A method for establishing a rat model of chronic obstructive pulmonary disease and its application.

Abstract

This invention provides a method for establishing a rat model of COPD to study the medicinal uses and pharmacological effects of Liuwei Buqi Decoction in the treatment of COPD. The traditional Chinese medicines in Liuwei Buqi Decoction are ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel, and cinnamon. This application provides a basis for the syndrome differentiation and treatment strategy of Liuwei Buqi Decoction by exploring the molecular mechanisms by which Liuwei Buqi Decoction regulates the COPD inflammatory immune network and the molecular mechanisms by which it regulates the lung-gut microecological axis and the inflammatory immune network. Taking the high-incidence syndromes of major diseases during the TCM-advantage stage as a starting point, and based on clinical needs, this study, building upon previous clinical research, uses various techniques such as scRNA-seq, CBA, and metagenomics to explore the molecular mechanism by which Liuwei Buqi Decoction improves airway immune inflammation by regulating the lung microecology in rats with COPD lung-kidney qi deficiency syndrome from the perspective of regulating the lung immune microenvironment and alveolar macrophage polarization. Its mechanism of action was further studied and verified through in vitro experiments.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for establishing a rat model of chronic obstructive pulmonary disease and its application. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a major global disease with high prevalence, disability, and mortality. Chronic inflammation is a core component of COPD progression. Common clinical treatments, primarily using corticosteroids, bronchodilators, and antibiotics, are prone to dependence, relapses, and are ineffective in halting the continued decline in lung function. Traditional Chinese medicine (TCM) plays a significant role in COPD treatment, believing that lung qi deficiency is the fundamental factor in the development of COPD and other pulmonary diseases. Prolonged illness depletes qi, leading to lung and kidney qi deficiency, resulting in impaired lung function, kidney dysfunction, blood stasis, and phlegm accumulation due to abnormal fluid metabolism. The disease manifests as a complex interplay of blood stasis, phlegm, and fluid retention. Initially located in the lungs, the disease progresses, with lung qi deficiency as the primary symptom gradually evolving into lung and spleen qi deficiency, and then lung and kidney qi deficiency. This application constructs an experimental animal model similar to human lung and kidney qi deficiency syndrome, and conducts clinical manifestations such as feeding status, hair, respiration, weight, and urination and defecation of rats in each group, as well as lung function tests, HE staining, and transmission electron microscopy to observe lung tissue pathology, in order to clarify the medicinal use and pharmacological effects of the drug in the treatment of COPD. Summary of the Invention

[0003] The technical problem solved by this invention is to provide an experimental animal model that is similar to human lung and kidney qi deficiency syndrome, so as to study the medicinal use and pharmacological effects of Liuwei Buqi Decoction in the treatment of COPD, thereby solving the problems mentioned in the background art.

[0004] The technical problem solved by this invention is achieved by the following technical solution: a method for establishing a rat model of chronic obstructive pulmonary disease (COPD) to study the medicinal uses and pharmacological effects of Liuwei Buqi Fang (a traditional Chinese medicine formula) in the treatment of COPD. The Liuwei Buqi Fang contains ginseng, astragalus, Alpinia oxyphylla, Polygonatum odoratum, tangerine peel, and cinnamon. The method includes the following steps:

[0005] Step 1: Select 60 SPF-grade SD rats, aged 5-8 weeks and weighing 180±20g, and randomly divide them into a normal group, a lung and kidney qi deficiency syndrome model group, and a Liuwei Buqi Decoction treatment group, with 20 rats in each group;

[0006] Step 2: Rats in the lung and kidney qi deficiency syndrome model group and the Liuwei Buqi Decoction treatment group were subjected to smoke fumigation combined with LPS tracheal instillation, forced swimming, and hormone injection to replicate the COPD lung qi deficiency syndrome rat model.

[0007] Step 3: Administration method. After LPS is administered via tracheal drip, the rats in the Liuwei Buqi Fang treatment group are given the drug by gavage at a dose of 4.95g / kg once a day after the model is established. The dosage is calculated based on the clinical equivalent dose until the model is established. The normal group rats and the rats in the lung and kidney qi deficiency syndrome model group are given an equal volume of physiological saline by gavage.

[0008] The specific operation method for replicating the COPD lung qi deficiency rat model in step two is as follows:

[0009] (1) Before smoking, rats were placed in a constant temperature water bath (43±1℃) and forced to swim for 30 minutes to deplete their lung qi. Then they were placed in a self-made smoking box and smoked with 50g of sawdust and 10 cigarettes. They were smoked once a day for 30 minutes each time for a total of 28 days.

[0010] (2) On day 1 and day 14, the trachea was surgically exposed and 200 μl LPS (1 mg / mL) was dripped into the trachea of ​​each rat. No smoke was used on the day of the incident. Starting from day 22, smoke was used 3 times a day. Hydrocortisone sodium succinate 0.3 ml / rat (25 mg / ml) was injected subcutaneously on the back once a day for a total of 8 days.

[0011] The application of a rat model of chronic obstructive pulmonary disease (COPD) and the related molecular mechanisms by which Liuwei Buqi Decoction regulates the COPD inflammatory immune network include the following aspects:

[0012] (1) Macroscopic condition detection and analysis of rats to clarify the effect of Liuwei Buqi Formula on the intervention of COPD rats with different syndromes;

[0013] (2) Detection of lung-gut axis microecological community to clarify the regulatory characteristics of specific lung-gut axis microecological community and Liuwei Buqi Formula;

[0014] (3) Detection of immune cell populations and cytokines to clarify the correlation between the regulation of specific microbiota in the lung-gut axis by Liuwei Buqi Decoction and the improvement of inflammatory immunity;

[0015] (4) Molecular detection of key metabolic pathways: the key immune metabolic pathways targeted by the Liuwei Buqi Decoction to exert its effects;

[0016] (5) Conduct relevant analysis on the results obtained from single-cell sequencing research;

[0017] (6) Flow cytometry was used to detect the expression of M1 markers CD80 and CD86 and M2 markers CD163 and CD206 in BALF;

[0018] (7) Immunohistochemical staining was used to detect the expression of M1 markers CD80, CD86, and iNOS and M2 markers CD163, CD206, and Arg-1 in lung tissue;

[0019] (8) Detect the expression of M1 markers CD80, CD86, iNOS expression cassette M2 markers CD163, CD206, and Arg-1 in rat AM;

[0020] (9) Western blot was used to detect the expression of M1 markers CD86 and iNOS proteins and M2 markers CD163, CD206 and Arg-1 proteins in the alveolar AM of rats.

[0021] (10) Based on the macrophage regulation-related genes obtained by scRNA-seq, we studied the relevant pathways of Liuwei Buqi Formula mediating alveolar macrophage polarization regulation of lung-gut axis microecological changes and targeting COPD inflammatory immunity.

[0022] (11) Using Co-IP technology, the interactions between key molecules targeted by the Gubenpeiyuan Formula (Liuwei Buqi Formula) based on the formula-symptom correspondence were clarified;

[0023] (12) The reversal experiment verified the molecular mechanism by which Liuwei Buqi Decoction regulates the COPD inflammatory immune network and the molecular mechanism by which it regulates the lung-gut microecological axis and the inflammatory immune network, further providing a basis for the syndrome differentiation and treatment strategy of Liuwei Buqi Decoction.

[0024] As a further aspect of the present invention: the macroscopic condition detection and analysis of rats, combined with the observation of syndrome elements, the effects of the Six-Ingredient Qi-Boosting Formula on the clinical manifestations of each group of rats, such as feeding status, hair, respiration, weight, and urination and defecation, as well as the detection of rat lung function, HE staining, and transmission electron microscopy to observe lung tissue pathology, are used to clarify the intervention effect of the Six-Ingredient Qi-Boosting Formula.

[0025] As a further aspect of the present invention: the lung-gut axis microecological community detection involves extracting DNA from rat bronchoalveolar lavage fluid and intestinal contents. After DNA integrity testing, qualified samples are amplified and sequenced using 16S rDNA to analyze biodiversity. Combined with metagenomic high-throughput sequencing technology, functional annotation analyses such as COG and KEGG are performed to clarify the regulatory characteristics of lung-gut axis specific microecological community transplantation and Liuwei Buqi Decoction on the lung-gut axis microecological community in COPD.

[0026] As a further aspect of the present invention: the detection of immune cell populations and cytokines involves using single-cell sequencing combined with spatial transcriptomics to analyze immune cell populations in rat bronchoalveolar lavage fluid samples, lung tissue, and intestinal tissue, screening for differentially expressed immune cell populations between groups, analyzing changes in immune cell populations and differentially expressed transcripts in rat bronchoalveolar lavage fluid, and using ELISA to detect the expression of cytokines in peripheral blood and bronchoalveolar lavage fluid to clarify the regulatory effect of Liuwei Buqi Fang on immune cell populations in COPD lungs. Furthermore, combined with WGCNA analysis, key molecules and pathways related to the target microecological community and immune cell populations are obtained to elucidate the correlation between Liuwei Buqi Fang's regulation of specific microbiota in the lung-gut axis and the improvement of inflammatory immunity.

[0027] As a further aspect of the present invention: the key metabolic pathway molecular detection is performed by using qPCR technology to detect the transcriptional expression level of key pathway metabolic molecules in rat lung tissue; and by using Western blot combined with immunohistochemistry to detect the expression of key proteins in rat lung tissue, so as to understand the effect of Liuwei Buqi Formula on the expression of targeted key metabolic pathway molecules and tissue localization.

[0028] As a further aspect of the present invention: the correlation analysis of the results obtained from single-cell sequencing includes the following aspects:

[0029] (1) Analyze the differential gene expression and cellular function changes of macrophage subsets among groups, and study the polarization characteristics and molecular mechanisms of macrophages in each group of rats;

[0030] (2) Macrophages were subdivided into subpopulations, and RNA rate analysis and time-fit analysis were performed on the subpopulations to explore the differentiation pathways and directions of different macrophage subtypes, and to find out the relationship between the changes in macrophage function in different differentiation states and the treatment of COPD inflammation by Liuwei Buqi Decoction.

[0031] (3) Perform cell communication analysis between macrophages and other immune cells and macrophage subpopulations to explore the interactions between immune cells, macrophage subpopulations and molecular interactions, and study the cell dynamics of macrophages and the relationships between immune cells during the treatment process of Liuwei Buqi Decoction.

[0032] Compared with existing technologies, the beneficial effects of this invention are as follows: This application provides a basis for the syndrome differentiation and treatment strategy of Liuwei Buqi Formula by exploring the molecular mechanisms by which Liuwei Buqi Formula regulates the COPD inflammatory immune network and the molecular mechanisms by which it regulates the lung-gut microecological axis and the inflammatory immune network. Taking the high-incidence syndromes of major diseases during the TCM advantageous stage as a starting point, and based on clinical needs, this study, building upon previous clinical research, utilizes various techniques such as scRNA-seq, CBA, and metagenomics to explore the molecular mechanisms by which Liuwei Buqi Formula improves airway immune inflammation by regulating the lung microecology from the perspective of regulating the lung immune microenvironment and alveolar macrophage polarization in rats with COPD lung-kidney qi deficiency syndrome. In vitro experiments further studied and verified its mechanism of action, comprehensively exploring the role of macrophages in the pathogenesis of COPD and the regulatory effect of Liuwei Buqi Formula on them. This helps to clarify the scientific connotation of the "strengthening the foundation and nourishing the source" theory, aiming to break through the treatment bottleneck of chronic inflammation and airflow limitation in COPD. The research objectives are clear and the approach is innovative. Attached Figure Description

[0033] Figure 1 This is a technical roadmap for the research content of this invention. Detailed Implementation

[0034] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific illustrations.

[0035] like Figure 1 As shown, this embodiment provides a method for establishing a rat model of COPD to study the medicinal uses and pharmacological effects of Liuwei Buqi Decoction in the treatment of COPD. The traditional Chinese medicines in Liuwei Buqi Decoction are ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel, and cinnamon. The method includes the following steps:

[0036] Step 1: Select 60 SPF-grade SD rats, aged 5-8 weeks and weighing 180±20g, and randomly divide them into a normal group, a lung and kidney qi deficiency syndrome model group, and a Liuwei Buqi Decoction treatment group, with 20 rats in each group;

[0037] Step 2: Rats in the lung and kidney qi deficiency syndrome model group and the Liuwei Buqi Decoction treatment group were subjected to smoke fumigation combined with LPS tracheal instillation, forced swimming, and hormone injection to replicate the COPD lung qi deficiency syndrome rat model.

[0038] Step 3: Administration method. After LPS is administered via tracheal drip, the rats in the Liuwei Buqi Fang treatment group are given the drug by gavage at a dose of 4.95g / kg once a day after the model is established. The dosage is calculated based on the clinical equivalent dose until the model is established. The normal group rats and the rats in the lung and kidney qi deficiency syndrome model group are given an equal volume of physiological saline by gavage.

[0039] The specific operation method for replicating the COPD lung qi deficiency rat model in step two is as follows:

[0040] (1) Before smoking, rats were placed in a constant temperature water bath (43±1℃) and forced to swim for 30 minutes to deplete their lung qi. Then they were placed in a self-made smoking box and smoked with 50g of sawdust and 10 cigarettes. They were smoked once a day for 30 minutes each time for a total of 28 days.

[0041] (2) On day 1 and day 14, the trachea was surgically exposed and 200 μl LPS (1 mg / mL) was dripped into the trachea of ​​each rat. No smoke was used on the day of the incident. Starting from day 22, smoke was used 3 times a day. Hydrocortisone sodium succinate 0.3 ml / rat (25 mg / ml) was injected subcutaneously on the back once a day for a total of 8 days.

[0042] Determining the syndrome attributes of model animals is a core step in constructing a disease-syndrome combined animal model. This study intends to use the following three methods to evaluate the construction of a disease-syndrome combined model of lung and kidney qi deficiency in COPD:

[0043] (1) Judging from the macroscopic signs and behavioral manifestations of the TCM model animals: The COPD kidney qi deficiency syndrome model is mainly due to kidney qi deficiency on the basis of lung qi deficiency, which cannot consolidate the body and manifests as curling up and arching back, slow movement, fatigue, weak grasping and resistance, dull hair, yellowing and easy shedding, and occasionally respiratory rales and polyuria (high bedding moisture).

[0044] (2) Based on the modeling factors, the syndrome attributes were inferred and determined from the perspective of traditional Chinese medicine etiology: The modeling factors for the COPD lung-kidney qi deficiency syndrome model were guided by the traditional Chinese medicine theory of "exertion depletes qi". Before each smoke fumigation to construct the COPD model, the model was forced to swim in a constant temperature water bath (43±1℃) for 30 minutes. Based on the clinical research results of patients who had been using hormones for a long time and then discontinued hormones, the model was designed to treat kidney deficiency. Starting from day 22, hydrocortisone sodium succinate 0.3ml / animal (25mg / ml) was continuously injected subcutaneously into the back once a day for 8 days.

[0045] (3) Judging syndrome attributes based on microscopic biological and physicochemical indicators: Some scholars have gradually referred to the basic research results of syndrome biology and incorporated clinical physicochemical indicators related to syndromes into the syndrome diagnostic criteria, thereby ensuring the construction of a disease-syndrome combination model. The lung tissue of the COPD lung-kidney qi deficiency syndrome rat model has certain characteristics under both light and electron microscopy. Secondly, in terms of biological indicators, the COPD lung-kidney qi deficiency syndrome rat model mainly shows changes in immune indicators such as decreased positive expression of CD4+ T cells and increased positive expression of CD8+ T cells in lung tissue.

[0046] The above-mentioned modeling and evaluation methods can be used to evaluate the rat model of COPD with lung and kidney qi deficiency, so as to ensure the reliability of the research results.

[0047] This embodiment provides an application of a rat model of chronic obstructive pulmonary disease (COPD) and demonstrates the molecular mechanisms by which Liuwei Buqi Decoction regulates the COPD inflammatory immune network, including the following aspects:

[0048] (1) Macroscopic condition detection and analysis of rats to clarify the effect of Liuwei Buqi Formula on the intervention of COPD rats with different syndromes;

[0049] (2) Detection of lung-gut axis microecological community to clarify the regulatory characteristics of specific lung-gut axis microecological community and Liuwei Buqi Formula;

[0050] (3) Detection of immune cell populations and cytokines to clarify the correlation between the regulation of specific microbiota in the lung-gut axis by Liuwei Buqi Decoction and the improvement of inflammatory immunity;

[0051] (4) Molecular detection of key metabolic pathways: the key immune metabolic pathways targeted by the Liuwei Buqi Decoction to exert its effects;

[0052] (5) Conduct relevant analysis on the results obtained from single-cell sequencing research;

[0053] (6) Flow cytometry was used to detect the expression of M1 markers CD80 and CD86 and M2 markers CD163 and CD206 in BALF;

[0054] (7) Immunohistochemical staining was used to detect the expression of M1 markers CD80, CD86, and iNOS and M2 markers CD163, CD206, and Arg-1 in lung tissue;

[0055] (8) Detect the expression of M1 markers CD80, CD86, iNOS expression cassette M2 markers CD163, CD206, and Arg-1 in rat AM;

[0056] (9) Western blot was used to detect the expression of M1 markers CD86 and iNOS proteins and M2 markers CD163, CD206 and Arg-1 proteins in the alveolar AM of rats.

[0057] (10) Based on the macrophage regulation-related genes obtained by scRNA-seq, we studied the relevant pathways of Liuwei Buqi Formula mediating alveolar macrophage polarization regulation of lung-gut axis microecological changes and targeting COPD inflammatory immunity.

[0058] (11) Using Co-IP technology, the interactions between key molecules targeted by the Gubenpeiyuan Formula (Liuwei Buqi Formula) based on the formula-symptom correspondence were clarified;

[0059] (12) The reversal experiment verified the molecular mechanism by which Liuwei Buqi Decoction regulates the COPD inflammatory immune network and the molecular mechanism by which it regulates the lung-gut microecological axis and the inflammatory immune network, further providing a basis for the syndrome differentiation and treatment strategy of Liuwei Buqi Decoction.

[0060] The macroscopic condition analysis of the rats, combined with the observation of syndrome elements, revealed the intervention effect of the Liuwei Buqi Formula on the clinical manifestations of each group of rats, such as feeding status, hair, respiration, weight, and defecation, as well as the rat lung function test, HE staining, and transmission electron microscopy to observe lung tissue pathology, thus clarifying the corresponding intervention effect of the Liuwei Buqi Formula.

[0061] The lung-gut axis microecological community detection involved extracting DNA from bronchoalveolar lavage fluid and intestinal contents from rats. After DNA integrity testing, qualified samples were amplified and sequenced using 16S rDNA to analyze biodiversity. Combined with metagenomic high-throughput sequencing technology, functional annotation analyses such as COG and KEGG were performed to clarify the regulatory characteristics of lung-gut axis specific microecological community transplantation and Liuwei Buqi Decoction on the lung-gut axis microecological community in COPD.

[0062] The detection of immune cell populations and cytokines involved analyzing immune cell populations in rat bronchoalveolar lavage fluid samples, lung tissue, and intestinal tissue using single-cell sequencing combined with spatial transcriptomics. Differential immune cell populations were screened between groups, and changes in immune cell populations and differentially expressed transcripts in rat bronchoalveolar lavage fluid were analyzed. ELISA was used to detect the expression of cytokines in peripheral blood and bronchoalveolar lavage fluid to clarify the regulatory effect of Liuwei Buqi Decoction on immune cell populations in COPD lungs. Furthermore, WGCNA analysis was used to identify key molecules and pathways related to the target microecological community and immune cell populations, elucidating the correlation between Liuwei Buqi Decoction's regulation of specific microbiota in the lung-gut axis and the improvement of inflammatory immunity.

[0063] The key metabolic pathway molecular detection was performed using qPCR to detect the transcriptional expression of key pathway metabolic molecules in rat lung tissue; and using Western blot combined with immunohistochemistry to detect the expression of key proteins in rat lung tissue, to understand the effect of Liuwei Buqi Formula on the expression of targeted key metabolic pathway molecules and their tissue localization.

[0064] The analysis of the results obtained from single-cell sequencing includes the following aspects:

[0065] (1) Analyze the differential gene expression and cellular function changes of macrophage subsets among groups, and study the polarization characteristics and molecular mechanisms of macrophages in each group of rats;

[0066] (2) Macrophages were subdivided into subpopulations, and RNA rate analysis and time-fit analysis were performed on the subpopulations to explore the differentiation pathways and directions of different macrophage subtypes, and to find out the relationship between the changes in macrophage function in different differentiation states and the treatment of COPD inflammation by Liuwei Buqi Decoction.

[0067] (3) Perform cell communication analysis between macrophages and other immune cells and macrophage subpopulations to explore the interactions between immune cells, macrophage subpopulations and molecular interactions, and study the cell dynamics of macrophages and the relationships between immune cells during the treatment process of Liuwei Buqi Decoction.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection of the present invention is defined by the appended claims and their equivalents. It should be noted that, in this document, the use of relational terms such as "first" and "second" is merely used to distinguish one entity or operation from another, and does not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

Claims

1. The application of a rat model of chronic obstructive pulmonary disease (COPD) to study the medicinal uses and pharmacological effects of Liuwei Buqi Fang (a traditional Chinese medicine formula) in the treatment of COPD. The traditional Chinese medicines in Liuwei Buqi Fang are ginseng, astragalus, alpinia oxyphylla, polygonatum odoratum, tangerine peel, and cinnamon. Its characteristics are: Includes the following steps: Step 1: Select 60 SPF-grade SD rats, aged 5-8 weeks and weighing 180±20g, and randomly divide them into a normal group, a lung and kidney qi deficiency syndrome model group, and a Liuwei Buqi Decoction treatment group, with 20 rats in each group; Step 2: Rats in the lung and kidney qi deficiency syndrome model group and the Liuwei Buqi Decoction treatment group were treated with smoke fumigation combined with LPS tracheal instillation, forced swimming, and hormone injection to replicate the rat model of chronic obstructive pulmonary disease. Step 3: Administration method. After LPS is administered via tracheal drip, the rats in the Liuwei Buqi Fang treatment group are given the drug by gavage at a dose of 4.95g / kg once a day after the model is established. The dosage is calculated based on the clinical efficacy dose until the model is established. The normal group rats and the rats in the lung and kidney qi deficiency syndrome model group are given an equal volume of physiological saline by gavage. The aforementioned application, through the molecular mechanisms by which Liuwei Buqi Formula regulates the COPD inflammatory immune network, includes the following aspects: (1) Macroscopic condition detection and analysis of rats to clarify the effect of Liuwei Buqi Formula on the intervention of COPD rats with different syndromes; (2) Detection of lung-gut axis microecological community to clarify the regulatory characteristics of specific lung-gut axis microecological community and Liuwei Buqi Formula; (3) Detection of immune cell populations and cytokines to clarify the correlation between the regulation of specific microbiota in the lung-gut axis by Liuwei Buqi Formula and the improvement of inflammatory immunity; (4) Molecular detection of key metabolic pathways: the key immune metabolic pathways targeted by Liuwei Buqi Decoction to exert its effects; (5) Conduct relevant analysis on the results obtained from single-cell sequencing research; (6) Flow cytometry was used to detect the expression of M1 markers CD80 and CD86 and M2 markers CD163 and CD206 in BALF; (7) Immunohistochemical staining was used to detect the expression of M1 markers CD80, CD86, and iNOS and M2 markers CD163, CD206, and Arg-1 in lung tissue; (8) Detect the expression of M1 markers CD80, CD86, and iNOS and M2 markers CD163, CD206, and Arg-1 in rat AM; (9) Western blot analysis was performed to detect the expression of M1 markers CD86 and iNOS proteins and M2 markers CD163, CD206 and Arg-1 proteins in the alveolar AM of rats. (10) Based on the macrophage regulation-related genes obtained by scRNA-seq, study the relevant pathways of Liuwei Buqi Formula mediating alveolar macrophage polarization regulation of lung-gut axis microecological changes and targeting COPD inflammatory immunity; (11) Using Co-IP technology, the interactions between key molecules targeted by Liuwei Buqi Formula based on the formula-symptom correspondence were clarified; (12) The reversal experiment verified the molecular mechanism by which Liuwei Buqi Formula regulates the COPD inflammatory immune network and the molecular mechanism by which it regulates the lung-gut microecological axis and the inflammatory immune network, and further provided a basis for the syndrome differentiation and treatment strategy of Liuwei Buqi Formula.

2. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The specific operation method for replicating the rat model of chronic obstructive pulmonary disease in step two is as follows: (1) Before smoking, rats were placed in a constant temperature water bath and forced to swim for 30 minutes to deplete their lung qi. Then they were placed in a self-made smoking box and smoked with 50g of sawdust and 10 cigarettes. They were smoked once a day for 30 minutes each time for a total of 28 days. (2) On day 1 and day 14, each rat was instilled with 200 μl LPS in the trachea. No smoke was used on the day. Starting from day 22, smoke was used 3 times a day. Hydrocortisone sodium succinate 0.3 ml / rat was injected subcutaneously on the back once a day for a total of 8 days.

3. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The macroscopic condition of the rats was analyzed, and the effects of the Liuwei Buqi Decoction on the rats' food intake, hair, respiration, weight, urination and defecation, pulmonary function, HE staining and transmission electron microscopy were observed to clarify the intervention effect of the Liuwei Buqi Decoction.

4. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The lung-gut axis microecological community detection involved extracting DNA from bronchoalveolar lavage fluid and intestinal contents from rats. After DNA integrity testing, qualified samples were amplified and sequenced using 16S rDNA to analyze biodiversity. Combined with metagenomic high-throughput sequencing technology, COG and KEGG functional annotation analyses were performed to clarify the regulatory characteristics of lung-gut axis specific microecological community transplantation and Liuwei Buqi Decoction on the lung-gut axis microecological community in COPD.

5. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The detection of immune cell populations and cytokines involved analyzing immune cell populations in rat bronchoalveolar lavage fluid samples, lung tissue, and intestinal tissue using single-cell sequencing combined with spatial transcriptomics. Differential immune cell populations were screened between groups, and changes in immune cell populations and differentially expressed transcripts in rat bronchoalveolar lavage fluid were analyzed. ELISA was used to detect the expression of cytokines in peripheral blood and bronchoalveolar lavage fluid to clarify the regulatory effect of Liuwei Buqi Decoction on immune cell populations in COPD lungs. Furthermore, WGCNA analysis was used to identify key molecules and pathways related to the target microecological community and immune cell populations, elucidating the correlation between Liuwei Buqi Decoction's regulation of specific microbiota in the lung-gut axis and the improvement of inflammatory immunity.

6. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The key metabolic pathway molecular detection was performed using qPCR to detect the transcriptional expression of key pathway metabolic molecules in rat lung tissue; and using Western blot combined with immunohistochemistry to detect the expression of key proteins in rat lung tissue, to understand the effect of Liuwei Buqi Formula on the expression of targeted key metabolic pathway molecules and their tissue localization.

7. The application of the rat model of chronic obstructive pulmonary disease according to claim 1, characterized in that: The analysis of the results obtained from single-cell sequencing includes the following aspects: (1) Analyze the differential gene expression and cellular function changes of macrophage subsets among groups, and study the polarization characteristics and molecular mechanisms of macrophages in each group of rats; (2) Macrophages were subdivided into subpopulations, and RNA rate analysis and time-fit analysis were performed on the subpopulations to explore the differentiation pathways and directions of different macrophage subtypes, and to find out the relationship between the changes in macrophage function in different differentiation states and the treatment of COPD inflammation by Liuwei Buqi Decoction. (3) Perform cell communication analysis between macrophages and other immune cells and macrophage subpopulations to explore the interactions between immune cells, macrophage subpopulations and molecular interactions, and study the cell dynamics of macrophages and the relationships between immune cells during the treatment process of Liuwei Buqi Decoction.

Images