New application of 8-aminoguanosine and injectable hydrogel prepared from 8-aminoguanosine

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to new application of 8-aminoguanosine and injectable hydrogel prepared from the 8-aminoguanosine. The invention finds that the compound 8-aminoguanosine has anti-inflammatory, anti-oxidation and anti-osteoclast effects. Furthermore, the injectable supramolecular hydrogel is constructed by using the 8-aminoguanosine, the 1, 4-benzene diboronic acid and the trihydroxymethyl aminomethane, and the hydrogel can inhibit the oxidative stress, the inflammatory phenotype and the osteoclast differentiation of the macrophages in vitro by releasing the 8-aminoguanosine. The hydrogel is applied to a mouse periodontitis model in a local injection mode, it is found that the hydrogel inhibits periodontitis infiltration and tissue damage, and the hydrogel has a good prospect in preparation of a periodontitis local treatment biological material.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a new use of 8-aminoguanosine and an injectable hydrogel prepared therefrom. Background Art

[0002] Periodontal disease is a chronic infectious disease of periodontal tissue caused by plaque microorganisms and is one of the most prevalent oral diseases worldwide. During the development of periodontitis, virulence factors and harmful metabolites produced by bacteria induce over-activation of the host immune system, thereby causing immune response disorders and oxidative stress, which ultimately lead to the destruction of periodontal tissues, followed by loosening and even loss of teeth. In the treatment of periodontitis, anti-inflammatory treatment targeting the host immune response has been considered an effective method to control the occurrence and progression of periodontal disease.

[0003] However, currently, the immunomodulatory drugs used clinically for periodontitis are mainly nonsteroidal anti-inflammatory drugs and antibiotics, which are usually applied to the whole body through oral and injection methods, and there are problems such as gastrointestinal reactions, allergic reactions, antibiotic resistance, and reduced effective drug concentrations caused by first-pass elimination in the liver. Therefore, the development of new immunomodulatory anti-inflammatory drugs and dosage forms with good biosafety is of great significance for the clinical treatment of periodontitis.

[0004] In order to avoid the side effects caused by systemic application of drugs, in the clinical treatment of periodontal disease, local delivery therapy is used in the form of a drug delivery system to deliver drugs directly to the subgingival or periodontal pocket to achieve therapeutic effects. It has become a common way to treat periodontal disease because of its characteristics of direct contact with periodontal tissues, maintaining high local drug concentrations through sustained release, prolonging the duration of action, and improving patient compliance. Among them, hydrogels are ideal drug delivery systems because of their similar structure and properties to the extracellular matrix. However, the particularity of the oral environment, including physiological activities such as gingival crevicular fluid flushing and chewing, makes drug-loaded hydrogels prone to dilution and drug leakage, affecting the efficacy of local treatment. Therefore, there are still challenges in developing hydrogels with high drug loading rates and stable drug encapsulation for the treatment of periodontitis.

[0005] Currently, there is a lack of injectable hydrogels with good injectability, stable drug encapsulation and high drug loading rate in the clinic, and their application in the preparation of periodontal anti-inflammatory local treatments that regulate host immune responses. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a new use of 8-aminoguanosine and an injectable hydrogel prepared therefrom.

[0007] The present invention provides use of 8-aminoguanosine in preparing a drug for anti-inflammation, anti-oxidation and / or anti-osteoclastogenesis.

[0008] Preferably, the drug is a drug for treating periodontitis.

[0009] The invention provides a pharmaceutical composition, which is prepared by taking 8-aminoguanosine as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0010] Preferably, the raw materials comprise the following molar parts:

[0011] 8-aminoguanosine 1.8-2.2 parts,

[0012] 0.675-1.375 parts of 1,4-phenylenediboronic acid,

[0013] 2.7-6.6 parts of tris(hydroxymethyl)aminomethane.

[0014] Preferably, the pharmaceutical composition is an injectable hydrogel comprising the following components in the following concentrations:

[0015] 8-aminoguanosine 0.025-0.15 mol / L,

[0016] 1,4-phenylenediboronic acid 0.008-0.1mol / L,

[0017] Tris(hydroxymethyl)aminomethane 0.03-0.5mol / L.

[0018] Preferably, the injectable hydrogel comprises the following components in the following concentrations:

[0019] 8-aminoguanosine 0.1 mol / L,

[0020] 1,4-phenylenediboronic acid 0.05 mol / L,

[0021] Tris(hydroxymethyl)aminomethane 0.15mol / L.

[0022] Preferably, the injectable hydrogel is prepared according to the following steps:

[0023] Mix 8-aminoguanosine, 1,4-phenylenediboronic acid and tris(hydroxymethyl)aminomethane) with water, heat until completely dissolved, and cool to obtain the product.

[0024] Preferably, the cooling is cooling to room temperature and standing for 5-30 minutes.

[0025] The present invention also provides a method for preparing the injectable hydrogel described in any one of the above items, which comprises the following steps:

[0026] Mix 8-aminoguanosine, 1,4-phenylenediboronic acid and tris(hydroxymethyl)aminomethane) with water, heat until completely dissolved, and cool to obtain the product.

[0027] The present invention also provides use of any of the above-mentioned pharmaceutical compositions in the preparation of drugs for anti-inflammatory and / or anti-oxidative and / or anti-osteoclastogenic effects.

[0028] The present invention found that the compound 8-aminoguanosine (8-AG) has anti-inflammatory, antioxidant and anti-osteoclast effects. Further, an injectable supramolecular hydrogel was constructed using 8-AG, 1,4-phenylenediboronic acid (PBA) and trishydroxymethylaminomethane (Tris), and the hydrogel can inhibit the oxidative stress, inflammatory phenotype and osteoclast differentiation of macrophages in vitro by releasing 8-AG. The hydrogel was applied to a mouse periodontitis model by local injection, and it was found that the hydrogel inhibited periodontal inflammatory infiltration and tissue destruction, showing good prospects for the preparation of biomaterials for local treatment of periodontitis.

[0029] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0030] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation of 8-AG-PBA hydrogel. a is a conceptual diagram of the preparation of 8-AG-PBA hydrogel. b is an image of the vial inversion experiment of 8-AG-PBA hydrogel (50mM) and an image after being placed at room temperature for 6 months. c is a rheological test of 8-AG-PBA hydrogel (50mM) on frequency scanning. d is a rheological test of 8-AG-PBA hydrogel (50mM) on amplitude scanning.

[0032] Figure 2 The shear-thinning injectability of 8-AG-PBA hydrogel. a is the rheological test result of 8-AG-PBA hydrogel (50mM) on self-healing. b is the rheological test result of 8-AG-PBA hydrogel (50mM) on shear thinning. c is the in vitro injection experimental image of 8-AG-PBA hydrogel (50mM). d is the subcutaneous injection experimental image of 8-AG-PBA hydrogel (50mM) in mice.

[0033] Figure 3Figure 8 shows the anti-oxidative stress, anti-inflammatory and anti-osteoclast effects of 8-AG and 8-AG-PBA hydrogels. a is the in vitro release image of 8-AG-PBA hydrogel (50mM). b is the in vitro release curve. c is a fluorescence image of RAW264.7 cells, LPS (final concentration of 500ng / mL) and 8-AG (final concentration of 1mM) or 8-AG-PBA hydrogel (final concentration of 500μM) after co-incubation for 24h, stained with ROS fluorescent dye (DCFH-DA). d is a statistical graph of ROS fluorescence intensity calculated by flow cytometry. e is a fluorescence image of iNOS protein expression obtained by immunofluorescence staining after co-incubation of RAW264.7 cells, LPS (final concentration of 500ng / mL) and 8-AG (final concentration of 1mM) or 8-AG-PBA hydrogel (final concentration of 500μM) for 24h. f is a graph of NO concentration in the culture medium measured by the Griess method. g is a fluorescence image of IL-6 protein expression obtained by immunofluorescence staining and enzyme-linked immunosorbent assay after RAW264.7 cells, LPS (final concentration of 500 ng / mL) and 8-AG (final concentration of 1 mM) or 8-AG-PBA hydrogel (final concentration of 500 μM) were co-incubated for 24 hours. f is a graph of IL-6 protein concentration in the culture medium. i is a representative image of osteoclasts characterized by tartrate-resistant acid phosphatase (TRAP) staining after RAW264.7 cells, RANKL (50 ng / mL) and 8-AG (final concentration of 1 mM) or 8-AG-PBA hydrogel (final concentration of 500 μM) were co-incubated for 6 days (medium was changed every other day). j is a statistical graph of the percentage of positive areas stained for tartrate-resistant acid phosphatase using ImageJ image processing software.

[0034] Figure 4 The in vivo application of 8-AG inhibits periodontitis tissue damage. a is a mouse periodontitis model treated with 8-AG-PBA hydrogel. The maxilla of 6-8 week-old C57BL / 6 mice was fixed after anesthesia, and silk thread (6 / 0) was used to ligature the bilateral second molars of the mice to induce periodontitis. 8-AG-PBA hydrogel (50mM) was injected locally into the second molar periodontium as the experimental group (8-AG-PBA hydrogel treatment group), PBS was used as the negative control (periodontitis group), and mice without periodontal ligature were used as blank controls (control group). b is a representative image of the mouse maxilla reconstructed by micro-CT three-dimensional reconstruction. c is a paraffin-embedded mouse maxilla sectioned after H&E staining. d is the statistics of vertical alveolar bone resorption (cementoenamel junction-alveolar crest distance) of the mouse maxilla analyzed by micro-CT and e is the statistics of alveolar bone mass (bone volume fraction) of the mouse maxilla analyzed by micro-CT. DETAILED DESCRIPTION

[0035] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0036] Example 1 An injectable hydrogel and its preparation method

[0037] like Figure 1 As shown in a, the injectable hydrogel of this embodiment is prepared by the following method:

[0038] 0.1 mmol 8-AG was dissolved in 1 mL deionized water, 0.05 mmol PBA and 0.15 mmol Tris were added, and after heating to completely dissolve the mixture, heating was stopped, the mixture was cooled to room temperature, and allowed to stand for 5-30 min to obtain 8-AG-PBA hydrogel.

[0039] Example 2 An injectable hydrogel and its preparation method

[0040] The preparation method described in Example 1 was followed, except that 8-AG was 0.025 mmol, PBA was 0.008 mmol, and Tris was 0.03 mmol.

[0041] Example 3 An injectable hydrogel and its preparation method

[0042] The preparation method described in Example 1 was followed, except that 8-AG was 0.15 mmol, PBA was 0.1 mmol, and Tris was 0.5 mmol.

[0043] The technical solution of the present invention is further described below through experiments. The samples tested in the following experimental examples are all prepared by the method of Example 1.

[0044] Experimental Example 1 Formation of 8-AG-PBA hydrogel

[0045] 1. Experimental Methods

[0046] 1. Vial inversion experiment

[0047] According to the preparation method of Example 1, a 200 μM 8-AG-PBA hydrogel was prepared in a transparent glass vial, and after the hydrogel was cooled and allowed to stand, the vial was inverted.

[0048] 2. Rheological test (amplitude sweep, frequency sweep)

[0049] 1 mL of 8-AG-PBA hydrogel was heated to form a clear transparent solution, transferred to a rotational rheometer test bench preheated to 65°C, cooled to 25°C to form a hydrogel, and tested using a 25 mm diameter plate. Amplitude scan: angular frequency (ω) was set to 1 rad / s, strain (γ) range: 0.1%-100%; frequency scan: γ = 0.1%, ω was 100 rad / s-0.1 rad / s, linear change. All tests were performed at 25°C.

[0050] 2. Experimental Results

[0051] The vial was inverted. Figure 1 As shown in b: After the vial was inverted, 8-AG-PBA did not flow down the vial wall, proving the formation of the hydrogel and remained stable after 6 months. The frequency sweep experiment results at a constant strain of 0.1% are shown in Figure 1 c: Throughout the measurement process, the storage modulus G' is always higher than the loss modulus G", indicating that the 8-AG-PBA hydrogel exhibits solid-like behavior within the applied frequency range. The strain sweep experiment is shown in Figure 1 As shown in (d), as the strain increases to about 6%, the behavior of 8-AG-PBA hydrogel changes from a quasi-solid state (G'>G") to a quasi-liquid state (G">G'), indicating that the supramolecular cross-linking in the hydrogel is destroyed, causing the originally stable hydrogel structure to transform into a sol.

[0052] The above results show that 8-AG, PBA and Tris can be used to prepare hydrogels and realize the transformation from gel to sol.

[0053] Experimental Example 2 Shear-thinning injectability of 8-AG-PBA hydrogel

[0054] 1. Experimental Methods

[0055] 1. Rheological tests (self-healing, shear thinning)

[0056] 1 mL of 8-AG-PBA hydrogel was heated to form a clear and transparent solution, which was transferred to a rotational rheometer test bench preheated to 65°C, cooled to 25°C to form a hydrogel, and tested using a 25 mm diameter plate. Self-healing test: ω = 1 rad / s, γ was set to 0.1% to keep the sample in a stable gel state, then the strain was rapidly increased from 0.1% to 100%, and after a certain period of time, the strain was reduced from 100% to 0.1%, and the cycle was repeated 3 times. Shear thinning test: Shear rate scanning was used to determine the fluidity of the hydrogel, ω = 1 rad / s, and the shear rate was gradually increased from 0.1 / s to 100 / s.

[0057] 2. In vitro injection experiment

[0058] Heat 1 mL of 8-AG-PBA hydrogel to form a clear and transparent solution, aspirate it into a 1 mL syringe, and let it stand at room temperature to form a gel. Inject the 8-AG-PBA hydrogel into a vial, and immediately invert the vial for observation. If the hydrogel does not flow down the vial wall, it can be considered that the hydrogel has been successfully injected.

[0059] 3. In vivo injection experiment

[0060] Female BALB / c mice aged 5-6 weeks were used for subcutaneous injection experiments. Six mice were kept in each cage. The mice were kept in an environment with a temperature of 22-26°C and a 12-hour day and night alternation. Water and food were provided for free drinking and eating. All mice were fed for 1 week before the experiment to adapt to the surrounding environment. After shaving the back of the mouse with a small animal shaver and depilatory cream, the pre-prepared 8-AG-PBA hydrogel was injected subcutaneously into the back of the mouse with a syringe, and 50 μL was injected into each mouse. The mice were killed by overdose of anesthesia at 0h, 24h, and 48h, respectively, the back skin was peeled off, and the injected hydrogel was observed.

[0061] 2. Experimental Results

[0062] Rheological test results are as follows Figure 2 As shown in Figures 2a and 2b: In three consecutive damage-repair cycles, 8-AG-PBA hydrogels stably and rapidly self-repaired with only a small loss of storage modulus, indicating that 8-AG-PBA hydrogels have good self-repair properties. When 8-AG-PBA hydrogels were squeezed out of the syringe, they changed from a gel state to a liquid-like state, which is consistent with the rheological test results, indicating that 8-AG-PBA hydrogels have shear-thinning properties. The results of the in vitro injection experiment are shown in Figure 2a. Figure 2 c: 8-AG-PBA hydrogel can be easily injected into the vial by a syringe. After the injection, the vial is immediately inverted and the hydrogel will not flow down the vial wall, which proves the rapid self-healing ability and good injectability of 8-AG-PBA hydrogel. Figure 2 As shown in d: The 8-AG-PBA hydrogel injected subcutaneously can be clearly observed with the naked eye. The volume of the hydrogel injected subcutaneously gradually decreases over time until it is completely unobservable after 48 hours.

[0063] The above results indicate that 8-AG-PBA hydrogel has rapid self-healing ability and good injectability, and can be injected into the body.

[0064] Experimental Example 3 In vitro anti-oxidative stress, anti-inflammatory and anti-osteoclast properties of 8-AG and 8-AG-PBA hydrogels

[0065] 1. Experimental Methods

[0066] 1. In vitro release experiment

[0067] Prepare 500 μL of 8-AG-PBA hydrogel in a vial, add 4.5 mL of PBS buffer, place in a 37°C 50 rpm shaker, extract 20 μL of supernatant at 0 h, 12 h, 24 h, 72 h, and 5 days, and store in a refrigerator at 4°C. The collected supernatant was diluted with 180 μL of methanol, and the concentration of 8-AG was determined by high performance liquid chromatography (HPLC). HPLC used a C18 reverse phase column, the mobile phase was 10 mM ammonium formate buffer containing 10% methanol, the flow rate was 1 mL / min, the temperature was 30°C, and the wavelength of the UV detector was 254 nm.

[0068] 2. Establishment of in vitro inflammation model

[0069] Mouse mononuclear macrophage leukemia cells (RAW264.7 cells) were used to establish an in vitro inflammatory model. RAW264.7 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum) overnight (37°C, 5% CO2), then replaced with fresh DMEM complete medium and stimulated with Escherichia coli lipopolysaccharide (LPS) at a final concentration of 500 ng / mL for 24 h (37°C, 5% CO2).

[0070] 3. Detection of reactive oxygen species in macrophages

[0071] The cells in the in vitro inflammation model were stimulated with LPS and 8-AG or 8-AG-PBA hydrogel at a final concentration of 1 mM or 500 μM was added to the cell culture medium as the treatment group.

[0072] (1) Detection of macrophage reactive oxygen species by DCFH-DA staining

[0073] After 24 h of LPS stimulation or 8-AG, 8-AG-PBA treatment, the cells were washed three times with PBS, and the DCFH-DA staining working solution was prepared using serum-free DMEM medium. The cells were added to the cells at a volume of 250 μL per well of a 24-well plate, incubated at 37°C for 1 h, and then the staining solution was removed and washed three times with PBS; the Hoechst 33342 staining working solution prepared in PBS was added (250 μL per well of a 24-well plate), incubated at 37°C for 10 min, and then the staining solution was removed. After washing twice with PBS, the cells were observed under a fluorescence microscope and images were collected.

[0074] (2) Semi-quantitative calculation of reactive oxygen species in macrophages by flow cytometry using DCFH-DA staining

[0075] After 24 hours of LPS stimulation or 8-AG, 8-AG-PBA treatment, the cells were collected into 1.5 mL centrifuge tubes by gently blowing with a pipette, and the supernatant was removed by centrifugation at 1500 rpm for 5 minutes. The cells were washed 3 times with PBS, and the DCFH-DA staining working solution was prepared using serum-free DMEM medium. The solution was added to the cells at a volume of 200 μL per centrifuge tube, incubated at 37°C for 1 hour, and then centrifuged to remove the dye solution, and washed 3 times with PBS; the cells were transferred into flow tubes, detected by flow cytometer, and the relative fluorescence intensity was calculated.

[0076] 4. Macrophage inflammatory activation detection

[0077] The cells in the in vitro inflammation model were stimulated with LPS and 8-AG or 8-AG-PBA hydrogel at a final concentration of 1 mM or 500 μM was added to the cell culture medium as the treatment group.

[0078] (1) Detection of iNOS expression in macrophages by immunofluorescence staining

[0079] After 24 hours of LPS stimulation or 8-AG or 8-AG-PBA treatment, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed 3 times with PBS; the membrane was permeabilized with 0.25% Tritonx-100 at room temperature for 15 minutes, and washed 3 times with PBS; after blocking with 10% FBS for 30 minutes, the cells were incubated with rabbit anti-mouse iNOS antibody (primary antibody) at 4°C overnight. The next day, the primary antibody was removed, and the cells were washed 3 times with PBS; the rabbit fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour, and washed 3 times with PBS; the DAPI was incubated at room temperature in the dark for 10 minutes, and washed once with PBS, and then observed and imaged under a fluorescence microscope.

[0080] (2) Detection of NO secretion by macrophages

[0081] After 24 hours of LPS stimulation or 8-AG, 8-AG-PBA treatment, 50 μL of cell culture medium was extracted, 50 μL of diazonium salt sulfonamide working solution was added, and then 50 μL of naphthyl vinyl diamine working solution was added, and the absorbance at 548 nm was immediately measured by microplate reader. The standard curve was drawn by detecting the 1mM, 500μM, 250μM, 125μM, 62.5μM, and 31.25μM standards obtained by multiple dilution with NaNO2 standard solution (1mM).

[0082] 5. Detection of macrophage inflammatory factor secretion

[0083] The cells in the in vitro inflammation model were stimulated with LPS and 8-AG or 8-AG-PBA hydrogel at a final concentration of 1 mM or 500 μM was added to the cell culture medium as the treatment group.

[0084] (1) Detection of IL-6 expression in macrophages by immunofluorescence staining

[0085] After 24 hours of LPS stimulation or 8-AG, 8-AG-PBA treatment, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed 3 times with PBS; the membrane was permeabilized with 0.25% Tritonx-100 at room temperature for 15 minutes, and washed 3 times with PBS; after blocking with 10% FBS for 30 minutes, the rabbit anti-mouse IL-6 antibody (primary antibody) was incubated at 4°C overnight. The next day, the primary antibody was removed, and the cells were washed 3 times with PBS; the rabbit fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour, and washed 3 times with PBS; the DAPI was incubated at room temperature in the dark for 10 minutes, and washed once with PBS, and then observed and collected under a fluorescence microscope.

[0086] (2) Detection of IL-6 secretion by macrophages

[0087] After 24 h of LPS stimulation or 8-AG or 8-AG-PBA treatment, 150 μL of cell culture medium was extracted and centrifuged at 2500 rpm at 4°C for 15 min. 100 μL of supernatant was extracted and the concentration of IL-6 in the culture medium was determined by an enzyme-linked immunosorbent assay kit.

[0088] 6. Macrophage osteoclast differentiation detection

[0089] RAW264.7 cells (mouse mononuclear macrophage leukemia cells) were used for in vitro osteoclast induction. RAW264.7 cells were cultured in DMEM complete medium (containing 10% fetal bovine serum) overnight (37°C, 5% CO2), then replaced with fresh α-MEM complete medium (containing 10% fetal bovine serum), and RANKL was added at a final concentration of 50 ng / mL, and cultured at 37°C, 5% CO2, and the medium was changed every other day. While RANKL was stimulated, 8-AG or 500 μM 8-AG-PBA hydrogel was added to the cell culture medium at a final concentration of 1 mM as the treatment group, and 8-AG or 8-AG-PBA hydrogel was updated with the medium change. On the 6th day, the culture medium was removed, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and washed 3 times with PBS; ethanol / acetone (50:50 v / v) was added, the membrane was broken for 1 min at -20°C, and washed twice with PBS; TRAP working solution was incubated at 37°C for 45 min, and washed twice with deionized water; methyl green dye was incubated at 37°C for 15 min, and washed twice with deionized water; multinucleated osteoclasts were observed under a microscope and images were collected. Osteoclasts were counted using ImageJ image processing software.

[0090] 2. Experimental Results

[0091] The results of in vitro release experiments were Figure 3As shown in a and 3b: At 37°C, 8-AG-PBA hydrogel incubated in PBS continuously releases 8-AG, with about 40% released at the 120h time point. Figure 3 c, 3d show: 8-AG and 8-AG-PBA hydrogels can effectively inhibit LPS-induced oxidative stress. Nitric oxide synthase (iNOS) and its product nitric oxide (NO) are markers of LPS-induced macrophage inflammatory activation. The results are shown in Figure 3d. Figure 3 e, 3f: 8-AG and 8-AG-PBA hydrogels have an inhibitory effect on LPS-induced iNOS overexpression and the production of product NO. At the same time, LPS stimulation causes macrophages to secrete the inflammatory factor interleukin-6 (IL-6). Figure 3 As shown in g and 3h, 8-AG and 8-AG-PBA hydrogels have an inhibitory effect on the secretion of IL-6 by macrophages. In addition, receptor activator of nuclear factor κB ligand (RANKL) is an important factor in regulating osteoclastogenesis and function. Figure 3 As shown in i and 3j: 8-AG and 8-AG-PBA hydrogels significantly inhibited the differentiation of macrophages into osteoclasts.

[0092] The above results prove that 8-AG has an inhibitory effect on LPS-induced macrophage oxidative stress, inflammatory activation and inflammatory factor secretion; it also shows a significant inhibitory effect on macrophage differentiation into osteoclasts. 8-AG-PBA hydrogel can continuously release 8-AG, and also has anti-inflammatory, antioxidant and anti-osteoclast effects.

[0093] Experimental Example 4: Use of 8-AG-PBA to inhibit periodontitis tissue damage in vivo

[0094] 1. Experimental Methods

[0095] 1. Establishment of mouse periodontitis model

[0096] Six-week-old male C57BL / 6 mice were used to establish the periodontitis model. Six mice were kept in each cage. The mice were kept in an environment with a temperature of 22-26°C and a 12-hour day and night alternation. Water and food were provided for free drinking and eating. All mice were fed for 1 week before the experiment to adapt to the surrounding environment. After the mice were anesthetized by subcutaneous injection of atropine sulfate and intramuscular injection of Shutai, silk thread (6-0) was ligated at the neck of the maxillary second molar of the mice to establish the periodontitis model. Starting from the second day of silk thread ligation, for 9 consecutive days, 5 μL of 8-AG-PBA hydrogel (8-AG-PBA treatment group) or PBS (periodontitis group) was injected into the four periodontal sites (mesial gingival, distal gingival, mesial buccal, distal buccal) of the maxillary second molar of the mice by microinjection after isoflurane inhalation anesthesia every day. Mice without a periodontitis model were injected with PBS according to the above injection method as a control group. On the tenth day, mice were sacrificed by overdose of anesthesia, and maxillary bone samples were collected for subsequent analysis.

[0097] 2. Micro-CT analysis

[0098] Mouse maxillary bone samples were fixed with 4% paraformaldehyde for 24 hours and scanned with micro-CT at a high resolution of 145 mA and 55 kVp, with a scan every 10 μm. Three-dimensional reconstruction was performed using micro-CT corresponding software, and statistics were performed on vertical alveolar bone resorption (cementoenamel junction-alveolar crest distance) and alveolar bone mass (bone volume fraction).

[0099] 3. H&E staining

[0100] Mouse maxillary bone samples were fixed with 4% paraformaldehyde for 24 hours, decalcified with 10% EDTA solution for 30 days, dehydrated, paraffin-embedded, and sliced. After the paraffin sections were dewaxed with xylene and hydrated with gradient alcohol, they were stained with hematoxylin solution for 10 seconds and then rinsed with running water for 10 minutes. Then they were stained with eosin solution for 1 minute and rinsed for 30 seconds. Finally, after gradient alcohol dehydration, xylene was made transparent, the sections were sealed with neutral resin, and the images were scanned and analyzed with an optical microscope.

[0101] 2. Experimental Results

[0102] The results are as follows Figure 4 As shown: 8-AG-PBA hydrogel significantly inhibited the vertical alveolar bone loss and alveolar bone volume reduction caused by ligation through periodontal injection. In the untreated group, silk ligation caused severe immune cell infiltration and epithelial destruction, and the treatment of 8-AG-PBA hydrogel alleviated this symptom.

[0103] The above results indicate that 8-AG-PBA hydrogel was applied to the mouse periodontitis model by local injection to inhibit periodontal inflammatory infiltration and tissue destruction.

[0104] It can be seen from the above embodiments and experimental examples that the present invention found that compound 8-AG has anti-inflammatory, antioxidant and anti-osteoclast effects. Further, an injectable supramolecular hydrogel was constructed using 8-AG, PBA and Tris, and the hydrogel can inhibit the oxidative stress, inflammatory phenotype and osteoclast differentiation of macrophages in vitro by sustained release of 8-AG. The hydrogel was applied to a mouse periodontitis model by local injection, and it was found that the hydrogel inhibited periodontal inflammatory infiltration and tissue destruction, showing good prospects for the preparation of biomaterials for local treatment of periodontitis.

Claims

1. Use of 8-aminoguanosine in the preparation of drugs for anti-inflammatory and / or anti-oxidative and / or anti-osteoclast effects.

2. The use according to claim 1, characterized in that: The medicine is a medicine for treating periodontitis.

3. A pharmaceutical composition, characterized in that: The invention is prepared by taking 8-aminoguanosine as an active ingredient and adding pharmaceutically acceptable auxiliary materials or supplementary ingredients.

4. The pharmaceutical composition according to claim 3, characterized in that Contains the following raw materials in molar proportions: 8-aminoguanosine 1.8-2.2 parts, 0.675-1.375 parts of 1,4-phenylenediboronic acid, 2.7-6.6 parts of tris(hydroxymethyl)aminomethane.

5. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is an injectable hydrogel comprising the following components in the following concentrations: 8-aminoguanosine 0.025-0.15 mol / L, 1,4-phenylenediboronic acid 0.008-0.1mol / L, Tris(hydroxymethyl)aminomethane 0.03-0.5mol / L.

6. The pharmaceutical composition according to claim 5, characterized in that The injectable hydrogel comprises the following components in the following concentrations: 8-aminoguanosine 0.1 mol / L, 1,4-phenylenediboronic acid 0.05 mol / L, Tris(hydroxymethyl)aminomethane 0.15mol / L.

7. The pharmaceutical composition according to claim 5, characterized in that The injectable hydrogel is prepared according to the following steps: Mix 8-aminoguanosine, 1,4-phenylenediboronic acid and tris(hydroxymethyl)aminomethane) with water, heat until completely dissolved, and cool to obtain the product.

8. The pharmaceutical composition according to claim 7, characterized in that: The cooling is cooling to room temperature and standing for 5-30 minutes.

9. The method for preparing the pharmaceutical composition according to any one of claims 5 to 8, characterized in that: The steps include: Mix 8-aminoguanosine, 1,4-phenylenediboronic acid and tris(hydroxymethyl)aminomethane) with water, heat until completely dissolved, and cool to obtain the product.

10. Use of the pharmaceutical composition according to any one of claims 3 to 8 in the preparation of anti-inflammatory and / or anti-oxidative and / or anti-osteoclast drugs.

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