g of polyvinylpyrrolidone and 131.7mg of potassium ferricyanide and add them to a beaker. Pour in 40ml of 0.01M hydrochloric acid solution and stir for 30min at 500rpm. Incubate in an 80℃ oven for 20h, cool to room temperature, and centrifuge to collect the mixture (4000rpm, 10min). Use an ethanol:water (2:1) system to centrifuge and wash the precipitate three times under the same centrifugation conditions to remove excess impurities, finally obtaining pure PBNPs. Subsequently, its physicochemical properties were systematically analyzed through a series of characterization techniques: Transmission electron microscopy (TEM) results showed that the synthesized PBNPs exhibited a regular morphology (cubic hexahedral structure) (…). Figure 1 The clear characteristic diffraction peaks in the X-ray diffraction (XRD) pattern confirm that PBNPs have a typical crystal structure. Figure 1 (B in the text); Dynamic light scattering (DLS) technology measured its hydrodynamic diameter to be approximately 400 nm, and it exhibits good monodispersity and excellent stability. Figure 1 (C in the text); Furthermore, Zeta potential analysis indicates that its surface carries a negative charge, suggesting good colloidal stability in solution (C). Figure 1 The characterization results above collectively confirm the successful preparation of PBNPs. The Prussian blue nanoparticles (PBNPs) exhibit a porous cubic microstructure and have the chemical formula [D]. .

[0018] Example 2. Evaluation of the cytocompatibility of PBNPs 1. This invention uses immortalized mouse cranial pre-osteoblasts (MC3T3-E1) (ATCC, CRL-2593, USA). TM () as a model.

[0019] MC3T3-E1 cells were seeded in 96-well plates and cultured for 24 hours with different concentrations of PBNPs (0, 5, 10, 15, 20, 25 μg / ml) when they were in the logarithmic growth phase. It was found that PBNPs concentrations below 10 μg / ml had no significant effect on cell viability. Figure 2 Therefore, PBNPs concentrations of 5 and 10 μg / mL were selected for subsequent experiments, and incubation was performed for 24, 48, and 72 hours, respectively. The results showed that when the effective concentrations of PBNPs were 5 μg / mL and 10 μg / mL, respectively, there was no significant difference in cell proliferation activity compared to the culture medium group alone. Figure 2 (B in the text). PBNPs at concentrations of 5 μg / mL and 10 μg / mL were selected as drug concentrations for subsequent experiments.

[0020] Subsequently, this invention employed live-dead staining to assess the cytotoxicity of PBNPs. MC3T3-E1 cells were seeded in 24-well culture plates. After cell attachment, different concentrations of PBNPs were added. The untreated cell group served as the control group (Con group). After incubation for 24 hours, the culture medium was slowly discarded, and the cells were washed once with PBS. An appropriate amount of Calcein-AM / PI staining working solution was added, and the cells were incubated in a cell culture incubator in the dark for 30 minutes. Finally, images were acquired using a fluorescence microscope. The experimental results showed that PBNPs did not produce significant toxic effects on MC3T3-E1 cells at concentrations of 5 μg / mL and 10 μg / mL. Figure 2 (C in the middle) 2. MC3T3-E1 cells were induced to form an inflammatory microenvironment with 2 μg / ml lipopolysaccharide (LPS) (LPS group), inflammatory MC3T3-E1 cells were treated with 5 μg / ml PBNPs solution (PBNPs (L) group), and inflammatory MC3T3-E1 cells were treated with 10 μg / ml PBNPs solution (PBNPs (H) group). Phalloidin cytoskeleton staining was performed on all four groups of cells. The results showed that the four groups of cells had similar morphology, all exhibiting a "fibroblast-like" flattened and extended shape, but the arrangement of actin microfilaments differed significantly. The LPS group showed a loose, irregularly diffuse arrangement, while the two PBNPs treatment groups showed a dense and regular arrangement, with no significant difference from the Con group. Figure 2 The results above indicate that PBNPs have good biocompatibility (D in the text).

[0021] Example 3. Effects of PBNPs on osteogenic differentiation of MC3T3-E1 cells Osteogenic induction was performed on the four groups of osteoblasts using osteogenic induction medium composed of α-MEM containing 10 mM β-glycerophosphate, 0.25 mM ascorbic acid, and 10 nM dexamethasone. Cells were first seeded in culture dishes and, after attachment, starved for 24 hours in serum-free medium. Then, 2 μg / ml LPS was added to the LPS group, PBNPs (L) group, and PBNPs (H) group, and the medium was replaced with osteogenic medium for 6 hours to obtain the inflammatory cell model. Subsequently, 0 μg / ml, 5 μg / ml, and 10 μg / ml PBNPs were added to the three groups of cells, respectively, with the medium changed every two days and LPS and PBNPs reintroduced for osteogenic induction. The Con group cells were cultured in osteogenic medium throughout without any other treatment. On day 7 of osteogenic induction, early osteogenic differentiation activity was assessed by ALP staining. The results showed that compared with the Con group, ALP activity in the LPS group was significantly weakened, as evidenced by a reduction in blue-purple deposits; while ALP activity in both PBNPs treatment groups was significantly enhanced, with significantly more blue-purple deposits generated than in the LPS group, and levels comparable to those in the Con group. Figure 3 (A) On day 21 of osteogenic induction, the formation of calcium nodules was assessed by alizarin red staining. The results showed that compared with the Con group, the number of red calcium nodules in the LPS group was significantly reduced, indicating that LPS significantly inhibited the mineralization capacity of cells. After intervention with PBNPs, the number of red calcium nodules formed in both treatment groups was greater than that in the control group, suggesting that PBNPs can not only alleviate LPS-induced osteogenic inhibition, but may also have a certain pro-mineralization effect. Figure 3(B in the text). RT-qPCR analysis of osteogenic-related gene expression levels revealed that, compared to the LPS group, both PBNPs treatment groups significantly increased the mRNA expression levels of Bmp2, Runx2, and Dmp1 in MC3T3-E1 cells. However, PBNPs treatment showed a slight inhibitory effect on the Opn gene, with its expression level slightly lower than that in the LPS group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Figure 3 CF in MC3T3-E1 cells). Western blot analysis showed that, compared with the LPS group, treatment with both concentrations of PBNPs significantly upregulated the expression levels of osteogenic-related proteins BMP2, Smad4, RUNX2, DMP1, and OPN in MC3T3-E1 cells (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Figure 3 (GH in the middle).

[0022] Example 4. Osteogenic therapeutic effect of PBNPs in mice with experimental periodontitis 1. A mouse model of experimental periodontitis was established using 5-week-old male C57BL / 6 mice. After successful model establishment, mice were injected locally with 1 mg / ml PBNPs solution into the gingival sulcus, while the control group was injected with an equal volume of PBS solution. One week after administration, local periodontal tissue was collected from the mice. After fixation, decalcification, dehydration, embedding, sectioning, and histological staining, it was found that compared with the control group, the local alveolar bone height was increased and the trabeculae were more dense in both treatment groups. Figure 4 AC in the middle.

[0023] 2. During osteoblast differentiation, BMP2 is an osteogenic induction signal. Its activation directly leads to the upregulation of RUNX2 expression and initiates the transcription and expression of a series of downstream osteogenic-related genes, playing a crucial role in maintaining bone mass and bone repair. In this invention, RT-qPCR and Western Blot experiments showed that BMP2 and RUNX2 were significantly upregulated at both the gene and protein levels in PBNP-treated inflamed MC3T3-E1 cells compared to the control group. Therefore, we preliminarily hypothesize that PBNPs can exert their osteogenic capacity by activating the BMP2 / RUNX2 signaling pathway.

[0024] To verify the above results, immunohistochemical staining was performed on local periodontal tissues. Sections were routinely dewaxed to water, subjected to high-temperature antigen retrieval with EDTA repair solution, and endogenous peroxidase in the tissues was blocked with 3% hydrogen peroxide. After blocking at room temperature for 15 minutes, the tissues were incubated with primary antibody overnight, followed by incubation with secondary antibody at 37°C for 30 minutes. Finally, color development and observation were performed. The results showed that in the Con group mice, positive expression of BMP2, Smad4, RUNX2, OPN, and DMP1 proteins was observed in the periodontal ligament and alveolar bone cells between the left maxillary first and second molars. In the Periodontitis+PBS group, the expression locations of BMP2, Smad4, RUNX2, OPN, and DMP1 proteins in periodontal tissues remained unchanged, but the quantity and intensity of protein expression decreased. Compared with the Periodontitis+PBS group, the Periodontitis+PBNPs treatment group showed increased quantity and intensity of protein expression. Figure 4 DG in Figure 5 This further verifies the above conjecture.

[0025] In summary, this invention has verified the osteogenic capacity of PBNPs in an in vitro inflammatory microenvironment and demonstrated the therapeutic effect of PBNPs on chronic periodontitis in a real in vivo inflammatory environment, and verified the relevant mechanisms.

Claims

1. Application of Prussian blue in the preparation of drugs for treating chronic periodontitis.

2. Application of Prussian blue in the preparation of drugs for the prevention of chronic periodontitis.

3. Application of Prussian blue in the preparation of health care products for the adjuvant treatment of chronic periodontitis.

4. The application according to any one of claims 1-3, characterized in that, The concentration of Prussian blue is 5 μg / mL to 10 μg / mL.

5. The application according to any one of claims 1-3, characterized in that, Preparation method of Prussian blue: Weigh 3g of polyvinylpyrrolidone and 131.7mg of potassium ferricyanide and add them to a beaker. Pour in 40ml of 0.01M hydrochloric acid solution and stir for 30min at 500rpm. Incubate in an oven at 80℃ for 20h. Cool to room temperature, centrifuge to collect the mixture, and wash the precipitate three times with 95% ethanol and then with anhydrous ethanol to obtain Prussian blue.

6. The application according to claim 5, characterized in that, Ethanol:water volume ratio = 2:

1.

7. The application according to any one of claims 1-3, characterized in that, The chemical formula of Prussian blue is .

8. The application according to any one of claims 1-3, characterized in that, The dosage form of the drug or health product is any one of tablets, capsules, granules, powders, or liquid preparations.

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