CN117426352B - Construction method and application of an animal model of interstitial lung disease - Google Patents

Abstract

The invention provides a construction method of an animal model and application thereof, wherein periodontal pathogens are applied to induce a variable animal model of interstitial lung diseases, and further type II collagen and an adjuvant are applied to improve the severity of interstitial lung lesions. The pathological features of the animal model are highly similar to those of human beings, and better simulate the joint performance and lung performance of environmental factors (such as smoking patients and periodontitis patients) in RA-ILD.

Description

Construction method and application of an animal model of interstitial lung disease

Technical field

The invention relates to the field of biomedicine technology, and in particular to a construction method and application of an animal model of interstitial lung disease.

Background technique

Interstitial lung disease (ILD) is based on diffuse lung parenchyma, alveolar inflammation and interstitial fibrosis as the basic pathological lesions. It is characterized by active dyspnea, diffuse infiltration shadow on chest X-ray, and restrictive ventilation disorder. , reduced diffusion (DLCO) function and hypoxemia are a general term for clinical-pathological entities composed of different types of disease groups with clinical manifestations.

ILD is usually not malignant and is not currently thought to be caused by a known infectious agent. However, it is the most common extra-articular symptom of rheumatoid arthritis (RA), occurring approximately between 40 and 40 years old. For 80% of RA patients, the mortality rate is 10 to 20%, and the average survival time is 5 to 8 years. ILD has a very poor prognosis and is one of the main causes of death in RA patients.

The establishment of experimental animal models is an important basis for studying pathogenesis and developing innovative drugs. Although RA animal models such as collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AA) can very well simulate the clinical symptoms and pathological characteristics of arthritis, the incidence of ILD is low. Tracheal injection of bleomycin is currently the most common method for ILD. However, although this method is simple to operate and low-cost, the scope of lung lesions is relatively limited, which is different from the diffuse distribution of human lesions, and it will cause irreversible damage to animals. Organic damage. Therefore, it is necessary to establish an RA-ILD animal model that not only has RA characteristics, but also has a high incidence of ILD and significant ILD symptoms.

According to the existing literature, there are five animal models of RA-ILD: CIA, AA, collagen induction combined with bleomycin (CIA+BLM), transgenic mice, and transgenic mice combined with zymosan. but:

(1) The most used CIA model and AA model are commonly used animal models of RA, and the CIA model and AA model have a lower incidence of interstitial lung disease on the basis of RA.

(2) The range of lung lesions in CIA+BLM models (such as CN110604098A, CN116076438A) is relatively limited, which is different from the diffuse distribution of human lesions, and will cause irreversible organic damage to animals.

(3) Transgenic mice and transgenic mice combined with zymosan animal models take a long time to take shape and cannot fully replicate the lung pathological manifestations related to UIP (common interstitial pneumonia), the main type of RA-ILD.

In addition, environmental factors including smoking and periodontitis are recognized environmental triggers that can cause lung inflammation and are also key risk factors for the development of ILD in RA patients. It may induce the formation of autoantibodies in RA patients, associated with high titers of anti-cyclic citrullinated peptide (CCP) antibodies. None of the above animal models involve environmental factors, so the mechanisms of generation of the above models and RA-ILD are different.

Contents of the invention

In order to overcome the deficiencies in the prior art, the present invention provides an RA-ILD animal model that has RA characteristics, high ILD incidence and significant ILD symptoms, established through periodontal pathogen infection combined with type II collagen. specific:

A first aspect of the present invention provides a method for constructing an animal model. The construction method includes administering periodontal pathogens, and the animal model is an interstitial lung disease animal model.

Preferably, the periodontal pathogens include but are not limited to Porphyromonas gingivalis (Pg), Tannerella forsythia , Treponema denticola , Actinomycetemcomitans One or more of Actinobacillus actinomycetemcomitans , Fusobacterium nucleatum , Prevotella intermedia , Prevotella nigrescens or Actinomyces viscosus combination.

In a specific embodiment, the periodontal pathogen may be Porphyromonas gingivalis . Or Porphyromonas gingivalis and other periodontal pathogens (such as Tannerella forsythia ), Treponema denticola ( Treponema denticola ), Actinobacillus actinomycetemcomitans ( Actinobacillus actinomycetemcomitans ), One or a combination of two or more of Fusobacterium nucleatum , Prevotella intermedia , Prevotella nigrescens or Actinomyces viscosus ).

Preferably, the periodontal pathogen is administered once every 1-7 (eg, 1, 2, 3, 4, 5, 6, 7) days.

Preferably, the amount of periodontal pathogen applied per time is 1×10 ⁷ -1×10 ¹⁰ (for example, 1×10 ⁷ , 2×10 7 , 3×10 ⁷ , 4×10 ^{7 ,} 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 7 , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ^{8 ,} 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ ) CFU.

Preferably, the total number of days of administration is 45-60 (for example, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60) days.

Preferably, the periodontal pathogen administration method includes but is not limited to smear administration, oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection. Further preferably, the administration method The method of administration is smearing, more preferably oral smearing, and even more preferably smearing on the gingival margins of the upper and lower jaws.

Preferably, the construction method further includes administering type II collagen. Preferably, type II collagen is applied at least once, such as once, 2 times, 3 times or more than 4 times; preferably, the interval between two applications of type II collagen is 15-25 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) days.

The interval between the first application of periodontal pathogens and the first application of type II collagen is 8-20 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) days, further An interval of 14 days is preferred. Preferably, the interval between the first application of periodontal pathogens and the first application of type II collagen and adjuvant combination is 8-20 (for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ,20 days.

Further preferably, administration of an adjuvant is also included. It is further preferred to administer type II collagen in combination with an adjuvant. It is further preferred that the adjuvant is administered simultaneously with type II collagen. Preferably, the adjuvant includes but is not limited to one or a combination of two or more of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, alum, heat shock protein or cytokines.

In a specific embodiment, the construction method is to administer only periodontal pathogens, type II collagen and adjuvants.

Preferably, the construction method includes first applying periodontal pathogens and then applying type II collagen (preferably in combination with an adjuvant).

Preferably, the administration methods of the type II collagen and/or adjuvant include but are not limited to smear administration, oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection.

Preferably, the type II collagen and adjuvant are administered in equal volumes.

In a specific embodiment, the modeling method includes applying 1×10 ⁷ -1×10 ¹⁰ CFU (for example, 1×10 ⁷ -1×10 ¹⁰ CFU/mL Pg of 2% CMC in PBS solution 10-1000 μL) of Porphyromonas gingivalis ( Porphyromonas gingivalis ), continued until the end of modeling; type II was used on days 8-20 and 30-40 days respectively. Immunization with a combination of collagen and adjuvant (equal volumes of type II collagen and adjuvant).

Preferably, the animal model is an animal model of arthritis combined with interstitial lung disease, and further preferably, the arthritis is rheumatoid arthritis.

Preferably, the animal model can be any mammal other than humans, such as horses, sheep, pigs, goats, camels, antelopes, dogs or rodents such as mice, rats, guinea pigs and hamsters. Rodents such as mice, rats, guinea pigs, and hamsters are particularly preferred.

Preferably, the animal model is an immune-susceptible rat or mouse.

In a specific embodiment, mouse species include, but are not limited to: ICR or DDY; BALB/cA, C57BL/6N, C3H/HeN, DBA/1J, DBA/2N or CBA/N; BDF1 (C57BL/6 x DBA /2), CDF1 (CBA/N x DBA/2) or B6C3F1 (C57BL6 x C3H/HeN); anything used as an experimental mouse can be used, such as BALB/c-nu or C,B- belonging to the mutant system 17SCID.

Preferably, the construction method also includes administering antibiotics before applying periodontal pathogens. Further preferably, the antibiotics include but are not limited to ampicillin, vancomycin, neomycin, metronidazole or tetracycline.

A second aspect of the present invention provides an animal model obtained by the above-mentioned construction method.

Preferably, the animal model exhibits symptoms of joint swelling, synovial inflammation, pannus, cartilage destruction, and bone destruction accompanied by alveolitis lesions, interstitial inflammatory cell infiltration, and alveolar wall thickening.

The third aspect of the present invention provides the above-mentioned construction method to obtain an animal model or the application of the above-mentioned animal model in screening or preparing drugs for treating interstitial lung disease or arthritis combined with interstitial lung disease.

This application uses the periodontal pathogen Porphyromonas gingivalis (Pg) combined with type II collagen-induced arthritis (Pg+CIA) to establish an RA-ILD animal model, and uses mouse arthritis index scores and HE staining of knee joints , anti-CCP antibody detection and immunoglobulin IgG results jointly confirmed the occurrence of RA. At the same time, the incidence and incidence of mice were counted, and the occurrence of ILD was confirmed by HE staining and Masson staining of mouse tissues. Compared with previous animal models of RA-ILD, the animal model of this application has the following advantages:

(1) The Pg+CIA model has a high incidence of ILD.

(2) Since Pg is the main risk factor for RA caused by periodontitis, the Pg+CIA model better simulates the joint manifestations and lung manifestations of RA-ILD in smoking patients and periodontitis patients.

(3) The HE and Masson staining pathology reports of the lungs of mice in the Pg+CIA model group showed a high degree of lesions, and the characteristics of the lesions were highly similar to those of humans.

The term "comprises" or "comprises" in the present invention is an open-ended description containing the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect.

Description of drawings

Figure 1: Clinical characteristics of DBA/1J mice. A is a representative photo of the hind paw; B is the arthritis score. in, P<0.01 vs Control group.

Figure 2: DBA/1J mouse autoantibody detection, where, P<0.01 vs Control group; ##P<0.01, #P<0.05 vs Pg+CIA group; &&P<0.01 vs CIA group.

Figure 3: Pathological staining (A) and pathological score (B) of the knee joint, where, P<0.01 vs Control group; ##P<0.01 vs Pg+CIA group; &&P<0.01 vs CIA group.

Figure 4: Micro-CT imaging test and osteometric parameter test results, among which, P<0.01 vs Control group; ##P<0.01, #P<0.05 vs Pg+CIA group; &&P<0.01, &P<0.05 vs CIA group; BMD is knee joint bone density, BV/TV is bone volume fraction, Tb .Th is the thickness of trabecular bone, and Tb.N is the number of trabecular bone.

Figure 5: Pathological staining and pathological scoring results of lung tissue. P<0.01 vs Control group, ##P<0.01, #P<0.05 vs Pg+CIA group.

Figure 6: Masson staining and collagen area ratio of lung tissue, P<0.01 vs Control group; ##P<0.01 vsPg+CIA group.

Figure 7: Expression levels of α-SMA, Col Ⅰ, and FN proteins in lung tissue and KL-6 mRNA expression (A) (B) Expression levels of α-SMA, Col Ⅰ, and FN proteins in lung tissue of mice in each group (immune Histochemistry, ×400); (C) α-SMA, Col Ⅰ, FN, and KL-6 mRNA expression levels; among them, P <0.01 vs Control group; # P <0.05 vs Pg+CIA group; ## P <0.01 vs Pg+CIA group.

Figure 8: Expression levels of TNF-α, IL-6, and IL-1β mRNA in lung tissue. P<0.01 vs Control group;&&P<0.01 vs CIA group; #P<0.05 vs Pg+CIA group; ##P<0.01 vs Pg+CIA group.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention. Not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

The materials, reagents, instruments, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example

1 Experimental methods and materials

1.1 Experimental animals

Forty male 6-week-old DBA/1J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. [License number: SYXK (Beijing) 2019-0008], experimental unit use license number: SYXK (Beijing) 2021- 0017. They were raised in an environment of 22±2°C, with free access to food and water. They were used for subsequent experiments after adapting to feeding for 7 days. The animal welfare and experimental procedures in this study complied with the relevant regulations of the Animal Ethics Committee of the China Academy of Chinese Medical Sciences.

1.2 Drugs and reagents

Pg ( Porphyromonas gingivalis , Pg) strain (ATCC#33277) was purchased from Beina Chuanglian Biotechnology Co., Ltd.; Columbia blood agar culture medium was purchased from Beekman Biotechnology Co., Ltd.; bovine type II collagen acetic acid solution (Product No.: NO. 2002-2) and complete Freund's adjuvant (Cat. No.: NO.7001) were purchased from Chondrex Company of the United States; mouse anti-cyclic citrullinated peptide antibody (Anti-CCP-antibody) ELISA kit (Cat. No.: CSB-EQ027743MO) Purchased from CUSBIO Company; Mouse Porphyromonas gingivalis antibody detection kit (Cat. No.: ml023665-J) and mouse immunoglobulin IgG (Cat. No.: ml057874) detection kit were purchased from Shanghai Enzyme Biotechnology Co., Ltd.; Masson's three-color staining solution kit (batch number: C210601) was purchased from Zhuhai Besuo Biotechnology Co., Ltd.; DEPC enzyme-free water was purchased from Beijing Lanjieke Technology Co., Ltd. (item number: BL510B), reverse transcription kit (item number: AE311) Purchased from Quanshijin Biotechnology Co., Ltd., Kangwei Century Ultra SYBR Mixture (High ROX), absolute ethanol (Cat. No.: 100092683), environmentally friendly dewaxing transparent liquid (Cat. No.: G1128), 20× citric acid antigen retrieval solution ( pH 6.0) (Cat. No.: G1202), 20×Tris-EDTA Antigen Retrieval Solution (pH9.0) (Cat. No.: G1203), 20×Tris-EDTA Antigen Retrieval Solution (pH 8.0) (Cat. No.: G1206), Hematoxylin Staining Solution (Cat. No.: G1203) Catalog No.: G1004), hematoxylin differentiation solution (G1039), hematoxylin blue-returning solution ( Catalog No.: G1040), and histochemistry kit DAB chromogen ( Catalog No.: G1212) were purchased from Wuhan Sevier Biotechnology Co., Ltd.

1.3 Experimental instruments

Mitsubishi 2.5LMGC anaerobic tank, anaerobic gas-generating bag (Mitsubishi Chemical Corporation, Japan); Multiskan MK3 microplate reader (Thermo Fisher Scientific). Baking machine (KPJ-1A), rotary microtome (QPJ-C), biological tissue embedding machine (BMJ-1B) (Tianjin Tianli Aviation Mechanical and Electrical Co., Ltd.); BX50 upright microscope (Olympus (Olympus) Japan) Company); micro-computed tomography Micro-CT (GE Healthcare Company, RS0800604-0063), the vortex mixer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., and the pre-cooling centrifuge was purchased from Shanghai Chubai Laboratory Equipment Co., Ltd., the homogenizer was purchased from Aika (Guangzhou) Instrument Equipment Co., Ltd., the PCR instrument (Jena, Germany), and the Thermo Fisher qPCR instrument.

1.4 Bacterial activation

Inject about 0.3-0.5ml of sterile water or liquid culture medium into the freeze-drying tube, and pipe gently to fully dissolve the bacteria into a suspension. Aspirate the bacterial suspension, evenly pour it onto the surface of two Columbia blood agar media (about 200uL/each), spread it evenly, and use 90% nitrogen, 5% carbon dioxide and A mixture of 5% hydrogen is in equilibrium. After activation is successful, relevant tests can be carried out. Determine the bacterial cell count using a spectrophotometer. The spectrophotometer at 600 nm is 1.0, which is equivalent to 1×10 ⁹ CFU/ml. Then collect 10 ⁹ CFU/ml bacteria and resuspend in PBS.

1.5 Modeling and grouping

The experimental animals were randomly divided into four groups, namely the normal control group (Control), the collagen induction group (CIA), the simple Porphyromonas gingivalis infection group (Pg), and the collagen induction combined with P. gingivalis infection group ( Pg+CIA). All mice were adaptively fed for 7 days. Within three days before adaptive feeding, all groups were fed ampicillin (10 mg/500mL) to eliminate endogenous bacteria that are not conducive to Pg colonization. The specific modeling methods of each group are as follows: (1) Control group: From the first day onwards, 200 μL of 2% CMC (carboxymethyl cellulose) in PBS was applied to the gingival margins of the upper and lower jaws of mice every day until the end of the modeling; (2) CIA group: From the first day, 200 μL of 2% CMC in PBS was applied to the gingival margins of the upper and lower jaws of mice every day until the end of the modeling. On days 14 and 35, emulsions of equal volumes (100 μl each) of CII (type II collagen, 2 mg/ml) and CFA (complete Freund's adjuvant, 4 mg/ml) were used to immunize mice subcutaneously from the tail. Injection; (3) Pg group: From the first day, 100 μL of 2% CMC in PBS containing 1×10 ⁹ CFU/mL Pg was applied to the gingival margins of the upper and lower jaws of mice every day until the end of the modeling; (4) Pg +CIA group: From the first day, 100 μL of 2% CMC in PBS containing 1×10 ⁹ CFU/mL Pg was applied to the gingival margins of the upper and lower jaws of mice every day until the end of the modeling. On days 14 and 35, they were immunized with emulsions of equal volumes (100 μl each) of CII and CFA, and injected subcutaneously from the tail of mice. The entire modeling period is 56 days. Among them, CII specifically uses bovine type II collagen acetic acid solution.

1.6 Morbidity and Arthritis Score

Statistics on the incidence of RA in mice, incidence rate = number of mice with disease in each group/total number of mice in each group × 100%. Refer to Table 1 for arthritis scores. The mice were evaluated at 4 ankle and wrist joints, 4 midfoot joints, and 12 digit joints.

Table 1: Arthritis Score

1.7 ELISA test

After the mice were sacrificed, the eyeballs were removed to collect blood, and the serum was obtained after standing and centrifuging. Take the 96-well plate prepared by the kit, add 50ul of the diluted standard into the reaction well, and add 50ul of the sample to be tested into the reaction well. Immediately add 50ul of biotin-labeled antibody to the reaction well, cover it with a sealing film on a 96-well plate, and incubate at 37°C for 60 minutes with shaking. Shake off and pat dry the liquid in the 96-well plate, then add 200ul of washing solution to each well, and repeat washing the plate 3 times. After washing the plate, pat it dry, add 80ul of streptavidin-HRP to each well, and incubate at 37°C for 30 minutes with shaking. Shake off and pat dry the liquid in the 96-well plate, then add 200ul of washing solution to each well, and repeat washing the plate 3 times. Add substrate A (the chromogen A in the kit (containing hydrogen peroxide) is the hydrogen donor (DH2)) and substrate B (the chromogen B (containing TMB) for color development) into the reaction well. Mix an equal volume of 100ul of the solution and incubate at 37°C for 10 minutes with shaking and avoid light. Finally, take out the 96-well plate, add 50ul of stop solution to the reaction well, and detect the absorbance in a Multiskan MK3 microplate reader immediately after the addition. At the specified wavelength of the kit (mouse anti-cyclic citrullinated peptide antibody (Anti-CCP-antibody) ELISA kit, mouse Porphyromonas gingivalis antibody detection kit, mouse immunoglobulin IgG detection kit) Determine the OD value of each well.

1.8 HE staining of knee joint

The knee joint was removed, fixed in paraformaldehyde, slowly decalcified with 10% EDTA, embedded in paraffin using a biological tissue embedding machine, and sectioned with a rotary microtome for later use. Place the slices on a baking machine at 60°C for 30 minutes. Dewax the slices in xylene and graded ethanol until they reach water. Place the slices in hematoxylin and eosin stains for staining. Rinse with running water and differentiate with differentiation solution. Finally, 0.5% ammonia water returns to blue, and finally rinsed with running water again. After the sections were dried slightly, they were sealed with neutral resin and observed and photographed under a BX50 upright microscope. The specific observation contents and scoring standards are as follows in Table 2. The higher the score, the higher the degree of the disease.

Table 2: Knee joint pathology score sheet

1.9 Osteometric analysis of Micro-CT scans of inflammatory knee joints

The knee joint was fixed in a 10% formaldehyde solution for 48 hours, and then a Micro-CT scan was performed at a resolution of 25 μm. The duration of a single scan of the knee joint tissue to be scanned was 72 minutes. The Micro-CT scan was followed by joint reconstruction. , and analyze osteometric parameters at the same time. The bone mineral density (BMD), bone volume fraction (bone volume/tissue volume, BV/TV), trabecular thickness (Tb.Th), trabecular number ( Trabecular number, Tb.N) and other osteometric parameters were statistically analyzed.

1.10 HE staining of lung tissue

Slice with rotary microtome and dewax to water, stain with hematoxylin for 10 minutes, wash fully with tap water, differentiate with solution for 5 seconds, wash fully with tap water, reverse blue with anti-blue solution for 5 seconds, wash fully with tap water, 0.1% eosin for 1 minute, 95% ethanol for 2 minutes , 95% ethanol for 2 min, 100% ethanol I for 3 min, 100% ethanol II for 3 min, xylene I for 7 min, xylene II for 7 min, fixed with neutral gum, and observed and photographed under a BX50 upright microscope. Pathological morphological changes (degree of alveolar inflammation, interstitial inflammatory cell infiltration, alveolar wall thickening) are marked as "+", "++", "+++" and "++++" according to severity, indicating mild and mild respectively. Severe, moderate, and severe, the corresponding scores are 1-4 points, no lesions are marked as 0 points, and missing marks are recorded as "none". The individual score is the sum of each lesion score (degree of alveolar inflammation, interstitial inflammatory cell infiltration, and alveolar wall thickening). The specific scoring standards are shown in Table 3.

Table 3: Lung tissue pathology scoring criteria

1.11 Masson staining

Bake the slices on a baking machine at 65°C for 1 hour. Dewax the slices in xylene and graded ethanol until they reach water. Place the slices in hematoxylin stain, Ponceau red, phosphomolybdic acid and aniline blue for staining. 1 % glacial acetic acid solution, gradient ethanol dehydration and xylene transparency. After the sections were dried slightly, they were sealed with neutral resin and observed and photographed under a BX50 upright microscope. Randomly select 3 visual fields of the same sample under a light microscope (10×10) to calculate the collagen area ratio. Use Image-ProPlus to measure the collagen area and the total area of the entire image, and calculate the collagen area ratio. Collagen area ratio (%) = (collagen area /Total area of the entire image)×100%. Specifically, Masson's tricolor staining solution kit was used for the experiment.

1.12 Immunohistochemistry of lung tissue

Dewax the paraffin sections to water and then perform antigen retrieval, block endogenous peroxidase, seal with serum, add primary antibody dropwise, add PBS on the sections, and add primary antibodies prepared in a certain proportion, and place the sections flat on the reagent Incubate overnight at 4°C in the box; add secondary antibody dropwise and incubate at room temperature for 50 minutes, then perform DAB color development, counterstain cell nuclei, and dehydrate and mount the slide for microscopic examination.

1.13 q-PCR detection of IL-1β, TNF-α, and IL-6 in lung tissue

Extract tissue RNA by TRIzol method, detect the concentration and purity, adjust the concentration, and reverse-transcribe into cDNA. Using cDNA as a template, RT-qPCR is used to detect the expression of IL-1β, TNF-α, and IL-6 mRNA. The upstream primer of GAPDH is AGTGGCAAAGTGGAGATT (SEQ ID NO: 1), and the downstream primer is GTGGAGTCATACTGGAACA (SEQ ID NO: 2); the upstream primer of IL-1β is TCCTTGTGCAAGTGTCTGAAGC (SEQ ID NO: 3), and the downstream primer is ATGAGTGATACTGCCTGCCTGA (SEQ ID NO :4); the upstream primer of TNF-α is CACCACCATCAAGGACTCAA (SEQ ID NO:5), and the downstream primer is AGGCAACCTGACCACTCTCC (SEQ ID NO:6); the upstream primer of IL-6 is CTCTGCAAGAGACTTCCATCCAGT (SEQ ID NO:7), and the downstream primer is GAAGTAGGGAAGGCCGTGG (SEQ ID NO: 8).

1.14 Data processing and statistical analysis

Data statistics and graphics were performed using GraphPad Prism 8.0.2 software and Image-ProPlus software. The results of measurement data were expressed as mean (Mean) ± standard deviation (SD).

2Experimental results

2.1 Symptoms after mouse modeling

To evaluate the effect of Pg infection combined with collagen induction on the occurrence of RA in mice, we first observed the arthritis index, incidence rate and disease-related conditions. The results showed that both the CIA group and the Pg+CIA group had symptoms such as obvious joint redness and swelling, toe deformation, skin congestion, and limited movement of some joints. The incidence rate was 100% (10/10), and the clinical arthritis scores were respectively 70 and 68, and there was no statistical difference in clinical scores between the two groups. See Figure 1 and Table 4 for details.

Table 4: Clinical characteristics of RA in different groups of DBA/1J mice

2.2 Serum antibody levels in model animals

The detection results of mouse serum antibodies showed that compared with the normal control group, the expression of anti-Pg antibodies in the Pg group was significantly increased ( P <0.01), the IgG antibody content in the CIA group was significantly increased ( P <0.01), and Pg + The anti-Pg antibodies and IgG antibodies in the CIA group were significantly increased, and there were statistical differences ( P <0.01). Compared with the CIA group, the IgG content in the Pg group was significantly reduced ( P <0.01). Compared with the Pg + CIA group, the expression levels of anti-CCP antibodies, anti-Pg antibodies, and IgG antibodies in the Pg group were significantly less ( P <0.01, 0.05), and the expression levels of anti-Pg antibodies in the CIA group were reduced ( P <0.05, 0.01). Specifically, as follows: As shown in Figure 2.

2.3 Histopathological examination of knee joint

The pathological characteristic of RA is chronic invasive arthritis. Histopathological observation of joints is currently the most important method to evaluate the pathogenesis characteristics and disease severity of RA model animals in experimental research. HE staining results of knee joints showed that the joint surfaces of mice in the normal group and Pg group were smooth, without pannus formation, and cells were arranged neatly. Both the CIA group and the Pg+CIA group showed obvious hyperplasia of synovial tissue around the ankle joint cavity and protruding into the joint cavity. The joint space narrowed. The proliferated synovial tissue invaded the subcutaneous tissue and partially formed fibrosclerotic lesions, accompanied by a large amount of inflammation in the synovium. There was infiltration of sex cells and proliferation of granulation tissue, formation of synovial pannus, and obvious damage to bone and cartilage, as shown in Figure 3. The pathological score is divided into four aspects: synovial inflammation, pannus, cartilage destruction, and bone destruction.

2.4 Micro-CT imaging detection

Micro-CT 3D reconstruction results showed that the knee joint of the Control group was smooth and complete, with clear bone contours. Changes such as slight bone erosion and joint cavity narrowing occurred in the Pg group. The surface of the knee joints of mice in the CIA group and Pg+CIA group was loose, rough, and uneven. The bone structure was eroded, and the bone tissue showed honeycomb-like erosion spots. Further osteometric statistical results showed that the BMD, BV/TV, Tb.Th, and Tb.N of the Pg+CIA group were statistically different from those of the Control group and Pg group (P<0.05), as shown in Figure 4.

2.5 Pathological detection of lung tissue by HE staining

The pathological characteristics of pulmonary interstitial lesions are infiltration of inflammatory cells in the alveoli and interstitium, severe alveolar wall thickening, fibrosis and nodule formation. Histopathological observation of the lungs is currently used in experimental studies to evaluate model animals for pulmonary interstitial lesions. The most important method is the characteristics of the onset and severity of the disease. HE staining results of lung tissue showed that the lung tissue structure of the mice in the normal group was clear, the alveolar surface was smooth, and there was no obvious deformation or rupture. The respiratory epithelial cells were arranged neatly, and there was no inflammatory cell infiltration in the interstitium; the mice in the Pg group had large areas of alveolar hemorrhage and edema. , necrosis, moderate or severe thickening of the alveolar septa, accompanied by mild or moderate infiltration of inflammatory cells, mainly lymphocytes and monocytes. There were significant differences compared with the normal group; a small amount of fibrous tissue proliferation could be seen in the interstitium and bronchi; the alveolar walls of mice in the CIA group were slightly or moderately thickened, accompanied by slight or mild inflammatory cell infiltration, mainly lymphocytes and mononuclear macrophages. Mainly, there is a significant difference compared with the normal group; there is a small amount of fibrous tissue proliferation in the interstitium and bronchi, and there is no statistical difference compared with the normal group; mice in the Pg+CIA group have large areas of alveolar hemorrhage, edema, and necrosis, and the alveolar interval is mild to severe. Thickening, moderate or severe granulomatous inflammatory cell infiltration can be seen in the interstitium, distributed in a clump shape, mainly lymphocytes and monocytes, with a small amount of neutrophils also gathering, and more fibrous tissue can be seen in the interstitium and bronchi. Hyperplasia was significantly more severe than that of the normal group, Pg and model groups (P<0.01). As shown in Figure 5, the pathological score is divided into three aspects: the degree of alveolar inflammation, interstitial inflammatory cell infiltration, and alveolar wall thickening.

The Pg group can induce symptoms of interstitial lung lesions, but after applying Pg for a certain period of time, such as starting from the first immunization (day 14) or the second immunization (day 28), Pg intervention cannot continue to significantly aggravate lung lesions. .

2.6 Masson staining of lung tissue

Masson's trichrome staining is the most classic method of connective tissue staining. It is also called Masson's staining. It is one of the main methods for displaying fibers in tissues. It is an authoritative and classic technical method for collagen fiber staining. As shown in Figure 6, the lung tissue structure of the mice in the normal group is clear, the alveolar surface is smooth, and there is no obvious deformation or rupture. The respiratory epithelial cells are arranged neatly, and there is no inflammatory cell infiltration in the interstitium. In the Pg group, large areas of alveolar hemorrhage and edema can be seen under the microscope. , necrosis, moderate or severe thickening of the alveolar septa, accompanied by mild or moderate infiltration of inflammatory cells, mainly lymphocytes and monocytes. There was a significant difference compared with the normal pair; a small amount of fibrous tissue proliferation could be seen in the interstitium and bronchi; under the microscope, the CIA group showed mild or moderate thickening of the alveolar walls, accompanied by slight or mild inflammatory cell infiltration, mainly lymphocytes and mononuclear macrophages. Mainly, there was a significant difference compared with the normal group; there was a small amount of fibrous tissue proliferation in the interstitium and bronchi, and there was no statistical difference compared with the normal group; in the CIA+Pg group, large areas of alveolar hemorrhage, edema, and necrosis were seen, and the alveolar interval was mild to severe. Thickening, moderate or severe granulomatous inflammatory cell infiltration can be seen in the interstitium, distributed in a clump shape, mainly lymphocytes and monocytes, with a small amount of neutrophils also gathering, and more fibrous tissue can be seen in the interstitium and bronchi. Hyperplasia was significantly more severe than that of the normal group, Pg model group and CIA model group (P<0.01).

2.7 Immunohistochemistry of lung tissue

Compared with the normal control group, the expression levels of α-SMA, ColⅠ and FN proteins in the lung tissue of mice in the Pg group, CIA group and Pg + CIA group were significantly higher than those in the normal group ( P <0. 01); compared with the Pg + Compared with the CIA group, only the Col Ⅰ protein expression level in the Pg group was significantly reduced ( P < 0. 01), and the CIA group α-SMA, Col Ⅰ and FN protein expression levels were all significantly reduced ( P < 0.05, 0. 01); the CIA group and There was no significant difference in the expression of the three proteins between the two groups in the Pg group. The results of fibrosis-related mRNA detection experiments showed that compared with the normal control group, the expression of α-SMA mRNA in the Pg group was significantly increased ( P <0.01), and the expression of α-SMA and FN mRNA in the CIA group was significantly increased ( P <0.01). , the mRNA expression levels of type I collagen (Collagen I, Col Ⅰ), α-SMA, FN and sialylated sugar chain antigen-6 (Correlation of Krebs von den Lungen-6, KL-6) in the Pg + CIA group were significantly increased. ( P <0.01); compared with the Pg + CIA group, the expression levels of α-SMA, FN and KL-6 mRNA in the Pg group and CIA group were significantly reduced ( P <0.01, 0.05). The results are shown in Figures 7A, B, and C.

2.8 Lung tissue q-PCR

The results of the inflammatory factor mRNA detection experiment showed that compared with the normal control group, the expression of tumor necrosis factor-α (TNF-α) mRNA in the Pg group was significantly increased ( P <0.01), and the expression of interleukin-1β in the CIA group was significantly increased ( P <0.01). (interleukin-6, IL-1β) mRNA expression levels were significantly increased ( P <0.01), and the mRNA expression levels of TNF- α, interleukin-6 (interleukin-6, IL-6), and IL-1β in the Pg + CIA group were all significantly increased ( P <0.01). significantly increased ( P <0.01); compared with the Pg + CIA group, the expression of IL-1β mRNA in the Pg group was significantly reduced ( P <0.01), and the expression of IL-6 and TNF-α mRNA in the CIA group was significantly reduced ( P <0.05 , 0.01); the TNF-α mRNA expression in the Pg group was higher than that in the CIA group ( P <0.01). The results are shown in Figure 8.

It can be seen from the above results that Pg alone can only induce mild ILD but not arthritis. Both CIA and Pg+CIA can induce arthritis and ILD, and the degree of arthritis lesions in the two groups is similar; compared with the alone Pg and CIA groups In comparison, the degree of ILD lesions of Pg+CIA, especially the degree of fibrosis, was significantly more serious, and the anti-CCP content was significantly increased, suggesting that Pg+CIA can induce significant RA-ILD. The degree of ILD and arthritis lesions is high, and the lesion characteristics are similar to those of Pg+CIA. It is highly similar to humans and better simulates the joint manifestations and lung manifestations of RA-ILD in smoking patients and periodontitis patients.

Although the invention has been described in detail by way of preferred embodiments, the invention is not limited thereto. Without departing from the spirit and essence of the invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the invention, and these modifications or substitutions should be within the scope of the invention/any Those skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention, and they should all be covered by the protection scope of the present invention.

Claims (10)

1. A method for constructing an animal model, characterized in that the method for constructing includes administering periodontal pathogens and type II collagen, and the animal model is an animal model of arthritis combined with interstitial lung disease, and the Periodontal pathogens include Porphyromonas gingivalis , and the animal is a mouse. 2. The construction method according to claim 1, characterized in that the periodontal pathogen is applied once every 1-7 days, and the amount of periodontal pathogen applied per time is 1×10 ⁷ -1×10 ¹⁰ CFU, the total number of days of administration is 45-60 days. 3. The construction method according to claim 1, wherein the periodontal pathogen administration method includes smear administration, oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection. injection. 4. The construction method according to claim 1, characterized in that the construction method includes applying type II collagen at least once. 5. The construction method according to claim 4, characterized in that the interval between two applications of type II collagen is 15-25 days. 6. The construction method according to claim 4, characterized in that the construction method includes administering an adjuvant and type II collagen simultaneously. 7. The construction method according to claim 6, wherein the adjuvant includes one of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, alum, heat shock protein or cytokines. one or a combination of two or more. 8. The construction method according to claim 6, characterized in that the administration method of the type II collagen and/or adjuvant includes smear administration, oral administration, intradermal injection, subcutaneous injection, and intramuscular injection. , intraperitoneal injection or intravenous injection. 9. The construction method according to claim 8, characterized in that the interval between the first application of periodontal pathogens and the first application of the combination of type II collagen and adjuvant is 8-20 days. 10. Application of the animal model obtained by the construction method of any one of claims 1 to 9 in screening or preparing drugs for treating interstitial lung disease or arthritis combined with interstitial lung disease.