CN116327762A - Application of coptisine hydrochloride in preparation of medicines for preventing and/or treating periodontitis - Google Patents
Application of coptisine hydrochloride in preparation of medicines for preventing and/or treating periodontitis Download PDFInfo
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- CN116327762A CN116327762A CN202310192682.XA CN202310192682A CN116327762A CN 116327762 A CN116327762 A CN 116327762A CN 202310192682 A CN202310192682 A CN 202310192682A CN 116327762 A CN116327762 A CN 116327762A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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Abstract
The invention provides an application of coptisine hydrochloride in preparing a medicine for preventing and/or treating periodontitis, and belongs to the field of biological medicines. The research of the invention discovers that the coptisine hydrochloride can effectively inhibit the burnt path mediated by macrophage NLRP3 inflammatory corpuscles and simultaneously can effectively inhibit the bone loss mediated by periodontitis, so that the coptisine hydrochloride can play a good role in preventing and/or treating periodontitis, is used for preparing medicines for preventing and/or treating periodontitis, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of coptisine hydrochloride in preparing medicines for preventing and/or treating periodontitis.
Background
The coptisine hydrochloride (Coptisine chloride) is an alkaloid of pale yellow needle-shaped crystals separated from coptis chinensis, and has the following structure:
the berberine hydrochloride has various biological activities, such as antibiosis and antiphlogosis, antimicrobial activity, gastric mucosa protection, anticancer, etc., and has stronger effect on resisting karst yeast than berberine, palmatine and africane stephanine. The coptisine hydrochloride has good medicinal value due to multiple effects. However, no research on the therapeutic effect of coptisine hydrochloride on periodontitis is seen.
Periodontitis (periodontis), also known as destructive periodontal disease, is a chronic inflammation caused by bacteria in dental plaque invading periodontal tissue, which can lead to destruction of periodontal supporting tissue (gums, periodontal membranes, alveolar bone and cementum), formation of periodontal pockets (small pockets formed by widening of gaps between teeth and gums), loss of adhesion, and resorption of alveolar bone, and gradual loosening of teeth as the course of disease progresses, and gingival recession can eventually lead to loss of teeth. The overall goal of periodontitis treatment is to control plaque, eliminate inflammation, prevent progression of disease and recurrence, and restore periodontal tissue function and physiological morphology. Therefore, the effect of general anti-inflammatory drugs on periodontitis is not ideal. Although the coptisine hydrochloride has a certain anti-inflammatory effect, whether the coptisine hydrochloride has a good treatment effect on periodontitis is unpredictable.
Disclosure of Invention
The invention aims to provide an application of coptisine hydrochloride in preparing medicines for preventing and/or treating periodontitis.
The invention provides an application of coptisine hydrochloride in preparing a medicine for preventing and/or treating periodontitis.
Further, the medicine is a medicine for inhibiting periodontitis bone resorption.
Further, the drug is a drug that inhibits alveolar bone resorption.
Further, the medicament is a medicament for inhibiting macrophage pyrosis.
Further, the drug is a drug that inhibits GSDMD cleavage;
and/or the drug is a drug that inhibits NLRP3 inflammatory body activation.
Further, the medicine is a preparation prepared by taking coptisine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;
preferably, the formulation is an injectable formulation.
Further, the injection preparation is a gingival local injection preparation;
preferably, the gingival local injection preparation is an injection preparation for local injection of the gingival on the palate side of a tooth of a patient suffering from periodontitis.
Further, when the coptisine hydrochloride injection preparation is used for treating periodontitis, the injection dose of a human is 1-2 mg/ml;
preferably, the injected dose in humans is 1.85mg/ml.
The invention also provides a medicine for preventing and/or treating periodontitis, which is a preparation prepared by taking coptisine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;
preferably, the formulation is an injectable formulation.
Further, the injection preparation is a gingival local injection preparation;
preferably, the gingival local injection preparation is an injection preparation for local injection of the gingival on the palate side of a tooth of a patient suffering from periodontitis;
more preferably, when the coptisine hydrochloride injection is used for treating periodontitis, the injection dose of the human is 1-2 mg/ml;
more preferably, the injected dose in humans is 1.85mg/ml.
Compared with the prior art, the invention has the beneficial effects that:
the research of the invention discovers that the coptisine hydrochloride can effectively inhibit the burnt path mediated by macrophage NLRP3 inflammatory corpuscles and simultaneously can effectively inhibit the bone loss mediated by periodontitis, so that the coptisine hydrochloride can play a good role in preventing and/or treating periodontitis, is used for preparing medicines for preventing and/or treating periodontitis, and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
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FIG. 1 shows the effect of local injection of coptisine hydrochloride on periodontitis bone resorption in mice.
FIG. 2 shows the result of the inhibitory effect of coptisine hydrochloride on the scorch of iBMDM cells.
Detailed Description
The materials and equipment used in the present invention are known products and are obtained by purchasing commercially available products, unless otherwise specified.
EXAMPLE 1 in vivo treatment of periodontitis with coptisine hydrochloride
1. Experimental method
1.1 modeling
6-8 weeks C57/BL6 mice were purchased from Chengdu Biotech, and after about one week adaptation, a model of mouse periodontitis was constructed by wire ligation. The model is a recognized model for periodontitis research in the field, and comprises the following specific operation steps: the 6-0 operation silk thread is used, silk threads are respectively put into the mouth of the mouse from the far middle gap of the first molar and the near middle gap of the third molar of the upper jaw of the mouse, the mouth of the mouse is confirmed to be pressed below the contact point, the mouth of the mouse is knotted at the palate side of the second molar, and the situation of the silk threads in the mouth of the mouse is checked every other day, so that the silk threads are ensured not to fall off halfway. After 7 days of ligation, the wire was removed and the modeling was determined to be successful by micro-CT scanning analysis.
1.2Micro-CT verifies success of periodontitis molding
Checking the condition of the mouth of the mouse every other day during the molding process, ensuring that the silk thread is not fallen off and knotted tightly, wherein local soft scale accumulation of the silk thread can be generally seen, and the situation of red and swollen gum around the affected teeth can be discharged from the research queue if the conditions of loose knots, silk thread falling off and the like occur. After 7 days of wire ligation induction, mice were sacrificed, maxillary samples were collected, 4% paraformaldehyde was fixed for 24 hours, micro-CT scan was performed to identify alveolar bone area as ROI (region of interest) for 3D reconstruction, and the distances (CEJ-ABC) from the cementum boundary (cementoenamel junc-tion) to the alveolar ridge crest (alveolar bone crest) and bone absorption areas (bone loss area) were statistically analyzed with ImageJ, and second molar and surrounding obvious alveolar bone absorption were seen, and modeling was successful.
1.3 local injection of coptisine hydrochloride-sodium chloride solution to intervene in periodontitis in mice
C57/BL6 mice were purchased from Chengdu Biotech for 6-8 weeks and were subjected to experiments after about one week of adaptation. Piptine hydrochloride (coptisine chloride) is purchased from Peprotech, CAS:6020-18-4, dissolved in DMSO, diluted in physiological saline, and prepared into a solution of Piptine hydrochloride-sodium chloride with a concentration of 20. Mu.M for use. The preparation method of the coptisine hydrochloride-sodium chloride solution comprises the following steps: 5mg of coptisine hydrochloride was dissolved in 0.702mL of DMSO and diluted 1000-fold to 20. Mu.M with physiological saline.
The mice were subjected to wire ligation according to the above 1.1 molding method, and the gum on the palate side of the patient was locally injected with coptisine hydrochloride-sodium chloride solution with an insulin needle once daily, 50 μl/time, starting from 3 days of wire ligation molding of the mice. After 7 days of modeling, the teeth Zhou Sixian are removed, and local treatment with coptisine hydrochloride-sodium chloride solution is continued for 7 days, and a control group is given with normal saline injection control. After the treatment is finished, the mice are killed by cervical spining, and a maxillary sample is collected for micro-CT analysis and section staining.
Grouping: A. sham surgery group (molding without thrombus, local injection of physiological saline at the same time, wherein the physiological saline contains 1%DMSO); B. physiological saline group (thrombosis moulding+injection physiological saline, the physiological saline used contains 1%DMSO); C. coptisine hydrochloride group (suppository molding + injection coptisine hydrochloride-sodium chloride solution). Each group was set with 6 replicates.
1.4 index detection
(1) Micro-CT: the detection method is the same as 1.2, a maxilla sample is collected, micro-CT scanning is carried out after 4% paraformaldehyde is fixed for 24 hours, an alveolar bone area ROI (region of interest) is identified for 3D reconstruction, and the distance (CEJ-ABC) from the cementum world (cementoenamel junction) to the crest of an alveolar ridge (alveolar bone crest) and the bone absorption area (bone loss area) are analyzed by using imageJ statistics.
(2) Staining of tissue sections:
the maxillary sample is subjected to decalcification and dehydration treatment, then is sectioned, the morphological difference of gingival tissues is observed by HE staining, CEJ-ABC is observed, and the secretion condition of inflammatory factors such as IL-1 beta and the like in periodontal local tissues is detected by immunofluorescence staining. The specific operation steps are as follows.
HE staining: dewaxing by xylene and gradient ethanol, hydrating tissue slices, staining by hematoxylin for 2min, and washing by PB for 3 times, each time for 5min; the ammonia water returns to blue. 0.5% eosin dye solution, transparent xylene, neutral resin sealing piece and observation under an optical microscope.
Immunofluorescent staining: xylene and ethanol dewax, hydrate tissue sections, an immunohistochemical pen circles around the tissue, tritonX-100 and BSA are closed for 1h at room temperature, primary antibody is incubated overnight at 4 ℃, PBS is washed for 5min for three times, secondary antibody is incubated for 1h at room temperature, PBS is washed for 5min for three times, DAPI is incubated for 5min, PBS is washed for 5min for three times, and fluorescent microscopy observation is carried out after water-based tablet sealing. The above detection methods are conventional in the art.
2. Experimental results
Fig. 1 shows the effect of the local injection of coptisine hydrochloride on the bone resorption of periodontitis of mice, and fig. 1 shows that: the maxillary dentition alveolar bone of the Sham surgery group (Sham) is complete and not obvious absorbed; a physiological saline group (Vehicle) exhibits an alveolar bone arc absorption centering on the second molar; the second molar of coptisine hydrochloride (COP) had slight bone resorption, which was significantly lighter than that of the normal saline group.
The results show that the invention can effectively inhibit alveolar bone absorption caused by periodontitis by using the coptisine hydrochloride for local administration, and has obvious treatment effect on periodontitis bone loss.
EXAMPLE 2 in vitro anti-inflammatory Studies of coptisine hydrochloride
1. Experimental method
The inhibition effect of coptisine hydrochloride with different concentrations on inflammatory reactions of mouse macrophage ibdm Jiao Wang and the like is verified.
1.1 cell culture and Induction
Immortalized mouse bone marrow derived macrophages (iBMDM) were cultured in DMEM supplemented with 10% fetal calf serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin, and placed at 37℃in 5% CO 2 Is provided. Before the induction of the pyrodeath model, 0, 5 mu M, 10 mu M and 20 mu M coptisine hydrochloride are respectively added into each component, the pretreatment is carried out for 24 hours, 1 mu M Lipopolysaccharide (LPS) is used for treating iBMDM cells for 5 hours, and then 50mg/ml adenine nucleoside triphosphate (ATP) is used for treating the cells for 30 minutes, so as to establish a macrophage pyrodeath cell model.
1.2 index detection
1)Western Blot
After the induction, the cell sample was lysed by RIPA containing 1% PMSF, left on ice for 5-10min, sonicated, centrifuged at 12000rpm at 4℃for 15min, and the supernatant was taken. Bradford row protein quantification. Protein samples were separated by electrophoresis on 10% SDS-PAGE polyacrylamide gel and transferred to PVDF membrane, (0.45 μm, millipore), 5% skim milk blocked at room temperature for 1h, primary antibody incubated overnight at 4 ℃, next day HRP-conjugated secondary antibody at room temperature for 1h, ECL development reagent exposure.
2) Immunofluorescence
ASC spot formation is one of the signs of inflammatory body assembly, and immunofluorescence staining of ASC can reflect the inhibition effect of coptisine hydrochloride with different concentrations on the activation of ibdm inflammatory body. Cells were inoculated in 24 well plate climbs, after the induction was completed, cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, permeabilized cells were incubated at 0.1% Triton X-100℃for 10 minutes, 5% BSA was blocked at room temperature for 1 hour, primary antibody was incubated overnight at 4℃and washed with PBS for 5 minutes three times, corresponding species were incubated at room temperature for 1 hour, washed with PBS for 5 minutes three times, stained with DAPI for 5-10 minutes at room temperature, washed with PBS for 5 minutes three times, water-based capper-capped patches were capped, and observed with a fluorescence confocal microscope.
2. Experimental results
FIG. 2 shows the result of the inhibition of the coke death of iBMDM cells by coptisine hydrochloride, as shown in FIG. 2: the coke apoptosis cell model is successfully established, and immunoblotting shows that the coptisine hydrochloride has concentration dependence on the NLRP3 expression and GSDMD shearing inhibition effect under the iBMDM coke apoptosis induction, and 20 mu M coptisine hydrochloride can effectively reduce the formation of GSDMD-N, thus showing that the coptisine hydrochloride has excellent inhibition effect on macrophage coke apoptosis. The immune fluorescent staining observes ASC spot formation, and can inhibit the inflammatory corpuscle assembly of iBMDM cells induced by LPS and ATP along with the increase of the concentration of coptisine hydrochloride, thereby proving that the coptisine hydrochloride plays a remarkable role in inhibiting the activation of inflammatory corpuscles of macrophages and the apoptosis of cell coke. The research of the invention finds that the coptisine hydrochloride can play a significant role in inhibiting NLRP3 inflammatory body activation, GSDMD shearing and cell apoptosis.
In conclusion, the research of the invention discovers that the coptisine hydrochloride can effectively inhibit the macrophage NLRP3 inflammatory body mediated pyrosis pathway and simultaneously can effectively inhibit periodontitis mediated bone loss, so that the coptisine hydrochloride can play a good role in preventing and/or treating periodontitis, and has a good application prospect in preparing medicines for preventing and/or treating periodontitis.
Claims (10)
1. Application of coptisine hydrochloride in preparing medicines for preventing and/or treating periodontitis.
2. Use according to claim 1, characterized in that: the medicine is a medicine for inhibiting periodontitis bone resorption.
3. Use according to claim 2, characterized in that: the medicine is a medicine for inhibiting alveolar bone absorption.
4. Use according to claim 1, characterized in that: the medicine is a medicine for inhibiting macrophage pyrosis.
5. Use according to claim 4, characterized in that: the medicine is used for inhibiting GSDMD shearing;
and/or the drug is a drug that inhibits NLRP3 inflammatory body activation.
6. Use according to any one of claims 1 to 5, characterized in that: the medicine is a preparation prepared by taking coptisine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;
preferably, the formulation is an injectable formulation.
7. Use according to claim 6, characterized in that: the injection preparation is a gingival local injection preparation;
preferably, the gingival local injection preparation is an injection preparation for local injection of the gingival on the palate side of a tooth of a patient suffering from periodontitis.
8. Use according to claim 7, characterized in that: when the coptisine hydrochloride injection preparation is used for treating periodontitis, the injection dose of a human is 1-2 mg/ml;
preferably, the injected dose in humans is 1.85mg/ml.
9. A medicament for preventing and/or treating periodontitis, characterized in that: the preparation is prepared by taking coptisine hydrochloride as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;
preferably, the formulation is an injectable formulation.
10. A medicament according to claim 9, characterized in that: the injection preparation is a gingival local injection preparation;
preferably, the gingival local injection preparation is an injection preparation for local injection of the gingival on the palate side of a tooth of a patient suffering from periodontitis;
more preferably, when the coptisine hydrochloride injection is used for treating periodontitis, the injection dose of the human is 1-2 mg/ml;
more preferably, the injected dose in humans is 1.85mg/ml.
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| CN202310192682.XA CN116327762A (en) | 2023-03-02 | 2023-03-02 | Application of coptisine hydrochloride in preparation of medicines for preventing and/or treating periodontitis |
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| CN202310192682.XA CN116327762A (en) | 2023-03-02 | 2023-03-02 | Application of coptisine hydrochloride in preparation of medicines for preventing and/or treating periodontitis |
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