Application of cimiprevir in preparation of bacteriostatic agent and medicine for preventing and treating periodontitis

Abstract

The invention discloses application of cimiprevir in preparation of a bacteriostatic agent and a medicine for preventing and treating periodontitis, and relates to the technical field of disease treatment by using cimiprevir. Studies find that cimiprevir can be combined with EF-Tu protein to inhibit adhesion of the EF-Tu protein to an interface, further inhibit adhesion of bacteria (especially oral streptococcus) to an oral interface and formation of a plaque biofilm, regulate proliferation and growth of the bacteria and play an effective bacteriostatic role, and cimiprevir can be used for preparing a bacteriostatic agent or a preparation for preventing and treating periodontitis; the composition can effectively reduce periodontal tissue damage, reduce alveolar bone absorption and reduce osteoclast infiltration, and is used for preparing a preparation for protecting periodontal tissue or alveolar bone.

Description

Technical Field

[0001] The invention relates to the technical field of disease treatment using simeprevir, and in particular to application of simeprevir in preparing antibacterial agents and drugs for preventing and treating periodontitis. Background Art

[0002] Periodontitis is a common chronic infectious inflammatory oral disease that affects the health of hundreds of millions of people worldwide. The proliferation of pathogenic microorganisms located in the gingival and periodontal tissues produces harmful metabolites, which is the direct cause of periodontitis. The early adhesion and colonization of oral Streptococcus oralis (S. oralis) on the tooth surface leads to the formation of subgingival plaque biofilm, which further leads to the body's immune response and inflammatory response, causing destruction and loss of periodontal tissues, and ultimately causing tooth loss, which has a serious impact on oral functions such as chewing and pronunciation. Periodontitis is also closely related to a series of systemic diseases, such as cardiovascular disease, cancer, diabetes and Alzheimer's disease. Subgingival plaque biofilm is considered to be the initiating factor of periodontitis.

[0003] At present, mechanical treatments such as supragingival scaling, subgingival scaling and root planing are the main means of treating periodontitis. However, in some areas that are difficult for instruments to reach, mechanical treatment methods cannot completely eliminate periodontal pathogens, and persistent chronic inflammation will still lead to the destruction of periodontal supporting tissues, playing a key role in the development of periodontitis. Therefore, anti-inflammatory drugs have become an important auxiliary means of treating periodontitis. Currently, the only drugs used for periodontitis are antibiotics, but antibiotics can only relieve acute inflammation and cannot protect alveolar bone and periodontal tissues. Summary of the invention

[0004] The present invention provides the use of simeprevir in the preparation of antibacterial agents and drugs for preventing and treating periodontitis, which is specifically achieved through the following technologies.

[0005] The first aspect of the present invention provides the use of simeprevir in preparing a preparation for preventing and treating periodontitis.

[0006] Furthermore, the periodontitis is caused by infection with oral Streptococcus oralis (S. oralis).

[0007] Furthermore, the preparation is used to inhibit alveolar bone resorption.

[0008] Furthermore, the preparation is used to inhibit the infiltration of osteoclasts.

[0009] The second aspect of the present invention provides the use of simeprevir in the preparation of an antibacterial agent, wherein the antibacterial agent is used to inhibit oral Streptococcus oralis (S. oralis).

[0010] The third aspect of the present invention provides a preparation for preventing and treating periodontitis, or for protecting periodontal tissue or alveolar bone; the preparation contains simeprevir as an ingredient.

[0011] Simeprevir is a hepatitis C virus NS3 / 4A protease inhibitor, currently mainly used for the treatment of chronic hepatitis in patients infected with HCV1 or HCV4. Elongation factor Tu (EF-Tu) is a protein translation elongation factor that plays an important role in the translation of proteins. EF-Tu can also be secreted to the surface of bacterial membranes to act as a moonlight protein, bind to cell surface fibronectin to promote bacterial adhesion and invasion of host cells, and activate immune responses. As a membrane surface protein of oral Streptococcus oralis (S. oralis), EF-Tu protein plays an important role in bacterial adhesion and immune activation.

[0012] The present invention discovered for the first time that simeprevir has the potential to bind to EF-Tu protein, and further verified through in vitro experiments that simeprevir can inhibit the adhesion of EF-Tu protein to the interface, inhibit the adhesion of bacteria to the oral interface and the formation of pathogenic bacteria biofilm, reduce periodontal tissue damage, reduce alveolar bone absorption, reduce osteoclast infiltration, and can also reduce inflammatory response, effectively preventing or treating periodontitis. Therefore, simeprevir can be used to prepare preparations for preventing and treating periodontitis, or antibacterial agents, or for preparing preparations for protecting periodontal tissue or alveolar bone.

[0013] The various preparations provided by the present invention have simeprevir as the main active ingredient. In addition, according to the needs of various types of preparations, pharmaceutically acceptable excipients such as excipients, binders, disintegrants, sustained-release agents, coating materials, etc. may also be included.

[0014] The various types of preparations provided by the present invention include, but are not limited to, tablets, capsules, caplets, gels, creams, films, and the like.

[0015] Compared with the prior art, the benefits of the present invention are as follows: for the first time, it is found that simeprevir can bind to EF-Tu protein, inhibit the adhesion of EF-Tu protein to the interface, thereby inhibiting the adhesion of bacteria (especially oral streptococci) to the oral interface and the formation of plaque biofilm, regulating the proliferation and growth of bacteria, and exerting an effective antibacterial effect, and can be used to prepare antibacterial agents or preparations for preventing and treating periodontitis; it can also effectively reduce the destruction of periodontal tissue, reduce the absorption of alveolar bone, and reduce the infiltration of osteoclasts, and can be used to prepare preparations for protecting periodontal tissue or alveolar bone. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Example 1: Effect of simeprevir on the adhesion ability of purified EF-Tu protein to the interface in vitro.

[0018] 1. Extraction and purification of EF-Tu protein

[0019] (1) The tuf gene was introduced into the pET28a plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) through homologous recombination, and then the pET28a-tuf recombinant plasmid was introduced into BL21 (DE3) competent Escherichia coli cells.

[0020] (2) Use LB solid culture medium with kanamycin to identify E. coli that have successfully introduced the pET28a plasmid; pick a single colony and culture it in LB liquid culture medium with kanamycin at 37°C until its absorbance OD 600 Reach 0.6-0.8.

[0021] (3) At 18°C, 0.5 mM IPTG was added to BL21 (DE3) E. coli to induce overexpression of EF-Tu protein.

[0022] (4) After 16 h, collect BL21 (DE3) Escherichia coli by centrifugation, add buffer (containing 20 mM Tris-HCl, 100 mM NaCl, 1 mM DNase I, 1 mM PMSF and 2 mM MgCl2), and mix thoroughly on ice; disrupt the cells by ultrasound, and centrifuge the bacterial cytoplasmic lysate at 4°C, 12,000 g for 30 min to obtain the protein supernatant.

[0023] (5) The obtained supernatant was incubated with Ni-NTA resin and eluted with buffer A (containing 20 mM Tris-HCl, 100 mM NaCl, 20 mM imidazole, pH = 7.5) and buffer B (containing 20 mM Tris-HCl, 100 mM NaCl, 100 mM imidazole, pH = 7.5) in sequence. The final EF-Tu protein was eluted with a buffer (containing 20 mM Tris-HCl, 100 mM NaCl, 300 mM imidazole) at pH = 7.5 to obtain purified EF-Tu protein. The protein concentration was determined using a protein quantification kit.

[0024] 2. Simiprevir inhibits the adhesion of EF-Tu protein to the interface

[0025] (1) Dilute the purified EF-Tu protein to a concentration of 4 mg / mL. Add 5-fluorescein isothiocyanate at a final concentration of 1 mg / mL to every 100 μL of protein solution. Incubate at 4°C for 8 h, and then add 50 mM NH4Cl to terminate the reaction.

[0026] (2) The obtained solution was centrifuged at 5000 rpm for 30 min at 4°C using a 10 kDa ultrafiltration tube (Millipore) to remove unbound FITC and obtain the fluorescently bound EF-Tu-FITC protein solution. The experiment was divided into 3 groups, one of which served as the control, and the other two groups were added with 1 μM and 5 μM simeprevir, respectively.

[0027] (3) Pipette 200 μL of the grouped EF-Tu-FITC protein solution onto the coverslip, use a TIRF microscope to take time-lapse photos of the protein's motion trajectory, use MATLAB and Origin to analyze the protein's motion, and calculate the displacement distribution frequency.

[0028] The results are shown in Table 1.

[0029] Table 1 Displacement distribution frequency results of EF-Tu protein

[0030]

[0031] As shown in Table 1, compared with the control group, the movement distance of EF-Tu protein treated with 1 μM or 5 μM simeprevir in the system increased, indicating that the adhesion ability of EF-Tu protein decreased. This further shows that simeprevir can inhibit the adhesion ability of EF-Tu protein to the interface.

[0032] Example 2: Effect of simeprevir on the adhesion ability of oral streptococci to interfaces

[0033] The test steps are as follows:

[0034] 1. Take a suspension of oral Streptococcus oralis (S. oralis, from ATCC, strain number ATCC 35037) cultured overnight at 37°C, centrifuge at 10,000 rpm for 1 min, discard the supernatant, resuspend with sterile PBS and add Dil dye with a final concentration of 5 μM, mix well and stain at 37°C for 30 min, and use PBS to wash away the Dil dye that is not bound to oral Streptococcus.

[0035] 2. Use TSB liquid culture medium to resuspend the stained oral streptococci. The experiment was divided into 3 groups, one of which was used as a control, and the other two groups were added with 1 μM and 5 μM simeprevir, respectively. The resuspended liquid culture medium was added to a laser confocal dish, and a Nikon Ti-E fluorescence microscope was used to continuously shoot for 10 min. The shooting interval of each photo was set to 0.22 s. MATALB was used to analyze the movement trajectory of oral streptococci and calculate the displacement distribution frequency.

[0036] The test results are shown in Table 2.

[0037] Table 2 Displacement distribution frequency results of oral streptococci

[0038]

[0039] As shown in Table 2, compared with the control group, the movement distance of oral streptococci treated with 1 μM or 5 μM simeprevir in the system increased, indicating that the adhesion ability of oral streptococci decreased. This further shows that simeprevir can inhibit the adhesion ability of oral streptococci to the interface.

[0040] Example 3: Effect of simeprevir on oral streptococcal biofilm formation

[0041] The test process is as follows:

[0042] 1. Use fresh TSB liquid medium to dilute the oral Streptococcus oralis (S. oralis, from ATCC, strain number ATCC 35037) cultured at 37°C overnight to obtain the absorbance (OD 600 ) was 0.1 bacterial solution. A control group (no additional reagents were added) and a simeprevir treatment group (1 μM and 5 μM simeprevir were added and mixed thoroughly) were set up.

[0043] 2. Pipette 1 mL of bacterial solution into a 24-well plate and culture in a 37°C incubator for 1 h, 2 h, 4 h, 8 h, 12 h and 24 h.

[0044] 3. After the incubation, fix with 2.5% glutaraldehyde at room temperature for 2 h. Gently discard the supernatant, add 500 μL of 10% crystal violet staining solution and let stand at room temperature for 15 min. Discard the supernatant, decolorize with 500 μL of 95% ethanol for 15 min, and measure the absorbance (OD 595 ) to quantify the biofilm content.

[0045] The results are shown in Table 3.

[0046] Table 3 Absorbance quantitative results of oral streptococcus biofilm cultured at different times

[0047] As shown in Table 3, compared with the control group, the absorbance of oral streptococci treated with 1 μM or 5 μM simeprevir in the system decreased, indicating that biofilm formation was reduced. This further indicates that simeprevir can inhibit the biofilm formation of oral streptococci.

[0048] Example 4: Effect of simeprevir on the proliferation and growth of oral streptococci

[0049] The test process is as follows:

[0050] 1. Use fresh TSB liquid medium to dilute the oral Streptococcus oralis (S. oralis, from ATCC, strain number ATCC 35037) cultured overnight at 37°C to obtain the absorbance (OD 600 ) was 0.1 bacterial solution. The experiment set up a control group (no additional reagents were added) and a simeprevir treatment group (1 μM and 5 μM simeprevir were added and mixed thoroughly), and cultured at 37°C and 220 rpm under shaking conditions;

[0051] 2. At 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h, 100 μL of bacterial solution was added to a 96-well plate and the absorbance (OD 600 ) to quantify the effect of simeprevir on the growth of oral streptococci.

[0052] The results are shown in Table 4.

[0053] Table 4 Absorbance quantitative results of oral streptococci cultured at different times

[0054] As shown in Table 4, compared with the control group, the absorbance of oral Streptococcus treated with 5 μM simeprevir was significantly reduced, indicating that 5 μM simeprevir significantly inhibited the proliferation and growth of oral Streptococcus; the absorbance of oral Streptococcus treated with 1 μM simeprevir did not decrease significantly, indicating that 1 μM simeprevir did not significantly inhibit the proliferation and growth of oral Streptococcus.

[0055] Example 5: Effect of simeprevir on the formation of periodontitis in mice infected with oral streptococci

[0056] In order to avoid the possible disturbance of flora caused by the antibacterial effect of simeprevir, this example uses 1 μM simeprevir for in vivo experiments in mice. This experiment uses healthy C57BL / 6 female mice, 8 weeks old, weighing about 20 g, provided by Hubei Experimental Animal Research Center. The experimental process is as follows:

[0057] 1. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution. A 5-0 suture was used to tie the neck of the maxillary second molars on both sides of the mice and a triple knot was used to fix the palate to establish a mouse periodontitis model.

[0058] 2. Twelve mice were randomly divided into 4 groups (n=3), namely, the control group, the oral streptococcus treatment group, the simeprevir treatment group, and the oral streptococcus and simeprevir co-treatment group.

[0059] In the control group, 10 μL of sterile PBS was dripped onto the bilateral ligatures for 7 consecutive days. In the oral streptococcus treatment group, oral streptococci cultured overnight were diluted to an absorbance of OD 600 ) to 0.1, then 10 μL of bacterial solution was added to the ligature. Simiprevir treatment group: 10 μL of sterile PBS with 1 μM simiprevir was added to the ligature for 7 consecutive days. Oral Streptococcus and Simiprevir co-treatment group: Oral Streptococcus cultured overnight was diluted to an absorbance of OD 600 ) was 0.1 and simeprevir was added at a final concentration of 1 μM, 10 μL of the mixture was drawn and dripped onto the ligature.

[0060] 3. Euthanize the mice, then remove the maxillary alveolar bones of the mice, fix them with 4% paraformaldehyde at 4°C for 48 hours, and then rinse them with running water overnight.

[0061] 4. MicroCT analysis: The maxillary alveolar bone of mice was scanned using a high-resolution MicroCT scanner with scanning conditions of 55 kV, 200 μA, and a resolution of 5 mm / pixel. The results were reconstructed in three dimensions using CTAn (Brucker) software, including parameters such as alveolar bone density (bone volume / tissue volume, BV / TV) and trabecular number (Tb.N). CTVox software was used for image analysis of the three-dimensional reconstruction results.

[0062] 5. Histological analysis: The alveolar bone tissue of mice was treated with 10% EDTA decalcification solution for 4 weeks to complete decalcification. The tissue sections were dehydrated with ethanol gradient and embedded in paraffin, and cut into 5 μm tissue sections. The tissue sections were stained with hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP). The staining kit was from Solarbio and was operated according to the instructions. The sections were sealed with neutral gum, observed under a microscope (Leica, Aperio VERSA 8) and the results were recorded. ImageScope and Image J software were used to analyze alveolar bone resorption and osteoclast infiltration.

[0063] The test results are shown in Tables 5-7.

[0064] Table 5 Bone density and trabecular number of alveolar bone in different groups of mice

[0065] As shown in Table 5, the mice in the oral streptococcus and simeprevir co-treated group had significantly increased bone density and increased number of trabeculae in the alveolar bone compared with the mice in the oral streptococcus treated group. Similarly, the bone density and number of trabeculae in the alveolar bone of the mice in the simeprevir treated group were also increased compared with the control group.

[0066] Table 6 The distance from the cementoenamel junction to the alveolar bone crest in different groups of mice

[0067] As shown in Table 6, the distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) was significantly reduced in the oral streptococcus and simeprevir co-treated group compared with the oral streptococcus treated group. Similarly, the distance from the cementoenamel junction to the alveolar bone crest was also reduced in the simeprevir treated group compared with the control group.

[0068] Table 7 Results of osteoclast infiltration in different groups of mice

[0069] As shown in Table 7, the osteoclast infiltration of mice in the oral streptococcus and simeprevir co-treatment group was reduced compared with the oral streptococcus treatment group. Similarly, the osteoclast infiltration of mice in the simeprevir treatment group was reduced compared with the control group.

[0070] From the results in Tables 5-7, we can see that 1 μM simeprevir can inhibit the resorption of alveolar bone in mice caused by oral streptococcal infection and reduce the infiltration of osteoclasts.

[0071] In summary, simeprevir can bind to EF-Tu protein, reduce the adhesion ability of EF-Tu protein to the interface, inhibit the adhesion of oral Streptococcus and biofilm formation, and effectively protect the alveolar bone. Therefore, simeprevir can be used in periodontitis and can provide a new option for the prevention or treatment of periodontitis.

[0072] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Application of simeprevir in the preparation of preparations for preventing and treating periodontitis.

2. The use of simeprevir according to claim 1 in preparing a preparation for preventing and treating periodontitis, characterized in that: The periodontitis is caused by oral streptococcal infection.

3. The use of simeprevir according to claim 1 in preparing a preparation for preventing and treating periodontitis, characterized in that: The preparation is used for inhibiting alveolar bone resorption.

4. The use of simeprevir according to claim 1 in preparing a preparation for preventing and treating periodontitis, characterized in that: The preparation is used to inhibit the infiltration of osteoclasts.

5. The use of simeprevir in the preparation of an antibacterial agent, characterized in that: The antibacterial agent is used for inhibiting oral streptococci.

6. A preparation, characterized in that The preparation is used for preventing and treating periodontitis, or for protecting periodontal tissue or alveolar bone; the preparation contains simeprevir as a component.

Images