Application of pyroptosis intervention agents in the preparation of drugs for regulating orthodontic tooth movement

Abstract

The present invention provides the use of a cell pyroptosis intervention agent in the preparation of a drug for regulating orthodontic tooth movement. The present invention establishes an animal tooth movement model and finds that a pyroptosis promoter can significantly accelerate orthodontic tooth movement. Histological staining shows that a pyroptosis promoter can activate osteoclasts in the alveolar bone and accelerate alveolar bone remodeling. It was also observed that a pyroptosis inhibitor can significantly slow down orthodontic tooth movement, and the tooth movement speed is significantly reduced after knocking out the key pyroptosis gene, which indicates that pyroptosis plays an important role in the tooth movement process. It was also found that a pyroptosis promoter can promote the secretion of osteoclast-related factors by acting on the stressed periodontal ligament stem cells, indicating that it can promote osteoclastogenesis, while a pyroptosis inhibitor inhibits the secretion of osteoclast-related factors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of a cell pyroptosis intervention agent in the preparation of a drug for regulating orthodontic tooth movement. Background Art

[0002] Orthodontics is a discipline that studies malocclusion, and mainly uses bone remodeling to move teeth. The principle of bone remodeling is that periodontal ligament stem cells (PDLSCs) secrete RANKL and other substances when they are under pressure in the periodontal ligament to promote osteoclast formation, thereby absorbing the alveolar bone on the pressure side. When PDLSCs on the tension side are subjected to tensile force, they differentiate into osteoblasts and promote new bone formation on the tension side, thus allowing the teeth to reach a new position.

[0003] However, in clinical practice, we often encounter patients whose teeth move slowly or even have difficulty moving. This may be due to low efficiency of bone remodeling and inability to activate osteoclasts, which will prolong the orthodontic treatment cycle and increase the risk of treatment. The extension of the orthodontic cycle will increase the risk of oral diseases such as caries and root resorption. At the same time, since it is difficult to clean teeth during the orthodontic process, long-term orthodontic treatment will also increase the risk of enamel demineralization, gingivitis and periodontitis. Therefore, how to move teeth healthily and quickly is one of the goals of orthodontic treatment.

[0004] At present, the main method for accelerating orthodontic treatment in clinic is cortical bone incision, but this operation requires cutting the cortical bone, which is very traumatic and will cause extra pain to the patient. Therefore, how to accelerate orthodontic tooth movement non-invasively and healthily is an urgent problem to be solved. Summary of the invention

[0005] Technical problem: In order to solve the defects of the prior art, the present invention provides the use of a cell pyroptosis intervention agent in the preparation of a drug for regulating orthodontic tooth movement.

[0006] Technical solution: In a first aspect, the present invention provides the use of a pyroptosis intervention agent in the preparation of a drug for regulating orthodontic tooth movement.

[0007] Furthermore, the pyroptosis intervention agent includes a pyroptosis promoter and a pyroptosis inhibitor; preferably, the pyroptosis promoter is selected from Paris polyphylla saponin VI (PPVI), and the pyroptosis inhibitor is selected from MCC950.

[0008] The pyroptosis promoter can promote orthodontic tooth movement, and the pyroptosis inhibitor can inhibit orthodontic tooth movement.

[0009] The cell pyroptosis intervention agent can affect osteoclast formation and the expression of osteoclast-related factors. Specifically, the pyroptosis promoter can promote osteoclast formation and the expression of osteoclast-related factors, and the pyroptosis inhibitor can inhibit osteoclast formation and the expression of osteoclast-related factors.

[0010] In a second aspect, the present invention provides a composition comprising a pyroptosis intervention agent.

[0011] The third aspect of the present invention provides the use of the composition in preparing a drug for regulating orthodontic tooth movement. The regulation method and regulation mechanism are as described above.

[0012] In a fourth aspect, the present invention provides the use of a pyroptosis intervention agent in the in vitro culture of periodontal ligament stem cells, wherein the pyroptosis intervention agent can affect osteoclastogenesis.

[0013] In a fifth aspect, the present invention provides a culture medium for in vitro culture of periodontal ligament stem cells capable of influencing osteoclastogenesis, wherein the culture medium contains a cell pyroptosis intervention agent.

[0014] In a sixth aspect, the present invention provides an in vitro culture method for periodontal ligament stem cells capable of influencing osteoclastogenesis, the method comprising culturing in a culture medium containing a cell pyroptosis intervention agent.

[0015] Preferably, an in vitro culture method of periodontal ligament stem cells to promote osteoclastogenesis comprises the following steps: culturing with a culture medium containing a pyroptosis promoter. The culture method is: adding a pyroptosis promoter to an α-MEM culture medium, culturing in a 37°C incubator with a 5% CO2 concentration, and culturing after 6 hours, the RANKL / OPG ratio secreted by PDLSCs increases significantly.

[0016] The present invention found that pyroptosis is highly involved in the acute inflammatory environment and can regulate bone remodeling. The pyroptosis promoter Polyphyllin VI (PPVI) has been shown to promote the activation of osteoclasts, suggesting that PPVI may activate osteoclasts during orthodontic tooth development, promote bone remodeling, and thus accelerate tooth movement.

[0017] Beneficial effects: The present invention provides the use of a cell pyroptosis intervention agent in the preparation of a drug for regulating orthodontic tooth movement, especially the use of a pyroptosis promoter to accelerate orthodontic tooth movement. In vivo studies have found that pyroptosis promoters can activate osteoclasts in the alveolar bone, accelerate alveolar bone remodeling, and thus promote orthodontic tooth movement. It was also found that pyroptosis promoters can promote periodontal ligament stem cells to secrete osteoclast induction-related factors, suggesting that they can promote osteoclastogenesis. The present invention also provides a method for culturing periodontal ligament stem cells to promote osteoclastogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To establish the tooth movement model, 7 days after observation, the pyroptosis promoter PPVI can significantly increase the tooth movement distance, while the pyroptosis inhibitor MCC950 can reduce the tooth movement speed.

[0019] Figure 2To establish the tooth movement model, 7 days after observation, the pyroptosis promoter PPVI can significantly increase the level of pyroptosis in the periodontal ligament. At the same time, TRAP staining shows that the pyroptosis promoter can significantly activate osteoclasts; while the pyroptosis inhibitor MCC950 can reduce the level of pyroptosis in the periodontal ligament and reduce the number of osteoclasts.

[0020] Figure 3 To establish a model of in vitro culture of clinical periodontal ligament stem cells after force application. By collecting orthodontic teeth of patients who have applied force during clinical orthodontic diagnostic tooth alignment, periodontal ligament stem cells subjected to force and non-forced periodontal ligament stem cells were extracted in vitro, and their differences were compared, which can better simulate the clinical force application environment.

[0021] Figure 4 In order to establish an in vitro force-applying model for periodontal ligament stem cells to simulate the force-applying environment of clinical periodontal ligament stem cells, periodontal ligament stem cells were extracted and, after the cells adhered to the wall, they were subjected to force using glass plates and steel balls to simulate the force-applying environment in clinical practice.

[0022] Figure 5 The changes in the pyroptosis level after pyroptosis promoters and pyroptosis inhibitors acted on PDLSCs under in vitro pressure. Among them, (a) is the expression changes of pyroptosis-related proteins detected by Western Blot, and it was found that pyroptosis promoters can significantly increase the expression of pyroptosis in PDLSCs, and pyroptosis inhibitors can inhibit the expression of pyroptosis. The right figure is a semi-quantitative statistical graph of Western Blot; (b) is the expression changes of pyroptosis-related genes detected by RT-qPCR, and it was found that pyroptosis promoters can significantly increase the expression of pyroptosis in PDLSCs, while pyroptosis inhibitor MCC950 will inhibit the expression level of pyroptosis;

[0023] Figure 6 The figure shows the changes in the expression of osteoclast induction factor RANKL and osteoprotegerin OPG. (a) Western Blot detected the expression changes of RANKL and OPG, and found that pyroptosis promoters can significantly increase the expression of RANKL / OPG in PDLSCs, and pyroptosis inhibitors can inhibit the expression of RANKL / OPG. The right figure is a semi-quantitative statistical graph of Western Blot; (b) RT-qPCR detected the expression changes of RANKL and OPG, and found that pyroptosis promoters can significantly increase the expression of RANKL / OPG in PDLSCs, and pyroptosis inhibitors can inhibit the expression of RANKL / OPG. DETAILED DESCRIPTION

[0024] Example 1

[0025] In order to verify the effect of pyroptosis promoter PPVI on the speed of tooth movement, we established a mouse tooth movement model.

[0026] In the experimental group, a spring was tied between the right incisor and the first molar of the mouse, so that the spring force was 20-30g. The mice were intraperitoneally injected with 0.9% NaCl, 5mg / kg PPVI, and 20mg / kg MCC950 every other day. After 7 days, the mouse jaws were fixed and then subjected to micro-CT scanning.

[0027] The results show:

[0028] Orthodontic force promotes tooth movement. The pyroptosis promoter PPVI can significantly increase the distance of tooth movement, while the pyroptosis inhibitor MCC950 can reduce the speed of tooth movement. Figure 1 ;

[0029] in conclusion:

[0030] Pyroptosis promoters can promote orthodontic tooth movement.

[0031] Example 2

[0032] In order to verify that pyroptosis promoters promote the level of pyroptosis and osteoclast formation in the periodontal ligament, immunohistochemical staining was used to observe the expression of pyroptosis-related proteins in the periodontal ligament, and TRAP staining was used to observe the morphology and number of osteoclasts. The specific experimental method of immunohistochemistry was as follows: the mouse alveolar bone was embedded and sliced ​​in paraffin, dewaxed with xylene and gradient alcohol, and then antigen repaired with proteinase K and trypsin; after adding 3% hydrogen peroxide, it was blocked with fetal bovine serum, and the primary antibody was incubated at 4°C overnight. The next day, the secondary antibody was added and incubated at room temperature for 30 minutes, and DAB was used for color development, and then counterstained with hematoxylin and sealed with gum. The experimental method of TRAP staining was as follows: the mouse alveolar bone was sliced, dewaxed with xylene and gradient alcohol, and then fixed with TRAP fixative, stained with Solebold TRAP incubation solution for 60 minutes, counterstained with methyl green, and sealed.

[0033] The results show:

[0034] Pyroptosis promoters can significantly increase the expression of pyroptosis-related proteins in the periodontal ligament, while pyroptosis inhibitors can reduce the expression of pyroptosis-related proteins in the periodontal ligament. TRAP staining showed that pyroptosis promoters can increase the number of osteoclasts in the periodontal ligament, and their morphology is more typical, while pyroptosis inhibitors can reduce the number of osteoclasts. Figure 2 .

[0035] in conclusion:

[0036] Pyroptosis promoters increase the level of pyroptosis in the periodontal ligament and increase the number of osteoclasts.

[0037] Example 3

[0038] In order to simulate the in vitro culture environment of periodontal ligament stem cells after clinical stress, a clinical stress-applied periodontal ligament stem cell model was established. By collecting orthodontic teeth of patients who had undergone stress during clinical orthodontic diagnostic tooth alignment, the periodontal ligament stem cells of stressed teeth and non-stressed teeth were extracted in vitro, and subsequent experiments were conducted when they were cultured to the second generation to compare the differences in the levels of pyroptosis of stressed and non-stressed cells in the same patient. The clinical stress-applied periodontal ligament stem cell model can better simulate the clinical stress-applied environment. Figure 3 .

[0039] in conclusion:

[0040] The orthodontic teeth of patients who have undergone orthodontic diagnostic tooth alignment have been stressed. The stressed periodontal ligament stem cells and non-stressed periodontal ligament stem cells can be extracted in vitro for culture and testing to simulate the periodontal ligament stem cells in the stressed environment in vivo.

[0041] Example 4

[0042] In order to simulate the state of periodontal ligament stem cells in the in vivo force environment, an in vitro force model for periodontal ligament stem cells was established. The premolars of orthodontic patients that needed to be extracted for treatment were collected, and 1 / 3 of the periodontal ligament in the root was scraped and digested with collagenase I to extract periodontal ligament stem cells. The cells were passaged and plated in a six-well plate at the fourth generation. After the cells grew to 80%, a glass plate was placed in the well plate, and a 15ml centrifuge tube cap filled with steel balls was added to the glass plate. In this way, force was applied to periodontal ligament stem cells to simulate the force environment in clinical practice. Figure 4 ;

[0043] in conclusion:

[0044] By extracting periodontal ligament stem cells, applying force to the cells with glass plates and steel balls after they adhere to the wall, the force environment in clinical practice can be simulated.

[0045] Example 5

[0046] In order to verify the promoting effect of pyroptosis promoter PPVI on pyroptosis of periodontal ligament stem cells, we observed the pyroptosis level by applying force to periodontal ligament stem cells in vitro.

[0047] Previous experiments have found that applying 1.5g / cm 2 The pyroptosis level increased most significantly after 6 hours of force action. In this experiment, 1.5 g / cm 2 The cells were treated with force for 6 hours, and the pyroptosis promoter PPVI (4 μM) and the pyroptosis inhibitor MCC950 (10 μM) were added 18 hours in advance. After sampling, the pyroptosis level was detected by Western Blot and RT-qPCR.

[0048] The results show:

[0049] Pyroptosis promoters can significantly increase the expression of pyroptosis-related proteins and genes, while pyroptosis inhibitors can inhibit the expression of pyroptosis. Figure 5 a and Figure 5 b.

[0050] in conclusion:

[0051] Pyroptosis promoters can significantly increase the expression of pyroptosis-related proteins and genes in PDLSCs.

[0052] Example 6

[0053] To verify the effect of pyroptosis promoters on PDLSCs-induced osteoclastogenesis, pressurized PDLSCs were treated with pyroptosis promoters and inhibitors.

[0054] In this experiment, 1.5g / cm 2 The cells were exposed to force for 6 hours, and the pyroptosis promoter PPVI (4 μM) and the pyroptosis inhibitor MCC950 (10 μM) were added 18 hours in advance. After sampling, the expressions of osteoclast-related factors RANKL and OPG were detected by Western Blot and RT-qPCR.

[0055] The results showed that pyroptosis promoters could increase the protein and gene expression of RANKL / OPG, while pyroptosis inhibitors could reduce the protein and gene expression of RANKL / OPG. Figure 6 a and Figure 6 b.

[0056] Conclusion: Pyroptosis promoters can increase the protein and gene expressions of RANKL / OPG, which contributes to the formation of osteoclasts.

Claims

1. Application of Paris polyphylla saponin VI in the preparation of drugs for promoting orthodontic tooth movement.

2. The use according to claim 1, characterized in that: The Paris polyphylla saponin VI can affect the formation of osteoclasts and the expression of osteoclast-related factors.

3. Use of a composition comprising Paris polyphylla saponin VI in the preparation of a drug for promoting orthodontic tooth movement.

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