Construction method and application of stomach fire flaming type chronic periodontitis animal model

Abstract

The invention discloses a construction method and application of a stomach fire flaming type chronic periodontitis animal model, and belongs to the technical field of animal model construction. The construction method comprises the following steps: S1, lavaging a rat with capsaicin liquid once a day, continuously lavaging for 13-15 days, and carrying out stomach fire flaming syndrome modeling; s2, on the basis of the stomach fire flaming type model established in the step S1, establishing a stomach fire flaming type chronic periodontitis animal model by using a periodontal ligation method; the concentration of the capsaicin liquid is 0.8 to 1.2 g / L. The animal model of the stomach fire flaming type chronic periodontitis is mainly applied to drug effect evaluation and mechanism discussion animal experiments of the stomach fire flaming type chronic periodontitis, and fills the blank of deficiency of a disease and symptom combination animal model of the chronic periodontitis and the stomach fire flaming type chronic periodontitis. Experiments can use multi-dimensional signs and molecular detection as evaluation and identification means, and the method has the advantages of simple and convenient test operation, prominent symptom characteristics, real-time dynamic detection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and in particular relates to a method for constructing an animal model of gastric fire inflammation type chronic periodontitis and application thereof. Background Art

[0002] Periodontitis is a chronic, multifactorial inflammatory disease that is closely related to the accumulation of dental plaque, which gradually causes damage to the tooth-supporting structure. The main pathological manifestations are periodontal pocket formation and alveolar bone loss. At present, the clinical efficacy of chronic periodontitis often fails to meet expectations. Traditional Chinese medicine is based on traditional Chinese medicine theory, emphasizing "holistic concept" and "differential diagnosis and treatment", showing unique advantages. However, current mechanism research is mostly based on the induction of a simple chronic periodontitis model, which fails to highlight the advantages and characteristics of traditional Chinese medicine in combining prevention and treatment of diseases and syndromes.

[0003] The capsaicin compound ligation model is mainly used in animal experiments for the efficacy evaluation and mechanism exploration of chronic periodontitis with gastric fire inflammation. Experimenters can use multidimensional signs and molecular detection as evaluation and identification methods, which has the advantages of simple experimental operation, prominent disease characteristics, and real-time dynamic detection. At present, there is no animal model for chronic periodontitis with gastric fire inflammation. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for constructing an animal model of chronic periodontitis of gastric fire inflammation type, the method comprising the following steps:

[0005] S1. Rats were gavaged with capsaicin solution once a day for 13-15 consecutive days to establish a model of gastric fire inflammation syndrome;

[0006] S2. Based on the gastric fire inflammation model established in step S1, an animal model of chronic periodontitis of gastric fire inflammation type was established by periodontal ligation;

[0007] The concentration of the capsaicin solution is 0.8-1.2 g / L.

[0008] Preferably, the concentration of the capsaicin solution is 1 g / L.

[0009] More preferably, the intragastric administration in step S1 is continued for 14 days.

[0010] More preferably, the solvent in the capsaicin solution is 35-45% ethanol aqueous solution.

[0011] More preferably, the solvent in the capsaicin solution is 40% ethanol aqueous solution.

[0012] The second purpose of the present invention is to provide the application of the animal model produced as above in simulating chronic periodontitis of gastric fire inflammation type in animals.

[0013] The third purpose of the present invention is to provide the application of the above-produced animal model in the pathological research of chronic periodontitis of gastric fire inflammation type.

[0014] A fourth object of the present invention is to provide a method for screening one or more candidate substances using the animal model generated as described above, the method comprising administering the one or more candidate substances to the animal model.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The capsaicin compound ligation model provided by the present invention is mainly used in animal experiments for the efficacy evaluation and mechanism exploration of chronic periodontitis with gastric fire inflammation. Experimenters can use multi-dimensional signs and molecular detection as evaluation and identification means, which has the advantages of simple experimental operation, prominent disease characteristics, and real-time dynamic detection. At present, there is no disease-syndrome combined animal model for chronic periodontitis with gastric fire inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Comparison of urine color between the blank control group and the gastric fire inflammation group after intragastric administration of the capsaicin solution in Example 1 for 2 weeks.

[0018] Figure 2 HE staining of the stomach of rats in Example 1, (A) blank control group (×100), (B) blank control group (×200), (C) gastric fire inflammation group (×100), (D) gastric fire inflammation group (×200).

[0019] Figure 3 The gingival conditions 12 weeks after ligation in Example 1, (A) blank control group, (B) periodontitis group (periodontitis non-gastric fire inflammation group (non-gastric fire group) and periodontitis gastric fire inflammation group (gastric fire group)).

[0020] Figure 4 This is a Micro-CT image of the alveolar bone defect in Example 1. DETAILED DESCRIPTION

[0021] Example 1

[0022] This embodiment provides a method for constructing and identifying an animal model of chronic periodontitis of gastric fire inflammation type, comprising the following steps:

[0023] S1. SPF healthy SD male rats were selected, aged between 6 and 8 weeks, weighing 250±20g. The ambient temperature was maintained at 23±2℃, the relative humidity was 60±5%, and the day-night cycle was maintained according to the 12-hour light and dark light treatment. They were housed in cages according to groups, and sufficient food and water were provided throughout the experiment, and the rats could eat and drink freely.

[0024] S2. After one week of adaptive feeding, the rats were randomly divided into: ① blank control group, ② periodontitis non-gastric fire inflammation group, and ③ periodontitis gastric fire inflammation group according to their body weight.

[0025] S3. The periodontitis group was gavaged with capsaicin solution once a day for 14 days to establish the gastric fire inflammation syndrome model. The control group was gavaged with an equal volume of carrier solution without capsaicin.

[0026] S31. Prepare a solution containing 1g / L capsaicin and 40% ethanol. Since capsaicin is not easily soluble in water, it is first dissolved in 200ml of anhydrous ethanol, and then pure water is added to make the volume 500ml, so that the mass concentration of capsaicin reaches 1g / L. The control group is prepared with a 40% ethanol solution in the same way. Rats are given a dose of 1ml of capsaicin per 100g of body weight by gavage every day, once a day, for 2 weeks.

[0027] S32. After two weeks, check the physical signs: the main symptoms include thirst and frequent drinking, and the daily water intake increases by more than 10%. The secondary symptoms include digestion and hunger, and the food intake increases by more than 10%; it also includes temperature rise, constipation and short yellow urine. The above symptoms are significantly different from those of the control group. If the above evaluation indicators are met, it can be determined that the rat model simulating human gastric fire inflammation syndrome has been successfully established.

[0028] After 14 days, the success of the model establishment was determined based on the animal's physical signs (see Table 1, Figure 1 , 2 ).

[0029] Table 1 Symptoms and signs of rats in the blank control group and the gastric fire inflammation group (n=5, x±s)

[0030]

[0031] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with before modeling, #P<0.05, ##P<0.01.

[0032] S4. The animals in the periodontitis group were subjected to periodontal ligation to establish a chronic periodontitis model. The control group was subjected to sham surgery (Tables 2 and 3, Figure 3 ).

[0033] S41. Rats were anesthetized with isoflurane gas, and then further anesthetized with intraperitoneal injection of sodium pentobarbital. Next, the ligation method was used to separate the gums of the left first molar of the rat using a spoon, and then a 3-0 surgical suture was ligated on the left first molar of the rat, and the suture was pushed toward the alveolar ridge until it was completely immersed in the gums. This periodontitis model was established for 3 months. The control group rats underwent sham surgery, and no other operations were performed on the rats after anesthesia.

[0034] S42. Evaluation of the establishment of a chronic periodontitis model based on probing depth (PD) and sulcus bleeding index (SBl). PD: The gingiva around the left maxillary first molar was examined using a periodontal probe. The examination area was divided into the buccal mesial, medial, distal and lingual gingival margins, and the scores of the four tooth surfaces were averaged. SBI: The examination was performed in the same manner as PD. The degree of visible inflammation was graded: 0 = healthy gums, no bleeding on probing; 1 = bleeding on probing, no edema and color change of the gingival papilla and marginal gingiva; 2 = bleeding on probing, color change of the gingival papilla and marginal gingiva, no edema; 3 = bleeding on probing, color change of the gingival papilla and marginal gingiva, mild edema; 4 = bleeding after probing, color change of the gingival papilla and marginal gingiva, obvious edema; 5 = bleeding on probing, spontaneous bleeding, color change and edema. There were significant differences between the evaluation indicators and the control group, which confirmed that a rat model simulating the clinical symptoms of human periodontitis has been established.

[0035] Table 2 PD and SBI observed 12 weeks after ligation (n=5, x±s)

[0036]

[0037] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0038] Table 3 Vertical distance between ABC and CEJ 12 weeks after ligation (n=3, x±s)

[0039]

[0040] Note: Compared with the blank control group, *P<0.05, **P<0.01.

[0041] S5. After treatment, the clinical indicators, alveolar bone resorption, bone density, cytokines, and periodontal pathological changes of rats in each group were evaluated to identify and evaluate the gastric fire inflammation type periodontitis model (Tables 4 and 5, Figure 4 ).

[0042] S51. Evaluate the physical signs of the rat as described in step S32.

[0043] S52. Detect PD and SBI scores as described in step S42.

[0044] S53. The method for evaluating alveolar bone resorption is micro-computed tomography (Micro-CT) images and bone density parameter analysis. The specific method is as follows. Under sodium pentobarbital anesthesia, rats were fixed by cardiac perfusion, and the left maxillary first molar and periodontal tissue were carefully dissected, and the excess tissue was removed and fixed in 4% paraformaldehyde. After 2 days, the bone samples were stored in 75% ethanol, and then washed in distilled water and soaked in 0.9% saline overnight for rehydration. The corresponding three-dimensional reconstructed images were observed by Micro-CT, which should include the distal root of the maxillary first molar and the mesial root of the maxillary second molar. The distance from the enamel-dentin junction (CEJ) to the alveolar bone crest (ABC) at three different locations (mesial, mesial, and distal) of each tooth was measured in the Micro-CT image, and the mean was calculated.

[0045] S54. The method for detecting the cytokine content in gingival crevicular fluid is as follows. After the gastric fire periodontitis rat model was established, the gingival crevicular fluid was extracted from the buccal side of the left maxillary first molar of each group of rats using high temperature and high pressure sterilized fingertips. Use sterilized tweezers to place each fingertip into the gingival sulcus for 30 seconds, and then place it in an EP tube. A total of 4 fingertips are placed in one EP tube. After taking the gingival crevicular fluid, immediately freeze it in a -80℃ refrigerator for subsequent experiments. Add 200μL of PBS to the ep tube containing the fingertips of gingival crevicular fluid, let it stand at room temperature for 1 hour, and then centrifuge it at 13000rpm for 15 minutes in a 4℃ environment to extract the supernatant. Use ELISA kits for Rat IL-1β, Rat IL-10 and Rat TNFα to determine the content of cytokines.

[0046] S55. Periodontal pathological changes were analyzed by HE staining as follows:

[0047] S551. Fixed tissue: Under sodium pentobarbital anesthesia, rats were fixed by cardiac perfusion using saline and 4% paraformaldehyde solution. Carefully remove the heart, liver, spleen, lung, kidney and stomach tissues of the rats, and immediately place these fresh tissues in 4% paraformaldehyde for more than 48 hours. Remove the alveolar bone of the left maxillary first molar, quickly place it in 4% paraformaldehyde for more than 48 hours, and then replace it with EDTA decalcification solution. Soak it for one month, and change the liquid 1-2 times a week to soften it to be pierced by the probe. Remove the tissue from the fixative, place it in a fume hood, use a scalpel to trim the target tissue, and place the trimmed tissue and the corresponding label in the embedding frame.

[0048] S552. Place the dehydration box in the dehydrator in sequence and perform dehydration in the following order: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes. Then perform alcohol benzene treatment for 5-10 minutes, xylene I for 10 minutes, and xylene II for 10 minutes. Finally, melt the paraffin wax at 65°C for 1 hour each time.

[0049] S553. Paraffin embedding: Place the wax-soaked tissue in the embedding machine for embedding. Before the wax solidifies, take the tissue out of the dehydration box, place it in the embedding frame as required, and label it. After cooling in a -20℃ freezer, take out the solidified wax block and trim it.

[0050] S554. Paraffin section: Place the trimmed wax block into a paraffin slicer and set the cutting thickness to 4 μm. Float the slices in 40°C warm water to flatten the tissue, then pick them up with a glass slide and place them in a 60°C oven to dry. After drying, take out the slices and store them at room temperature for later use.

[0051] S555. Dewax the paraffin sections to water: Place the sections into environmentally friendly dewaxing liquid I (20 minutes), environmentally friendly dewaxing liquid II (20 minutes), anhydrous ethanol I (5 minutes), anhydrous ethanol II (5 minutes), and 75% alcohol (5 minutes) in sequence, and then rinse with tap water.

[0052] S556. Rewarming and fixing of frozen sections: Take out the frozen sections from the -20℃ refrigerator and let them return to room temperature. Then fix them with tissue fixative for 15 minutes, then rinse with running water. Place the sections in high-definition constant stain pretreatment solution for 1 minute.

[0053] S557. Hematoxylin staining: Soak the slices in hematoxylin stain for 3-5 minutes, then rinse with tap water. Perform differentiation treatment and rinse again. Finally, the slices are blued in blueing solution and then rinsed with running water.

[0054] S558. Eosin staining: Dehydrate the sections in 95% alcohol for 1 minute and then stain them in eosin solution for 15 seconds.

[0055] S559. Dehydration, sealing and image acquisition: Place the slices in the dehydration solution in order, namely, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, n-butanol I, n-butanol II, xylene I, and xylene II, and dehydrate for 2 minutes each. Then, place the slices in transparent neutral gum to seal the slices. Microscope inspection and image acquisition and analysis.

[0056] S56. Tartrate-resistant acid phosphatase (TRAP) staining was used to assess osteoclast activity in periodontitis as follows:

[0057] S561. Working solution preparation: Preparation of TRAP incubation solution

[0058] S562. Incubation and staining: circle the slices with a brush and place them in a humidified box. Incubate them with distilled water at 37°C for 2 hours. Pour off the distilled water, place the slices back in the humidified box, add filtered TRAP incubation solution, and incubate them in a 37°C oven for 20 minutes.

[0059] S563. Hematoxylin staining: Pour off the staining solution, wash with water, re-stain the nucleus with hematoxylin staining solution for 15 seconds, differentiate with differentiation solution, and re-blue with bluing solution.

[0060] S564. Dehydration and sealing: conventional dehydration, transparent, neutral gum sealing.

[0061] S565. Measure the number of TRAP-positive cells between the maxillary first molar and the maxillary second molar using a microscope.

[0062] Table 4 Clinical index observation results of each group (n=5, x±s)

[0063]

[0064] Note: Compared with the blank control group, *P<0.05; compared with non-gastric periodontitis, #P<0.05; compared with gastric periodontitis, ◇P<0.05.

[0065] Table 5 Analysis of vertical distance between ABC and CEJ in each group (n=3, x±s)

[0066]

[0067] Note: Compared with the blank control group, *P<0.05; compared with non-gastric periodontitis, #P<0.05; compared with gastric periodontitis,

[0068] ◇P<0.05.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an animal model of chronic periodontitis of gastric fire inflammation type, characterized in that: The construction method comprises the following steps: S1. Rats were gavaged with capsaicin solution once a day for 13-15 consecutive days to establish a model of gastric fire inflammation syndrome; S2. Based on the gastric fire inflammation model established in step S1, an animal model of chronic periodontitis of gastric fire inflammation type was established by periodontal ligation; The concentration of the capsaicin solution is 0.8-1.2 g / L.

2. The construction method according to claim 1, characterized in that: The concentration of the capsaicin solution is 1 g / L.

3. The construction method according to claim 2, characterized in that: In step S1, intragastric administration was continued for 14 days.

4. The construction method according to any one of claims 1 to 3, characterized in that: The solvent in the capsaicin solution is 35-45% ethanol aqueous solution.

5. The construction method according to claim 4, characterized in that: The solvent in the capsaicin solution is 40% ethanol aqueous solution.

6. Use of the animal model produced by any one of claims 1-3 or 5 in simulating chronic periodontitis of gastric fire inflammation type in animals.

7. Use of the animal model produced according to claim 4 in simulating chronic periodontitis of gastric fire inflammation type in animals.

8. Use of the animal model produced by any one of claims 1-3 or 5 in the study of the pathology of chronic periodontitis of gastric fire inflammation type.

9. Use of the animal model produced by claim 4 in the study of the pathology of chronic periodontitis of gastric fire inflammation type.

10. A method for screening one or more candidate substances using the animal model produced by any one of claims 1 to 3 or 5, characterized in that: The method includes administering the one or more candidate substances to the animal model.

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