Application of artesunate in preparation of oral disease treatment drugs or oral care products

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of artesunate in preparation of oral disease treatment medicines or oral care products. The artesunate can promote migration of the dental pulp stem cells, promote osteogenesis / dentin differentiation of the dental pulp stem cells and promote repair and regeneration of inflammatory dental pulp. Research finds that artesunate can promote the mineralization process by reducing inflammatory cell infiltration, inducing DPSCs dentin / osteogenic differentiation and promoting expression of dentin matrix protein 1 (DMP1) and dentin salivary phosphoprotein (DSPP) related genes of odontogenic differentiation, so as to promote formation of a repairable dentin bridge and preserve dental pulp. Animal experiments also find that when artesunate is applied to treatment of inflammatory dental pulp, repair and regeneration of the inflammatory dental pulp can be effectively promoted, and a new way is provided for clinical live pulp treatment. The new application has a great application prospect in oral cavity marrow activating treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of artesunate in preparation of oral disease treatment drugs or oral care products. BACKGROUND

[0002] The health of dental pulp tissue plays an important role in the structure and function of teeth. On the one hand, the preservation of pulp activity can promote the continuous development of permanent teeth in the early stage; on the other hand, as a biological receptor, the pulp can produce a defensive response to pathological stimulation and induce dentin regeneration. For a perforation with a diameter less than 1 mm caused by mechanical or traumatic factors, direct pulp capping is often used in clinical practice to preserve the vital pulp. The success of direct pulp capping is closely related to the pulp capping material. Calcium hydroxide, MTA, iRoot BP and other pulp capping materials are often used in clinical practice, but so far, all the pulp capping materials used have various drawbacks. Long-term clinical studies have shown that the success rate of direct pulp capping is about 80% to 90%. Therefore, it is worth trying to find a new and better pulp capping material.

[0003] Artesunate (ART) is a semi-synthetic derivative of artemisinin. It is one of the most effective drugs for treating malaria in the world due to its high efficiency, low toxicity and non-resistance. More and more research reports at home and abroad show that artesunate has various drug activities in addition to its anti-malarial activity. For example, artesunate has obvious anti-inflammatory effect. Some studies have found that artesunate protects against acute lung injury induced by LPS by inhibiting the expression of TLR4 and inducing the activation of Nrf2. In addition, artesunate has been confirmed to have antibacterial effect. In specific applications, some studies have shown that artesunate can be used in oral care products to prevent and treat periodontitis and gingivitis. For example, Chinese Patent Application No. CN202310562552.0, entitled "Gum-protecting and hemostatic toothpaste and preparation method thereof", and Chinese Patent Application No. CN201811538066.0, entitled "Toothpaste composition containing artesunate and preparation method thereof". However, before the present application, there has been no report on the application of artesunate in pulp capping agents. The present application has found that artesunate can promote the migration of dental pulp stem cells, promote the osteogenic / odontogenic differentiation of dental pulp stem cells, and promote the repair and regeneration of injured inflammatory dental pulp in an inflammatory microenvironment. Artesunate has great application prospects as a pulp capping agent, root canal flushing agent, root canal sealing agent, periodontal flushing and sealing agent in the treatment of oral diseases, especially in the treatment of vital pulp. SUMMARY

[0004] The present application aims at the above-mentioned problems, and provides an application of artesunate in preparation of a drug for treating oral diseases or an oral care product.

[0005] The present application adopts the following technical solutions:

[0006] The application of artesunate in preparation of a drug for treating oral diseases or an oral care product.

[0007] Further, the drug for treating oral diseases is a drug for treating vital pulp in oral cavity.

[0008] The application of artesunate in preparation of a drug for promoting migration of dental pulp stem cells or a product.

[0009] The application of artesunate in preparation of a drug for promoting osteogenic / odontogenic differentiation of dental pulp stem cells or a product.

[0010] The application of artesunate in preparation of a drug for promoting repair and regeneration of inflammatory dental pulp or a product.

[0011] The artesunate is prepared into a preparation for use; the preparation forms include tablets, injections, pastes, pastes, semi-solid and fluids.

[0012] Further, the drug is a dental pulp cap agent, a root canal flushing agent, a root canal sealing agent, a periodontal flushing and sealing agent.

[0013] Further, the oral care product is a mouthwash.

[0014] By adopting the above technical solutions, the present application has at least the following beneficial effects compared with the prior art:

[0015] The inventor team of the present application found in early research that artemether can treat SD rats with periodontitis associated with diabetes to some extent. Through continuous research by the inventor team, it was found that artemether can promote the formation of reparative dentin bridge, save the dental pulp by reducing inflammatory cell infiltration, inducing dental pulp stem cell odontoblast / osteogenic differentiation, promoting the expression of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) related to odontogenic differentiation, and promoting the mineralization process. In the present application, artemether has great application prospects in the preparation of oral treatment drugs, especially vital pulp treatment drugs. It can be used as an active ingredient in drugs such as dental pulp cap agents, root canal flushing agents, root canal sealing agents, and periodontal flushing and sealing agents. It can also be used as an active pharmaceutical ingredient in mouthwash. It has good biological safety, can promote inflammation relief and resolution by reducing inflammatory cell infiltration, and promote the formation of dentin-pulp complex, thereby saving the vitality of dental pulp. It has an "anti-inflammatory and regenerative" dual functional mechanism, and the effect is remarkable. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the experimental result graph of experiment one CCK-8 experiment of the present application;

[0017] Figure 2 is the experimental result graph of experiment two scratch experiment of the present application;

[0018] Figure 3 is the experimental result graph of experiment three qRT-PCR experiment of the present application;

[0019] Figure 4 is the experimental result graph of experiment four WB experiment of the present application;

[0020] Figure 5 is the experimental result graph of experiment five animal experiment Micro-CT of the present application;

[0021] Figure 6 is the experimental result graph of experiment six animal experiment HE staining experiment of the present application. DETAILED DESCRIPTION

[0022] The present application will be further described below in combination with examples and experiments.

[0023] EMBODIMENT

[0024] Application of artemether in the preparation of oral disease treatment drugs or oral care products. Specifically, it can be applied to oral vital pulp treatment drugs.

[0025] In terms of drug efficacy, artemether can promote the osteogenic / odontogenic differentiation of dental pulp stem cells.

[0026] In the efficacy of the drug, the artesunate can promote the repair and regeneration of inflammatory dental pulp.

[0027] In a specific application, the artesunate can be prepared into a pharmaceutical preparation for use; the pharmaceutical preparation forms are tablets, injections, pastes, pastes, semi-solid and fluids, of course, not limited to these several pharmaceutical preparation forms, and others.

[0028] The specific drug name can be dental pulp agent, root canal flushing agent, root canal sealing agent, periodontal flushing and sealing agent, etc.

[0029] The specific oral care product can be a mouthwash.

[0030] In order to prove the effect and mechanism of artesunate for oral disease treatment drugs or oral care products, the present application provides the following experimental evidence:

[0031] Experiment one: CCK-8 experiment

[0032] Experimental materials: cck-8 kit, P3 generation of human dental pulp stem cells (hDPSC) cultured by tissue block method.

[0033] Experimental method:

[0034] 1. Use 96-well plates, 4000 cells in each well, and culture for 24 hours.

[0035] 2. Grouping

[0036] (1) blank control group;

[0037] (2) 0.1 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM different concentrations of artesunate were added, and a total of 7 experimental groups.

[0038] 3. After 1 day, 3 days, 5 days and 7 days of culture, add CCK-8 liquid, and incubate in the dark for 2 h.

[0039] 4. The enzyme label instrument detects the absorbance value of each well at 450 nm wavelength, and determines the effect of culture medium containing different concentrations of artesunate on the proliferation of hDPSCs.

[0040] The experimental results are shown in Figure 1 , and it is found that 1 μM artesunate has no toxic effect on dental pulp stem cells.

[0041] Experiment two: scratch test

[0042] Experimental materials: DMEM culture medium, serum, double antibody, 6-well plate.

[0043] Experimental method:

[0044] (1) Markers and ruler draw 3 equidistant parallel horizontal lines on the bottom of the well plate as the fixed point mark for the photo.

[0045] (2) hDPSCs were inoculated into 6-well plates and cultured in an incubator. When they grew to about 90%, they were replaced with high-sugar DMEM medium without serum and starved for 24 hours.

[0046] (3) Make a scratch, wash with PBS for 3 times, and then add different conditions of 2% FBS-containing medium according to the experimental grouping.

[0047] (4) Take out the well plate at 0h and 24h respectively, observe under an inverted microscope, and take pictures. Record 6 fields of view for each group.

[0048] (5) Use Image J software to measure the scratch area of each group at different time points. Cell migration rate = (0h scratch area-24h scratch area) / 0h scratch area x 100%, and use t test for statistical analysis of the results.

[0049] The experimental results are shown in Figure 2 , Figure 2 , where A is the scratch test graph (i.e. the actual experimental result graph), and B is the statistical analysis graph made according to graph A. It can be seen from Figure 2 that artemether can promote the migration of dental pulp stem cells in an inflammatory microenvironment.

[0050] Experiment three: qRT-PCR

[0051] Experimental materials: DEPC water, Trizol reagent, HiScript III qRT SuperMix for qPCR, 2x premixed real-time fluorescence quantitative rapid PCR reaction system, RT-PCR primer.

[0052] Experimental method:

[0053] Take the third generation of human dental pulp stem cells, inoculate them into 6-well plates at 2x10 5 cells per well, and replace the culture medium after the cells grow to 80%. Use complete medium containing 10% FBS to detect inflammation-related genes, and use osteogenic medium to detect dentin / osteogenic-related genes.

[0054] Inflammatory-related genes IL-1β, IL-6, IL-4, IL-10, TNF-α and TGF-β were detected at 1d and 3d. Osteogenic / dentinogenic-related genes DSPP and DMP1 were detected at 7d, 14d and 21d.

[0055] (1) Total RNA extraction: After the hDPSCs were cultured for the corresponding time, the six-well plates were taken out and the total RNA of the cells was extracted.

[0056] (2) Reverse transcription: Reverse transcription was performed using Ⅲ RT SuperMix for Qpcr(+gDNA wiper) kit. The resulting product can be used immediately for RT-PCR reaction or stored at -80°C for up to six months.

[0057] (3) Real-time quantitative PCR reaction: Real-time RT-PCR reaction was performed using ChamQ Universal SYBR qPCR Master Mix kit.

[0058] (4) The expression level of the gene was detected by the ΔCt method with the expression level of the internal reference β-actin as the standard. Each data was repeated three times, and the standard error was calculated.

[0059] The experimental results are shown in Figure 3 , artemether has anti-inflammatory effect, can up-regulate anti-inflammatory factors and down-regulate pro-inflammatory factors, in addition to being used as a dental pulp cap, it can also be applied to, such as the preparation of mouthwash, root canal irrigation fluid, root canal sealant, etc.; artemether can up-regulate DMP1 and DSPP under inflammatory microenvironment, thereby promoting the differentiation of human dental pulp stem cells into osteoblasts / dentinoblasts, indicating that it can promote the formation of reparative dentin as a dental pulp cap.

[0060] Experiment four: WB experiment

[0061] Experimental materials:

[0062] Rabbit anti-DMP1 antibody (Shanghai Abmart, China);

[0063] Rabbit anti-DSPP antibody (Shanghai Abmart, China);

[0064] Mouse anti-β-actin (Proteintech, China);

[0065] Goat anti-rabbit IgG (Thermo ScientificTM, USA);

[0066] Goat anti-mouse IgG (Thermo ScientificTM, USA).

[0067] Experimental method:

[0068] (1) Total protein extraction: According to the grouping (same as experiment three), the medium of experiment three was replaced with osteogenic induction medium. On the 21st day after osteogenic induction culture, total protein was extracted.

[0069] (2) Protein electrophoresis: prepare separation gel and concentration gel, and pour the gel and insert the comb. This process should avoid the formation of bubbles. After the gel solidifies, transfer it to the electrophoresis tank, load each well, and add pre-stained protein markers to the preset lanes. Set the voltage to 80V and electrophorese for about 30min. After the marker bands are aligned, change to constant voltage 120V and electrophorese for 60min. When the bromophenol blue reaches the bottom of the electrophoresis tank, stop the electrophoresis.

[0070] (3) Transfer membrane: assemble the transfer membrane clamp in the order of "black plate-sponge-gel-PVDF membrane-sponge-white plate", insert it into the transfer membrane slot, and add the transfer membrane solution. Transfer the membrane at a constant current of 400mA for 45min.

[0071] (4) Blocking: after the transfer is completed, remove the PVDF membrane, and shake it in TBST containing 5% skimmed milk at room temperature for 1-2h.

[0072] (5) Incubate the primary antibody: dilute the primary antibody according to the proportion: DSPP (1:3000), DMP1 (1:3000), β-catenin (1:1000), APC (1:1000), and β-actin (1:10000). After blocking, wash the PVDF membrane with TBST for 3 times, each for 5-10min, and then place it in the primary antibody at 4°C overnight.

[0073] (6) Incubate the secondary antibody: the next day, recover the primary antibody. Remove the PVDF membrane, wash it with TBST for 3 times, each for 10min, and then immerse it in the diluted secondary antibody. Incubate it on the shaker at room temperature for 1h, and then wash it with TBST for 3 times, each for 5-10min.

[0074] (7) Soak the PVDF membrane in the luminescent working solution for 30s, and then place the membrane in the imaging system for exposure and development to collect the image.

[0075] (8) Gray value analysis: use Image J software to measure the gray values of the target protein band and the corresponding internal reference protein band, and calculate the relative expression amount of the target protein.

[0076] The experimental results are shown in Figure 4 : The expression levels of DSPP and DMP1 dentinogenesis-related proteins in the inflammatory microenvironment treated with artesunate were significantly higher than those in the simple inflammatory environment group. DSPP reached a similar expression level to the control group (normal), and the expression amount of DMP1 was even higher than that of the control group. This indicates that artesunate has a promoting effect on osteogenic / odontogenic differentiation, and can promote the formation of reparative dentin as a dental pulp agent.

[0077] Experiment five: animal experiment-Micro-CT

[0078] (1) Experimental animal grouping: After 1 week of adaptive feeding, the bilateral first maxillary molars of SD rats were selected as experimental teeth. The inclusion criteria for experimental teeth were: complete crown, no dental and periodontal disease. Ten rats were included, of which, 4 were randomly selected as the normal group without any treatment. The remaining 6 rats randomly selected one side of the maxillary first molar as the experimental group, and the contralateral side as the control group.

[0079] (2) Model establishment: The experimental objects were fasted for 6 hours before anesthesia. Preoperative weight, intraperitoneal injection of 10% chloral hydrate with 0.3ml / 100g for general anesthesia. A 1 / 4 diamond drill was used to drill a hole on the mesial surface of the rat's first maxillary molar, with a diameter of about 1mm. Keep the physiological saline flowing to avoid overheating and negative pressure suction. When the near-pulp area is slightly pink, use a sterile probe to gently penetrate the pulp cavity and expand to 40#K file. Both before and after changing the file, the pulp opening needs to be flushed with physiological saline to remove dentin debris. A sterile cotton ball is gently pressed into the pulp hole to stop bleeding. After stopping bleeding, 10mg / ml LPS is injected into the cavity, soaked for 15 minutes. Then rinse with a large amount of physiological saline to remove LPS, and dry with a sterile paper tip. The experimental group uses a 0.5mm3 size of gelatin sponge as a carrier, which is immersed in artemether with a concentration of 1mg / ml. When the gelatin sponge is saturated, it is transported to the pulp penetration hole for direct pulp capping, and the top is covered with iRoot BP Plus for self-etching bonding, and the cavity is filled with flowable resin. The control group is to immerse the same volume of gelatin sponge in sterile physiological saline, and the rest of the method is the same as the experimental group.

[0080] (3) After modeling, the rats were sacrificed on the 21st day, and the maxilla was carefully separated and fixed in 4% paraformaldehyde solution for 24 hours. The micro-CT was taken at the appropriate time.

[0081] The experimental results are shown in Figure 5 , after 21 days of direct pulp capping with 1μM artemether, obvious reparative dentin bridge can be seen near the injured pulp, which can almost isolate the injured area from the normal pulp area. Through Mimics software, each experimental tooth was reconstructed in three dimensions, and the volume of newly formed reparative dentin was calculated. At the same observation time point, the volume of reparative dentin formed in the artemether group was much larger than that in the control group, and the difference was statistically significant (P<0.5). This shows that in vivo, artemether as a dental pulp capping agent can promote the formation of reparative dentin.

[0082] Experiment six animal experiment-HE staining

[0083] The animal modeling process and sample collection are the same as experiment five.

[0084] (1) Decalcification: After the maxillary bone specimen was fixed with 4% paraformaldehyde for 24 h, it was washed with running water for 24 h, and then was placed in EDTA decalcification solution, with the volume of the solution being about 20 times that of the specimen. The specimen was sealed and placed in a constant-temperature shaker for decalcification. The decalcification solution was replaced every 2-3 days. The degree of decalcification was observed every 3 days. When a needle could pass through the teeth without resistance, the decalcification was considered to be completed.

[0085] (2) Embedding: After the decalcification was completed, the specimen was washed with running water for 24 h, and then was subjected to gradient ethanol dehydration, xylene transparency, wax immersion, and paraffin embedding in sequence.

[0086] (3) Sectioning: The paraffin specimen was sectioned in a direction parallel to the tooth long axis, and the sectioning was performed in a mesial-distal direction. The thickness of the section was 4 μm. The section was baked in a 60°C oven and was stored at 4°C for standby use.

[0087] (4) De-waxing: The section stored at 4°C was re-baked for 60 min, and then was placed in xylene I for 20 min and xylene II for 20 min.

[0088] (5) To water: The section was subjected to gradient alcohol to water in sequence: anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and running water for 5 min.

[0089] (6) Hematoxylin staining: The section was stained with hematoxylin staining solution for 3-5 min, was washed with tap water, was differentiated with differentiation solution, was washed with tap water, was returned to blue with return blue solution, and was washed with running water.

[0090] (7) Eosin staining: The section was dehydrated with 95% alcohol for 1 min, was stained with eosin staining solution for 15 s, and was washed with tap water.

[0091] (8) Dehydration and mounting: The section was dehydrated with 95% alcohol I for 1 min, 95% alcohol II for 1 min, anhydrous ethanol I for 1 min, anhydrous ethanol II for 1 min, xylene I for 2 min, and xylene II for 2 min, and was mounted with neutral balsam.

[0092] (9) Observation under an inverted microscope and image acquisition and analysis.

[0093] The experimental results are shown in Table 1. Figure 6 (N, neutrophil; RD, reparative dentin), and the histological performance at 21 days after the operation: In the Control group, there were still a large number of inflammatory cells, and the entire mesial root canal and coronal pulp was severely calcified. No normal pulp-like structure was observed in the mesial root canal. In the ART group, a dense reparative dentin structure was formed around the perforation hole, and the damage area was completely sealed. A single layer of arranged and neat odontoblasts was observed around the reparative dentin, and the remaining coronal pulp and root pulp were normal pulp tissue structures. The blood vessels in the mesial root canal were dilated and rich, which might provide strong circulation support for the pulp repair process.

[0094] This result fully shows that, in vivo, ART, as a dental pulp agent, not only can curb the excessive activation and amplification of inflammatory response, but also has the repair and regeneration ability of dentin-pulp complex, of course, the preparation of artemether mouthwash, root canal irrigation fluid, root canal sealant and other drugs or products has the same effect.

[0095] The research results of the above-mentioned experiments 1 to 6 of the present application show that 1 μM artemether has good biocompatibility for human dental pulp stem cells. Artemether reduces inflammatory cell infiltration, induces DPSCs dentin / osteogenic differentiation, promotes the expression of dentin-derived differentiation-related genes dentin matrix protein 1 (DMP1) and dentin sialoprotein (DSPP), promotes mineralization process, thereby promotes the formation of reparative dentin bridge and saves dental pulp. The animal experiment results show that the application of artemether to the treatment of inflammatory dental pulp can effectively promote the repair and regeneration of damaged inflammatory dental pulp, and provide a new way for the clinical treatment of vital pulp.

[0096] The above description is a detailed description of the preferred embodiments of the present application, but the embodiments are not intended to limit the scope of the patent application of the present application. Any equivalent changes or modifications made under the technical spirit of the present application should be included in the scope of the patent.

Claims

1. Application of artesunate in the preparation of oral disease treatment drugs or oral care products.

2. The application as described in claim 1, characterized in that, The oral disease treatment drug is an oral pulp therapy drug.

3. The application as described in claim 1, characterized in that, The use of artesunate in the preparation of drugs or products that promote the migration of dental pulp stem cells.

4. The application as described in claim 1, characterized in that, The application of artesunate in the preparation of drugs or products that promote osteogenic / odontogenic differentiation of dental pulp stem cells.

5. The application as described in claim 1, characterized in that, The application of artesunate in the preparation of pharmaceuticals or products that promote the repair and regeneration of inflammatory dental pulp.

6. The application as described in any one of claims 1-5, characterized in that, Artesunate is prepared into formulations for use; the formulations include tablets, injections, ointments, pastes, semi-solids, and fluids.

7. The application as described in any one of claims 1-5, characterized in that, The medications mentioned are pulp capping agents, root canal irrigators, root canal sealing agents, and periodontal irrigators and sealing agents.

8. The application as described in any one of claims 1-5, characterized in that, The oral care product mentioned is a mouthwash.

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