Pharmaceutical composition, application thereof and medicine for preventing or treating pulpitis

Abstract

The invention relates to the field of biological medicine research, in particular to a medicine composition and application thereof and a medicine for preventing or treating pulpitis, thymosin alpha 1, nisin and ciprofloxacin are used in a combined mode and play a synergistic role, bacteria can be effectively killed, and the effect of preventing or treating pulpitis is achieved. Moreover, the composition can obviously improve inflammatory environment, enhance cellular immunity, obviously reduce dental pulp tissue inflammatory cell infiltration and recover dental pulp tissue, so that the composition has a good treatment effect on pulpitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical research, and particularly to a pharmaceutical composition, its use, and a drug for preventing or treating pulpitis. Background Art

[0002] Pulpitis is a common oral disease, which can be divided into acute pulpitis and chronic pulpitis according to the course of the disease, accompanied by spontaneous pain or pain on thermal or cold stimulation, affecting the quality of life of patients. Microbial infections, including Enterococcus faecalis, are the main causes of pulpitis. Bacterial infections and infiltration of inflammatory cells cause dentin destruction.

[0003] Root canal treatment is the most commonly used treatment method clinically at present, but the treated teeth have increased brittleness and are prone to fracture, limiting its widespread use. Calcium hydroxide paste is the current mainstream drug. Sealing it into the root canal can assist in reducing the inflammatory response. At present, the treatment of pulpitis still mainly relies on mechanical methods to remove the infected dental pulp. The existing drugs have limited effects and only play an auxiliary role. After treatment, the affected tooth loses its dental pulp and the tooth strength decreases.

[0004] Pulp capping is also one of the current methods for treating pulpitis and preserving the vital pulp. It is divided into direct pulp capping and indirect pulp capping. It uses a preparation with the effect of restoring pulp lesions to cover the exposed pulp wound surface or dentin near the pulp, and is applicable to smaller mechanical pulp perforations, deep caries approaching the pulp, and reversible pulpitis. An ideal pulp capping agent should have good biocompatibility, the ability to persistently inhibit bacteria, suppress inflammation, and promote the regeneration of dental pulp tissue. iRoot BP Plus is one of the commonly used pulp capping agents clinically, but its antibacterial ability is limited and it cannot improve the autoimmunity of dental pulp tissue.

[0005] Therefore, at present, finding a drug for treating pulpitis with good curative effect, good antibacterial effect, few side effects, and the ability to improve pulp immunity is still a technical problem that needs to be solved urgently 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 curative effect, good antibacterial effect, few side effects, and the ability to improve pulp immunity, its use, and a drug for preventing or treating pulpitis.

[0007] To solve the above technical problems, the present invention is realized through the following technical solutions:

[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 thymus tissue, which can reduce the levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-18 produced by macrophages, reduce macrophage phagocytosis and migration, and reduce the differentiation of pro-inflammatory M1 macrophages, increase the differentiation of anti-inflammatory M2 macrophages, and improve the dental pulp inflammatory environment. In addition, Tα1 can enhance the activity of natural killer cells, induce the differentiation and maturation of T cells, and enhance cellular immunity.

[0010] Nisin is an antibacterial peptide derived from lactic acid bacteria with extremely low toxicity. Nisin has high antibacterial activity against a variety of Gram-positive bacteria and is even effective against some drug-resistant pathogens. Nisin can significantly improve the antibacterial and anti-biofilm activities of a variety of antibiotics including ciprofloxacin, which is beneficial to the clearance of pathogenic microorganisms in the infected dental pulp.

[0011] Ciprofloxacin is a synthetic third-generation quinolone antibacterial drug with broad-spectrum antibacterial activity, good bactericidal effect, low toxicity, not easy to produce 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-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating pulpitis.

[0015] Furthermore, the drug can promote macrophages to clear intracellular bacteria and / or kill the pathogenic bacteria of pulpitis.

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

[0017] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for inhibiting inflammatory reactions and / or antibacterial.

[0018] The present invention also provides a drug for preventing or treating pulpitis, which comprises the above-mentioned pharmaceutical composition and further comprises 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 the conventional production methods in the pharmaceutical field.

[0021] Furthermore, the pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, emulsifiers, colorants, binders, disintegrants, solubilizers, cosolvents, fillers, lubricants, wetting agents, glidants, flavoring agents, preservatives, suspending agents, coating materials, pH regulators, plasticizers, thickeners, clathrates, humectants, flocculants or deflocculants. The above excipients can all be conventional excipients in the art.

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

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

[0024] On the one hand, the bacteria invading the dental pulp can produce a variety of harmful substances to directly damage cells, and on the other hand, they can trigger an inflammatory response leading to damage of the dental pulp tissue. The pharmaceutical composition provided by the present invention, through the combined use of thymosin α1, nisin and ciprofloxacin, the three play a synergistic role, not only can effectively kill bacteria, but also can significantly improve the inflammatory environment and enhance cellular immunity, significantly reduce the infiltration of inflammatory cells in the dental pulp tissue, and restore the dental pulp tissue, thereby exerting a good therapeutic effect on pulpitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

[0027] Figure 2 It is the test result of macrophages clearing intracellular bacteria (compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0028] Figure 3 It is the result diagram of the bactericidal experiment of clinical samples;

[0029] Figure 4 It is the HE staining diagram;

[0030] Figure 5 Results of the inflammatory score of dental pulp tissue (compared with the inflammation group; *p < 0.05, **p < 0.01, ***p < 0.001);

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

[0032] Figure 7 Immunofluorescence images of CD80 and CD163 in dental pulp tissue;

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

[0034] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0035] For those experimental steps or conditions not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified for the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0036] Example 1

[0037] This example provides a pharmaceutical composition, which includes thymosin α1, nisin, and ciprofloxacin with a mass ratio of 1:200:2.

[0038] This example also provides a solution, which includes dissolving the above pharmaceutical composition in physiological saline to obtain a mixed solution. Among them, 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 Ikang Biotechnology Co., Ltd., ciprofloxacin was purchased from Shanghai Macklin Biochemical Co., Ltd., and thymosin α1 (Tα1) was from SciClone Pharmaceuticals (China) Co., Ltd.

[0042] Preparation of thymosin α1 stock solution: 1.6 mg of thymosin α1 was dissolved in 1 ml of sterile water to obtain the thymosin α1 stock solution.

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

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

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

[0046] 2. Cell grouping and drug administration

[0047] Dental pulp stem cells (DPSCs) were obtained from healthy impacted third molars of patients aged 16 to 25 years old with informed consent from the Stomatological Hospital of Sun Yat-sen University. Human dental pulp stem cells were isolated by enzymatic digestion method. The isolated cells were cultured in complete medium in a humid environment at 37°C and 5% CO₂. The cells began to grow within 2 weeks. When the cells reached 90% confluence, they were passaged and regarded as primary cells. Cells of passages 3 to 5 (P3-5) were used in this study. When the cells reached 90% confluence, they were treated. The cells were resuspended with complete medium and the cell concentration was adjusted to 25,000 / ml. They were seeded into 96-well plates, 200 μl was added to each well, and 4 replicate wells were inoculated in each group. Then they were placed in an incubator at 37°C for culture. When the cells reached about 50% confluence, in the wells corresponding to each group in turn, the original medium was aspirated, and 200 μl of the above-mentioned test drug solutions with different concentrations was added for treatment. Among them, the control group was treated with an equal volume replacement. The liquid was aspirated and 200 μl of fresh complete medium was added. The plate was placed back in the incubator at 37°C and continued to be cultured for 6 h, 12 h or 24 h. After culturing for 6 h, 12 h or 24 h, the liquid in the medium was aspirated, and a solution prepared by mixing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (CCK8) solution and complete medium in a volume ratio of 1:10 was added to each well and placed in an incubator at 37°C for continued culture for 2 h. Then the 96-well plate was taken out and the absorbance was measured at 450 nm.

[0048] 3. Experimental results

[0049] The results are shown in Figure 1 As can be seen from the results, nisin at 200 μg / ml, ciprofloxacin at 2 μg / ml and the combination agent at 1 μg / ml of Tα1 and below had no effect on the viability of dental pulp cells.

[0050] Experimental Example 2

[0051] 1. Reagents

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

[0053] Preparation of BHI culture medium: Weigh 3.7 g of BHI medium (Difco, USA) using an electronic balance, dissolve it in 95 mL of ultrapure water, heat it until completely dissolved, and add water to reach 100 mL. Sterilize it at 121 °C for 15 min under high temperature and high pressure, seal it after cooling to room temperature, and store it at 4 °C. BHI agar medium: Prepare 100 mL of the above BHI culture medium, add 1.5 g of technical agar powder (Difco, USA), and dissolve it completely. Sterilize it at 121 °C for 15 min under high temperature and high pressure, and cool it to 50 °C. Pour about 20 mL into each petri dish, seal it after cooling to room temperature under sterile conditions, and store it at 4 °C.

[0054] Dissolve 2 mg of nisin and 20 μg of ciprofloxacin in 10 ml of 1640 basal medium and add 6.25 μl of the thymosin α1 mother liquor prepared in Experimental Example 1 to obtain a test drug solution containing 200 μg / ml of nisin, 2 μg / ml of ciprofloxacin, and 1 μg / ml of Tα1.

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

[0056] (1) The experimental research object in this study is the human monocyte macrophage leukemia cell line (THP-1) (Cell Bank of the Chinese Academy of Sciences). Take out the cell cryopreservation tube from the -80 °C refrigerator, quickly place it in a 37 °C water bath to completely melt it within 1 min. Under sterile conditions, mix 1 mL of cell suspension with 9 mL of complete medium in a 15 mL centrifuge tube, centrifuge at 37 °C and 1000 rpm for 5 min, carefully aspirate the supernatant, resuspend it with 3 mL of complete medium, gently pipette and mix well, and then inoculate it into a 25 cm 2 culture flask, and place it in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity for culture. When the cells reach more than 90% confluence, centrifuge at 1000 rpm for 5 min, resuspend the cell pellet with 1640 basal medium, adjust the concentration to 50,000 cells / ml, spread it into a 12-well plate, add 1 ml of cell suspension to each well, and add 200 ng / mL of phorbol myristate acetate PMA (GIBCO, USA) to each well. After inducing for 24 h, replace it with fresh 1640 basal medium and continue to culture for 24 h to induce M0 macrophages.

[0057] (2) Another experimental research object in this study is the Enterococcus faecalis OG1RF strain (Guangdong Institute of Microbiology, Culture Collection Center). Take out the frozen Enterococcus faecalis standard strain OG1RF from the -80 °C refrigerator, streak inoculate it on the surface of BHI agar medium and place it in a 37 °C incubator for 24 h. Select a monoclonal colony and inoculate it into 5 mL of fresh BHI culture medium, and continue to culture for 18 h for standby.

[0058] (3) The stationary-phase Enterococcus faecalis OG1RF bacterial solution was rinsed with PBS and resuspended in complete medium to obtain a bacterial suspension. The OD value of the bacterial suspension was measured, and the colony-forming units (CFU) were counted by gradient dilution and plating.

[0059] (4) The macrophages obtained in step (1) were carefully aspirated to remove the supernatant. The macrophages were resuspended with the bacterial suspension obtained in step (3) at a multiplicity of infection (MOI) = 100 and cultured in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity for 6 h.

[0060] 3. Grouping and drug administration

[0061] After the macrophages and Enterococcus faecalis were co-cultured for 6 h, the extracellular bacteria were removed by washing with PBS. They were randomly divided into two groups, an experimental group and a control group. The experimental group was given 1 ml of the test drug solution containing 200 μg / ml nisin, 2 μg / ml ciprofloxacin, and 1 μg / ml Tα1 per well and cultured at 37 °C and 5% CO2 for 2 h, 4 h, 8 h, 12 h, or 24 h. The control group was added with 1 ml of 1640 basal medium and cultured at 37 °C and 5% CO2 for 2 h, 4 h, 8 h, 12 h, or 24 h. Three replicate wells were set for each culture time in each group.

[0062] After the corresponding time, the cells were lysed with RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the colonies were counted by the plate colony counting method 48 h later.

[0063] 4. Experimental results

[0064] The results are shown in Figure 2 As can be seen from the results, compared with the control group, the number of intracellular bacteria in the experimental group was significantly reduced, indicating that the combination of thymosin α1, nisin, and ciprofloxacin significantly promoted the macrophages to clear intracellular bacteria.

[0065] Experimental example 3

[0066] 1. Reagents

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

[0068] Dissolve 2 mg of nisin and 20 μg of ciprofloxacin in 10 ml of the above-mentioned serum-containing BHI culture medium, and add 6.25 μl of the thymosin α1 mother liquor prepared in Experimental Example 1 to obtain a test drug solution containing 200 μg / ml of nisin, 2 μg / ml of ciprofloxacin, and 1 μg / ml of Tα1.

[0069] 2. Collection of clinical pulpitis samples

[0070] Collect the dental pulps of 3 teeth that require root canal treatment after pulp exposure due to pulpitis in the Department of Conservative Dentistry and Endodontics of the Affiliated Stomatological Hospital of Sun Yat-sen University. Under local anesthesia with articaine hydrochloride (Primacaine company), use a KAVO high-speed handpiece to expose the pulp, and use sterile instruments to clamp a part of the infected dental pulp and store it in a sterile ep tube containing 1 ml of serum-containing BHI medium.

[0071] Collect 2 impacted wisdom teeth extracted due to pulpitis in the Department of Alveolar Surgery of the Affiliated Stomatological Hospital of Sun Yat-sen University. Use sterile instruments to remove the infected dental pulp and store it in a sterile ep tube containing 1 ml of serum-containing BHI medium.

[0072] 3. Sample grouping and treatment

[0073] Divide each pulpitis sample into 4 parts and place them in sterile ep tubes containing 1 ml of serum-containing BHI medium, and divide them into 4 groups: Group ①: conventional culture control group; Group ②: anaerobic culture control group; Group ③: conventional culture after adding 1 mL / tube of the test drug solution (i.e., conventional culture drug treatment group); Group ④: anaerobic culture after adding 1 mL / tube of the test drug solution (i.e., anaerobic culture drug treatment group). Anaerobic culture: Use an anaerobic jar (Mitsubishi company) and an anaerobic gas-generating pack (Mitsubishi company) to create an anaerobic environment, and then incubate at 37 °C for 2 h. Conventional culture: Directly place it in an incubator at 37 °C, 5% CO2, and saturated humidity for 2 h.

[0074] After culturing for 2 h, in an anaerobic workbench (SHELLAB / Bactrox EZ-2), take 100 μl of the samples in Groups ② and ④ in the anaerobic jar and inoculate them on Columbia blood agar plates (Huankai Microbial). Use a sterile spreading rod (Wuhan Sevier Biotechnology Co., Ltd.) to spread the plates and incubate anaerobically for 48 h. In a biosafety cabinet (Thermo Fisher Scientific), treat the samples in Groups ① and ③ in the same steps, and then invert the plates and incubate them in an incubator at 37 °C, 5% CO2, and saturated humidity for 48 h. Then use an interscience Scan500 colony counter to take pictures.

[0075] 4. Experimental results

[0076] The results are shown in Figure 3As shown, the results showed that the combined use of ciprofloxacin, nisin and Tα1 had a good bactericidal effect on the pathogenic bacteria of pulpitis, and most of the bacteria were killed after 2 h.

[0077] Experimental Example 4

[0078] 1. Animal information

[0079] SD rats, male, weighing 150 - 250 g per rat. This experiment was approved by the Experimental Animal Ethics Committee of Sun Yat-sen University, and the approval number was SYSU-IACUC-2024-002911.

[0080] 2. Preparation of test drug solutions

[0081] Using normal saline as the solvent, prepare a mixed solution containing 200 μg / ml of nisin, 2 μg / ml of ciprofloxacin and 1 μg / ml of Tα1, a mixed solution containing 200 μg / ml of nisin and 2 μg / ml of ciprofloxacin, and a solution containing 1 μg / ml of Tα1 respectively.

[0082] 3. Experimental method

[0083] (1) Animal grouping and drug administration

[0084] Sealing drug for 3 days experiment: Select 25 male SD rats (about 150 - 250 g per rat), anesthetize them by intraperitoneal injection of 1% pentobarbital sodium (0.1 mL / 10 g), open the pulp chamber of the right maxillary first molar with a high-speed 1 / 4 round bur, and use a No. 8 file to detect and dredge it. Expose the pulp chamber to the oral cavity for 10 min, with the left maxillary first molar as the blank control.

[0085] Rats were randomly divided into five groups. For the rats in groups ① - ④, the pulp cavities were injected with the corresponding drugs or pulp capping treatment was performed, and they were raised in a suitable environment for 3 days; the rats in group ⑤ were directly filled with resin. ① BP group, 5 rats: After pulp capping with iRoot BP Plus material, the pulp cavity was sealed with resin; ② Nisin + ciprofloxacin (N + C) group, 5 rats: After injecting 5 μL of a mixed solution containing 200 μg / ml of nisin and 2 μg / ml of ciprofloxacin into the pulp cavity, the pulp cavity was sealed with resin; ③ Tα1 group (T group), 5 rats: After injecting 5 μL of 1 μg / ml Tα1 solution into the pulp cavity, the pulp cavity was sealed with resin; ④ Tα1 + nisin + ciprofloxacin group (T + N + C group), 5 rats: After injecting 5 μL of a mixed solution containing 1 μg / ml of Tα1, 200 μg / ml of nisin and 2 μg / ml of ciprofloxacin into the pulp cavity, the pulp cavity was sealed with resin. ⑤ Inflammatory group: After the pulp cavity was exposed to the oral cavity for 10 min, it was directly filled with resin. Three days after drug sealing, 2 rats were randomly selected and sacrificed from each group, and the first maxillary right molar was taken, fixed with 4% paraformaldehyde (White Shark Biotechnology Co., Ltd.) for 24 h, decalcified with EDTA decalcifying solution (White Shark Biotechnology Co., Ltd.) at room temperature for 2 weeks, and then paraffin-embedded and sectioned.

[0086] According to the experimental results of drug sealing for 3 days, another 20 rats were taken for the drug sealing experiment for 7 days. The operation was the same as the above operation of drug sealing for 3 days, with the only difference being that in this experiment, the nisin + ciprofloxacin (N + C) group was deleted, and 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] The sections were baked at 60 °C for 2 h, dewaxed with 200 ml of xylene for 30 min, rehydrated with 200 ml of absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol in gradient for 10 min each. Washed with water for 10 min, treated with 200 ml of hematoxylin staining solution (Wuhan Sevier Biotechnology Co., Ltd.) for 3 min, differentiated with 1 ml of acidic ethanol differentiation solution (the volume ratio of concentrated hydrochloric acid to ethanol is 1:99, Wuhan Sevier Biotechnology Co., Ltd.) for 3 s, treated with 1 ml of blueing solution for 10 s (specifically: hematoxylin blueing solution, product number G1040, Wuhan Sevier Biotechnology Co., Ltd.), dehydrated with 200 ml of 75% ethanol and 85% ethanol for 2 min each, treated with 200 ml of eosin staining solution (Wuhan Sevier Biotechnology Co., Ltd.) for 30 s, dehydrated with 200 ml of 95% ethanol and absolute ethanol for 5 min each, cleared with 200 ml of xylene for 10 min, and sealed with 100 μL of neutral gum (Wuhan Sevier Biotechnology Co., Ltd.). The sections were scanned and observed with a Leica Aperio AT2 slide scanner.

[0090] HE staining was used to observe the degree of pulp inflammation, and inflammation scores were performed according to ISO 7405:2018(E):Dentistry-Evaluation ofbiocompatibility 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] The slices were baked at 60℃ for 2h, dewaxed in 200ml xylene for 30min, rehydrated in 200ml anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol for 10min each, and washed with 200ml PBS (phosphate buffer, Wuhan Sewell Biotechnology Co., Ltd.) for 10min. Antigen repair solution (specifically: antigen repair solution (pepsin method, Fuzhou Maixin Biotechnology Development Co., Ltd.) 50μL was used for antigen repair at room temperature for 30min, endogenous peroxide scavenger (Hunan Aifang Biotechnology Co., Ltd.) 50μL was used for treatment at room temperature for 30min; 50μL 0.1% trition X-100 (Beijing Solebao Technology Co., Ltd.) was used for membrane permeabilization for 10min. 5% goat serum (GIBCO, USA) was used for blocking at room temperature for 30min. Monoclonal anti-mouse NLRP3 antibody (Abcam, USA) (1:200) 30 μL was treated at 4℃ for 18h, secondary antibody (Hunan Aifang Biotechnology Co., Ltd.) was treated at room temperature for 30min, DAB (3,3'-diaminobenzidine tetrahydrochloride) colorimetric solution (Beijing Solebao Technology Co., Ltd.) was added for 3min. 200ml hematoxylin staining solution (Wuhan Seville Biotechnology Co., Ltd.) was treated for 3min, 1ml acid ethanol differentiation solution (concentrated hydrochloric acid and ethanol volume ratio of 1:99, Wuhan Seville Biotechnology Co., Ltd.) was differentiated for 3s, and 1ml blueing solution (specifically: hematoxylin blueing solution, item number G1040, Wuhan Seville Biotechnology Co., Ltd.) was treated for 10s. 200ml 75% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol were dehydrated for 5min each, 200ml xylene was transparent for 10min, and 100μL neutral gum (Wuhan Seville Biotechnology Co., Ltd.) was used to seal the slides. The slides were scanned and observed by Leica Aperio AT2 slide scanner.

[0096] 3) Macrophage typing

[0097] The sections were baked at 60°C for 2 hours, dewaxed with 200 ml of xylene for 30 minutes, and rehydrated in gradients of 200 ml of absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 10 minutes each, and then washed with 200 ml of PBS (Wuhan Sevier Biotechnology Co., Ltd.) solution on a shaker for 10 minutes. Antigen retrieval solution (specifically: antigen retrieval solution (pepsin method), Fuzhou Maixin Biotechnology Development Co., Ltd.) 50 μL was used for antigen retrieval at room temperature for 30 minutes; 50 μL of 0.1% trition X-100 (Beijing Solarbio Science & Technology Co., Ltd.) was used for membrane permeabilization for 10 minutes; 5% goat serum (GIBCO, USA) was used for blocking at room temperature for 30 minutes. 30 μL of monoclonal anti-mouse CD80 / CD163 antibody (Abcam, USA) (1:200) was mixed and incubated at 4°C for 18 hours, and then 30 minutes of treatment was performed after mixing with a fluorescence secondary antibody (Abcam, USA) (1:400) in the 488 nm / 594 nm channel at room temperature; DAPI (4',6-diamidino-2-phenylindole) staining solution (Beijing Solarbio Science & Technology Co., Ltd.) was used for treatment at room temperature for 5 minutes, and then sealed with 100 μL of anti-quenching mounting medium (specifically: anti-fluorescence quenching mounting medium, Wuhan Sevier Biotechnology Co., Ltd.). Images were taken using a Zeiss LSM 980 super-resolution confocal microscope.

[0098] 4. Experimental Results

[0099] 1) HE staining

[0100] The results are shown in Figure 4 and Figure 5 As shown, after sealing the medicine for 3 days, HE staining showed that in the inflammation group, a large number of inflammatory cells infiltrated the coronal pulp, partial pulp necrosis occurred, and abscesses formed. A large number of inflammatory cells infiltrated the coronal pulp in the BP group. Inflammatory cell infiltration was seen in the coronal pulp of the nisin + ciprofloxacin group. A large number of inflammatory cells infiltrated the coronal pulp in the Tα-1 group, and a small number of abscesses formed. A small number of scattered inflammatory cells infiltrated the coronal pulp in the Tα-1 + nisin + ciprofloxacin group. Compared with other medicated groups, the ciprofloxacin + nisin + Tα1 group significantly reduced the infiltration of inflammatory cells in the pulp tissue.

[0101] After sealing the medicine for 7 days, HE staining showed that in the inflammation group, a large number of inflammatory cells infiltrated the coronal pulp, partial pulp necrosis occurred, and abscesses formed. Inflammatory cell infiltration was seen in the coronal pulp of the BP group. A large number of inflammatory cells infiltrated the coronal pulp in the Tα-1 group, and a small number of abscesses formed. A small number of scattered inflammatory cells infiltrated the coronal pulp in the Tα-1 + nisin + ciprofloxacin group, and a small amount of reparative dentin formation was observed. Compared with other medicated groups, the ciprofloxacin + nisin + Tα1 group significantly reduced the infiltration of inflammatory cells in the pulp tissue and promoted the formation of reparative dentin.

[0102] 2) Immunohistochemistry

[0103] The results are shown in Figure 6As shown, after medicament sealing for 3 days, the inflammation group expressed a relatively high level of Nlrp3. The Nlrp3 levels in the BP group, the nisin + ciprofloxacin group, the Tα-1 group, and the Tα-1 + nisin + ciprofloxacin group all decreased to varying degrees; among them, the decrease in the Tα-1 group and the Tα-1 + nisin + ciprofloxacin group was more obvious.

[0104] After medicament sealing for 7 days, the inflammation group expressed a relatively high level of Nlrp3. The Nlrp3 levels in the BP group, the Tα-1 group, and the Tα-1 + nisin + ciprofloxacin group all decreased to varying degrees; among them, the decrease in the Tα-1 + nisin + ciprofloxacin group was more obvious.

[0105] 3) Immunofluorescence

[0106] The immunofluorescence results of CD80 (M1) / CD163 (M2) are shown in Figure 7 and 8 As shown, after medicament sealing for 3 days, the macrophages in the dental pulp of the inflammation group and the 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. The Tα-1 group and the Tα-1 + nisin + ciprofloxacin group were mainly anti-inflammatory M2 macrophages.

[0107] After medicament sealing for 7 days, 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. 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 reduced the infiltration of inflammatory cells in the dental pulp tissue, restored the dental pulp tissue, and had the best therapeutic effect on pulpitis.

[0110] Obviously, the above-mentioned embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. A pharmaceutical composition, characterized in that, It includes thymosin α1, nisin and ciprofloxacin.

2. The pharmaceutical composition according to claim 1, wherein In the said pharmaceutical composition, the mass ratio of thymosin α1, nisin and ciprofloxacin is 1 - 4:100 - 400:0.5 - 2.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, In the said pharmaceutical composition, the mass ratio of thymosin α1, nisin and ciprofloxacin is 1:200:

2.

4. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a medicament for preventing or treating pulpitis.

5. The use according to claim 4, characterized in that, The said medicament can promote macrophages to clear intracellular bacteria and / or kill the pathogenic bacteria of pulpitis.

6. The use according to claim 4 or 5, characterized in that, The said medicament can promote the formation of reparative dentin.

7. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a medicament for inhibiting inflammatory reaction and / or antibacterial.

8. A medicament for preventing or treating pulpitis, which comprises the pharmaceutical composition according to any one of claims 1 - 3, and further comprises one or more pharmaceutically acceptable excipients.

9. The medicament for preventing or treating pulpitis according to claim 8, wherein, The dosage form of the said medicament is selected from tablets, capsules, pills, solutions, powders, granules, suspensions, gels, ointments, sprays, patches or granules.

10. The drug for preventing or treating pulpitis according to claim 8 or 9, characterized in that, The said thymosin α1, nisin and ciprofloxacin are respectively in different preparation units, or three or any two of the said thymosin α1, nisin and ciprofloxacin are in the same preparation unit.

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