Application of immunosuppressive agents in pulmonary arterial hypertension and a model of pulmonary arterial hypertension in preterm infants

Abstract

The present application relates to the technical field of medicine, and specifically includes the application of immunosuppressants in the preparation of drugs for treating pulmonary hypoperfusion type congenital heart disease, and further provides a premature infant pulmonary hypoperfusion animal model, and results show that lung development is significantly improved after using immunosuppressants. The present application verifies the Wnt pathway which is critical for lung development, indicates that immunosuppressants can improve the Wnt pathway changes caused by pulmonary hypoperfusion, confirms that immunosuppressants have a therapeutic effect on the pulmonary hypoperfusion type congenital heart disease caused by pulmonary hypoplasia, provides a new method and idea for improving lung development of such children, and thereby improves the long-term survival quality of congenital heart disease children in China.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of immunosuppressants in congenital heart disease with pulmonary hypoperfusion and a model of pulmonary hypoperfusion in premature infants. Background Technology

[0002] Although life expectancy in children with congenital heart disease (CHD) has significantly improved with advancements in cardiac surgery and perioperative techniques, their exercise capacity remains significantly lower than that of healthy controls. Exercise capacity is a crucial determinant of survival in children with CHD and is associated with their quality of life. Recent clinical observations have shown that patients with CHD exhibiting reduced pulmonary blood flow (RPF) have a more significant reduction in exercise capacity compared to patients with normal or increased pulmonary blood flow. Therefore, the potential mechanisms by which RPF impairs exercise capacity may contribute to improving the long-term quality of life in children with CHD.

[0003] An individual's motor abilities depend to a great extent on their respiratory and cardiovascular performance and the profound, functional, and anatomical interactions between the two. A child's respiratory performance depends on the development of blood vessels and alveoli in the lungs, with 95% of alveoli forming in the first seven years of human life. Fetal studies have shown that pulmonary blood flow regulates the development of the fetal lung parenchyma and blood vessels, but whether pulmonary blood flow regulates postnatal lung development remains unclear. Furthermore, although a clinical study of adult patients with congenital pulmonary valve stenosis (leading to recurrent lung pulmonary fibrosis) showed that their lungs were smaller than those of healthy controls, it is unclear whether this reduction in lung volume is caused by RPF. Pulmonary dysplasia is a major feature of bronchopulmonary dysplasia (BPD) and one of the most common complications in preterm infants, affecting approximately 40% of extremely preterm infants (born at <28 weeks of gestation). Epidemiological studies indicate that 58% of extremely preterm infants have pulmonary hypertension, which leads to RPF. However, the most prevalent hyperoxia-induced BPD animal models currently available do not mimic the symptoms of RPF in preterm infants, and the efficacy of targets derived from these models in clinical treatment is limited.

[0004] Patent document CN101940501A, published on January 12, 2011, discloses a method for constructing an animal model of congenital heart disease with reduced pulmonary blood flow. The method involves making an incision in the right anterolateral chest at the 4th intercostal space, opening and suspending the pericardium, and directly dilating the atrial septum through the right atrium under ultrasound guidance using a puncture needle and balloon dilator to create an atrial septal defect with a diameter of 1.0 cm. An artificial vascular band is used to encircle the main pulmonary artery, and peripheral arterial blood pressure and the pressure gradient across the constriction are monitored. The band is gradually tightened until the pressure gradient across the constriction is 20–30 mmHg in the mild to moderate pulmonary stenosis group, or ≥30 mmHg in the severe pulmonary stenosis group, and the arterial blood pressure remains stable. However, the method does not provide any implications for the treatment of related diseases.

[0005] Immunosuppressants are drugs that inhibit the body's immune response. They can suppress the proliferation and function of cells related to the immune response (such as T cells and macrophages like B cells) and reduce antibody immune responses. Commonly used immunosuppressants mainly fall into five categories: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tebufenozide; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents, such as cyclophosphamide. Among them, cyclosporine (CsA) is suitable for preventing rejection reactions in allogeneic organ or tissue transplants such as kidney, liver, heart, and bone marrow; and for preventing and treating graft-versus-host disease in bone marrow transplants. It is often used in combination with immunosuppressants such as adrenocortical hormones to improve efficacy. The literature (Dai Yongyue, Zhu Renwu, Ni Shirong, et al. Effects of cyclosporine A on apoptosis in rat lung ischemia / reperfusion injury [J]. Chinese Journal of Applied Physiology, 2010(4):5.) reported the effects of cyclosporine (CsA) on apoptosis in rat lungs after normothermic ischemia / reperfusion. The results showed that cyclosporine may reduce lung tissue cell apoptosis by inhibiting MPTP opening and reducing the release of mitochondrial CytC after ischemia / reperfusion. Currently, there are no literature reports on the therapeutic effect of cyclosporine on pulmonary hypoplasia in children with pulmonary hypoperfusion type congenital heart disease. At the same time, no reports have been found on the pulmonary hypoperfusion model of premature infants as described in this application. Summary of the Invention

[0006] The first objective of this invention is to provide a use of an immunomodulator, addressing the shortcomings of the prior art.

[0007] The second objective of this invention is to provide an animal model of reduced pulmonary blood flow in premature infants.

[0008] To achieve the first objective mentioned above, the technical solution adopted by the present invention is as follows:

[0009] Application of immunosuppressants in the preparation of drugs for treating congenital heart disease with reduced pulmonary blood flow.

[0010] As a preferred example, the immunosuppressant is cyclosporine.

[0011] As a preferred example, immunomodulators improve lung development by modulating the Wnt pathway.

[0012] As a preferred example, the congenital heart disease with reduced pulmonary blood flow is Tetralogy of Fallot, a congenital heart disease characterized by pulmonary atresia and severe pulmonary stenosis, which often leads to pulmonary dysplasia.

[0013] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows:

[0014] A method for constructing an animal model of reduced pulmonary blood flow in premature infants.

[0015] As a preferred example, the animal models include pigs and rats.

[0016] As a preferred example, the animal model is a pulmonary artery banding surgery performed on piglets and rats.

[0017] As a preferred example, the described pulmonary artery circumduction surgery for piglets involves placing a circumduction band through the bottom of the main pulmonary artery.

[0018] As a preferred example, the described pulmonary artery circumduction surgery in rats involves quantitative pulmonary artery contraction.

[0019] As a preferred example, the successful construction of the model is marked by pulmonary hypoplasia, specifically manifested in alveolar simplification, reduced number of blood vessels, and decreased epithelial density of type II alveoli.

[0020] The advantages of this invention are:

[0021] 1. This invention provides a method for improving lung development in children with congenital heart disease characterized by pulmonary hypoperfusion. Pulmonary hypoperfusion is one of the most common congenital heart abnormalities, frequently occurring in children with tetralogy of Fallot, pulmonary atresia, and severe pulmonary stenosis. These children often exhibit underdeveloped lungs and decreased lung function. This invention provides a method for improving lung development in these children, which will improve the long-term quality of life for children with congenital heart disease in my country.

[0022] 2. The animal models provided in this invention use human, piglet, and rat RPF lung samples to demonstrate that RPF causes pulmonary hypoplasia in patients with congenital heart disease. This invention reveals for the first time that the potential mechanism of reduced exercise tolerance in children with CHD suffering from RPF is inflammatory / immune-induced pulmonary hypoplasia, and suggests that cyclosporine may be a candidate drug for improving the exercise capacity of children with CHD. Furthermore, RPF is used in pulmonary hypoplasia animal models as a supplement to currently used animal models of preterm pulmonary hypoplasia. Attached Figure Description

[0023] Appendix Figure 1 Image showing the results of RPF-induced pulmonary dysplasia in human infants: (A) Representative HE staining of lung tissue from RPF and control (CON) lungs. Scale bar: 100 μm in 10× images, 50 μm in 20× images; (B) Quantification of MLI. n = 60 fields of view from 12 samples; (C) Representative CD31 staining of lung tissue from RPF and CON lungs. CD31 (green), DAPI (blue). Scale bar: 75 μm; (D) Quantification of CD31 intensity. n = 60 fields of view from 6 samples; (E) Representative AT2 cells, Sftpc (red), DAPI (blue) from RPF and CON lungs; (F) Quantification of Sftpc intensity. n = 60 fields of view from 12 samples. (G): Transthoracic echocardiography of RPF and control (CON) lungs. Scale bar: 75 μm in 63× images, 5 μm in magnified images. Similar scale bars are shown in the following images.

[0024] Appendix Figure 2 Images showing the results of RPF-induced pulmonary dysplasia in piglets: (A) Typical echocardiograms of PAB (pulmonary artery banding) and Sham (sham surgery) piglets; (B) Quantitative MPG of PAB and Sham piglets. n = 5 samples; (C) Quantitative pulmonary blood flow of PAB or sham piglets. n = 5 samples; (D) Representative HE staining of lung tissue from PAB and Sham piglets; (E) Quantitative MLI. n = 50, from 10 samples; (F) Representative CD31 staining of lung tissue from PAB and Sham piglets. CD31 (green), DAPI (blue); (G) Quantitative analysis of CD31 intensity in lung tissue from PAB and Sham piglets. n = 50 fields of view from 10 samples; (H) Representative AT2 cells from PAB and Sham piglets. Sftpc (red), DAPI (blue); (I) Quantitative Sftpc intensity. n = 50 fields of view from 10 samples.

[0025] Appendix Figure 3 Construction of the RPF model in newborn rats: (A) Flowchart of the rat experiment; (B) Diagram of the PAB procedure; (C) Typical transthoracic echocardiography of the PA. White arrows indicate stenosis, irregularity, and color Doppler flow signals, representing high-velocity blood flow in the stenotic region. (D) Quantification of MPG in PAB and Sham rats. n = 5 samples; (E) Typical transthoracic echocardiography of the left ventricular outflow tract; (F) Quantification of pulmonary blood flow. n = 5 samples. Abbreviations: RAA, right atrial appendage; aorta; PA, pulmonary artery; left atrial appendage; RPA, right pulmonary artery.

[0026] Appendix Figure 4RPF-induced lung hypoplasia in rats: (A) Representative H&E staining of lung tissue from PAB and Sham rats; (B) Quantification of MLI. n=50, from 5 samples; (C) Representative vWF staining of lung tissue from PAB and Sham rats. vWF (red), DAPI (blue); (D) Quantification of vWF intensity in lung tissue from PAB and Sham rats. n=50, from 5 lungs; (E) Representative AT2 cells from PAB and Sham lungs. (red), DAPI (blue); (F) Quantification of Sftpc intensity. n=50 field-view images from 5 lungs; (G) Gross images of the whole lungs of PAB and Sham rats; (H) Quantification of lung volume in Sham and PAB rats. n=5 lungs, (I) Representative PAB and Sham rats. Note the cyanosis and weight loss in PAB rats; (J) Quantification of body weight in PAB and Sham rats. n=5 rats.

[0027] Appendix Figure 5 For RPF interference with late alveolar development: (A) Volcano plot of differentially expressed genes (DEG). Alveolar surfactant proteins (Sftpa, Sftpb, Sftpc, Sftpd) are all in the DEG list; (B) Heatmap of DEG; (C) PCA plot of DEG; (D) Top 20 enriched metabolism-related GO terms; (E) Top 20 enriched metabolism-related KEGG terms; (F) Top 20 enriched migration and development-related GO terms; (G) Top 20 enriched migration and development terms associated with KEGG.

[0028] Appendix Figure 6 To evaluate RPF-induced lung cell apoptosis and inflammation: (A) The most significantly enhanced immune response was associated with GO terms; (B) The top 20 KEGG terms associated with enhanced immune responses; (C) Representative flow cytometry diagrams of CD4+ / CD8+ cells from the Sham and PAB groups; (D) Quantification of the percentage of CD4+ and CD8+ cells and the CD4+ / CD8+ ratio; (E) The most abundant GO terms for cell apoptosis / death; (F) The most significantly abundant KEGG terms for cell apoptosis / death; (G) Representative TUNEL-positive cells from the Sham and PAB groups; (H) Quantification of the percentage of TUNEL-negative cells from the Sham and PAB groups. n=50 images from 5 lung fields.

[0029] Appendix Figure 7To demonstrate how the immunosuppressant CsA (cyclosporine) rescues RPF-induced BPD by activating the Wnt signaling pathway: (A) Representative H&E staining of rat lung tissue treated with Sham, PAB, or CsA; (B) Quantification of MLI. n=50 images from 5 lungs; (C) Representative vWF staining of rat lung tissue treated with Sham, PAB, or CsA. vWF (green), DAPI (blue); (D) Quantification of vWF intensity in lung tissue. n=50 images from 5 lungs; (E) Gross images of the lungs of rats treated with Sham, PAB, or CsA; (F) Quantification of lung volume in rats treated with Sham, PAB, or CsA. n=5 lungs; (G) Representative AT2 cells from lungs treated with PAB and CsA. Sftpc (red), DAPI (blue); (H) Quantification of Sftpc intensity. n = 50 field-view images from 5 lungs; (I) Representative Wnt3a / β-catenin blots from rats treated with Sham, PAB, or CsA. (J) Quantification of Wnt3a. n = 5 samples; (K) Quantification of β-catenin. n = 5 samples.

[0030] It should be noted that the colors in the above-described figures are the colors from the actual experimental results, while in this invention they are all colors that have undergone grayscale processing. Detailed Implementation

[0031] This invention first elucidates, using children with tetralogy of Fallot, that reduced pulmonary blood flow leads to alveolar and pulmonary vascular dysplasia. Subsequently, animal model experiments were conducted on pigs and rats undergoing pulmonary artery (PA) circumduction (PAB) surgery to further demonstrate whether reactive lung disease (RPF) causes pulmonary dysplasia in large animals and rodents. Then, RNA sequencing (RNA-seq) was performed on lung tissue from PAB rats to reveal possible pathways regulating RPF-induced pulmonary dysplasia. Finally, relevant pathways were tested to examine their therapeutic effects on RPF-induced pulmonary dysplasia. It should be noted that, unless otherwise specified, all cells, materials, and reagents used in the examples are commercially available, and all sequencing results are uploaded to the GEO public database under the number GSE201522.

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0033] Example 1: Application of immunomodulators in models of congenital heart disease with pulmonary hypoperfusion and pulmonary hypoperfusion in premature infants

[0034] 1. Materials

[0035] 1.1 Reagents

[0036] Isoflurane (isofluorane / oxygen, 5%); phosphate-buffered saline (PBS); tissue fixative (0.2 mL / 10 g, 5 cmH2O); 4% paraformaldehyde (pH 7.4); paraffin; 7.5% goat serum and 0.5% Trixon X-100; primary rabbit anti-vWF (ab6994; dilution, 1:200; Abcam, Cambridge, UK); anti-CD31 (A4900; dilution, 1:100; Abclonal, Wuhan, China); anti-Sftpc (PA5-71680; dilution, 1:25; Thermo Fisher Scientific, Waltham, MA, USA); Alexa Fluor® 488 goat anti-rabbit fluorescent secondary antibody (ab150077; dilution, 1:500; Abcam, Cambridge, UK); 4',6-diamino-2-phenylindole (DAPI) (sample number: P0131; Beyotime, Shanghai, China); TUNEL apoptosis detection kit (sample number C1089; Beyotime, Shanghai, China); H&E staining kit (Sorapio, Shanghai, China); erythrocyte lysis buffer (eBiosciences, Waltham, MA, USA); CD4 or CD8 antibody (dilution, 1:100; eBi Osciences, Waltham, MA, USA) antibody Wnt3a (ab219412; dilution, 1:1000; Abcam, Cambridge, UK); horseradish peroxidase conjugated secondary antibody (dilution, 1:5000; Beyotime, Shanghai, China); stripping buffer (Beyotime Biotechnology, Shanghai, CHN); β-actin (ab8229; dilution, 1:500; Abcam, Cambridge, UK); bis(octanoic acid) protein assay kit (P0012; Beyotime, Shanghai, China).

[0037] 1.2 Instruments

[0038] 25 MHz transducer (MS400 microscanning transducer; Visual Sonics); Leica M205 FA stereo microscope (Leica Microsystems, Wetzlar, Germany); Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA); confocal microscope; TruSeq PE version 3-cBot-HS cluster kit (Illumina, San Diego, CA, USA); Vevo 2100 echocardiography system with 25 MHz transducer (Visual Sonic, Toronto, Ontario, Canada).

[0039] 1.3 Case Data

[0040] Between January and December 2021, 12 tiny lung tissue samples (5 mm × 5 mm × 5 mm) were collected from patients during routine cardiothoracic surgeries at Shanghai Children's Medical Center. These included 6 patients with recurrent lung failure (RPF) and 6 patients with normal pulmonary blood flow. All procedures were performed in accordance with the requirements of the Animal Welfare and Human Research Committee of Shanghai Children's Medical Center. Written informed consent was obtained from the children's parents. Patient clinical information is shown in Table 1.

[0041] Table 1 Patient Clinical Information

[0042]

[0043] TOF: Tetralogy of Fallot; COA: Coarctation of the aorta

[0044] 2 Research Methods

[0045] 2.1 Processing of animal RPF lung samples

[0046] PAB surgery was performed on newborn rats and piglets. The animal experiments in this invention were approved by the Ethics Committee of Shanghai Children's Medical Center. Wild-type Sprague-Dawley rats at timed gestation (day 18 of embryonic development) were obtained from Shanghai Bikai Experimental Animal Co., Ltd., China, and housed in pairs per cage until farrowing. Piglets were purchased from Jiagan Biotechnology Co., Ltd. (Shanghai, China) and housed one per cage until two months of age. Animals had free access to food and water under identical conditions and maintained a 12- / 12-hour diurnal cycle. Pups were randomly assigned to either the PAB group or the Sham group, the latter receiving the same procedure as the former except for the circumduction step.

[0047] 2.2 Pulmonary artery banding (PAB) surgery

[0048] Newborn mice were anesthetized on ice for approximately 3 minutes using hypothermia, and then transferred to an ice bed to maintain anesthesia during surgery. The surgery, including sternal clipping, pleural opening, pericardiotomy, quantitative systolic arteriosclerosis (PA), and pleural closure, was performed under a stereomicroscope in a supine position. The surgical procedure is as follows: Figure 3 As shown in B. After suturing the thorax, the pup was removed from the ice bed, placed on a heating plate for heating, and then returned to its mother.

[0049] For piglet PAB surgery, simply put, under general anesthesia and mechanical ventilation, a left thoracotomy is performed in the fourth or fifth intercostal space. The PA is exposed through a partial pericardial incision. A constricting band is placed through the base of the main MPA and then constricted to produce transesophageal echocardiography.

[0050] 2.3 Transthoracic echocardiography

[0051] On day 14 postnatally (P14), transthoracic echocardiography was performed by an experienced echocardiologist to verify aortic pulmonary artery stenosis and quantify pulmonary blood flow. Rats were anesthetized in the induction chamber with isoflurane (isofluorane / oxygen, 5%) for 3–5 minutes, and then placed in a heated plate in a supine position using a nasal cone. Anesthesia was maintained by inhalation of 1.5%–2% isoflurane / oxygen via the nasal cone. Echocardiography was performed using a Vevo 2100 echocardiography system equipped with a 25 MHz transducer. To assess PA stenosis, blood flow images in color Doppler mode, mean pressure gradient (MPG), and velocity-time integral (VTI) data in pulsed wave Doppler mode were acquired from the long axis view of the MPA. To calculate pulmonary blood flow, heart rate (HR), VTI, and aortic root diameter (AoD) were obtained from the long axis view of the aortic outflow tract. According to the law of continuity, pulmonary blood flow was calculated as: Pulmonary blood flow = (AoD / 2)² * π * VTI * HR.

[0052] 2.4 Morphological examination and tissue preparation

[0053] Lung morphometric analysis was performed at P14. After euthanasia, the ribs along the mid-axillary line of the rats were incised to expose the thoracic cavity. The lungs were removed and washed with phosphate-buffered saline (PBS). Gross morphology of each lung was photographed under a Leica M205 FA stereomicroscope. For histological and immunohistochemical studies, rats and pigs were euthanized at P14. In short, the thoracic cavity was exposed, a small incision was made in the left atrium, and blood in the pulmonary vascular bed was cleared by slow perfusion of PA with PBS. The alveoli were fixed and inflated by intratracheal injection of tissue fixative (0.2 mL / 10 g, 5 cmH2O). The lung tissue was then removed, rinsed with PBS solution, and fixed overnight at room temperature with 4% paraformaldehyde (pH 7.4). Subsequently, the tissue was dehydrated by a series of ethanol solutions, embedded in paraffin, and sectioned into 5 µm sections. In RNA sequencing, lung tissue is cut into 1-2 cm slices, rapidly frozen in liquid nitrogen, and then stored in a freezer at -80°C.

[0054] 2.5 Immunofluorescence

[0055] This study investigated immunofluorescence staining for Von Willebrand factor (vWF), CD31, surfactant protein C (Sftpc), and terminal transferase-mediated dUTP nick-end labeling (TUNEL). For vWF, CD31, and Sftpc staining, paraffin-embedded lung sections were dewaxed in xylene, hydrated using an alcohol gradient, and antigens were recovered. After blocking with PBS containing 7.5% goat serum and 0.5% Trixon X-100 for 1 hour, sections were incubated overnight at 4°C with primary rabbit anti-, anti-CD31, and anti-Sftpc antibodies. The next day, primary antibodies were washed with PBST (PBS containing 1% Tween), and sections were incubated with AlexaFluor® 488 goat anti-rabbit fluorescent secondary antibody for 1 hour at room temperature. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI), and sections were fixed with anti-quenching retention medium and sealed with nail polish. TUNEL staining was performed using a 1-step TUNEL apoptosis detection kit according to the manufacturer's instructions. In short, the sections were incubated with 20 μg / mL protein K at room temperature for 30 minutes, and then hydrated as described above. Next, 50 µL of TUNEL assay solution (consisting of 5 µL terminal deoxynucleotidyl transferase and 45 µL fluorescein labeling solution) was added to each tissue, and the sections were incubated at 37°C.

[0056] 2.6 Assessment of alveolar and vascular development

[0057] According to standard protocols, hematoxylin and eosin (H&E) staining was performed using a staining kit to assess alveolar polarization, and the images were imaged under an optical microscope. The degree of alveolarization was quantified using mean linear intercept (MLI) with ImageJ software (www.rsb.info.nih.gov / ij; National Institutes of Health, Bethesda, Maryland, USA). Simply put, to assess MLI, a grid of horizontal and vertical lines was placed on the image, and the total number of intersections between the lines and alveoli was recorded. MLI was defined as: MLI = (total length of horizontal and vertical lines / total number of intersections), in μm. Immunofluorescence staining was also performed on the alveolar type 2 (AT2) cell marker Sftpc to assess lung growth. The results were quantified by mean Sftpc density. To determine pulmonary vascular density, immunofluorescence staining was performed on the endothelial markers vWF and CD31, and the images were imaged under a confocal microscope. Pulmonary vascular density was defined as the mean intensity of CD31.

[0058] 2.7 RNA Sequence and Enrichment Analysis

[0059] High-throughput sequencing was performed at P14 to detect differentially expressed genes (DEGs) in the lungs (n = 5 / group). RNA was first extracted from lung tissue and reverse transcribed into complementary DNA (cDNA) using M-MuLV reverse transcriptase and DNA polymerase I. Libraries were constructed using the NEBNext Ultra RNA Library Preparation Kit for Illumina®. Library and RNA quality were assessed using an Agilent 2100 Bioanalyzer. RNA integrity number (RIN) values ​​were all greater than 9.

[0060] Subsequently, following the manufacturer's instructions, clustering was performed on the cBot clustering system using the TruSeq PE version 3-cBot-HS clustering kit. The Novaseq platform (Illumina) was used for library preparation and sequencing.

[0061] The raw data in .fastq format was then processed to obtain clean data by removing reads containing N bases, adapters, or low-quality information. Q20, Q30, and GC were calculated simultaneously. Sequence reads were aligned to a rat reference genome (rnor_6.0) using HISAT2 version 2.5.0. New transcription predictions were performed using StringTie (version 1.3.3b) (MihaelaPertea et al., 2015). The number of reads for each gene was then counted using featureCounts version 1.5.0-p3. Based on gene length and the number of reads mapped to said gene, the expected number of fragments per million base pairs of transcribed sequence per thousand bases for each gene was calculated. Differential expression analysis: Enrichment analysis of DEG using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was subsequently performed using the clustering profile R package. GO terms with a corrected p-value < 0.05 were designated as significantly enriched. Based on the KEGG database, KEGG pathways are enriched from DEG (http: / / www.genome.jp / kegg / ).

[0062] 2.8 Flow cytometry

[0063] Lung digestion was performed to obtain a single-cell suspension for flow cytometry. In short, each lung was exposed at 37°C and perfused with 1 mL of PBS to remove red blood cells, then rinsed and aerated for 45 min with a digestion solution containing elastase (4 U / mL in PBS), dispase II (1 U / mL in PBS), DNase (200 μg / mL in water), and liberase (5 mg / mL in PBS). The lung was then cut into small pieces and transferred to a 70 μm cell filter to collect the cell suspension. Remaining red blood cells were removed by incubation with red blood cell lysis buffer for 5 min. The cells were then washed, resuspended three times in 2% FPBS, and blocked with 2% normal rabbit serum. The cells were then stained with CD4 or CD8 antibodies at 4°C for 30 min. After three washes, the cells were resuspended and analyzed by flow cytometry (FACSAria).

[0064] 2.9 Apoptosis Assessment

[0065] Apoptosis was assessed by immunofluorescence staining of TUNEL cells. Images were captured under a confocal microscope, and cell nuclei co-stained with the target antibody and DAPI were considered positive. The proportion of positive cells to the total number of cells was used as a quantitative indicator of apoptosis. Ten slides were randomly selected from each tissue sample for counting of positively stained cells.

[0066] 2.10 Immunoblotting

[0067] Western blotting was used to detect the expression of Wnt3a and β-catenin proteins in 14-day-old rats treated with Sham, RPF, and CsA. To extract total protein, lung tissue was homogenized with benzyl sulfonyl fluoride (1 mM) in radioimmunoprecipitation assay lysis buffer (25 mg / mL) and centrifuged at 3000 × g at 4°C. The supernatant was then collected, and protein concentration was quantified using a standard bis(octanoic acid) protein assay kit. 20 μg of protein sample was then mixed with 8 μL of loading buffer and diluted to 24 μL with ddH2O. The mixture was subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 20 μL per lane, and transferred to a nitrocellulose membrane. The membrane was then blocked for 1 hour at room temperature with 5% skim milk. Subsequently, the membrane was washed three times in TBST (150 mM saline and 10 mM Tris containing 0.05% Tween-20) and incubated at 4°C with primary antibodies against Wnt3a and β-catenin. The primary antibody was detected using a horseradish peroxidase-conjugated secondary antibody. The blot was scanned using an AmerSham Imager 600. The primary and secondary antibodies were then removed using stripping buffer. Following the same procedure, the primary and secondary antibodies were re-incubated with β-actin, followed by scanning. Expression levels were quantified using ImageJ software.

[0068] 3. Statistical Analysis

[0069] Quantitative data are expressed as mean ± standard deviation. If the variable is normally distributed, statistical analysis is performed using unpaired, two-tailed Student's t test or one-way ANOVA and Student Newman-Keuls post-hoc test; otherwise, the rank-sum test is used for comparison. P < 0.05 is considered statistically significant. All statistical analyses were performed using SAS version 9.2 software (SAS Institute, Cary, NC, USA).

[0070] 4 Results

[0071] 4.1 RPF causes maldevelopment of the lungs in human infants

[0072] As shown in Table 1 and Figure 1As shown in Figure G, compared with the control group, the pulmonary artery diameter was significantly smaller in the RPF group (0.51±0.11 cm vs. 1.05±0.20 cm, P=0.0002), while the PA velocity was significantly increased (4.52±0.74 vs. 1.10±0.37, P<0.0001). Correspondingly, compared with the control group, the PA flow rate was significantly reduced in the RPF group (0.09377±0.03436 L / s vs. 0.15537±0.05279 L / s, P=0.0376). H&E staining showed that compared with the control group, the MLI (indicating alveolar simplification) was significantly increased in the RPF group (P<0.0001). Figure 1 AB). Immunostaining showed that, compared with the control group, the intensity of the vascular marker CD31 in the RPF group was significantly reduced (P<0.0001). Figure 1 CD). Type 2 alveolar epithelial cells (AT2) are crucial for alveoli because they not only secrete surfactant proteins to maintain alveolar sac stability but also function as stem cells, differentiating into type 1 alveolar epithelial cells during injury repair. Compared to the control group, the expression of the AT2 cell marker Sftpc was significantly downregulated in the RPF group (P<0.0001). Figure 1 These results indicate that RPF leads to impaired alveolarization and vascularization in human infants.

[0073] 4.2 RPF induces pulmonary dysplasia in piglets and rats

[0074] To confirm the results obtained from human infants, we performed the PAB procedure to generate RPF in newborn piglets and rats. Figure 2 As shown in AC, PAB significantly increased MPG in piglets and led to RPF. Therefore, compared with the Sham group, the MLI in the PAB group was significantly increased (P<0.01). Figure 2 DE), while the intensities of CD31 and Sftpc were significantly reduced (P<0.01, Figure 2 FI), indicating that RPF leads to poor lung development in newborn piglets.

[0075] Due to the limited surgical space in newborn rats, surgery is difficult. We provide illustrations of neonatal PAB surgery for researchers to learn this skill. Figure 3 AB). PAB significantly increased the PA rate in rats ( Figure 3 CD) and induced RPF ( Figure 3 Similar to humans and piglets, RPF also leads to incomplete lung development in newborn rats. Figure 4 AF). Figure 4 As shown in GJ, RPF leads to a decrease in lung volume and weight loss. Figure 4 GJ)

[0076] 4.3 RPF activates apoptosis-induced inflammation

[0077] There are 2013 DEGs between the Sham and PAB groups, of which 936 are revised upwards and 1077 downwards. Figure 5 A). Cluster analysis based on heatmaps showed that samples within the sham surgery group or PAB group were more similar than samples between groups. Figure 5 B). PCA plots are used to analyze variability between and within groups. For example... Figure 5 As shown in Figure C, the reproducibility of each group was good, and there were significant differences between the groups. Analysis of the GO and KEGG pathways downregulated by DEGs indicated abundant cell migration and metabolism, characteristic of late alveolarization (P9 to P18). Figure 5 DG). Cell migration is essential for alveolar morphogenesis, and metabolism is essential for the production of alveolar surfactant and homeostasis. These results further confirm that RPF leads to lung dysplasia. GO and KEGG pathway analysis of DEGs also showed enrichment of upregulated immune responses (DGs). Figure 6 Flow cytometry confirmed the upregulation of the immune response, showing that RPF increased the ratio of CD4+ cells and CD4+ / CD8 cells (AB). Figure 6 CD). These results indicate that RPF activates lung inflammation. Multifactorial analysis showed that almost all inflammatory factors were significantly upregulated, similar to the previous results.

[0078] Next, we explored why inflammation is activated by RPF. GO and KEGG pathway analysis of DEGs revealed a wealth of cell death / apoptosis terms. Figure 6 EF). Immunostaining results showed that RPF increased the percentage of TUNEL-positive cells in the lungs (EF). Figure 6 (GH), consistent with enrichment analysis. These results suggest that inflammation in RPF lungs may be induced by cell death / apoptosis. Given the crucial role of inflammation in preterm BPD, it may be a potential mechanism of RPF-induced BPD.

[0079] 4.4 Immunosuppressant CsA rescues RPF-induced pulmonary dysplasia

[0080] To further confirm the role of inflammation in RPF-induced pulmonary dysplasia, we treated PAB rats with the immunosuppressant CsA (10 mg / kg / day). Figure 7 As shown in the AD diagram, MLI significantly decreased after CsA treatment, while the number of pulmonary vessels significantly increased after CsA treatment, indicating that CsA salvaged RPF-induced alveolar and vascular damage. Consequently, the RPF-induced lung volume reduction was salvaged ( Figure 7 EF). Furthermore, AT2 cell expression was significantly increased in the CsA treatment group ( Figure 7(GH), (P<0.05). These results confirm that inflammation plays a key role in RPF-induced pulmonary dysplasia and suggest that CsA has a therapeutic effect on RPF-induced pulmonary dysplasia.

[0081] 4.5 CsA upregulates the Wnt signaling pathway

[0082] The expression of Wnt3b and β-catanin was examined to investigate whether the therapeutic effect of CsA is related to the Wnt signaling pathway. Figure 7 As shown in IK, compared with sham-operated rats, RPF significantly downregulated Wnt3a and β-catanin (P<0.05). These results suggest that CsA may improve exercise tolerance in children with RPF-related CHD by activating the Wnt signaling pathway.

[0083] In summary, this invention, using samples from children with tetralogy of Fallot, newborn pigs with reduced pulmonary blood flow, and rats, elucidates that reduced pulmonary blood flow leads to alveolar and pulmonary vascular dysplasia. Transcriptome sequencing results indicate that reduced pulmonary blood flow significantly alters biological processes related to lung development, particularly lung tissue metabolism and migration, suggesting pulmonary dysplasia. Furthermore, transcriptome sequencing results also indicate a significant upregulation of the immune response. This immune response was subsequently validated by flow cytometry. In addition, intervention with immunosuppressants in rats significantly improved lung development, demonstrating that immunosuppressants can mitigate Wnt pathway changes caused by reduced pulmonary blood flow. It can be inferred that reduced pulmonary blood flow leads to pulmonary dysplasia, with the immune response playing a crucial role, and immunosuppressants can improve lung development by modulating the Wnt pathway.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of immunosuppressants in the preparation of drugs for treating congenital heart disease with reduced pulmonary blood flow, characterized in that, The immunosuppressant mentioned is cyclosporine, and the congenital heart disease with reduced pulmonary blood flow refers to tetralogy of Fallot, pulmonary atresia, and severe pulmonary stenosis.

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