A pharmaceutical composition, use thereof, and a drug for preventing or treating pulpitis

Abstract

The present application relates to the field of biological medicine research, and in particular to a kind of pharmaceutical composition and its use and the drug for preventing or treating pulpitis, by thymus peptide alpha 1, streptococcal lactase and ciprofloxacin are combined, three play synergies, not only can effectively kill bacteria, but also can significantly improve inflammatory environment and enhance cellular immunity, significantly reduce pulp tissue inflammatory cell infiltration, restore pulp tissue, thereby exerting good therapeutic effect on pulpitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical research, specifically to a pharmaceutical composition and its uses, and to a drug for the prevention or treatment of pulpitis. Background Technology

[0002] Pulpitis is a common oral disease, which can be divided into acute pulpitis and chronic pulpitis according to its course. It is accompanied by spontaneous pain or pain on hot or cold stimuli, affecting the patient's quality of life. Microbial infection, including Enterococcus faecalis, is the main cause of pulpitis. Bacterial infection and infiltration of inflammatory cells cause dentin destruction.

[0003] Root canal treatment is currently the most commonly used clinical treatment method, but the increased brittleness and fracturing of teeth after treatment limits its widespread use. Calcium hydroxide paste is currently the mainstream medication; sealing it into the root canal can help reduce inflammation. Currently, the treatment of pulpitis still primarily relies on mechanical removal of the infected pulp; existing medications have limited effectiveness and only play a supplementary role. After treatment, the affected tooth loses its pulp and its strength is reduced.

[0004] Pulp capping is one of the current methods for treating pulpitis and preserving vital pulp. It is divided into direct and indirect pulp capping. It involves covering the exposed pulp wound or dentin near the pulp with a preparation that has a restorative effect on pulp lesions. It is suitable for small mechanical perforations, deep caries near the pulp, and reversible pulpitis. An ideal pulp capping agent should have good biocompatibility, long-lasting antibacterial properties, the ability to inhibit inflammation, and the ability to promote pulp tissue regeneration. iRoot BP Plus is one of the commonly used pulp capping agents in clinical practice, but its antibacterial ability is limited and it cannot improve the autoimmunity of the pulp tissue.

[0005] Therefore, finding a drug for treating pulpitis that is effective, has good antibacterial properties, few side effects, and can improve pulp immunity remains a technical challenge that needs to be addressed by those skilled in the art. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating pulpitis with good efficacy, excellent antibacterial effect, few side effects, and the ability to improve pulp immunity, as well as its uses and a drug for preventing or treating pulpitis.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0008] The present invention provides a pharmaceutical composition comprising thymosin α1, nisin, and ciprofloxacin.

[0009] Among them, thymosin α1 is a physiologically active polypeptide secreted by thymic tissue. It can reduce the levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-18 produced by macrophages, decrease macrophage phagocytosis and migration, reduce the differentiation of pro-inflammatory M1 macrophages, increase the differentiation of anti-inflammatory M2 macrophages, and improve the inflammatory environment of dental pulp. In addition, Tα1 can enhance natural killer cell activity and induce T cell differentiation and maturation, thereby enhancing cellular immunity.

[0010] Nisin is an antimicrobial peptide derived from lactic acid bacteria with extremely low toxicity. Nisin exhibits high antimicrobial activity against a wide range of Gram-positive bacteria, and is even effective against some drug-resistant pathogens. Nisin can significantly enhance the antimicrobial and antibiofilm activity of various antibiotics, including ciprofloxacin, which is beneficial for clearing pathogenic microorganisms from infected dental pulp.

[0011] Ciprofloxacin is a synthetic third-generation quinolone antibacterial drug with broad-spectrum antibacterial activity, good bactericidal effect, low toxicity, low likelihood of developing drug resistance, and strong permeability.

[0012] Furthermore, in the pharmaceutical composition, the mass ratio of thymosin α1, nisin, and ciprofloxacin is 1-4:100-400:0.5-2.

[0013] Furthermore, in the pharmaceutical composition, the mass ratio of thymosin α1, nisin, and ciprofloxacin is 1:200:2.

[0014] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention or treatment of pulpitis.

[0015] Furthermore, the drug can promote macrophage clearance of intracellular bacteria and / or kill pathogens causing pulpitis.

[0016] Furthermore, the drug can promote the formation of reparative dentin.

[0017] The present invention also provides the use of the pharmaceutical composition described above in the preparation of medicaments for inhibiting inflammatory responses and / or for antibacterial purposes.

[0018] The present invention also provides a medicament for the prevention or treatment of pulpitis, comprising the pharmaceutical composition described above, and further comprising one or more pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage form of the drug is selected from tablets, capsules, pills, solutions, powders, granules, suspensions, gels, ointments, sprays, patches, or granules.

[0020] Furthermore, the drug can be prepared according to conventional pharmaceutical manufacturing methods.

[0021] Furthermore, the pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, emulsifiers, colorants, binders, disintegrants, solubilizers, cosolvents, fillers, lubricants, wetting agents, flow aids, flavoring agents, preservatives, suspending agents, coating materials, pH adjusters, plasticizers, thickeners, inclusion agents, humectants, flocculants, or deflocculators. All of the above excipients can be conventional excipients in the art.

[0022] Furthermore, the thymosin α1, nisin, and ciprofloxacin are in different formulation units, or three or any two of the thymosin α1, nisin, and ciprofloxacin are in the same formulation unit.

[0023] The technical solution of this invention has the following advantages:

[0024] Bacteria invading the dental pulp can produce various harmful substances that directly damage cells, and can also trigger inflammatory responses leading to pulp tissue damage. The pharmaceutical composition provided by this invention, through the combined use of thymosin α1, nisin, and ciprofloxacin, works synergistically to not only effectively kill bacteria but also significantly improve the inflammatory environment and enhance cellular immunity, significantly reduce inflammatory cell infiltration in the pulp tissue, and restore pulp tissue, thereby achieving a good therapeutic effect on pulpitis. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 Results of dental pulp cell viability test (compared with control group; *p<0.05, **p<0.01, ***p<0.001); where C is ciprofloxacin, N is nisin, and T is thymosin α1.

[0027] Figure 2 Results of macrophage clearance of intracellular bacteria (compared with control group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0028] Figure 3 This is a graph showing the results of a sterilization experiment on clinical samples.

[0029] Figure 4 HE staining diagram;

[0030] Figure 5 The results of the pulp inflammation score (compared with the inflammation group; *p<0.05, **p<0.01, ***p<0.001);

[0031] Figure 6 The expression of Nlrp3 in dental pulp tissue is shown in Figure A, which is an immunostaining image, and Figure B is the percentage of Nlrp3 expression area in dental pulp tissue of each group (compared with the inflammation group; *p<0.05, **p<0.01, ***p<0.001).

[0032] Figure 7 Immunofluorescence image of CD80CD163 in dental pulp tissue;

[0033] Figure 8 Statistical results of CD80 (M1) and CD163 (M2) fluorescence intensity in dental pulp tissue (compared with the inflammation group; *p<0.05, **p<0.01, ***p<0.001). Detailed Implementation

[0034] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0035] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0036] Example 1

[0037] This embodiment provides a pharmaceutical composition comprising thymosin α1, nisin, and ciprofloxacin in a mass ratio of 1:200:2.

[0038] This embodiment also provides a solution, comprising dissolving the above-mentioned drug composition in physiological saline to obtain a mixed solution, wherein the concentration of nisin in the mixed solution is 200 μg / ml, the concentration of ciprofloxacin is 2 μg / ml, and the concentration of Tα1 is 1 μg / ml.

[0039] Experimental Example 1

[0040] 1. Test drugs and reagents

[0041] Nisin was purchased from Shanghai Yika Biotechnology Co., Ltd., ciprofloxacin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and thymosin α1 (Tα1) was purchased from SciSheng Pharmaceutical (China) Co., Ltd.

[0042] Preparation of thymosin α1 stock solution: Dissolve 1.6 mg of thymosin α1 in 1 ml of sterile water to prepare thymosin α1 stock solution.

[0043] Weigh out 1 mg, 2 mg, 3 mg and 4 mg of nisin powder; and 10 μg, 20 μg, 30 μg and 40 μg of ciprofloxacin powder, respectively, for later use.

[0044] 1 mg of nisin and 10 μg of ciprofloxacin were dissolved in 10 ml of α-minimum essential medium (αMEM; Gibco) and 3.125 μl of thymosin α1 stock solution were added to prepare a test drug solution containing 100 μg / ml nisin, 1 μg / ml ciprofloxacin, and 500 ng / ml Tα1. 2 mg of nisin and 20 μg of ciprofloxacin were dissolved in 10 ml of αMEM basal medium and 6.25 μl of thymosin α1 stock solution were added to prepare a test drug solution containing 200 μg / ml nisin, 2 μg / ml ciprofloxacin, and 1000 ng / ml Tα1. 3 mg of nisin and 30 μg of ciprofloxacin were dissolved in 10 ml of αMEM basal medium, and 9.375 μl of thymosin α1 stock solution was added to prepare a test drug solution containing 300 μg / ml nisin, 3 μg / ml ciprofloxacin, and 1500 ng / ml Tα1. 4 mg of nisin and 40 μg of ciprofloxacin were dissolved in 10 ml of αMEM basal medium, and 12.5 μl of thymosin α1 stock solution was added to prepare a test drug solution containing 400 μg / ml nisin, 4 μg / ml ciprofloxacin, and 2000 ng / ml Tα1.

[0045] Complete culture medium: α-minimum essential medium (αMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco BRL), 100 U / ml penicillin G and 100 U / ml streptomycin (Sigma-Aldrich).

[0046] 2. Cell grouping and drug administration

[0047] Dental pulp stem cells (DPSCs) were obtained from healthy impacted third molars aged 16 to 25 years from patients at the Stomatological Hospital of Sun Yat-sen University with informed consent. DPSCs were isolated using enzymatic digestion. The isolated cells were cultured in complete culture medium at 37°C in a humidified environment with 5% CO2. Cells began to grow within 2 weeks. When the cells reached 90% confluence, they were passaged and considered primary cells. This study used cells from passages 3 to 5 (P3-5). After the cells reached 90% confluence, they were resuspended in complete culture medium and the cell concentration was adjusted to 25,000 / ml. The cells were then seeded into 96-well plates, with 200 μl per well and four replicates per group. The plates were then incubated at 37°C. When the cells reached approximately 50% confluence, the original culture medium was aspirated from each well, and 200 μl of the test drug solution at different concentrations was added for treatment. The control group underwent an equal volume medium replacement. After aspirating the liquid, 200 μl of fresh complete culture medium was added, and the plates were incubated at 37°C for 6 h, 12 h, or 24 h. After 6 h, 12 h, or 24 h of incubation, the liquid in the culture medium was aspirated, and a solution of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (CCK8) solution and complete culture medium mixed at a volume ratio of 1:10 was added to each well. The plates were then incubated at 37°C for another 2 h. The absorbance of the 96-well plate was then measured at 450 nm.

[0048] 3. Experimental Results

[0049] See results Figure 1 As shown in the results, the combination of nisin 200 μg / ml, ciprofloxacin 2 μg / ml and Tα 11 μg / ml and below has no effect on dental pulp cell viability.

[0050] Experimental Example 2

[0051] 1. Reagents

[0052] Complete culture medium: 1640 basal medium containing 10% FBS.

[0053] Preparation of BHI culture medium: Weigh 3.7g of BHI medium (Difco, USA) using an electronic balance, dissolve it in 95mL of ultrapure water, heat until completely dissolved, and add water to a final volume of 100mL. Autoclave at 121℃ for 15min, cool to room temperature, seal, and store at 4℃. BHI agar medium: Prepare 100mL of the above BHI culture medium, add 1.5g of technical agar powder (Difco, USA), and dissolve completely. Autoclave at 121℃ for 15min, then cool to 50℃. Pour approximately 20mL into each petri dish, cool to room temperature under aseptic conditions, seal, and store at 4℃.

[0054] 2 mg of nisin and 20 μg of ciprofloxacin were dissolved in 10 ml of 1640 basal medium and 6.25 μl of thymosin α1 stock solution prepared in Experiment 1 was added to obtain a test drug solution of nisin 200 μg / ml, ciprofloxacin 2 μg / ml and Tα1 1 μg / ml.

[0055] 2. Co-culture of cells and bacteria

[0056] (1) The experimental subject of this study was the human mononuclear macrophage leukemia cell line (THP-1) (Chinese Academy of Sciences Cell Bank). Cell cryopreservation tubes were removed from the -80℃ freezer and quickly placed in a 37℃ water bath to thaw completely within 1 minute. Under aseptic conditions, 1 mL of cell suspension was mixed with 9 mL of complete culture medium in a 15 mL centrifuge tube, centrifuged at 37℃ and 1000 rpm for 5 minutes, the supernatant was carefully aspirated, and the cells were resuspended in 3 mL of complete culture medium. After gentle pipetting and mixing, the cells were inoculated into 25 cm... 2 Culture flasks were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. When the cells reached confluence of over 90%, they were centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in 1640 basal medium, and the concentration was adjusted to 50,000 cells / ml. The cells were then seeded into 12-well plates, with 1 ml of cell suspension added to each well. 200 ng / mL phorbol ester PMA (GIBCO, USA) was added to each well for induction for 24 h. The medium was then replaced with fresh 1640 basal medium, and the cells were cultured for another 24 h to induce M0 macrophages.

[0057] (2) Another research subject in this study was Enterococcus faecalis strain OG1RF (Guangdong Provincial Institute of Microbiology Culture Collection Center). Enterococcus faecalis standard strain OG1RF, which was frozen at -80℃, was streaked on the surface of BHI agar medium and incubated at 37℃ for 24h. Single colonies were selected and inoculated into 5mL of fresh BHI medium and cultured for another 18h for later use.

[0058] (3) Take the quiescent Enterococcus faecalis OG1RF bacterial suspension, wash it with PBS, suspend it in complete culture medium to obtain bacterial suspension, and determine the OD value of bacterial suspension and the colony-forming unit (CFU) of graded dilution bacterial plate count.

[0059] (4) Carefully aspirate the supernatant from the macrophages obtained in step (1), resuspend the macrophages in the bacterial suspension obtained in step (3) with a multiplicity of infection (MOI) of 100, and incubate them in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for 6 hours.

[0060] 3. Grouping and Dosing

[0061] After co-culturing macrophages with Enterococcus faecalis for 6 hours, extracellular bacteria were removed by PBS washing. Macrophages were randomly divided into two groups: an experimental group and a control group. In the experimental group, each well was incubated with 1 ml of the test drug solution containing 200 μg / ml nisin, 2 μg / ml ciprofloxacin, and 11 μg / ml Tα, and cultured at 37℃ and 5% CO2 for 2 h, 4 h, 8 h, 12 h, or 24 h. In the control group, 1 ml of 1640 basal culture was added, and the cells were cultured at 37℃ and 5% CO2 for 2 h, 4 h, 8 h, 12 h, or 24 h. Three replicates were set for each incubation time in each group.

[0062] Cells were lysed using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) at the corresponding time, and colonies were counted using the plate count method after 48 hours.

[0063] 4. Experimental Results

[0064] See results Figure 2 As shown in the results, the number of intracellular bacteria in the experimental group was significantly lower than that in the control group, indicating that the combined use of thymosin α1, nisin, and ciprofloxacin significantly promoted the clearance of intracellular bacteria by macrophages.

[0065] Experimental Example 3

[0066] 1. Reagents

[0067] Preparation of serum-containing BHI culture medium: Weigh 3.7g of BHI medium (Difco, USA) using an electronic balance, dissolve it in 95mL of ultrapure water, heat until completely dissolved, and add water to a final volume of 100mL. Autoclave at 121℃ for 15min, cool to room temperature, add 10% fetal bovine serum (FBS; Gibco BRL), seal, and store at 4℃.

[0068] 2 mg of nisin and 20 μg of ciprofloxacin were dissolved in 10 ml of the above-mentioned serum-containing BHI culture medium, and 6.25 μl of the thymosin α1 stock solution prepared in Experiment 1 was added to obtain a test drug solution of 200 μg / ml nisin, 2 μg / ml ciprofloxacin and 1 μg / ml Tα1.

[0069] 2. Collection of clinical pulpitis samples

[0070] The pulp of three teeth that required root canal treatment due to pulpitis were collected from the Department of Endodontics at the Affiliated Stomatological Hospital of Sun Yat-sen University. Under local anesthesia with articaine hydrochloride (Primacaine), the pulp was opened with a Kavo LASIK machine, and a portion of the infected pulp was extracted and preserved in a sterile ep tube containing 1 ml of serum-containing BHI culture medium.

[0071] Two wisdom teeth that had been extracted due to pulpitis were collected from the Department of Oral Surgery at the Affiliated Stomatological Hospital of Sun Yat-sen University. The infected pulp was removed using sterile instruments and preserved in a sterile ep tube containing 1 ml of serum-containing BHI culture medium.

[0072] 3. Sample grouping and processing

[0073] Each pulpitis sample was divided into four aliquots and placed in a sterile EP tube containing 1 ml of serum-containing BHI medium. The aliquots were divided into four groups: Group ①: conventional culture control group; Group ②: anaerobic culture control group; Group ③: conventional culture with 1 mL / tube of the test drug solution added (i.e., conventional culture drug treatment group); Group ④: anaerobic culture with 1 mL / tube of the test drug solution added (i.e., anaerobic culture drug treatment group). Anaerobic culture: An anaerobic environment was created using an anaerobic chamber (Mitsubishi Corporation) and an anaerobic gas generator (Mitsubishi Corporation), and then incubated at 37°C for 2 hours. Conventional culture: Directly incubated in an incubator at 37°C, 5% CO2, and saturated humidity for 2 hours.

[0074] After 2 hours of incubation, 100 μL of samples from groups ② and ④ in the anaerobic workbench (SHELLAB / Bactrox EZ-2) were inoculated onto Columbia blood agar plates (Huankai Microbiology) and spread using sterile spreaders (Wuhan Sewell Biotechnology Co., Ltd.). The plates were then anaerobically incubated for 48 hours. In a biosafety cabinet (Thermo Fisher Scientific), samples from groups ① and ③ were processed using the same procedure. The plates were then inverted and incubated for 48 hours in an incubator at 37°C, 5% CO2, and saturated humidity. Finally, images were taken using an Interscience Scan500 colony counter.

[0075] 4. Experimental Results

[0076] See results Figure 3As shown in the results, the combined use of ciprofloxacin, nisin, and Tα1 has a good bactericidal effect on the pathogenic bacteria of pulpitis, and most of the bacteria were killed after 2 hours.

[0077] Experiment Example 4

[0078] 1. Animal Information

[0079] Male SD rats, weighing 150-250g each, were used in this experiment, which was approved by the Laboratory Animal Ethics Committee of Sun Yat-sen University, approval number SYSU-IACUC-2024-002911.

[0080] 2. Preparation of the test drug solution

[0081] A mixed solution containing 200 μg / ml nisin, 2 μg / ml ciprofloxacin and 11 μg / ml Tα was prepared using physiological saline as the solvent. A mixed solution containing 200 μg / ml nisin and 2 μg / ml ciprofloxacin and a solution containing 11 μg / ml Tα were also prepared.

[0082] 3. Experimental Methods

[0083] (1) Animal grouping and administration

[0084] 3-day drug sealing experiment: 25 male SD rats (approximately 150-250g / rat) were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.1mL / 10g). The pulp chamber of the right maxillary first molar was opened with a high-speed 1 / 4 ball drill and probed and cleared with an 8-size file. The pulp chamber was exposed in the oral cavity for 10 minutes. The left maxillary first molar was used as a blank control.

[0085] Rats were randomly divided into five groups. Groups ①-④ received intramedullary injection of the corresponding drugs or medullary capping and were housed in a suitable environment for 3 days. Group ⑤ received direct resin filling. ① BP group (n=5): medullary cavity capped with iRoot BP Plus material followed by resin sealing. ② Nisin + Ciprofloxacin (N+C) group (n=5): medullary cavity sealed with resin after intramedullary injection of 5μL of a mixed solution containing 200μg / ml nisin and 2μg / ml ciprofloxacin. ③ Tα1 group (T group) (n=5): medullary cavity sealed with resin after intramedullary injection of 5μL of a Tα1 1μg / ml solution. ④ Tα1 + Nisin + Ciprofloxacin group (T+N+C group) (n=5): medullary cavity sealed with resin after intramedullary injection of 5μL of a mixed solution containing 1μg / ml Tα1, 200μg / ml nisin, and 2μg / ml ciprofloxacin. ⑤ In the inflammation group, the pulp cavity was exposed in the oral cavity for 10 minutes before direct resin filling. Three days after sealing the drug, two rats from each group were randomly sacrificed, and the right maxillary first molar was taken, fixed in 4% paraformaldehyde (White Shark Biotechnology Co., Ltd.) for 24 hours, decalcified in EDTA decalcification solution (White Shark Biotechnology Co., Ltd.) at room temperature for 2 weeks, and then embedded in paraffin for sectioning.

[0086] Based on the results of the 3-day drug-sealing experiment, another 20 rats were selected for a 7-day drug-sealing experiment. The procedure was the same as the 3-day drug-sealing procedure, except that the nisin + ciprofloxacin (N+C) group was removed in this experiment. The other groups were the same. After 7 days, all rats were sacrificed for sectioning and testing.

[0087] (2) Detection method

[0088] 1) HE staining

[0089] Sections were baked at 60℃ for 2 hours, dewaxed in 200ml xylene for 30 minutes, and rehydrated in gradients of 200ml anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 10 minutes each. After washing with water for 10 minutes, they were treated with 200ml hematoxylin staining solution (Wuhan Sewell Biotechnology Co., Ltd.) for 3 minutes, differentiated in 1ml acidic ethanol differentiation solution (concentrated hydrochloric acid to ethanol volume ratio 1:99, Wuhan Sewell Biotechnology Co., Ltd.) for 3 seconds, treated with 1ml hematoxylin blueing solution (specifically, hematoxylin blueing solution, catalog number G1040, Wuhan Sewell Biotechnology Co., Ltd.) for 10 seconds, dehydrated in 200ml 75% ethanol and 85% ethanol for 2 minutes each, treated with 200ml eosin staining solution (Wuhan Sewell Biotechnology Co., Ltd.) for 30 seconds, dehydrated in 200ml 95% ethanol and anhydrous ethanol for 5 minutes each, cleared in 200ml xylene for 10 minutes, and mounted with 100μL neutral resin (Wuhan Sewell Biotechnology Co., Ltd.). The Leica Aperio AT2 slide scanner was used to scan and observe the slides.

[0090] HE staining was used to observe the degree of pulp inflammation, and the inflammation score was determined according to ISO 7405:2018(E): Dentistry - Evaluation of biocompatibility of medical devices used in dentistry. Specifically:

[0091] Table 1 Inflammation Scoring Criteria

[0092]

[0093]

[0094] 2) Immunohistochemistry and immunofluorescence observation of inflammatory factors

[0095] Sections were baked at 60℃ for 2 hours, dewaxed in 200ml xylene for 30 minutes, rehydrated in gradients of 200ml absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 10 minutes each, and washed with 200ml PBS (phosphate-buffered saline, Wuhan Sewell Biotechnology Co., Ltd.) for 10 minutes. Antigen retrieval was performed at room temperature for 30 minutes with 50μL of antigen retrieval solution (specifically, antigen retrieval solution (gastric enzyme method, Fuzhou Maixin Biotechnology Development Co., Ltd.), followed by treatment with 50μL of endogenous peroxide scavenger (Hunan Aifang Biotechnology Co., Ltd.) at room temperature for 30 minutes; membrane permeation was then performed with 50μL of 0.1% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd.) for 10 minutes. Finally, the membrane was blocked with 5% goat serum (GIBCO, USA) at room temperature for 30 minutes. Monoclonal anti-mouse NLRP3 antibody (Abcam, USA) (1:200) 30 μL was treated at 4℃ for 18 h, followed by secondary antibody treatment (Hunan Aifang Biotechnology Co., Ltd.) at room temperature for 30 min, and then DAB (3,3'-diaminobenzidine tetrahydrochloride) chromogenic solution (Beijing Solarbio Science & Technology Co., Ltd.) was added for 3 min. 200 ml hematoxylin staining solution (Wuhan Sewell Biotechnology Co., Ltd.) was used for 3 min, followed by differentiation in 1 ml acidic ethanol differentiation solution (concentrated hydrochloric acid to ethanol volume ratio 1:99, Wuhan Sewell Biotechnology Co., Ltd.) for 3 s, and then 1 ml hematoxylin blueing solution (specifically, hematoxylin blueing solution, catalog number G1040, Wuhan Sewell Biotechnology Co., Ltd.) for 10 s. The slides were dehydrated in 200 ml each of 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 min, cleared in 200 ml xylene for 10 min, and mounted with 100 μL neutral resin (Wuhan Sewell Biotechnology Co., Ltd.). The slides were scanned and observed using a Leica Aperio AT2 slide scanner.

[0096] 3) Macrophage typing

[0097] Sections were baked at 60℃ for 2 hours, dewaxed in 200ml xylene for 30 minutes, rehydrated in a gradient of 200ml absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 10 minutes each, and washed with 200ml PBS (Wuhan Sewell Biotechnology Co., Ltd.) for 10 minutes. Antigen retrieval was performed at room temperature for 30 minutes with 50μL of antigen retrieval solution (specifically: antigen retrieval solution (gastric enzyme method), Fuzhou Maixin Biotechnology Development Co., Ltd.); membrane permeation was performed with 50μL of 0.1% Trition X-100 (Beijing Solarbio Science & Technology Co., Ltd.) for 10 minutes; and blocking was performed at room temperature for 30 minutes with 5% goat serum (GIBCO, USA). Monoclonal anti-mouse CD80 / CD163 antibody (Abcam, USA) (1:200) was mixed in 30 μL and treated at 4°C for 18 h. Then, a 488 nm / 594 nm channel fluorescent secondary antibody (Abcam, USA) (1:400) was mixed and treated for 30 min at room temperature. DAPI (4',6-diamidinyl-2-phenylindole) staining solution (Beijing Solarbio Science & Technology Co., Ltd.) was used for 5 min at room temperature. The slides were then mounted with 100 μL of anti-quenching mounting medium (specifically, anti-fluorescence quenching mounting medium, Wuhan Sewell Biotechnology Co., Ltd.). Images were taken using a Zeiss LSM 980 super-resolution confocal microscope.

[0098] 4. Experimental Results

[0099] 1) HE staining

[0100] See results Figure 4 and Figure 5 As shown, after 3 days of medication, HE staining revealed extensive inflammatory cell infiltration in the coronal pulp of the inflammation group, with partial pulp necrosis and abscess formation. Extensive inflammatory cell infiltration was also observed in the coronal pulp of the BP group. Inflammatory cell infiltration was also seen in the coronal pulp of the nisin + ciprofloxacin group. Extensive inflammatory cell infiltration and a small number of abscesses were observed in the Tα-1 group. A small number of scattered inflammatory cell infiltrations were observed in the coronal pulp of the Tα-1 + nisin + ciprofloxacin group. Compared to other medication groups, the ciprofloxacin + nisin + Tα1 group significantly reduced inflammatory cell infiltration in the dental pulp tissue.

[0101] Seven days after medication, HE staining revealed extensive inflammatory cell infiltration in the coronal pulp of the inflammation group, along with partial pulp necrosis and abscess formation. Inflammatory cell infiltration was also observed in the coronal pulp of the BP group. Extensive inflammatory cell infiltration and a small number of abscesses were observed in the Tα-1 group. A small number of scattered inflammatory cells and minimal reparative dentin formation were observed in the coronal pulp of the Tα-1 + nisin + ciprofloxacin group. Compared to other medication groups, the ciprofloxacin + nisin + Tα1 group significantly reduced inflammatory cell infiltration in the dental pulp and promoted reparative dentin formation.

[0102] 2) Immunohistochemistry

[0103] See results Figure 6As shown, after 3 days of medication, the inflammation group expressed a relatively high level of Nlrp3. The Nlrp3 levels were decreased to varying degrees in the BP group, the nisin + ciprofloxacin group, the Tα-1 group, and the Tα-1 + nisin + ciprofloxacin group; the decrease was more significant in the Tα-1 group and the Tα-1 + nisin + ciprofloxacin group.

[0104] Seven days after medication, the inflammation group showed high levels of Nlrp3 expression. The Nlrp3 levels were decreased to varying degrees in the BP group, Tα-1 group, and Tα-1 + nisin + ciprofloxacin group; the decrease was more significant in the Tα-1 + nisin + ciprofloxacin group.

[0105] 3) Immunofluorescence

[0106] The immunofluorescence results of CD80(M1) / CD163(M2) are shown in the figure. Figure 7 and 8 As shown, after 3 days of medication, the macrophages in the pulp of the inflammation group and BP group were mainly pro-inflammatory M1 macrophages. The nisin + ciprofloxacin group was mainly pro-inflammatory M1 macrophages, with a small number of anti-inflammatory M2 macrophages scattered throughout. The Tα-1 group and the Tα-1 + nisin + ciprofloxacin group were mainly anti-inflammatory M2 macrophages.

[0107] Seven days after medication, the macrophages in the dental pulp of the inflammation group were mainly pro-inflammatory M1 macrophages. The BP group was mainly pro-inflammatory M1 macrophages, with a small number of anti-inflammatory M2 macrophages scattered throughout. The Tα-1 group and the Tα-1 + nisin + ciprofloxacin group were mainly anti-inflammatory M2 macrophages.

[0108] 5. Experimental Conclusions

[0109] The combined use of ciprofloxacin, nisin, and Tα1 significantly reduces inflammatory cell infiltration in dental pulp tissue, restores dental pulp tissue, and has the best therapeutic effect on pulpitis.

[0110] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A pharmaceutical composition for treating pulpitis, characterized in that, It is composed of thymosin α1, nisin, and ciprofloxacin; the mass ratio of thymosin α1, nisin, and ciprofloxacin is 1:200:

2.

2. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for the prevention or treatment of pulpitis.

3. The use according to claim 2, characterized in that, The drug can promote macrophages to clear intracellular bacteria and / or kill pathogens causing pulpitis.

4. The use according to claim 2 or 3, characterized in that, The drug can promote the formation of reparative dentin.

5. A medicament for the prevention or treatment of pulpitis, comprising the pharmaceutical composition of claim 1, and further comprising one or more pharmaceutically acceptable excipients.

6. The medicament for preventing or treating pulpitis according to claim 5, characterized in that, The dosage form of the drug is selected from solutions.

7. The medicament for preventing or treating pulpitis according to claim 5 or 6, characterized in that, The thymosin α1, nisin, and ciprofloxacin are in different formulation units, or three or any two of the thymosin α1, nisin, and ciprofloxacin are in the same formulation unit.

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