Application of glycoursodeoxycholic acid in prevention and treatment of periodontitis

Abstract

The invention discloses an application of glycine ursodeoxycholic acid in prevention and treatment of periodontitis. Experiments such as RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) and the like on a cellular level prove that the GUDCA can inhibit inflammatory reaction of bone marrow-derived macrophages; meanwhile, TRAP dyeing, RT-qPCR and other experiments prove that the GUDCA can inhibit osteoclastic differentiation of the BMDM. The traditional Chinese medicine composition can inhibit macrophage inflammatory response and osteoclast differentiation, and inhibit mouse periodontitis inflammatory response and bone resorption in vivo, so that occurrence and development of periodontitis are prevented and treated, and the traditional Chinese medicine composition can become a novel medicine for clinically preventing and treating periodontitis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, relates to a new application of glycoursodeoxycholic acid, and in particular to an application of glycoursodeoxycholic acid in preventing and treating periodontitis. Background Art

[0002] Periodontitis is a chronic inflammatory disease caused by bacteria that occurs in periodontal supporting tissues, including the gums, periodontal ligament, alveolar bone, and cementum. As the sixth most common disease in the world, it is mainly manifested by alveolar bone loss, which eventually leads to loose teeth and loss of chewing function. In addition, more and more studies have confirmed that periodontitis can participate in the occurrence and development of a variety of systemic diseases through metabolic pathways and ectopic bacterial colonization, such as atherosclerosis, chronic gastritis and gastric ulcers, aspiration pneumonia, systemic lupus erythematosus, rheumatoid arthritis, diabetes, obesity, etc. Given the severity and danger of periodontitis, it is of great significance to conduct research on the prevention and treatment of periodontitis.

[0003] The pathogenesis of periodontitis is very complex. Local inflammatory response and osteoclast differentiation are important pathological manifestations of periodontitis. Targeted inhibition of inflammatory response and osteoclast differentiation has become the focus of research on the prevention and treatment of periodontitis. Commonly used auxiliary drugs in clinical practice include antibiotics and non-steroidal anti-inflammatory drugs, and long-term use will produce adverse reactions such as drug resistance in the human body. Therefore, further research is needed to seek new human metabolites to prevent and treat periodontitis.

[0004] Glycinursodeoxycholic acid (GUDCA) is a secondary bile acid, which is formed by the catalytic binding of ursodeoxycholic acid (UDCA) with glycine. It has anti-apoptotic, antioxidant and anti-inflammatory properties. Previous studies have shown that GUDCA can effectively improve obesity, atherosclerosis and other related diseases; however, there are no reports on whether GUDCA can inhibit inflammation and prevent and treat periodontitis. At the same time, whether GUDCA can inhibit osteoclast differentiation and alleviate alveolar bone resorption in periodontitis has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an application of glycoursodeoxycholic acid in preventing and treating periodontitis in view of the deficiencies of the prior art.

[0006] The objective of the present invention is achieved through the following technical scheme: application of glycoursodeoxycholic acid in preventing and treating periodontitis.

[0007] Furthermore, the glycoursodeoxycholic acid prevents and treats periodontitis by inhibiting macrophage inflammation and osteoclast differentiation.

[0008] Furthermore, the glycoursodeoxycholic acid is used to reduce the inflammatory response of periodontitis.

[0009] Furthermore, the glycoursodeoxycholic acid is used to reduce alveolar bone resorption.

[0010] The invention also provides the use of glycoursodeoxycholic acid in preparing medicine for preventing and treating periodontitis.

[0011] Furthermore, the drug consists of glycoursodeoxycholic acid as an active ingredient and a pharmaceutically acceptable carrier and / or additive.

[0012] Furthermore, the dosage form of the drug is a pharmaceutically acceptable oral dosage form, dressing dosage form or injection dosage form.

[0013] Furthermore, the oral dosage forms include granules, tablets, capsules, and liquids.

[0014] Furthermore, the effective dose of the drug is 50 mg / kg body weight.

[0015] The invention also provides a medicine for preventing and treating periodontitis, which contains glycoursodeoxycholic acid as an active ingredient.

[0016] The beneficial effect of the present invention is that the present application provides a new application of glycoursodeoxycholic acid, that is, the application of glycoursodeoxycholic acid in preventing and treating periodontitis, providing a new idea for the prevention or treatment of periodontitis. Among them, glycoursodeoxycholic acid prevents and treats periodontitis mainly through the following two aspects: first, glycoursodeoxycholic acid inhibits lipopolysaccharide-induced macrophage inflammation, inhibits the expression of IL-6 and IL-1β, and slows down the inflammatory response of periodontitis; second, glycoursodeoxycholic acid can inhibit osteoclast differentiation, and in vitro experiments have verified that glycoursodeoxycholic acid inhibits periodontitis inflammation and alveolar bone resorption, thereby preventing and treating the occurrence and development of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0018] Figure 1 The statistical results of the effect of GUDCA on cell activity, ns, no statistical significance;

[0019] Figure 2 The results of IL-6 and IL1β PCR and statistical analysis were shown in the co-culture of macrophages with GUDCA and lipopolysaccharide (LPS) for 6 h, **P<0.01, ***P<0.001;

[0020] Figure 3The results of tartrate acid phosphatase (TRAP) staining show that GUDCA inhibits RANKL-induced osteoclast differentiation;

[0021] Figure 4 PCR results and statistical analysis of nfatc1, ctsk, and mmp9 for GUDCA inhibiting RANKL-induced osteoclast differentiation, **P<0.01, ***P<0.001;

[0022] Figure 5 A in the figure is the sagittal micro-CT image of the in vivo experiment. Figure 5 B in the figure is the statistical chart of alveolar bone density. Figure 5 C is the ratio of alveolar bone volume to total volume (BV / TV). DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the features of the following embodiments and implementations can be combined with each other.

[0024] Example 1: GUDCA is not cytotoxic

[0025] S1. Isolation of mouse bone marrow-derived macrophages (BMDM)

[0026] Wild-type (WT) mice of 6-8 weeks were killed by CO2, and the femur and tibia were stripped. After washing with sterile PBS containing gentamicin, the epiphyseal end of the bone was cut off, and α-MEM medium was sucked with a 5ml syringe and inserted into the bone marrow cavity. The bone marrow was blown out until the bone tissue turned white. The flushed cell suspension was collected into a centrifuge tube, and red blood cell lysis solution was added. After centrifugation, the cells were cultured with α-MEM complete medium containing 50ng / ml colony stimulating factor and placed in a constant temperature incubator at 37℃ and 5% CO2 for 7 days. The medium was changed every other day. The adherent cells visible under an inverted microscope are BMDMs.

[0027] The effect of GUDCA on the proliferation of BMDM cells was tested using CCK8 assay. Figure 1 As shown in the figure, at 72h, GUDCA had no inhibitory effect on BMDM cells at a concentration of 0-100 μmol, and ns was not statistically significant, indicating that GUDCA was not cytotoxic.

[0028] Example 2: GUDCA inhibits macrophage inflammatory response

[0029] Experimental groups:

[0030] (1) Control group: BMDM cells were cultured in normal culture medium

[0031] (2) LPS group: BMDM cells were treated with LPS (1 μg / ml) for 6 h

[0032] (3) GUDCA group: BMDM cells were cultured in normal culture medium containing GUDCA (50 μmol)

[0033] (4) LPS+GUDCA group: BMDM cells, LPS (1 μg / ml), and GUDCA (50 μmol) were cultured simultaneously for 6 h.

[0034] The expression of inflammatory genes was detected by RT-qPCR. Figure 2 It was shown that under LPS stimulation, IL6 and IL1β inflammatory genes were significantly upregulated, and GUDCA reduced the expression of IL6 and IL1β inflammatory genes. The upregulation of macrophage inflammatory response caused by LPS was specifically manifested by a significant increase in inflammatory-related genes, indicating that the inflammatory model was successfully established. In addition, GUDCA alleviated LPS-induced macrophage inflammation, indicating the anti-inflammatory effect of GUDCA.

[0035] Example 3: GUDCA inhibits osteoclast differentiation of macrophages

[0036] Experimental groups:

[0037] 1) Control group: BMDM cells were cultured in normal culture medium for 7 days, and the medium was changed every other day.

[0038] 2) RANKL group: BMDM cells were induced with 50 ng / mL RANKL for 7 days, and the medium was changed every other day.

[0039] 3) GUDCA group: BMDM cells were cultured in normal culture medium containing GUDCA (50 μmol) for 7 days, and the medium was changed every other day.

[0040] 4) RANKL+GUDCA group: BMDM were cultured with culture medium containing GUDCA (50 μmol) and 50 ng / mL RANKL for 7 days, and the medium was changed every other day.

[0041] The expression of osteoclast-related genes was detected by RT-qPCR, and osteoclast differentiation was detected by TRAP staining. Figure 3 As shown in the figure, the RANKL component alone differentiated a large number of large multinucleated osteoclasts. After GUDCA intervention, the number and size of osteoclasts were reduced. Figure 4 The results showed that under RANKL stimulation, nfatc1, ctsk and mmp9 osteoclast genes were significantly upregulated, and GUDCA reduced the expression of osteoclast differentiation genes. The upregulation of macrophage osteoclast differentiation response caused by RANKL was specifically manifested as an increase in osteoclasts, indicating that the osteoclast differentiation model was successfully established. In addition, GUDCA alleviated RANKL-induced osteoclast differentiation, indicating the inhibitory effect of GUDCA on osteoclast differentiation.

[0042] Example 4. Preparation of animal model of periodontitis

[0043] 6-8 weeks old SPF grade male C57BL / 6J experimental mice were purchased from Beijing Weitonglihua Experimental Animal Center and then housed in the SPF grade animal room barrier of Zhejiang University. The establishment of the periodontitis animal model has been approved by the ethics committee of the Zhejiang University Animal Center.

[0044] Male mice aged 6-8 weeks were first fed adaptively for one week, and periodontitis models were established on the 8th day. Atropine, 0.04 mg / kg, was first used intramuscularly to reduce tracheal secretions, and then anesthesia was performed with Shutai 50, 50 mg / kg intramuscularly. After anesthesia, the mice were fixed, the mouth was opened, and the surgical area was cleaned and disinfected with 75% alcohol. The second molars on both sides of the mice were ligated with 5-0 silk thread, and penicillin was used after surgery to prevent infection. Check whether the silk thread of the mouse is in place every other day. If it is not in place, re-ligate it and keep the silk thread in place for 14 days. On the 22nd day, the mice were killed and alveolar bone samples were collected. 18 C57BL / 6J mice were randomly divided into 3 groups, with 6 mice in each group. The groups were as follows:

[0045] C: Normal control group.

[0046] P: Ligature-induced periodontitis group.

[0047] P+GUDCA (50mg / kg): ligature-induced periodontitis + GUDCA (50mg / kg) drug intervention group.

[0048] Starting from the 8th day of modeling of periodontitis mice, the GUDCA drug intervention group was given drug intervention once a day until death.

[0049] Example 5: GUDCA relieves alveolar bone resorption in periodontitis

[0050] The maxilla of the mouse was quickly obtained, and most of the muscle tissue was separated from the mid-palatal suture. The maxilla was placed in 4% paraformaldehyde with a volume 10 times that of the sample, fixed for 48 hours, and then rinsed with running water overnight. The alveolar bone resorption was measured by Micro-CT after the soft tissue attached to the alveolar bone was removed from the right maxilla.

[0051] The results of mirco-CT showed Figure 5 In the P group, the distance between the cementum-enamel junction and the enamel-glandular junction significantly increased, and the bone density decreased significantly. Compared with the P group, the distance between the cementum-enamel junction and the enamel-glandular junction in the P+GUDCA group was significantly reduced, and the bone density increased. The results showed that GUDCA treatment can alleviate alveolar bone resorption in mice with periodontitis.

[0052] The above embodiments are only used to illustrate the design ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, any equivalent changes or modifications made based on the principles and design ideas disclosed by the present invention are within the protection scope of the present invention.

Claims

1. Application of glycoursodeoxycholic acid in the prevention and treatment of periodontitis.

2. The use according to claim 1, characterized in that: The glycoursodeoxycholic acid prevents and treats periodontitis by inhibiting macrophage inflammation and osteoclast differentiation.

3. The use according to claim 1, characterized in that: The glycoursodeoxycholic acid is used to reduce the inflammatory response of periodontitis.

4. The use according to claim 1, characterized in that: The glycoursodeoxycholic acid is used to reduce alveolar bone resorption.

5. Application of glycoursodeoxycholic acid in the preparation of drugs for the prevention and treatment of periodontitis.

6. The use according to claim 5, characterized in that: The medicine consists of glycoursodeoxycholic acid as an active ingredient and a pharmaceutically acceptable carrier and / or additive.

7. The use according to claim 5, characterized in that: The dosage form of the drug is a pharmaceutically acceptable oral dosage form, dressing dosage form or injection dosage form.

8. The use according to claim 7, characterized in that: The oral dosage forms include granules, tablets, capsules, and liquids.

9. The use according to claim 5, characterized in that: The effective dose of the drug is 50 mg / kg body weight.

10. A drug for preventing and treating periodontitis, characterized in that: Contains the active ingredient glycoursodeoxycholic acid.

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