A method for preparing a rat model of chronic obstructive pulmonary disease combined with respiratory failure and application of lung rehabilitation in the model

Abstract

The present application aims to develop an animal model which can focus on the classic symptoms of chronic obstructive pulmonary disease combined with respiratory failure, and can well evaluate the efficacy of chronic obstructive pulmonary disease combined with respiratory failure. The present application induces a short-time high-efficiency, good operability and repeatability, stable and unified, more close to clinical rat model of chronic obstructive pulmonary disease combined with respiratory failure by sodium periodate and p-hydroxybenzyl alcohol injection + benzene sulfonic acid gavage + low oxygen restraint + cold and hot stimulation. The chronic obstructive pulmonary disease combined with respiratory failure induced by multiple factors can better simulate the signs of human chronic obstructive pulmonary disease combined with respiratory failure. The modeling method is short-time high-efficiency, simple operation, stable and reliable, good universality, close to clinical.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction for diseases, specifically relating to a method for preparing a rat model of COPD complicated with respiratory failure and the application of pulmonary rehabilitation in this model. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a progressive chronic respiratory disease characterized by persistent respiratory symptoms and airflow limitation, usually caused by airway and alveolar lesions resulting from exposure to toxic particles or gases. It is associated with an enhanced chronic inflammatory response of the airways and lungs to nitrogen oxide particles or harmful gases. Chronic airflow limitation is caused by the combined effects of small airway obstruction, chronic bronchitis, and lung parenchymal destruction (emphysema), which can affect daily activities and overall quality of life. Globally, approximately 328 million people are affected, with a higher prevalence rate among the elderly; about 10% are middle-aged and 25% are over 70 years old. COPD is currently the third leading cause of death, causing more than 3 million deaths annually. Due to an increasingly aging population and historically and currently high smoking rates, the global mortality rate from COPD is rising annually, and the World Health Organization predicts that the overall prevalence and mortality rates of COPD will increase by 30% by 2030. In my country, the prevalence of COPD among people aged 40 and above is 13.7%, with nearly 100 million patients. It is highly prevalent among adults and ranks as the third cause of death among Chinese residents, clearly becoming an important health problem in today's society.

[0003] The pathogenesis of COPD is highly complex and not yet fully understood. Besides abnormal inflammatory responses and prolonged immune responses, oxidative stress, protease / anti-protease imbalance, microbial infection, apoptosis / anti-apoptosis imbalance, accelerated aging of lung tissue, and vascular endothelial cell dysfunction also contribute to the occurrence and development of COPD. Currently, the exact mechanisms of COPD occurrence and development remain unclear, and effective therapies to halt or reverse disease progression require further research. Therefore, basic experimental research on COPD is of great significance. Animal models are important tools for disease research and serve as a bridge between basic and clinical research. To further explore the mechanisms of traditional Chinese medicine (TCM) in treating respiratory diseases and evaluate the efficacy of TCM drugs, research using animal models with higher clinical concordance is needed. The ability to quickly establish stable animal models that exhibit COPD markers is fundamental to experimental success. Currently developed animal models include sheep, dogs, pigs, rabbits, rodents, and primates, all of which can reflect the pathophysiological state of human COPD to some extent. Among these, rodents (represented by mice and rats) are the most commonly used. Both mice and rats share advantages such as small size, low feeding cost, short breeding cycle, and relatively thorough biological information; however, rats are stronger than mice, and their trachea and bronchi are wider, which is more conducive to performing procedures such as intratracheal instillation of reagents, bronchoalveolar lavage, and lung function testing, and they are more likely to survive in experiments.

[0004] Numerous methods for establishing COPD animal models have been reported, including single-factor methods (passive smoking, sulfur dioxide (SO2) inhalation, lipopolysaccharide tracheal instillation, recurrent respiratory infections (mainly bacterial), protease induction, and genetic engineering) and multi-factor methods (passive smoking + LPS, passive smoking + protease, passive smoking + respiratory infection, etc.). Despite the variety of methods, a unified standard is lacking, and induction and modeling are time-consuming, with poor model formation results. Furthermore, maintaining animal condition during rearing is difficult, and the absence of standardized animal models remains a key obstacle to COPD research. The pathogenesis of COPD is not yet fully understood, and establishing COPD animal models is crucial for understanding its mechanisms. An ideal COPD animal model should resemble human COPD in terms of pathogenesis, pathological changes, lung function impairment, and systemic complications. However, currently known animal models only meet some of these criteria. Therefore, exploring and establishing animal models that are less time-consuming, standardized, and consistent with the development and pathological characteristics of COPD is of great significance for in-depth research into the pathogenesis of COPD. Summary of the Invention

[0005] This study aimed to successfully establish a rat model of COPD complicated with respiratory failure, observe the pathological changes in the lungs of the animals, and detect relevant indicators in lung tissue and blood to evaluate the stability of the model. The goal was to create a short-term, efficient, operable, reproducible, stable, and more clinically relevant rat model of COPD complicated with respiratory failure, which would facilitate further research on the pathogenesis and pathophysiological changes of COPD complicated with respiratory failure, and provide a basis for the implementation of lung protection interventions.

[0006] This invention aims to develop an animal model that effectively targets the classic symptoms of chronic obstructive pulmonary disease (COPD) complicated with respiratory failure, and can provide a good evaluation of the efficacy of drugs targeting this condition. The rat COPD-associated respiratory failure model constructed in this invention demonstrates high success rate and stability in inducing classic symptoms (chronic bronchitis, emphysema, airflow limitation, and small airway remodeling) in rats with COPD-associated respiratory failure. This study can effectively simulate the clinical pathogenesis of COPD-associated respiratory failure in rats, laying a solid foundation for rapid screening of therapeutic drugs and interventions. The modeling method is short-term, efficient, simple to operate, stable, reliable, universally applicable, and closely resembles clinical practice.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a rat model of COPD complicated with respiratory failure includes the following steps:

[0009] (1) Temporary rearing: Select 6-8 week old SPF-grade male SD rats with normal development and good vitality, weighing 190-210g, and raise them at a temperature of 20℃-24℃ and a humidity of 50%-70%. Provide them with normal food and water, and provide them with a half-day and half-night lighting cycle. Adapt to rearing for 7 days. All operating procedures and rearing conditions comply with the regulations for the management and rearing of experimental animals and follow humanitarian principles.

[0010] (2) Sodium periodate and p-hydroxybenzyl alcohol injection: After 7 days of acclimatization, the rats were transferred to the modeling cage to start the modeling process. During the modeling process, the rats drank water and ate normally. On the first day of modeling, 6 mg / kg of sodium periodate (based on the rat's body weight) was injected into the abdominal cavity of the rats in the morning and evening. 30 minutes after each injection of sodium periodate, 8 mg / kg of p-hydroxybenzyl alcohol (based on the rat's body weight) was injected into the rats intramuscularly.

[0011] (3) Benzenesulfonic acid gavage: On the 2nd-3rd day of modeling, benzenesulfonic acid aqueous solution was administered to rats by gavage once a day, with a dose of 0.1 ml / rat each time. The concentration of the benzenesulfonic acid aqueous solution was 40%.

[0012] (4) Hypoxia restraint: On the 4th day of modeling, the rats were transferred into the restraint tube of the restraint cage. The restraint tube was set in the restraint cage and the diameter of the restraint tube matched the size of the rat, that is, the rat could easily enter but could not turn around or back after entering. Then the restraint cage was placed in a hypoxia incubator with an oxygen concentration of 12% O2. After 30 minutes of hypoxia restraint, the rats were transferred back to the modeling cage. Hypoxia restraint was performed once in the morning and once in the afternoon on the 4th day of modeling.

[0013] (5) Hot and cold stimulation: On the 5th day of modeling, the rats were transferred to a hot and cold stimulation box, which consists of a cold box and a hot box. The temperature of the cold box is 12℃ and the temperature of the hot box is 38℃. The rats were first transferred to the cold box, then to the hot box after 10 minutes, and then back to the cold box after 10 minutes. This cycle was repeated for 1 hour. The rats were then transferred back to the modeling cage. The rat model of COPD with respiratory failure was obtained on the 6th day of modeling.

[0014] After modeling, the following methods were used to investigate the effects on rat behavior: grip strength test, open field test, cognitive ability assessment, pulmonary function test, lung tissue pathological observation, TUNEL staining to observe lung tissue apoptosis, transmission electron microscopy, peripheral blood T lymphocyte and subset detection, bronchoalveolar lavage fluid collection and counting, serum inflammatory factor levels detected by ELISA, protein expression levels of NF-κB, CD83, IFN-γ and IL-4 in lung tissue detected by immunohistochemistry, TLR4, MyD88, NF-κB and TNF-α protein expression in lung tissue detected by Western blot, and TLR4, MyD88, NF-κB and casepase-3 in lung tissue detected by RT-qPCR. The model was evaluated in terms of mRNA expression and other aspects. The results showed that the rats exhibited typical symptoms of chronic obstructive pulmonary disease (COPD) complicated with respiratory failure (chronic bronchitis, emphysema, airflow limitation and small airway remodeling, etc.), which met the criteria for the reliability and effectiveness of the animal model of COPD complicated with respiratory failure, namely the pathogenesis, symptoms and pathophysiology of the disease. According to the construction method of the present invention, the rat model of COPD complicated with respiratory failure can be successfully prepared.

[0015] The technical solution of the present invention has the following advantages:

[0016] 1. Current chronic obstructive pulmonary disease (COPD) models suffer from drawbacks such as long processing time, inconsistent standards, poor model formation, and poor controllability and reproducibility. There is a lack of efficient and ideal standard animal models for studying the mechanisms of COPD complicated with respiratory failure and for drug screening. The present invention provides a method for preparing a rat model of COPD complicated with respiratory failure. This model is induced by injection of sodium periodate and p-hydroxybenzyl alcohol, gavage with benzylsulfonic acid, hypoxia restraint, and thermo-cold stimulation. This induces a short-term, efficient, operable, reproducible, stable, and clinically relevant rat model of COPD complicated with respiratory failure. This multi-factor-induced COPD complicated with respiratory failure better simulates the symptoms of human COPD complicated with respiratory failure.

[0017] 2. The non-single-factor induced COPD complicated with respiratory failure animal model of this invention is an ideal animal model from both a pathophysiological perspective and from the perspective of simulating typical clinical symptoms and predicting treatment. It will support the exploration of the pathogenesis of COPD complicated with respiratory failure and provide a theoretical basis for developing new treatment options for COPD complicated with respiratory failure. The COPD complicated with respiratory failure animal model constructed by this invention is highly efficient, time-saving, has stable effects, and exhibits small individual differences; the synergistic interaction among multiple factors leads to a more accurate disease model. This model demonstrates high success rate and stability in inducing classic symptoms of COPD complicated with respiratory failure (chronic bronchitis, emphysema, pulmonary airflow limitation, and small airway remodeling, etc.).

[0018] 3. In this invention, on the first day of modeling, rats were intraperitoneally injected with sodium periodate (6 mg / kg) in the morning and intramuscularly injected with p-hydroxybenzyl alcohol (8 mg / kg) in the evening. Sodium periodate can inhibit the activity of endogenous antiproteases and recruit and activate alveolar macrophages and neutrophils, which release large amounts of neutrophil elastase, cathepsins, etc. These proteases can degrade the extracellular matrix, elastic fibers, and collagen, causing lung tissue damage. p-Hydroxybenzyl alcohol can induce the binding of thioredoxin-interacting protein (TXNIP) to NLRP3 in rats, mediating the assembly and activation of the inflammasome complex, initiating apoptosis, damaging respiratory and lung tissue cells, triggering a series of oxidative stress and inflammatory responses, and promoting the progression and deterioration of COPD. The intraperitoneal injection of sodium periodate followed by intramuscular injection of p-hydroxybenzyl alcohol synergistically induces lung tissue damage in rats to a greater extent, increasing the tissue infiltration of inflammatory cells and endothelial cell dysfunction.

[0019] Oral administration of benzylsulfonic acid to rats activates the pro-inflammatory cell signaling pathway NF-κB, promoting NF-κB translocation to the cell nucleus and facilitating the transcription and expression of inflammatory mediators, chemokines, and proteases. This induces the recruitment of inflammatory cells such as macrophages, neutrophils, and lymphocytes to the lungs, amplifying and prolonging the chronic inflammatory response. Oral administration of benzylsulfonic acid to rats following in vivo injection of sodium periodate and p-hydroxybenzyl alcohol not only consolidated and enhanced the induction effect of the injection but also further amplified the inflammatory response and exacerbated lung tissue damage, significantly contributing to the induction of COPD complicated by respiratory failure in rats.

[0020] Hypoxic restraint in rats stimulates airway epithelial cells to recruit and activate large amounts of PMNs, releasing ROS, pro-inflammatory cytokines, and proteases, inducing an innate immune response and activating T lymphocytes, thus triggering an acquired immune response. These cytokines further stimulate the massive activation of inflammatory cells, creating a cascade effect that leads to a continuous and abnormal inflammatory response in lung tissue, accompanied by various biochemical effects such as oxidative stress and damage, protease / anti-protease imbalance, and apoptosis / anti-apoptosis imbalance. In vivo injection of sodium periodate and p-hydroxybenzyl alcohol, followed by benzylsulfonic acid gavage, further exacerbates the pulmonary inflammatory response, oxidative stress, and lung tissue damage in rats under hypoxic restraint.

[0021] This invention involves subjecting rats to thermal stimulation on day 5 of model establishment. Thermal stimulation induces overexpression of IL-6 in rats, thereby activating JAK family proteins, which subsequently phosphorylate and activate STAT3 (a signal transduction and transcription activator). Activated STAT3 enters the cell nucleus and promotes the transcription of inflammation-related genes, such as IL-8, IL-1β, and TNF-α, exacerbating pulmonary inflammation and reducing lung function. In vivo injection of sodium periodate and p-hydroxybenzyl alcohol, followed by benzylsulfonic acid gavage and hypoxia restraint, followed by thermal stimulation, activates oxidative stress in rats, further damaging airways and lung tissue. It also promotes mucus secretion from goblet cells in the airway epithelium, causing airway obstruction, and induces alveolar epithelial cell apoptosis, leading to emphysema.

[0022] After intraperitoneal injection of sodium periodate and intramuscular injection of p-hydroxybenzyl alcohol, rats were administered benzylsulfonic acid by gavage and hypoxia restraint. Simultaneously, the rats were subjected to hot and cold stimulation in the later stage of modeling. Multiple steps worked together. Drug injection caused lung inflammation and lung tissue damage. Benzylsulfonic acid gavage not only consolidated and strengthened the induction effect of drug injection, but also further amplified the inflammatory response and aggravated lung tissue damage. Hypoxia restraint could further aggravate the inflammatory response, oxidative stress response and lung tissue damage in rats. The hot and cold stimulation based on the above treatment further induced and aggravated lung tissue inflammation, oxidative stress and emphysema in rats, aggravating the pathological response in the rat lungs. Multiple steps worked together to ensure a stable rat COPD combined with respiratory failure model.

[0023] 4. This study provides strong theoretical support and a model reference for in-depth analysis of the immune mechanisms in humans with COPD complicated by respiratory failure and for evaluating new treatment methods. The research team has repeatedly validated the model, with reliable results. The model has also been used to evaluate the efficacy of various drugs, validating its application value. This animal model exhibits typical characteristics of COPD complicated by respiratory failure: chronic bronchitis, emphysema, airflow limitation, and small airway remodeling. This model can be used for basic research in the field of COPD complicated by respiratory failure, laying an experimental animal foundation for exploring the pathogenesis of COPD complicated by respiratory failure. This study provides a reliable animal model for evaluating the efficacy of drugs for COPD complicated by respiratory failure, with advantages such as shorter time consumption, safety and controllability, good operability and reproducibility, stability and uniformity, good universality, and closer relevance to clinical practice. Attached Figure Description

[0024] Figure 1 This section describes the clinical observation of rats in the control group and the model group.

[0025] Figure 2 HE staining results of lung tissues from the control group and the model group.

[0026] Figure 3 Masson staining images of the control group and the model group.

[0027] Figure 4 Apoptosis in the lung tissue of rats in each group.

[0028] Figure 5 The ultrastructure of rat lung tissue cells.

[0029] Figure 6 This study describes the detection of peripheral blood T lymphocytes and their subsets in rats.

[0030] Figure 7 The expression of NF-KB, CD83, IFN-γ and IL-4 in the lung tissue of two groups of rats was determined.

[0031] Figure 8 The image shows the protein bands of TLR4, MyD88, NF-κB, and TNF-α in rat lung tissue. Detailed Implementation

[0032] I. A method for preparing a rat model of COPD complicated with respiratory failure, comprising the following steps:

[0033] (1) Temporary rearing: Select 6-8 week old SPF-grade male SD rats with normal development and good vitality, weighing 190-210g, and raise them at a temperature of 20℃-24℃ and a humidity of 50%-70%. Provide them with normal food and water, and provide them with a half-day and half-night lighting cycle. Adapt to rearing for 7 days. All operating procedures and rearing conditions comply with the regulations for the management and rearing of experimental animals and follow humanitarian principles.

[0034] (2) Sodium periodate and p-hydroxybenzyl alcohol injection: After 7 days of acclimatization, the rats were transferred to the modeling cage to start the modeling process. During the modeling process, the rats drank water and ate normally. On the first day of modeling, 6 mg / kg of sodium periodate (based on the rat's body weight) was injected into the abdominal cavity of the rats in the morning and evening. 30 minutes after each injection of sodium periodate, 8 mg / kg of p-hydroxybenzyl alcohol (based on the rat's body weight) was injected into the rats intramuscularly.

[0035] (3) Benzenesulfonic acid gavage: On the 2nd-3rd day of modeling, benzenesulfonic acid aqueous solution was administered to rats by gavage once a day, with a dose of 0.1 ml / rat each time. The concentration of the benzenesulfonic acid aqueous solution was 40%.

[0036] (4) Hypoxia restraint: On the 4th day of modeling, the rats were transferred into the restraint tube of the restraint cage. The restraint tube was set in the restraint cage and the diameter of the restraint tube matched the size of the rat, that is, the rat could easily enter but could not turn around or back after entering. Then the restraint cage was placed in a hypoxia incubator with an oxygen concentration of 12% O2. After 30 minutes of hypoxia restraint, the rats were transferred back to the modeling cage. Hypoxia restraint was performed once in the morning and once in the afternoon on the 4th day of modeling.

[0037] (5) Hot and cold stimulation: On the 5th day of modeling, the rats were transferred to a hot and cold stimulation box, which consists of a cold box and a hot box. The temperature of the cold box is 12℃ and the temperature of the hot box is 38℃. The rats were first transferred to the cold box, then to the hot box after 10 minutes, and then back to the cold box after 10 minutes. This cycle was repeated for 1 hour, and then the rats were transferred back to the modeling cage. The rat model of COPD combined with respiratory failure can be obtained on the 6th day of modeling.

[0038] After modeling, the model was evaluated through the following methods: behavioral observation of rats, grip strength test, open field test, cognitive ability assessment, pulmonary function test, lung tissue pathological observation, TUNEL staining to observe lung tissue apoptosis, transmission electron microscopy, peripheral blood T lymphocyte and subset detection, bronchoalveolar lavage fluid collection and counting, ELISA to detect serum inflammatory factor levels, immunohistochemistry to detect the protein expression levels of NF-κB, CD83, IFN-γ and IL-4 in lung tissue, Western blot to detect the protein expression of TLR4, MyD88, NF-κB and TNF-α in lung tissue, and RT-qPCR to detect the mRNA expression of TLR4, MyD88, NF-κB and casepase-3 in lung tissue (the control group received corresponding saline injection, gavage and routine feeding). The results are as follows:

[0039] 1. Behavioral observation

[0040] Daily observations were conducted on the coughing, wheezing, and shortness of breath in each group of rats, as well as their fur, food and water intake, nasal and oral secretions, mental state, and mortality. Rats in the control group were in good mental condition, with glossy white fur, moderate body size, active and alert behavior, and stable breathing; no coughing, sputum production, or sneezing was observed. Compared to the control group, rats in the model group were lethargic, with dull, yellowish fur, frequent coughing and sneezing, slow responses, increased nasal secretions, pronounced rattling sounds in the throat, decreased food intake, and increased water intake. No rat deaths occurred during the experiment. Figure 1 The clinical observation results of rats in each group are as follows.

[0041] 2. Grip test

[0042] After modeling, gripping force tests were performed on both groups of rats. Using a gripping force testing device, the rats were guided to grasp the front bar of the device with their forepaws, and then pulled backward. Each rat was tested three times, and the average value was recorded as the statistical data. If a rat lost its grip, turned its head, or struggled from side to side, the data was not recorded, and the measurement was repeated. The average of the maximum gripping force values ​​of the two groups of rats was compared.

[0043] The results showed that the average maximum gripping force of the control group was 7288.32 ± 1101.02 (g); (n=10), the average gripping force of the model group is 5368.26±1016.45 (g); (n=10), the maximum gripping force of the model group was significantly lower than that of the control group (P<0.05).

[0044] 3. Open field experiment

[0045] A 100cm×100cm×40cm black opaque open box was placed in the center of the experimental field, with 25 equally sized squares at the bottom. A camera was fixed to the top of the box, its field of view covering the entire box. Rats from each group were placed in the central square, and the experiment began, with each animal timed individually for 6 minutes. After the experiment, all images recorded by the camera were analyzed using an animal behavior analysis system, and behavioral indicators related to total walking distance and average walking speed (total open field distance, average open field speed) were analyzed.

[0046] The results showed no significant difference in open field distance among the rat groups before modeling. Six days after modeling, the total open field distance in the control group was 1582.25 ± 332.82 cm. (n=10) The average velocity of the open field is 6.45±1.56 (cm / s); (n=10), the total open field distance of the model group is 435.65±134.45 (cm); (n=10) The average velocity in the open field is 1.76 ± 0.52 (cm / s). (n=10). It can be seen that the total walking distance and average speed of the model group were significantly lower than those of the control group (P<0.05).

[0047] 4. Cognitive Ability Assessment

[0048] The Morris water maze test was used to assess the cognitive abilities of two groups of rats. The movement trajectories of the experimental animals were collected and analyzed using a camera tracking system and SMART 3.0 software. Visual platform training phase: Rats were first placed in water, starting in the quadrant opposite to the visual platform. The time required for them to reach the platform (i.e., escape latency) was recorded. If a rat did not find the platform within 60 seconds, it was guided to the visible platform; the latency in this case was recorded as 60 seconds. Spatial reference memory test phase: This phase consisted of a training period and an exploration period. 24 hours after the training period ended, an exploration test was conducted. The platform was removed, and all rats underwent a 60-second exploration test. The time and number of times the rats crossed the original platform location were recorded, and the swimming paths were compared for subsequent analysis.

[0049] The results showed that, compared with the control group, the rats in the model group crossed the platform in the target area less frequently and for less time, and swam a longer distance; on the 5th day of training, the escape latency of the rats in the model group was significantly prolonged compared with the control group (P<0.05).

[0050] 5. Lung function test

[0051] Rats were anesthetized with 3% sodium pentobarbital (60 mg / kg, intraperitoneal injection), and forced vital capacity (FVC) and forced expiratory volume in 0.1 seconds (FEV1) were measured by endotracheal intubation. 0.1 ) and FEV 0.1 / FVC.

[0052] The results are shown in the table below. (n=10) Compared with the control group, the lung function value of the model group rats was significantly reduced (P<0.01), and FEV1 was also significantly reduced. 0.1 and FEV 0.1 / FVC was significantly lower than that of the control group (P<0.01).

[0053] Lung function of rats in each group ( n=10)

[0054]

[0055] 6. Pathological observation of lung tissue

[0056] The left and right lungs of rats in each group were separated, the right main bronchus was ligated, and the left lung was connected to the perfusion system. The lungs were perfused and fixed with 4% formaldehyde solution for 20 min, dehydrated, fixed, embedded in paraffin, sectioned, and stained with HE and Masson to observe the structural changes of the alveoli, bronchioles, and small blood vessels in rats.

[0057] HE staining results are as follows Figure 2 As shown, the alveoli in the control group were intact and uniform in size, with a small number of inflammatory cells around the airways. In the model group, rats exhibited significant dilation of alveolar ducts, alveolar sacs, and alveoli; disordered alveolar structure; thinning and rupture of the alveolar walls, with some alveolar walls merging into bullae; ciliary cell degeneration and necrosis, as well as ciliary collapse, breakage, adhesion, and shedding; epithelial cell shedding; goblet cell metaplasia; and increased mucus secretion. The pulmonary artery walls were thickened, the lumen narrowed, and there was varying degrees of peripheral neutrophil, lymphocyte, and mononuclear macrophage infiltration.

[0058] Masson staining, such as Figure 3 As shown, the lung tissue of the control group rats showed no obvious accumulation of inflammatory cells, the airway epithelium and alveolar structure were intact, and no fibrosis was observed in the bronchial walls. In the model group, there was extensive inflammatory cell infiltration in the small airways and pulmonary vessels, partial alveolar septal rupture, irregular enlargement of alveolar cavities, disordered alveolar structure, bronchial wall epithelial hyperplasia, thickening of the smooth muscle layer, and increased and elongated mucosal folds, leading to bronchial stenosis; a large amount of blue collagen deposition was observed in the lung tissue, indicating severe pulmonary fibrosis.

[0059] 7. TUNEL staining method for observing lung tissue apoptosis

[0060] Lung tissue paraffin sections were routinely dewaxed to water, and antigen retrieval was performed using sodium citrate. The sections were then incubated with DNase-free proteinase K for 20 min, Triton for 8 min, and TUNEL reaction solution for 1 h. After washing with PBS, DAB was added and reacted for 10 min. Following washing, the sections were mounted with anti-fluorescence quenching mounting medium and observed under a fluorescence microscope. Green fluorescence indicated positive apoptotic cells, and blue fluorescence indicated cell nuclei. Three fields of view were randomly selected from each slide, and the average optical density of apoptotic cells was recorded using ImageJ software.

[0061] Compared with the control group, the model group showed increased green fluorescence (see...). Figure 4 The number of TUNEL-positive cells increased significantly (P<0.05); the percentage of positive cells in the blank group was (0.23±0.02)%, while that in the model group was (22.85±0.57)%. n = 3).

[0062] 8. Observation using transmission electron microscopy

[0063] Take rats ≤1mm from each group 3 Fresh lung tissue blocks of various sizes were fixed in electron microscopy fixative at 4°C for 4 hours, rinsed with PBS, and then fixed with osmium tetroxide (OsO4) for 2 hours. After dehydration with graded ethanol, the blocks were infiltrated, embedded in pure epoxy resin 812, sectioned, stained with lead citrate, and observed under a transmission electron microscope. Images were then acquired.

[0064] The results are as follows Figure 5 As shown, the control group had intact and uniform alveolar structure, and no obvious inflammatory cell aggregation in the lung tissue. In the model group, the alveolar walls were ruptured, the alveolar cavities were fused, bullae appeared, and inflammatory cells infiltrated the alveolar cavities and the peritracheal airways.

[0065] 9. Detection of peripheral blood T lymphocytes and their subsets

[0066] Six days after modeling, blood was collected from the orbital sinus of rats in each group. 50 μL of each blood sample was added, along with 2 μL each of flow cytometry antibodies CD3-APC, CD4-FITC, and CD8-PE. The mixture was incubated in the dark for 20 min. 1 mL of PBS was added, and the mixture was incubated at 1100 rpm. -1 Centrifuge for 5 min. Discard the supernatant, add 1 mL of 1× erythrocyte lysis buffer, and lyse for 10 min at room temperature (1100 rpm). -1 Centrifuge for 5 min; discard the supernatant, add 1 mL PBS, and centrifuge at 1100 rpm. -1 Centrifuge for 5 min; discard the supernatant, add 300 μL PBS to resuspend, filter with a cell sieve, and then use a microarray to detect the proportion of T lymphocytes (T cells), helper T cells (Th, CD4+), and cytotoxic T cells (Tc, CD8+) in peripheral blood T lymphocytes.

[0067] The results are as follows Figure 6 As shown, compared with the control group, the proportion of T cells and Th cells and the Th / Tc ratio in the peripheral blood of rats in the model group decreased significantly, while the proportion of Tc cells increased significantly.

[0068] 10. Collection and counting of bronchoalveolar lavage fluid

[0069] Rats were euthanized by cervical dislocation after pulmonary function testing. The left thoracic cavity was opened, and the left main bronchus was dissected and ligated upwards along the lung lobe. A 22G intravenous catheter cannula was inserted through the tracheotomy, and the trachea was ligated. A 1mL syringe was attached to the end of the intravenous catheter, and 0.5mL of sterile PBS was rapidly injected for unilateral BALF. Immediately after PBS injection, the fluid was slowly recovered under low negative pressure. This process was repeated three times, with a recovery rate of 90%–92%. The collected BALF was mixed and 20μL was aspirated onto a hemocytometer for cell counting. The remaining BALF was centrifuged at 4℃ and 1000r / min for 10min. The precipitate was resuspended in PBS, and three cell smears were prepared. Wright-Giemsa staining was used for cell classification and counting.

[0070] The results are shown in the table below. (n=10) The BALF recovery rates in the normal control group and the model group were (91.6±1.2)% and (90.8±1.5)%, respectively, with no statistically significant difference in BALF recovery amount and rate between the two groups (P>0.05). The total number of BALF cells in the model group was higher than that in the control group, and the number of macrophages (AM), neutrophils (N), and the proportion of neutrophils (N%) were also higher in the model group than in the normal control group, with statistically significant differences (P<0.05).

[0071] Comparison of cell count and classification results in rat BALF ( n=10)

[0072]

[0073] 11. Detection of serum inflammatory factor levels using ELISA method

[0074] Serum from each group of rats was collected, and the levels of serum inflammatory factors TNF-α, IL-1β, IL-6, IL-13, TGF-β1, MDA, and SOD activity were detected by ELISA according to the kit instructions.

[0075] The results are shown in the table below. (n=8) Compared with the blank control group, the serum SOD activity of rats in the model group was decreased (P<0.01), and the levels of TNF-α, IL-1β, IL-6, IL-13, TGF-β1 and MDA were increased (P<0.01).

[0076] Rat serum inflammatory factor expression levels ( n=8)

[0077]

[0078] 12. Immunohistochemistry was used to detect the protein expression levels of NF-κB, CD83, IFN-γ, and IL-4 in lung tissue.

[0079] Rat lung tissue sections were dewaxed and dehydrated before antigen retrieval and blocking. They were then incubated overnight at 4°C with rabbit anti-rat NF-κB antibody (1:500), CD83 antibody (1:400), IFN-γ antibody (1:200), and IL-4 antibody (1:200). After rewarming, staining was performed using a rat secondary antibody ultrasensitive two-step immunohistochemical assay kit, followed by DAB staining and hematoxylin and violet counterstaining. Sections were then differentiated, dehydrated, dried, mounted, and examined under a microscope. IPWIN60 software was used to analyze selected sections, and five high-power fields (×400) were randomly selected from areas of significant positive expression. The average integrated optical density (IOD) was calculated as an indicator of the target protein expression intensity in the sample.

[0080] The results are as follows Figure 7 As shown in the table below, the integrated optical density (IOD) of NF-κB and IFN-γ in the lung tissue of the model group rats was higher than that of the control group (P<0.05), while the integrated optical density (IOD) of CD83 and IL-4 was lower than that of the control group (P<0.05).

[0081] Expression of NF-κB, CD83, [FN-γ and IL-4 in lung tissue of two groups of rats ( n=10)

[0082]

[0083] 13. Western blot analysis of TLR4, MyD88, NF-κB, and TNF-α protein expression in lung tissue.

[0084] 60 mg of lung tissue was weighed and added to RIPA lysis buffer, then lysed on ice. Total protein was extracted from the tissue, and protein concentration was determined using the BCA method. Related proteins were separated by electrophoresis, transferred to a membrane at low temperature, blocked with 5% skim milk powder solution for 1 h, and then incubated overnight at 4°C with primary antibody dilution buffer (TLR4, MyD88, NF-κB, TNF-α, and β-actin diluted 1:500 each). After thorough washing with TBST, secondary antibody (1:1000) was added and incubated at room temperature for 1 h. After thorough washing with TBST, ECL was added for color development and scanning. β-actin was used as an internal control. ImageJ software was used to analyze the optical density (OD) value of the target protein band and calculate the expression level of the target protein.

[0085] The results are as follows Figure 8 As shown, compared with the blank control group, the expression of TLR4, MyD88, NF-κB and TNF-α proteins in the lung tissue of rats in the model group was increased (P<0.01).

[0086] 14. RT-qPCR method for detecting the expression of TLR4, MyD88, NF-κB, and casepase-3 mRNA in lung tissue.

[0087] Total RNA was extracted from rat lung tissue using the Trizol one-step method and reverse transcribed into cDNA. Primers were designed and synthesized by Sangon Biotech Co., Ltd. The PCR reaction program consisted of 40 cycles of amplification: 94℃ for 10 min, 94℃ for 30 s, and 72℃ for 30 s; the reaction was terminated at 72℃ for 10 min. β-actin was used as an internal control. Results were analyzed using 2... -ΔΔCt The expression of each target gene can be calculated.

[0088] The results are shown in the table below. Compared with the blank group, the expression of TLR4, MyD88, NF-κB and casepase-3 mRNA in the lung tissue of rats in the model group was increased (P<0.01).

[0089] Expression of TLR4, MyD88, NF-κB, and casepase-3 mRNA in rat lung tissue ( n=6)

[0090]

[0091] II. Selection of Modeling Methods

[0092] Male SPF-grade SD rats aged 6-8 weeks with normal development and good vitality, weighing 190-210g, were selected and, after one week of acclimatization feeding, divided into eight groups: control group, model group, control group 1, control group 2, control group 3, control group 4, control group 5, and control group 6. Control group rats were raised under routine conditions (receiving corresponding saline injections and gavage). Model group rats were raised according to the model preparation method of this invention, i.e., injection of sodium periodate and p-hydroxybenzyl alcohol + gavage with benzylsulfonic acid + hypoxia restraint + hot and cold stimulation. Control group 1 did not receive sodium periodate injection, i.e., injection of p-hydroxybenzyl alcohol + gavage with benzylsulfonic acid + hypoxia restraint + hot and cold stimulation. Control group 2 did not receive p-hydroxybenzyl alcohol injection, i.e., injection of sodium periodate + gavage with benzylsulfonic acid + hypoxia restraint + hot and cold stimulation. Control group 3 did not receive sodium periodate and p-hydroxybenzyl alcohol injection, i.e., injection of saline + gavage with benzylsulfonic acid + hypoxia restraint + hot and cold stimulation. Group 4 was compared without benzyl sulfonate gavage; the treatment consisted of p-hydroxybenzyl alcohol injection + saline gavage + hypoxia restraint + hot and cold stimulation. Group 5 was compared without hypoxia restraint; the treatment consisted of sodium periodate and p-hydroxybenzyl alcohol injection + benzyl sulfonate gavage + hot and cold stimulation. Group 6 was compared without hot and cold stimulation; the treatment consisted of sodium periodate and p-hydroxybenzyl alcohol injection + benzyl sulfonate gavage + hypoxia restraint. Six days after rats were induced to develop the model, lung tissue was collected for HE staining, Masson staining, TUNEL staining, and transmission electron microscopy. Serum inflammatory factor levels were detected using ELISA.

[0093] The results showed that the control group rats had normal physiological behavior and lung function. The alveolar structure in the control group was intact and uniform in size, with no obvious inflammatory cell aggregation and fewer positive apoptotic cells in the lung tissue. The model group rats were lethargic, with dull, yellowish fur, and frequent coughing and sneezing; their alveolar structure was disordered, the pulmonary artery walls were thickened, the lumen was narrowed, and there was varying degrees of peripheral neutrophil, lymphocyte, and monocyte / macrophage infiltration; severe pulmonary fibrosis; increased positive apoptotic cells; pulmonary bullae appeared; and inflammatory cell infiltration was observed in the alveolar cavity and around the airways. Serum levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-13, TGF-β1, and MDA were significantly higher than in the control group, while SOD activity was significantly lower. All six control groups exhibited similar behaviors and symptoms to the model group, but the severity of symptoms was significantly lower. Serum levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-13, TGF-β1, and MDA were lower than in the model group, while SOD activity was higher in all control groups.

[0094] Therefore, the COPD model with respiratory failure established in this invention exhibits multiple key pathological features of COPD with respiratory failure, including chronic inflammatory cell infiltration in the airways and bronchi at all levels, increased mucus secretion, significant airway remodeling, restricted airflow, bullae formation, and emphysema. Both the pathological changes in tissue structure and the expression levels of inflammatory and cell transformation-related proteins at the lung tissue and cellular levels demonstrate that the preparation method of this invention can effectively replicate the clinicopathological features of human COPD with respiratory failure.

[0095] III. Application of Pulmonary Rehabilitation in a Rat Model of COPD Complicated with Respiratory Failure

[0096] Pulmonary rehabilitation (PR) refers to a series of individualized exercise training, breathing exercises, nutritional support, and psychological interventions to help patients with chronic respiratory diseases improve respiratory function, alleviate symptoms, enhance exercise tolerance, and improve quality of life. Functional exercise is the core of PR; it does not mean strenuous exercise, but rather scientific and gradual exercise to progressively enhance lung function and overall endurance. Aerobic exercise can increase respiratory and cardiovascular activity, effectively improving cardiopulmonary endurance and exercise tolerance, and improving lung function, exercise capacity, and mental state in COPD patients, especially for elderly COPD patients and those with cardiovascular disease, muscle dysfunction, and obesity. Regular aerobic exercise can improve lung function and exercise capacity by regulating immune and inflammatory responses, thereby improving the quality of life and prognosis of COPD patients. Regular aerobic exercise intervention can promote the balance of oxidation and antioxidation in the body, thereby improving the occurrence and development of CS-induced COPD. Long-term adherence to aerobic exercise not only helps improve airway remodeling after the onset of the disease but also prevents remodeling and inflammatory processes during disease progression. Previous studies by our research group have also shown that aerobic exercise can delay lung tissue structural damage and improve airflow limitation in COPD patients, and is superior to resistance exercise and high-intensity interval exercise in improving lung hyperinflation in COPD rats.

[0097] SPF-grade male SD rats aged 6-8 weeks with normal development and good vitality, weighing 190-210g, were selected and, after one week of acclimatization, divided into three groups: control group, model group, and pulmonary rehabilitation group. The control group rats were raised under routine conditions (including corresponding saline injections and gavage). The model group and pulmonary rehabilitation group were raised according to the model preparation method of this invention, namely, injection of sodium periodate and p-hydroxybenzyl alcohol + gavage with benzyl sulfonic acid + hypoxia restraint + hot and cold stimulation. Six days after modeling, the control group and model group received no treatment, while the pulmonary rehabilitation group rats underwent a moderate-intensity aerobic exercise program, which included acclimatization exercise in week 1 and formal exercise in weeks 2-9. Maximum capacity testing was conducted on the last day of week 1. The testing method was as follows: the treadmill incline was 0°, the warm-up speed was 5 m / min for 5 minutes, and then the treadmill speed was gradually increased by 2 m / min every 3 minutes until the rats were exhausted. This was used to measure the rats' maximum exercise speed. Week 1: Day 1: 10 min; Days 2-6: increasing by 10 min / day until reaching 60 min on Day 6, with an exercise speed of 5 m / min; Weeks 2-9: moderate-intensity aerobic treadmill exercise, 55% of maximum exercise speed, 6 days / week, once / day, 60 min / session. After 9 weeks, lung function was tested in each group of rats. Lung tissue was collected for HE staining, Masson staining, TUNEL staining, and transmission electron microscopy. Serum inflammatory factor levels were detected using ELISA.

[0098] The results showed that the control group rats had normal physiological behavior and lung function, intact alveolar structure, no obvious inflammatory cell accumulation in lung tissue, and fewer positive apoptotic cells. The model group rats were lethargic, had dull, yellowish fur, and frequent coughing and sneezing; their alveolar structure was disordered, with varying degrees of peripheral neutrophil, lymphocyte, and monocyte / macrophage infiltration; severe pulmonary fibrosis; increased positive apoptotic cells; inflammatory cell infiltration in the alveolar cavity and airway peribronchial region; and serum levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-13, TGF-β1, and MDA were significantly higher than those in the control group. The pulmonary rehabilitation group showed significantly reduced physiological behavior and pulmonary symptoms compared to the model group, with improved lung function and FEV1. 0.1 and FEV 0.1 The FVC ratio was significantly higher in the control group than in the model group, while the serum levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-13, TGF-β1, and MDA were significantly lower in the control group than in the model group.

[0099] Therefore, moderate-intensity aerobic exercise can effectively reduce airway resistance in rats with COPD and respiratory failure, prevent lung overinflation, alleviate symptoms of dyspnea and airflow limitation, and improve lung function in rats with COPD and respiratory failure. Pathological examination of lung tissue also shows that exercise training can improve emphysema and airway remodeling, increase lung tissue elasticity and compliance, and reduce the destructive process of lung tissue structure in rats with COPD and respiratory failure. At the molecular biological level, exercise training can reduce the secretion of inflammatory cells in lung tissue, such as TNF-α and IL-1β, and lower the level of inflammation in lung tissue.

Claims

1. A method for preparing a rat model of COPD complicated with respiratory failure, characterized in that, Includes the following steps: (1) Temporary rearing: Select 6-8 week old SPF-grade male SD rats with normal development and good vitality, weighing 190-210g, and raise them at a temperature of 20℃-24℃ and a humidity of 50%-70%. Provide them with normal food and water, and provide them with a half-day and half-night lighting cycle. Adapt to rearing for 7 days. All operating procedures and rearing conditions comply with the regulations for the management and rearing of experimental animals and follow humanitarian principles. (2) Sodium periodate and p-hydroxybenzyl alcohol injection: After 7 days of acclimatization, the rats were transferred to the modeling cage to start the modeling process. During the modeling process, the rats drank water and ate normally. On the first day of modeling, 6 mg / kg of sodium periodate was injected into the abdominal cavity of the rats in the morning and evening. 30 minutes after each injection of sodium periodate, 8 mg / kg of p-hydroxybenzyl alcohol was injected into the muscle of the rats. (3) Benzenesulfonic acid gavage: On the 2nd-3rd day of modeling, benzenesulfonic acid aqueous solution was administered to rats by gavage once a day, with a dose of 0.1 ml / rat each time. The concentration of the benzenesulfonic acid aqueous solution was 40%. (4) Hypoxia restraint: On the 4th day of modeling, the rats were transferred into the restraint tube of the restraint cage. The restraint tube was set in the restraint cage and the diameter of the restraint tube matched the size of the rat, that is, the rat could easily enter but could not turn around or back after entering. Then the restraint cage was placed in a hypoxia incubator with an oxygen concentration of 12% O2. After 30 minutes of hypoxia restraint, the rats were transferred back to the modeling cage. Hypoxia restraint was performed once in the morning and once in the afternoon on the 4th day of modeling. (5) Hot and cold stimulation: On the 5th day of modeling, the rats were transferred to a hot and cold stimulation box, which consists of a cold box and a hot box. The temperature of the cold box is 12℃ and the temperature of the hot box is 38℃. The rats were first transferred to the cold box, then to the hot box after 10 minutes, and then back to the cold box after 10 minutes. This cycle was repeated for 1 hour. The rats were then transferred back to the modeling cage. The rat model of COPD with respiratory failure was obtained on the 6th day of modeling.

2. The application of pulmonary rehabilitation in a rat model of COPD complicated with respiratory failure, characterized by: The rat model of COPD complicated with respiratory failure is a rat model prepared by the preparation method described in claim 1.

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