Methods, formulations, and uses for promoting exosome endocytosis, bone metabolism, and tooth movement.

Abstract

This invention provides a method, formulation, and use for promoting exosome endocytosis, bone metabolism, and tooth movement. Experiments have demonstrated that ANXAⅢ protein can promote exosome endocytosis, promote osteocyte formation, and tooth movement, thereby more effectively promoting orthodontic tooth movement.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method, formulation, and use for promoting exocytosis, bone metabolism, and tooth movement. Background Technology

[0002] ANXAⅢ protein (also known as lipocortin 3 or placental anticoagulant protein 3, PAP-Ⅲ) is a member of the annexin family. Current research mainly focuses on its role in tumors, and ANXAⅢ protein can be used as a biomarker for lung cancer (patent application number: CN201811065192.9). It is closely related to liver cancer (Guo, Chunmei et al. "33-kDa ANXA3 isoform contributes to hepatocarcinogenesis via modulating ERK, PI3K / Akt-HIF and intrinsic apoptosis pathways." Journal of Advanced Research vol. 30 85-102. 8 Nov. 2020) and pancreatic cancer (Wan, Xiaohui et al. "microRNA-382 suppresses the progression of pancreatic cancer through the PI3K / Akt signaling pathway by inhibition of Anxa3." American Journal of Physiology. Gastrointestinal and Liver Physiology vol. 319, 3 (2020)). However, no specific applications of it in other areas have been found yet.

[0003] Exosomes are nanovesicles with a diameter of 30–150 nm secreted by various cell types (Johnstone, 1992). They play a crucial role in intercellular communication by transferring bioactive molecules (such as lipids, proteins, and genetic information) between cells (Tkach and Thery, 2016). Regulation of exosome biogenesis is essential for intercellular communication (Zhang et al., 2015). Various microenvironmental factors, including hypoxia, inflammatory cytokines, and infectious agents, can promote exosome biogenesis (Colombo et al., 2014).

[0004] Orthodontic tooth movement (OTM) is induced by mechanical forces and facilitated by remodeling of the periodontal ligament (PDL) and alveolar bone. The PDL is a dense fibrous connective tissue between the tooth root and alveolar bone that contributes to bone homeostasis and remodeling under mechanical stimulation. The PDL is exposed to intrinsic mechanical stimuli from mastication and is also subjected to extrinsic mechanical loads, such as occlusal interferences and orthodontic appliances. These forces can be tensile, fluid shear, compressive, and hydrostatic. During this process, the PDL generates various components that modulate osteoblast- and osteoclast-related signaling, thereby remodeling periodontal tissue (Li et al., 2018). Periodontal ligament stem cells (PDLSCs) have been isolated and identified as the major MSCs in the PDL (Huang et al., 2018; Seo et al., 2004). With immunomodulatory functions and the potential to generate cementum / periodontal ligament-like complexes (Seo et al., 2004; Wada et al., 2009), PDLSCs may be sensitive to mechanical stress and play a key role in alveolar bone remodeling during orthodontic tooth movement. During their response to mechanical forces, PDLSCs produce a variety of inflammatory cytokines and chemokines to regulate osteoblast- and osteoclast-related signaling, thereby achieving alveolar bone remodeling (Liu et al., 2017; Zhang et al., 2016). However, whether mechanical forces induce PDLSCs to secrete exosomes to promote alveolar bone remodeling remains to be determined. Furthermore, whether ANXAⅢ protein is associated with exosomes and orthodontic tooth movement (OTM) also requires further investigation. Summary of the Invention

[0005] The purpose of this invention is to provide a method, formulation, and use for promoting exocytosis, bone metabolism, and tooth movement.

[0006] To achieve the above objectives, the present invention provides a method for promoting exosome endocytosis, the method comprising the step of using ANXAIII protein.

[0007] According to some specific embodiments of the present invention, the concentration of the ANXAIII protein is 4-6 μg / mL, which is the concentration used in in vitro experiments.

[0008] According to some specific embodiments of the present invention, the step of using the ANXAIII protein includes applying the ANXAIII protein to osteoclasts RAW264.7.

[0009] The present invention also provides a method for promoting bone metabolism, the method comprising the steps of using ANXAIII protein as defined above.

[0010] According to some specific embodiments of the present invention, the promotion of bone metabolism includes inducing osteoclast formation.

[0011] The present invention also provides a method for promoting tooth movement, the method comprising the steps of using ANXAIII protein as defined above.

[0012] The present invention also provides an formulation that promotes exocytosis, bone metabolism, or tooth movement, wherein the formulation comprises ANXAⅢ protein.

[0013] According to some specific embodiments of the present invention, the dosage form of the preparation is selected from any one of oral liquid, pill, powder, tablet, granule, capsule, decoction and injection.

[0014] According to some specific embodiments of the present invention, the ANXAⅢ protein is an unmodified natural protein.

[0015] The present invention also provides the use of ANXAIII protein in the preparation of formulations that promote exosome endocytosis, bone metabolism, or tooth movement.

[0016] This invention further confirms through experiments with ANXAⅢ protein (MedChemExpress; HY-P7516) that ANXAⅢ protein can promote exocytosis, promote the formation of osteocytes, and orthodontic tooth movement. Attached Figure Description

[0017] Figure 1 This is a fluorescence staining image of ANXAⅢ regulating exocytosis in Example 1 of the present invention;

[0018] Figure 2 This is a diagram showing the relative intensity of red fluorescence in Example 1 of the present invention, which illustrates the regulation of exocytosis by ANXAⅢ.

[0019] Figure 3 This is a TRAP staining image of cells in Example 2 of the present invention that promote osteoclast formation by ANXAⅢ;

[0020] Figure 4 Example 2 of the present invention is a TRAP that promotes osteoclast formation by ANXAⅢ. + Number of osteoclasts;

[0021] Figure 5 This is a TRAP-stained image of a tissue section from Example 2 of the present invention that promotes osteoclast formation using ANXAⅢ.

[0022] Figure 6 Example 2 of the present invention is a TRAP that promotes osteoclast formation by ANXAⅢ. + Number of osteoclasts;

[0023] Figure 7 This is a diagram showing the improvement of orthodontic tooth movement rate by ANXAⅢ in Embodiment 3 of the present invention (gross and CT results);

[0024] Figure 8 The OTM distance for improving the orthodontic tooth movement rate (gross and CT results) in Embodiment 3 of the present invention is ANXAⅢ. Detailed Implementation

[0025] To more clearly understand the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in further detail. It should be understood that the following specific embodiments are merely exemplary, and the technical solution of the present invention is not limited to the specific embodiments listed below.

[0026] The micro-computed tomography (micro-CT) analysis and tartrate-resistant acid phosphatase (TRAP) staining procedures used in the following examples are as follows:

[0027] Micro-CT Analysis

[0028] The maxillae of mice were scanned using a miniature CT system (SkyScan, Bruker, Germany, 860 μA, 55 kV, 12.5 μm pixel size). The radiographs were reconstructed and analyzed using CTan and CTvox software (Bruker Corp).

[0029] Tartrate-resistant acid phosphatase (TRAP) staining

[0030] Dewax the same sections used for histological studies and then perform TRAP staining. Fix cells in 4% paraformaldehyde and wash with PBS. Perform TRAP staining on sections and cells using a leukocyte acid phosphatase kit (387A-1KT; Sigma, USA). Add 0.5 ml of Fast Garnet GBC basal solution and 0.5 ml of sodium nitrite solution to a test tube according to the manufacturer's instructions, gently invert to mix for 30 seconds, and let stand for 2 minutes. Add the diazotized Fast Garnet GBC solution to 45 ml of deionized water at 37°C. Add the following solutions to the deionized water: 0.5 ml of naphthol AS-Bl phosphate solution, 2.0 ml of acetate solution, and 1.0 ml of tartrate solution. Transfer the solution from the beaker to a suitable Coplin jar. Heat the solution in the jar to 37°C in a water bath. Add a slide to the Coplin jar and incubate in a light-protected 37°C water bath for 1 hour. One hour later, the slides were thoroughly rinsed in deionized water and then counterstained for 2 minutes in hematoxylin (Soleb, G1140) solution, Gill No. 3. The slides were then rinsed in tap water for several minutes until blue nuclei were observed. The slides were air-dried and evaluated under a microscope. The mean number of TRAP-positive multinucleated cells (>3 nuclei) per well in the PDL region near the alveolar bone or in RAW264.7 macrophages was [not specified]. 2Number them.

[0031] Example

[0032] Unless otherwise specified, the experimental methods and apparatus used in the following examples and comparative examples are conventional methods and apparatus.

[0033] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available.

[0034] Example 1

[0035] This embodiment uses periodontal ligament stem cell exosomes as a control (i.e., no treatment) and force-treated (applied via sterile glass slides and stainless steel beads at 1 g / cm²). 2 Periodontal ligament stem cell exosomes were subjected to pressure for 12 hours to increase the expression of ANXAⅢ protein in exosomes. Three types of exosomes were stained red and co-cultured with macrophages (RAW264.7, culture conditions: RMPI 1640 medium, fetal bovine serum concentration 15%, culture temperature: 37℃, CO2 concentration: 5%) for 3 hours (exosome concentration 25 μg / ml). The phagocytosis of different exosomes by macrophages was observed under a 40x laser confocal microscope. The results showed that ANXAⅢ protein can regulate the endocytosis of exosomes. Figure 1 and Figure 2 It was found that increased ANXAⅢ protein levels led to increased exosome phagocytosis, while knockdown of ANXAⅢ protein using siRNA reduced exosome phagocytosis. The results were statistically significant (*P<0.05, **P<0.01, ***P<0.001). This indicates that ANXAⅢ protein can promote exosome endocytosis.

[0036] Example 2

[0037] This embodiment describes the observation of osteoclast formation by directly adding 5 μg / ml of ANXAⅢ protein (MedChemExpress; HY-P7516) to macrophage (RAW264.7) culture in vitro. This was verified in mice (female, 8-10 weeks old, C57BL / 6 mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Mice were divided into a control group (no treatment on the right teeth), a force-only group (30g orthodontic force applied between the right incisors and first molars for 7 days, followed by a local injection of 200 μL PBS solution), and a force-only group combined with local injection of ANXAⅢ-containing exosomes (force-only plus a local injection of 100 μg of ANXAⅢ protein-containing exosomes dissolved in 200 μL PBS into the maxillary first molar region). After 7 days, local jawbone tissue from the maxillary first molar region was collected, and osteoclast formation was observed through in vitro cell experiments. Figure 3 and Figure 4 As shown in the image, dark red represents osteoclasts, which, through... Figure 3 and Figure 4 It can be seen that the addition of ANXAⅢ protein increased osteoclast formation, and this was statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0038] Further, the local jawbone tissue in the first molar region of each group was decalcified for 2 weeks, sectioned, and stained with TRAP. Figure 5 and Figure 6 As can be seen from TRAP staining of mouse tissue sections, compared with the control group, more osteoclasts were formed in the force-applied group, and the group with exosomes containing ANXAⅢ protein added at the same time as force application showed the highest osteoclast formation, which was statistically significant (*P<0.05, **P<0.01, ***P<0.001). Here, AB represents alveolar bone; R represents root; and PDL represents periodontal ligament.

[0039] Example 3

[0040] In this embodiment, a mouse orthodontic tooth movement model was used. The experiment was divided into three groups: a control group (mouse right teeth were not treated), a simple force application group (30g orthodontic force was applied between the right incisor and first molar of the mouse for 7 days, followed by a local injection of 200 μL PBS solution), and a force application combined with local injection of exosomes containing ANXAⅢ (i.e., simple force application + local injection of 100 μg of exosomes containing ANXAⅢ protein dissolved in 200 μL PBS in the maxillary first molar region of the mouse). After 7 days of experiment, the maxilla of each group of mice was scanned using a mini CT system to observe the differences in tooth movement distance among the different groups.

[0041] pass Figure 7 and Figure 8A mouse orthodontic tooth movement model showed that the control group had no tooth movement, while the force-applied group exhibited significant tooth movement and interdental gaps. The group receiving both orthodontic force and local injection of exosomes containing ANXAⅢ showed greater tooth movement and larger interdental distances than the force-applied group, which was statistically significant (*P<0.05, **P<0.01, ***P<0.001). Figure 7 In the diagram, M1 represents the first molar, M2 represents the second molar, and the blue arrows indicate the distance of orthodontic tooth movement (OTM). The results show that local injection of exosomes containing ANXAⅢ protein during force application can accelerate orthodontic tooth movement.

[0042] The above description is merely a preferred embodiment of the present invention. It should be noted that, without departing from the spirit and essence of the present invention, those skilled in the art can make various modifications, combinations, alterations, or substitutions to the details and features of the present invention. These modifications, combinations, alterations, or substitutions should also be understood to be included within the scope of protection claimed by the present invention.

Claims

1. Use of ANXA III protein in the preparation of a preparation for promoting orthodontic tooth movement.

2. Use according to claim 1, the dosage form of the preparation being selected from any one of oral liquid, pill, powder, tablet, granule, capsule, decoction and injection.

3. Use according to claim 1 or 2, the ANXA III protein being a natural protein without modification.

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