Application of bmp4 gene regulation product in preparation of prevention and treatment product for chronic obstructive pulmonary disease

Abstract

This invention discloses the application of the BMP4 gene and its regulatory products in the preparation of products for the prevention and treatment of COPD, belonging to the field of biomedical technology. This invention found that BMP4 expression is reduced in the lung tissue of COPD patients; animal experiments demonstrated that after CS exposure, BMP4 expression... + / ‑ Mice showed decreased body weight, significantly impaired lung function, more pronounced emphysema and inflammatory infiltration, a significantly increased mean alveolar intercept, and a significantly increased number of inflammatory cells, predominantly neutrophils, in bronchoalveolar lavage fluid. CS exposure combined with airway infusion of AAV9-BMP4 revealed that airway infusion of AAV9-BMP4 (1×10⁻⁶) significantly reduced lung function. 11 PFU / mouse significantly improved decreased lung function, emphysema, inflammatory infiltration, increased mean alveolar intercept, and increased number of inflammatory cells in bronchoalveolar lavage fluid in mice after CS exposure. This suggests that BMP4 may be a potential target for COPD treatment and could be applied to the development of new drugs and technologies for COPD.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of BMP4 gene and a regulation product thereof in preparation of a chronic obstructive pulmonary disease prevention and treatment product. BACKGROUND

[0002] Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous lung condition characterized by persistent, progressive airflow limitation, usually accompanied by symptoms of chronic bronchitis and emphysema, of which emphysema is a key pathological feature. COPD has a high morbidity and mortality rate worldwide, and cigarette smoke (CS) exposure is the main risk factor for COPD. Lung tissue has a certain regenerative capacity, and when lung tissue is damaged, it can be repaired through the activation and differentiation of stem / progenitor cells. Alveoli are mainly composed of flat alveolar type I epithelial cells (AT1) and cuboidal alveolar type II epithelial cells (AT2), and AT2 cells are the main progenitor cells in the alveoli, which have the ability to self-replicate and differentiate into AT1 cells to repair the alveolar structure. However, there is a defect in the repair of lung tissue in COPD patients. Bone Morphogenetic Protein 4 (BMP4) is a member of the TGF-beta superfamily, which is composed of two polypeptide chains covalently bound by disulfide bonds, and is involved in regulating cell proliferation, differentiation and maturation. During lung development, BMP4 regulates the formation and maturation of airways and alveoli, and promotes normal lung development. Studies have shown that in emphysema, abnormalities in the BMP signaling pathway may lead to decreased lung tissue repair capacity and affect lung epithelial cell regeneration and differentiation. However, the exact role of BMP4 in emphysema repair induced by cigarette smoke (CS) is still not completely clear. SUMMARY

[0003] To solve the above technical problems, the application provides, for the first time, application of BMP4 gene and a regulation product thereof in preparation of a chronic obstructive pulmonary disease prevention and treatment product. The application detects the expression of BMP4 protein in lung tissue of 17 COPD patients and 17 control group by Western Blots (WB) experiment, and finds that the expression of BMP4 in lung tissue of COPD patients is reduced. Animal experiments prove that after exposure to cigarette smoke, the expression of BMP4 in lung tissue of mice is reduced, and the expression of BMP4 in lung tissue of mice is increased after treatment with BMP4, which proves that BMP4 can promote the repair of emphysema induced by cigarette smoke. + / -Mice showed weight loss, significantly decreased lung function, and more pronounced emphysema and inflammatory infiltration in lung pathology, with a significantly increased mean alveolar intercept. Simultaneously, the bronchoalveolar lavage fluid also showed a significant increase in inflammatory cells, predominantly neutrophils. Three months after tobacco smoke exposure, mice were administered AAV9-BMP4 (1×10⁻⁶) via airway infusion. 11 In mice exposed to PFU (phenylephrine urealyticum) for three months, subsequent intratracheal instillation of AAV9-BMP4 significantly improved lung function decline, emphysema, inflammatory infiltration, increased mean alveolar intercept, and increased number of inflammatory cells in bronchoalveolar lavage fluid following tobacco smoke exposure. This result suggests that BMP4 may be a potential therapeutic target for COPD. This invention can be applied to the development of new drugs and technologies for COPD treatment.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] One of the objectives of this invention is to provide the application of BMP4 gene regulation products in the preparation of COPD prevention and treatment products, wherein the regulation is positive regulation.

[0006] Preferably, the BMP4 gene regulation product is AAV9-BMP4.

[0007] More preferably, the BMP4 gene regulation product is administered via inhalation.

[0008] In a further preferred embodiment, the dosage of the BMP4 gene regulation product is 0.5–1.5 × 10⁻⁶. 11 PFU.

[0009] More preferably, the amount of the BMP4 gene regulation product used is 1.0 × 10⁻⁶. 11 PFU.

[0010] The second objective of this invention is to provide a product for the prevention and treatment of COPD, wherein the product contains the BMP4 gene regulation product described in any of the above applications.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) The present invention found that the expression level of BMP4 in the lung tissue of COPD patients is reduced, and the immunofluorescence results show that BMP4 expression is reduced in the lung tissue of COPD patients, the fluorescence intensity is weakened, and it is mainly expressed in type II alveolar epithelial cells.

[0013] (2) This invention found that, compared with wild-type littermates, BMP4 exposed to CS... + / -Mice showed significant weight loss and more severe decline in lung function, manifested by increased functional residual capacity (FRC) and resistance index (RI), as well as decreased dynamic lung compliance (Cchord), 20-millisecond forced expiratory volume (FEV20) / FVC ratio, and 50-millisecond forced expiratory volume (FEV50) / FVC ratio. (BMP4) + / - In mice, HE-stained sections revealed disrupted and disordered alveolar structure, thinning or rupture of alveolar walls, alveolar fusion, enlarged alveolar cavities, and inflammatory cells infiltrating the alveolar septa and alveolar cavities, as well as submucosal and mucosal inflammatory cell infiltration in the alveolar septa. Mice with BMP4 gene deletion showed no significant changes in lung function under normal air exposure. Six months of CS exposure led to an increase in the total number of cells and differential cell count in the BALF of wild-type mice, with a significant increase in BMP4 knockout mice. These results indicate that BMP4 knockout exacerbates CS-induced emphysema.

[0014] (3) This invention found that, compared with mice exposed to tobacco smoke alone, airway instillation of AAV9-BMP4 improved the decline in lung function in mice, manifested as a decrease in functional residual capacity (FRC) and a decrease in the 50-millisecond forced expiratory volume (FEV50) / FVC ratio. After airway instillation of AAV9-BMP4, the alveolar structure induced by CS exposure remained relatively intact with less damage, only a few alveolar septal breaks, alveolar fusion, and significantly reduced inflammatory cell infiltration. The number of inflammatory cells in the bronchoalveolar lavage fluid of the CS+AAV9-GFP group was significantly increased, mainly neutrophils, and BMP4 overexpression improved the increase in inflammatory cells after CS exposure. These results indicate that airway instillation of overexpression can improve emphysema induced by CS exposure.

[0015] (4) This invention found that, compared with wild-type littermates, BMP4 exposed to CS... + / - Immunofluorescence results of mouse lung tissue sections showed that the expression of AQP5, a surface marker of type I alveolar epithelial cells, was significantly reduced, while the expression of SFTPC, a surface marker of type II alveolar epithelial cells, was increased. These results suggest that exposure to tobacco smoke may impair the transdifferentiation of type II alveolar epithelial cells into type I alveolar epithelial cells.

[0016] (5) The present invention found that after stimulating hAT2 cells with 2% CSE for 36 h, the WB results showed that the expression of AQP5, p-SMAD159 and β-catenin were all reduced, while the expression of AQP5, p-SMAD159 and β-catenin were all increased after stimulating with 2% CSE and administering 20 ng BMP4 at the same time.

[0017] In summary, this invention is the first to discover that BMP4 overexpression can improve emphysema caused by CS exposure. This may be a potential target for COPD treatment. This invention can be applied to the research and development of new drugs and technologies for COPD treatment. Attached Figure Description

[0018] Figure 1 This document describes the expression and distribution of BMP4 in lung tissue samples from COPD patients and normal controls in Example 1 of this invention. The results show: (A) BMP4 protein expression level in human lung tissue; (B) grayscale values ​​of protein expression level; (C) representative immunofluorescence images of BMP4 in human lung tissue sections; (D) representative immunofluorescence images of BMP4 and SFTPC in human lung tissue sections; and (E) representative immunofluorescence images of BMP4 and FN in human lung tissue sections. For the Western blot experiment, N=17 per group; for the immunofluorescence experiment, N=5. *P<0.05, **P<0.01, ***P<0.001.

[0019] Figures 2-3 In Example 1 of this invention, the deletion of the BMP4 gene led to decreased lung function and worsened alveolar cavity enlargement in mice exposed to cigarette smoke, wherein: ( Figure 2 A) FRC value; Figure 2 B) IC value; Figure 2 C) Cydn value; Figure 2 D) RI value; ( Figure 2 E) FEV20 / FVC value; Figure 2 F) FEV50 / FVC value; Figure 2 G) HE staining of lung sections; Figure 2 H) Statistical graph of mean linear intercept of lung sections; Figure 2 A) HE staining of bronchoalveolar lavage fluid; Figure 3 B) Total cell count in bronchoalveolar lavage fluid; Figure 3 C) Number of macrophages; Figure 3 D) Lymphocyte count; Figure 3 E) Neutrophil count; *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 3 The values ​​are: (A) BMP4 protein expression level in lung tissue after exposure to tobacco smoke with BMP4 knocked out in Example 1 of the present invention; (B) grayscale value of protein expression level; *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 4 This refers to the changes in lung function and alveolar space in mice exposed to cigarette smoke after airway overexpression of BMP4 in Example 1 of the present invention; wherein: ( Figures 5-6 A) FRC value;Figure 5 B) IC value; Figure 5 C) Cydn value; Figure 5 D) RI value; ( Figure 5 E) FEV50 / FVC value; Figure 5 F) FEV100 / FVC value; Figure 5 G) BMP4 protein expression level in lung tissue; Figure 5 H) Gray value of protein expression level; Figure 5 I) HE staining of lung sections; Figure 5 J) Statistical graph of mean linear intercept of lung sections; Figure 5 A) HE staining of bronchoalveolar lavage fluid; Figure 6 B) Total cell count in bronchoalveolar lavage fluid; Figure 6 C) Number of macrophages; Figure 6 D) Lymphocyte count; Figure 6 E) Neutrophil count; *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 6 This is an immunofluorescence image of GFP expression in mouse lung tissue after airway overexpression of BMP4 in Example 1 of the present invention.

[0023] Figure 7 The image shows the immunofluorescence staining of lung tissue sections after exposure to tobacco smoke following BMP4 knockout in Example 1 of this invention. Figures 8-9 A representative image of mouse lung tissue sections co-stained with BMP4 and AQP5 immunofluorescence; Figure 8 A representative image of mouse lung tissue sections co-stained with BMP4 and SFTPC immunofluorescence.

[0024] Figure 9 This invention relates to the effect of BMP4 on transdifferentiation of human type II alveolar epithelial cell line (hAT2 cells) under CSE stimulation, as described in Example 1 of this invention. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0026] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0027] Example 1

[0028] 1 Experimental Methods

[0029] 1.1 Research Subjects

[0030] This study consecutively enrolled patients who underwent surgical treatment for pulmonary nodules or lung transplantation for COPD at the First Affiliated Hospital of Guangzhou Medical University between January 2023 and March 2024. Twenty healthy controls and 20 COPD patients were included. Patients were aged 40-80 years, with no statistically significant differences in sex ratio or age between the groups. Patients in the healthy control group had normal lung function and no other underlying lung diseases besides pulmonary nodules. Normal lung tissue adjacent to the pulmonary nodules was used as the healthy control group. The lung tissue used for the healthy control group showed normal structure and no macrophage neutrophil infiltration in the alveoli. Patients in the COPD group showed obstructive ventilatory dysfunction and no other underlying lung diseases besides COPD. All participants were informed and signed informed consent forms.

[0031] Inclusion criteria are: (1) COPD patients: 1. Population: Han Chinese; 2. Age: 40 to 80 years old; 3. Confirmed COPD according to GOLD guidelines, stable patients; 4. Lung function classification: GOLD I-IV. Normal population: 1. Population: Han Chinese; 2. Age: 40 to 80 years old; 3. Normal lung function. Basic information approximate ratio (e.g., gender). "Chronic" is defined as symptoms lasting more than 3 months, or symptoms that recur every 3 months. Respiratory symptoms mainly include cough, sputum, wheezing, and dyspnea.

[0032] Exclusion criteria: 1. Pregnant women; 2. History of acute exacerbation of COPD within 6 weeks; 3. History of lung diseases: asthma, bronchiectasis, cystic fibrosis, panbronchiolitis, pulmonary fibrosis, pneumonia, lung cancer during or in the advanced stage of treatment, suspected lung cancer, history of lung surgery, and other parenchymal lung diseases; 4. Patients with acute myocardial infarction or other acute-phase heart diseases; patients with other serious organ dysfunction such as heart failure, chronic renal failure, liver failure, etc.; 5. Patients who are unable to cooperate in completing the examination or enrollment due to organic diseases of the nervous system, mental illness, or any other reason.

[0033] 1.2 COPD mouse model

[0034] BMP4 gene knockout (BMP4) + / - ) mice and wild-type (BMP4) mice with C57BL / 6J background + / +The fetal mice were provided by the Jackson Laboratory in Barthon, Maine, USA. Animals were housed in a specific pathogen-free facility, and all experimental protocols were approved by the Animal Protection and Use Committee of Guangzhou Medical University. All methods were performed in accordance with the guidelines and regulations approved by the Animal Protection and Use Committee of Guangzhou Medical University. The cigarettes used to establish the mouse COPD model were Red Rose brand filtered cigarettes (Guangdong Tobacco Industry Corporation, China), each containing 11 mg of tar, 1.0 mg of nicotine, and 13 mg of carbon monoxide. BMP4 levels were measured at 6–8 weeks of age. + / - Mice and their littermates (wild-type mice) were exposed to CS using a whole-body exposure system. Simply put, mice were exposed to CS (12 doses / hour, 1 hour / time, 4 times / day, 6 days / week) for 24 weeks. The control group was exposed only to filtered room air (RA). At the end of the experiment, all mice underwent lung function testing and were euthanized.

[0035] 1.3 Lung function test in mice

[0036] Lung function was measured on day 181 after CS exposure. The specific procedures were as follows: Mice were anesthetized with afodin (17.5 μl / g). After anesthesia, the limbs of the mice were fixed to a plastic board with medical tape. After alcohol disinfection, the skin in the midline of the mouse's neck was cut open and the trachea was bluntly dissected with forceps to expose it. A small incision was made in the trachea, and a tracheostomy cannula was inserted. The tracheostomy cannula was connected to a pulmonary function instrument (BUXCO PFT system), and mechanical ventilation was initiated. The experimental instrument settings were as follows: ventilator pressure ±10 cmH2O, inspiratory airflow rate 0.8 ml / s, expiratory airflow rate 0.5 ml / s, maximum inspiratory and expiratory pressure ±40 cmH2O, and respiratory rate 150 breaths / min. After the mice's respiratory rhythm became synchronized with the ventilator, lung function tests were performed to measure the mice's forced vital capacity (FVC), airway resistance index, dynamic lung compliance (Cydn), 20-millisecond FEV / FVC (FEV20%), and 50-millisecond FEV / FVC (FEV50%).

[0037] 1.4 Analysis of Bronchoalveolar Lavage Fluid (BALF)

[0038] After the mouse lung function test, the mouse was fixed to the sampling plastic board with its abdomen facing upward. After opening the mouse's chest cavity, the left main bronchus was clamped with a vascular clamp. An indwelling needle was inserted through the tracheal incision and a slipknot was tied at the distal end of the opening to fix the indwelling needle. A 1mL syringe was connected to the indwelling needle, and 0.4mL of physiological saline was used for irrigation each time, for a total of 6 times. The irrigation fluid from the first three times was collected and placed in the first 1.5mL EP tube (tube 1). The irrigation fluid from the last three times was mixed and placed in the second 1.5mL EP tube (tube 2). The tubes were then placed on ice. The collected BALF was centrifuged at 1000rpm and 4℃ for 15 minutes. The supernatant was aliquoted into two tubes and stored at -80℃ for subsequent experiments. The cell pellet was resuspended in 1mL of cold PBS, counted, and smeared using a cell smearer to ensure that each cell smear contained 1.5×10^5 cells. Then, HE staining was performed to classify and count neutrophils, macrophages, and lymphocytes.

[0039] 1.5 Mouse lung tissue pathology

[0040] Hematoxylin-eosin (HE) staining. The left lung of mice was fixed with 10% formaldehyde solution for 24 h, then embedded in paraffin. The paraffin block was cut into 5 μm thick sections. The dewaxed tissue sections were histologically examined using an HE staining kit (Saiwell, Wuhan).

[0041] 1.6 Western blotting

[0042] Anti-β-actin antibody (66009-1-Ig) was obtained from Proteintech (Wuhan Sanying, China). Anti-BMP4 antibody (sc12721) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and (A11405, A21065) were obtained from Abclonal (Abclonal, China). Anti-SFTPC antibody (A23181) was obtained from Abclonal (Abclonal, China). Anti-AQP5 antibody (A23292) was obtained from Abclonal (Abclonal, China). Peroxidase-labeled secondary antibodies were purchased from Anti-Mouse IgG (H+L) antibody (5220-0341) and Anti-Rabbit IgG (H+L) antibody (5220-0336), which were obtained from Seracare (USA). The binding antibody signal was detected using the Immun-Star HRP chemiluminescence kit (Horseradish peroxidase). Western blot images were acquired using a Tanon 5200 chemiluminescence imaging system (Shanghai Tanon Science & Technology, Shanghai, China). Semi-quantitative analysis of the immunoblot was performed using ImageJ.

[0043] 1.7 Immunofluorescence assay of mouse lung tissue

[0044] Antibody FN (A12977) was obtained from Abclonal (Abclonal, China). Anti-SFTPC antibody (A23181) was obtained from Abclonal (Abclonal, China). Anti-AQP5 antibody (A23292) was obtained from Abclonal (Abclonal, China). Anti-BMP4 antibodies (A11405, A21065) were obtained from Abclonal (Abclonal, China). TSA multiplex immunofluorescence kit (Aifang Biotechnology, China).

[0045] 1.8 Preparation of Cigarette Smoke Extract (CSE)

[0046] CSE was derived from Red Rose brand cigarettes (Guangdong Tobacco Industry Company) and prepared within 30 minutes before cell treatment.

[0047] 1.9 Cell Culture

[0048] The specialized cell culture medium (TM0482) was purchased from the Chinese branch of Applied Biomaterials (ABM) Zhenjiang Aibimeng Biotechnology Co., Ltd. Human type II alveolar epithelial cell line (hAT2) was collected from ABM's Chinese branch, Zhenjiang Aibimeng Biotechnology Co., Ltd., and cultured in hAT2 cell-specific culture medium in a humidified incubator at 37°C, 95% (v / v) air, and 5% (v / v) CO2.

[0049] 1. Treatment of recombinant human protein BMP4

[0050] hAT2 cells were seeded in 6-well culture dishes and cultured for 24 hours. After treating the cells with 20 ng BMP4, 50 ng BMP4 recombinant human protein and 2% CSE for 36 hours, the cell proteins were collected for Western blot experiments.

[0051] 1.11 Statistical Analysis

[0052] All statistical analyses were performed using GraphPadprism software (version 10.0, USA). Normally distributed continuous data were expressed as (Mean ± SD). If the continuous data were uncorrected for skewness, Median (IQR) was used. The t-test was used to compare the means between two groups. For samples from multiple groups that were normally distributed and had homogeneous variances, one-way ANOVA was used to compare the population means, followed by pairwise comparisons using the LSD method. If the samples were not normally distributed or had unequal variances, the data were transformed (sqrt, lg, exp, etc.) before normality and homogeneity of variance tests were performed. If homogeneity of variance or normality was still not met, nonparametric tests were used. A p-value < 0.05 was considered statistically significant between the groups.

[0053] 2 Experimental Results

[0054] 2.1 Expression and distribution of BMP4 in lung tissue of COPD patients

[0055] Western blot (WB) results showed that BMP4 protein expression was decreased in the lung tissue of COPD patients, and immunofluorescence results showed that BMP4 expression was reduced and fluorescence intensity was weakened in the lung tissue of COPD patients, and it mainly co-localized with type II alveolar epithelial cells. Figure 10 ).

[0056] 2.2 BMP4 gene deletion exacerbated CS-induced lung function decline and emphysema in mice.

[0057] To understand the role of BMP4 in the development and progression of COPD, we established a mouse COPD model through 6 months of continuous CS exposure and compared the effects of BMP4. + / -BMP4 of mice and their wild-type littermates + / + The mice's performance and the detection of BMP4 + / - BMP4 of mice and their wild-type littermates + / + Lung function in mice was assessed, along with lung pathology. Results were as follows: Figure 1 As shown.

[0058] The specific experimental method is as follows: BMP4 + / - and BMP4 + / + Mice were continuously exposed to CS for 6 months (12 doses per hour, 4 times per group, 1 hour each time, 6 days a week), and lung function parameters were measured using a pulmonary function analyzer (BUXCO PFT system). Figures 2-3 A- Figure 2 F), including FRC, IC, Cydn, RI, FEV20 / FVC and FEV50 / FVC values, 6 in each group; Figure 2 G-2H represents HE staining of lung sections and statistical analysis of the mean linear intercept; N=6 per group. HE staining of bronchoalveolar lavage fluid cell smears, total cell count, and cell differential count were also performed. Figure 2 (A-3E), N=6 animals per group.

[0059] Lung function results showed that, compared with wild-type littermates, Muc1 mice exposed to CS had significantly lower lung function. - / - Mice experienced a more severe decline in lung function, manifested in functional residual capacity (FRC). Figure 3 A) Inspiratory volume (IC) Figure 2 B) Resistance Index (RI) Figure 2 D) Increased and dynamic lung compliance (Cydn) Figure 2 C) Forced expiratory volume in 20 milliseconds (FEV20) / FVC value ( Figure 2 E), 50-millisecond forced expiratory volume (FEV50) / FVC ( Figure 2 The F) value decreased. HE results showed BMP4. + / + Lung tissue sections from CTL group mice showed normal, continuous alveolar structures and no obvious inflammatory infiltration; BMP4 + / - Lung tissue sections from CTL group mice showed partial alveolar septal rupture and alveolar fusion, but no obvious inflammatory infiltration; while BMP4... + / - CS and BMP4 + / + In CS mice, alveolar structure was disrupted and disordered, with thinning or rupture of alveolar walls, alveolar fusion, enlargement of alveolar cavities, and varying alveolar sizes. Inflammatory cells were present in the alveolar septa and alveolar cavities. Bronchial walls were thickened, bronchial lumens were narrowed, bronchial mucosal epithelium was sloughed off, and inflammatory cells infiltrated the submucosa and mucosa of the alveolar septa. BMP4 was also present. + / - The CS group showed more severe symptoms. Figure 2G); Calculation of mean alveolar intercept (MLI) shows that BMP4 + / - CTL group compared to BMP4 + / + The mean alveolar intercept increases in CTL, and significantly increases after CS, BMP4 + / - CS vs. BMP4 + / + CS increased significantly ( Figure 2 H); The number of inflammatory cells in the bronchoalveolar lavage fluid was significantly increased after CS, including BMP4. + / - CS group compared to BMP4 + / + The CS group mice showed a more significant increase in inflammatory cells in their bronchoalveolar lavage fluid, with an increase in neutrophils being the most prominent. Figure 2 A-3E).

[0060] Western blot analysis was used to detect the expression level of BMP4 protein in lung tissue. The results showed that BMP4 expression level decreased after CS exposure. Figure 3 A-4B).

[0061] In conclusion, BMP4 knockout exacerbates emphysema caused by CS exposure.

[0062] 2.3 Intratracheal instillation of AAV9-BMP4 improves CS-induced decline in lung function and emphysema in mice.

[0063] To understand the role of BMP4 in the development and progression of COPD, we analyzed BMP4... + / + Three months after CS exposure, mice were given AAV9-BMP4 via airway infusion, followed by another three months of CS exposure to establish a BMP4-treated COPD model. The performance of mice in the CS+AAV9-GFP group and the CS+AAV9-BMP4 group was compared. Lung function was assessed in both groups, and lung pathology was also evaluated. Results are as follows: Figure 4 As shown.

[0064] The specific experimental method is as follows: BMP4 + / + Mice were randomly divided into two groups and continuously exposed to CS for 3 months (12 doses per hour, 4 times per group, 1 hour each time, 6 days a week). One group of mice was then given an intratracheal infusion of AAV9-BMP4 (1 x 10^6 mmol / L). 11 PFU / mouse was administered to mice, while the control group received the same amount of AAV9-GFP. Simultaneously, mice continued CS exposure for 3 months. Pulmonary function parameters were measured using a pulmonary function analyzer (BUXCO PFT system). Figures 5-7 A- Figure 5 F), including FRC, IC, Cydn, RI, FEV20 / FVC and FEV50 / FVC values, 5 animals in each group; the expression of BMP4 protein in lung tissue was detected by Western blotting. Figure 5G-5H; N = 6 per group. Figure 5 I-5J shows HE staining of lung sections and statistical analysis of the mean linear intercept; N=6 per group. HE staining of bronchoalveolar lavage fluid smears, total cell count, and cell differential count were also performed. Figure 5 (A-6E), N=6 animals per group.

[0065] Lung function results showed that after CS, the CS+AAV9-GFP group had a significantly lower lung function compared to the CTL group, and FRC ( Figure 6 A), IC ( Figure 5 B) significantly increased, FEV20 / FVC ( Figure 5 E), FEV50 / FVC ( Figure 5 F) decreased significantly, while airway instillation of AAV9-BMP4 improved lung function in mice after CS, while FRC and IC increased and FEV20 / FVC and FEV50 / FVC decreased after CS improvement; HE results showed no obvious abnormalities in the lung tissue of the CTL group, while CS+AAV9-GFP showed obvious destruction and disorder of the alveolar structure in mice, thinning or rupture of the alveolar walls, some of which fused into bullae, and inflammatory cell infiltration, while the alveolar structure of the CS+AAV9-BMP4 group was relatively intact, with less destruction, only a few alveolar septal breaks, alveolar fusion, and significantly reduced inflammatory cell infiltration (5I); calculation of mean alveolar intercept (MLI) showed that the increase in mean alveolar intercept was significantly reduced after airway instillation of AAV9-BMP4 (5I). Figure 5 J). The number of inflammatory cells in the bronchoalveolar lavage fluid of the CS+AAV9-GFP group was significantly increased, with neutrophils being the predominant type. BMP4 overexpression improved the increase of inflammatory cells after CS exposure. Figure 5 (A-6E). Western blot results showed that BMP4 expression was decreased in the CS+AAV9-GFP group compared to the CTL group, while BMP4 expression was increased in the CS+AAV9-BMP4 group compared to the CS+AAV9-GFP group. Figure 6 G-5H). Meanwhile, frozen section results of lung tissue showed a significant increase in GFP in mouse lung tissue 10 weeks after airway infusion of the AAV9-GFP vector. Figure 5 ).

[0066] 2.4 Next, we investigated the effect of BMP4 on transdifferentiation of human type II alveolar epithelial cell line (hAT2 cells) under CSE stimulation. Immunofluorescence co-staining experiments using mouse lung tissue sections showed that BMP4... + / - AQP5 expression was significantly reduced in mice after CS exposure. Figure 7 SFTPC expression increased ( Figure 8Meanwhile, Western blot analysis (WB) results after 36 hours of treatment with 20 ng BMP4 and 50 ng BMP4, in addition to 2% CSE stimulation of hAT2 cells, showed that 20 ng BMP4 promoted the expression of p-SMAD159, β-catenin, and AQP5. This result indicates that recombinant BMP4 protein can alleviate the CSE-induced restricted transdifferentiation of hAT2 cells. Figure 9 A- Figure 10 Figure 10 B).

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of BMP4 gene positive regulation products in the preparation of COPD treatment products, characterized in that, The positive regulation product of the BMP4 gene is AAV9-BMP4.

2. The application according to claim 1, characterized in that, The AAV9-BMP4 is administered via inhalation.

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