Method for constructing plateau chronic obstructive pulmonary disease rodent model

Abstract

The invention relates to the technical field of disease animal model construction, in particular to a method for constructing a plateau chronic obstructive pulmonary disease rodent model. The method includes exposing a rodent to a low pressure hypoxia cabin and tobacco smoke (CSE) environment. Through the model, the obvious difference between the pathophysiology characteristics of the plateau chronic obstructive pulmonary disease and the pathological characteristics caused by pure anoxia or CSE exposure can be conveniently researched, so that assistance is provided for the increase of the morbidity of the plateau chronic obstructive pulmonary disease and the acceleration of the disease progress.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing a rodent model of high-altitude COPD. Background Technology

[0003] The low-pressure, low-oxygen environment at high altitudes may influence the development of COPD. At high altitudes, reduced atmospheric pressure is one of the most prominent features, leading to decreased oxygen levels. As altitude increases, the partial pressure of oxygen decreases, resulting in hypoxic conditions that affect human physiology and activity. Hypoxia-induced changes in organismal function, metabolism, and structure form the basis of the pathology of high-altitude diseases. Chronic hypoxia causes changes in lung structure, leading to pulmonary vasoconstriction and pulmonary vascular remodeling, resulting in pulmonary hypertension. Furthermore, hypoxia exacerbates airway inflammation and mucus production, hallmarks of COPD. Studies by Mikami et al. have shown that chronic airway epithelial hypoxia leads to excessive mucus secretion, causing airway obstruction and further impairing gas exchange, ultimately increasing respiratory symptoms such as dyspnea and respiratory distress in COPD patients. Therefore, the low-pressure, low-oxygen environment at high altitudes may exacerbate the occurrence and development of COPD. Studies have shown that after adjusting for relevant risk factors, high altitude remains an independent risk factor for COPD, with a higher prevalence at higher altitudes.

[0004] The prevalence of COPD at high altitudes is unclear, whether it is higher or lower than at low altitudes. Therefore, there is an urgent need to study the effects of low-pressure, low-oxygen environments at high altitudes on COPD using animal models with controlled single-variable methods, and to establish a high-altitude COPD model to further investigate its molecular mechanisms. Summary of the Invention

[0005] This invention covers the following technical solutions: One aspect of the present invention relates to a method for constructing a rodent model of high-altitude COPD, comprising: exposing rodents to a low-pressure, low-oxygen chamber and a tobacco smoke environment.

[0006] Another aspect of the present invention relates to the application of animal models prepared by the method described above in screening therapeutic targets, candidate prevention and treatment methods, or candidate prevention and treatment drugs for high-altitude COPD.

[0007] This invention establishes and validates a rodent model for COPD at high altitudes. This model allows for convenient study of the pathophysiological characteristics of COPD at high altitudes, which differ significantly from those caused by simple hypoxia or CSE exposure. This provides valuable insights into the increased incidence and accelerated progression of COPD in high-altitude regions. Attached Figure Description

[0008] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0009] Figure 1 This invention relates to a method for creating a mouse model of high-altitude COPD.

[0010] Figure 2 This invention relates to the detection of inflammatory factors in the bronchoalveolar lavage fluid of mice with COPD at high altitudes.

[0011] Figure 3 This invention relates to the lung function testing of a high-altitude COPD mouse model; the corresponding abbreviations in the accompanying drawings represent: FEV1 50 Forced expiratory volume (FVC): The volume of air exhaled within 50 milliseconds of the start of exhalation; Forced expiratory volume (FVC): The maximum volume of air that can be exhaled as quickly as possible after a maximal inhalation; Residual volume / Total lung capacity (RV / TLC): The ratio of residual volume to total lung capacity; Cchord: Lung compliance.

[0012] Figure 4 This invention provides pathological examination of lung tissue in a mouse model of high-altitude COPD. Detailed Implementation

[0013] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0014] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0015] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to animal behavior, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0016] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this invention, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0017] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0018] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0019] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0020] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0021] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0022] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0023] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0024] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0025] In this invention, the terms “prevention” and its variations or “improvement” refer to preventive or therapeutic treatments aimed at preventing, reversing, alleviating, improving, inhibiting, slowing or stopping the progression or severity of a disease (e.g., high-altitude COPD), its related symptoms and / or conditions.

[0026] In this invention, the term "high-altitude COPD" refers to a chronic obstructive pulmonary disease that occurs in high-altitude areas and is induced or aggravated by long-term exposure to a hypoxic environment. It is usually characterized by chronic airway inflammation, airflow limitation, and progressive decline in lung function, and may be accompanied by complications such as pulmonary hypertension, increased right ventricular load, and respiratory failure. The high-altitude COPD can be caused by factors such as smoking, dust, indoor and outdoor air pollution, genetic susceptibility, and the unique climate and environment of high-altitude areas, either alone or in combination. It covers the acute exacerbation and stable phases caused by single or multiple etiologies, and includes various pathological types and their variants characterized by lung tissue structure destruction, decreased alveolar elasticity, increased mucus secretion, and airway remodeling.

[0027] This invention relates to a method for constructing a rodent model of high-altitude COPD, comprising: exposing rodents to a low-pressure hypoxic chamber and a tobacco smoke (CSE) environment.

[0028] In some embodiments, the parameters of the low-pressure hypoxic chamber are set to simulate an altitude of 3500-4500 meters (e.g., 3200 meters, 3300 meters, 4000 meters, 4200 meters, 4300 meters), an atmospheric pressure of 56 kPa-66 kPa (e.g., 58 kPa, 60 kPa, 61 kPa, 62 kPa, 64 kPa), and an oxygen content of 12.2%-13.2% (e.g., 12.4%, 12.7%, 13%).

[0029] In some embodiments, cigarette smoke extract (CSE) is prepared using a vacuum extraction method.

[0030] In some embodiments, the rodent is a mouse ( Mus musculus Mouse strains can be selected from BALB / c, C57BL, C3H / He, Kunming mice, ICR, NIH, CFW, LACA, nude mice, or Scid mice, etc.

[0031] In some embodiments, the rodent is male.

[0032] In some implementations, the mice are 6-8 weeks old.

[0033] In some implementations, during modeling, the mice are administered CSE via nasal instillation.

[0034] In some embodiments, the preparation of the CSE includes: absorbing the smoke from a lit cigarette with a buffer solution, wherein each milliliter of the solution contains unfiltered smoke from the burnt-out cigarettes of two medium-tar cigarettes.

[0035] As used herein, the term "buffer solution" refers to an aqueous solution that resists changes in pH when an acid or base is added. This resistance to pH changes is due to the buffering properties of such solutions. Therefore, solutions or compositions exhibiting buffering activity are called buffers or buffer solutions. Buffers generally do not have an unlimited capacity to maintain the pH of a solution or composition. Instead, they are generally able to maintain a pH within a specific range, such as pH 7–pH 9. Generally, buffers are able to maintain pH within their pKa and the next logarithm (see, for example, Mohan, Buffers, A guide for the preparation and use of buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffer salts or, preferably, nonionic buffer components. Common buffer solutions are selected from phosphate buffers, phosphate-buffered saline (PBS), 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, TBS buffered saline (TBS), and TIS / EDTA (TE). Phosphate buffers are preferred for biosafety considerations.

[0036] In cigarettes with a medium tar content, each cigarette typically contains between 0.6 mg and 1.1 mg of nicotine, 6 mg and 10 mg of tar, and 7 mg and 13 mg of carbon monoxide. It's easy to understand why the cigarette filter should be removed when collecting the smoke.

[0037] In some implementations, the treatment of rodents with CSE and / or hypobaric chambers can be continuous or intermittent (but the cumulative time should be sufficient to help induce the onset of symptoms of high-altitude COPD). The term "interval" refers to the time interval during which hypoxia and / or CES are not applied.

[0038] In some implementations, the CSE is used 4-7 times (e.g., 5, 6) per week, with each administration of 20µL-40µL (e.g., 25µL, 30µL, 35µL) of CSE.

[0039] In some implementations, CSE is used for 10-14 weeks (e.g., 11, 12, 13).

[0040] In some implementations, the low-pressure hypoxia chamber is used continuously for 10-14 weeks (e.g., 11, 12, 13).

[0041] In some embodiments, the CSE is administered in a low-pressure, low-oxygen chamber.

[0042] According to another aspect of the invention, the application of animal models prepared by the method described above in screening therapeutic targets, candidate prevention and treatment methods, or candidate prevention and treatment drugs for high-altitude COPD is also involved.

[0043] Methods for screening therapeutic targets can be combined with commonly used techniques in the field, depending on the objective. Exemplary methods also include microarrays, single-cell transcriptome sequencing (SCRNA-seq), quantitative PCR (qPCR) / high-throughput PCR arrays, whole-genome sequencing (WGS), transcriptome sequencing, exome sequencing (WES), methylation arrays / whole-genome methylation sequencing (WGBS), proteomics by mass spectrometry, tissue microarrays (IHC arrays), protein microarrays, and single-cell proteomics (such as CyTOF). Furthermore, the above methods can be combined with bioinformatics methods such as cluster analysis, differential expression analysis (DESeq2, edgeR, limma, etc.), functional enrichment analysis (GO, KEGG, Reactome, GSEA, etc.), network biology analysis (PPI analysis, regulatory networks, etc.), and machine learning modeling screening (LASSO regression, random forest, SVM-RFE) for further analysis and screening.

[0044] "Candidate drugs for prevention and treatment" refer to potential drugs that can prevent and treat COPD at high altitudes. These can be chemical or biological therapeutic agents, such as bronchodilators, hormones (e.g., glucocorticoids), mucolytics, anti-infectives, pulmonary hypertension symptomatic drugs, pathogen vaccines (e.g., influenza vaccines), or any combination thereof. The alveolar macrophages obtained by the method according to the present invention enable the determination of whether candidate prevention and treatment methods or candidate drugs have activity in preventing and treating COPD at high altitudes, the magnitude of that activity, and the extent of drug side effects. Suitable candidate prevention and treatment methods and candidate drugs for combined use can be screened as needed.

[0045] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0046] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0047] Example 1 Male C57BL / 6J mice (purchased from Beijing Spefol Biotechnology Co., Ltd.) aged 6-8 weeks were housed in a specific pathogen-free environment. The environment was maintained at room temperature (23±2)℃ and relative humidity between 45% and 55%, using a light-dark cycle. Mice had free access to food and water during the rearing period. After one week of acclimatization, experiments began. Mice were randomly assigned using a random number table to four groups: control group (CON), tobacco smoke extract group (CL), hypoxia-hypotension group (Hy), and hypoxia-hypotension combined with tobacco smoke extract treatment group (HyCL). Each experimental group contained 8 mice.

[0048] 2. Extraction method of tobacco smoke extract Take two purple cigarettes (0.7 mg nicotine, 8 mg carbon monoxide, and 6 mg tar), remove the cigarette filters, and connect them to a glass bottle containing 1 ml of phosphate-buffered saline (PBS). Light the cigarettes and inhale using a bulb syringe attached to the other end of the bottle to obtain cigarette smoke extract (CSE). Prepare the CSE within half an hour before the experiment.

[0049] 3. Methods for establishing a high-altitude COPD mouse model Mice in the CON group were exposed to normal indoor air and simultaneously administered an equal volume of PBS. Mice in the CL group received 60 mg / kg PBS. -1 After anesthesia with sodium pentobarbital, 30 µL of CSE was administered nasally once daily. In addition to the same treatment as the CL group, the HyCL group mice were placed in a hypobaric hypoxic chamber (24 hours, for 3 months). The chamber parameters were set to simulate an altitude of 4000 meters, an atmospheric pressure of 61 kPa, and an oxygen content of 12.7%. Lung function was assessed and tissue samples were collected from all mice after 3 months.

[0050] 4. Mouse lung function test Mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine (0.2 ml / 10 g body weight, Nanjing Aibei Biotechnology Co., Ltd., M2920). Tracheostomy was then performed on the mice, and they were connected to a DSIBuxco-PFT Controller (DATA SCIENCES INTERNATIONAL, INC., St. Paul, Minnesota, USA) via endotracheal intubation. Pulmonary function tests were then performed according to the instrument's instruction manual. After the pulmonary function tests were completed, the mice were euthanized by inhalation of an overdose of isoflurane.

[0051] 5. Detection of inflammatory cells in mouse bronchoalveolar lavage fluid (BALF) Mice were euthanized by inhalation of isoflurane. Bronchoalveolar lavage fluid (BALF) was collected three times. Each time, 0.6 mL of physiological saline was slowly injected into the lungs via an endotracheal cannula until the lung margins were fully inflated. This process was repeated slowly and repeatedly. The three BALF collections were then combined and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was collected. The cell pellet was then mixed with erythrocyte lysis buffer and incubated (on ice for 5 minutes). After centrifugation at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded. The cell pellet was resuspended in 1 mL of physiological saline, and cell counting was performed using an automated cell counter (Countess 3, Invitrogen).

[0052] 6. Pathological examination of lung tissue in mice with COPD at high altitude According to the quantitative assessment statement on lung structure published by ATS / ERS, pressure fixation and morphological analysis of the lungs were performed. After fully opening and exposing the mouse thoracic cavity, the left atrial appendage was removed, and physiological saline was slowly injected into the right ventricle of the mouse using a syringe to thoroughly flush out the blood in the pulmonary vessels until the fluid flowing from the left atrial appendage became clear. Then, the mouse lung tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, and the lung tissue sections were cut into 4-micrometer thick slices for subsequent HE staining and Masson staining. Six fields of view were selected from the lung tissue sections of each mouse for alveolar counting, small airway epithelial cell thickening, and collagen deposition around the small airways. Specifically, a cross line was drawn in the center of the pathological section of the mouse lung tissue, the number of alveolar septa passing through the cross line (Ns) was counted, and the total length of the cross line (L) was measured. The mean linear intercept (MLI) of the mouse lung tissue was obtained by MLI = L / Ns. For the assessment of small airway remodeling in mice, the total area of ​​the small airway walls (Wat) and the perimeter of the basement membrane (Pbm) were measured using image analysis software (Image-Pro Plus 6.0), and airway remodeling was assessed using the Wat / Pbm ratio. Additionally, the area of ​​collagen deposition around the airway (Cdaaa) and the perimeter of the basement membrane (Pbm) were measured using image analysis software (Image-Pro Plus 6.0), and airway remodeling was assessed using the Cdaaa / Pbm ratio.

[0053] II. Experimental Results 1. Compared with tobacco smoke exposure alone, the lung function decline was more significant in the high-altitude COPD mouse model. Compared to the CON group mice, the CL group mice showed a significantly lower FEV50 / FVC ratio in the first 50 milliseconds, while exhibiting an increased quasi-static lung compliance (Cchord). Compared to the CL group, the HyCL group mice showed an even more significant decrease in the FEV50 / FVC ratio in the first 50 milliseconds, and an even more significant increase in quasi-static lung compliance (Cchord); similar trends were observed in total lung capacity (TLC) and residual volume / total lung capacity (RV / TLC). This indicates that CL reduces lung function in the mouse model, and this reduction is further exacerbated under hypobaric and hypoxic conditions.

[0054] 2. Compared with tobacco smoke exposure alone, the high-altitude COPD mouse model showed significantly greater airway inflammatory cell infiltration. Bronchoalveolar lavage fluid (BALF) cell count in COPD patients is an important indicator for assessing pulmonary inflammation and infection. Compared to the CON group, the CL group mouse model showed a significant increase in BALF cells. Furthermore, compared to the CL group, a hypobaric and hypoxic environment to some extent increased the BALF cell count in the HyCL group mice.

[0055] 3. Compared with exposure to tobacco smoke alone, the lung tissue pathological damage in the high-altitude COPD mouse model was more significant. Compared to the CON group, CL treatment increased alveolar diameter in mice; the hypobaric and hypoxic environment further increased alveolar diameter in the CL-induced mouse model. Compared to the CON group, CL treatment increased small airway epithelial thickness and surrounding collagen deposition; simultaneously, the hypobaric and hypoxic environment further amplified these pathological changes in the lungs induced by CL. This indicates that CL promotes emphysema and small airway remodeling in the mouse model, and that hypobaric and hypoxic conditions further exacerbate these changes. Thus, we have established a CL-induced COPD mouse model and, based on this, successfully established a hypobaric and hypoxic combined with CL-induced COPD mouse model in high-altitude areas.

[0056] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method of constructing a high altitude chronic obstructive pulmonary disease rodent model, comprising: Exposing rodents to low pressure and low oxygen cabin and tobacco smoke (CSE) environment.

2. The method of claim 1, wherein the low pressure and low oxygen cabin is set to simulate an altitude of 3500-4500 meters, an atmospheric pressure of 56-66 kPa, and an oxygen content of 12.2-13.2%.

3. The method of claim 1, wherein the cigarette smoke extract (CSE) is prepared by vacuum extraction.

4. The method of any one of claims 1-3, wherein the rodents are mice.

5. The method of claim 4, wherein the rodents are mice; preferably 6-8 weeks old.

6. The method of claim 5, wherein the mice are administered CSE by intranasal instillation during modeling.

7. The method of any one of claims 3, 5, 6, wherein the preparation of the CSE comprises:

7. A method for preparing an animal model for high altitude chronic obstructive pulmonary disease, comprising: a) exposing rodents to low pressure and low oxygen cabin and tobacco smoke (CSE) environment; and b) administering CSE to the rodents by intranasal instillation.

8. The method of claim 7, wherein the CSE is administered 4-7 times per week, and each administration is 20-40 µL of CSE; preferably the CSE is administered for 10-14 weeks.

9. The method of any one of claims 3, 5, or 6, wherein the low pressure and low oxygen cabin is administered for 10-14 weeks.

10. Use of the animal model prepared by the method of any one of claims 1-9 in screening therapeutic targets, or candidate prevention and treatment methods, or candidate prevention and treatment drugs for high altitude chronic obstructive pulmonary disease.

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