Application of WSB1 as a target in HPH disease

Abstract

The present invention discloses an application of WSB1 as a target in HPH disease. The present invention discovers a new use of WSB1. WSB1 is a damaging factor that promotes the progression of HPH disease. WSB1 participates in hypoxia-induced endothelial dysfunction by promoting the accumulation of HIF-2α in endothelial cells. Based on the research on the role and molecular mechanism of WSB1 in HPH disease, it provides a new target and new choice for the treatment of hypoxic pulmonary hypertension, and provides a target for the development of drugs for alleviating or / and treating HPH disease. WSB1 inhibitors can be used to prepare drugs for alleviating or / and treating HPH disease, and targeting WSB1 inhibitors to endothelial cells is a new choice for HPH treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of WSB1 as a target in HPH disease. Background Art

[0002] Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by pulmonary vascular remodeling and progressive increase in pulmonary vascular resistance. If not effectively treated, right heart failure or even death may eventually occur. The current international hemodynamic definition of PH is that the mean pulmonary artery pressure is higher than 20 mmHg when measured by right cardiac catheter at rest. In the past two decades, with the advent of targeted drugs such as endothelin receptor antagonists, prostacyclins, and phosphodiesterase inhibitors, the prognosis of PH patients has improved to a certain extent. However, the targeted drugs currently used in clinical practice mainly work by causing vasodilation. Their ability to improve vascular remodeling is very limited and generally cannot reverse the course of the disease. Pulmonary hypertension caused by lung disease and / or hypoxia, also known as hypoxic pulmonary hypertension (HPH), is classified as group 3 PH in the World Symposium on Pulmonary Hypertension (WSPH) classification system. Combined pulmonary hypertension is an independent risk factor for hypoxic lung disease and is closely associated with worse prognosis and higher mortality. With the increasing prevalence of chronic lung diseases, HPH has received increasing attention. However, many clinical studies have shown that HPH patients are unlikely to benefit from currently used targeted drugs, and there is still a huge unmet medical need in this field. In recent years, supported by strong preclinical data, many new pharmacological targets for PH have emerged, such as BMPR2 (bone morphogenetic protein type II receptor), TGF-β (transforming growth factor-β), IL-6 (interleukin-6), etc. However, due to limited efficacy or adverse side effects, they ultimately failed in clinical trials. Therefore, it is of great clinical significance to explore new targets for HPH vascular remodeling and develop new treatment strategies.

[0003] The endothelial cells in the innermost layer of blood vessels are the first barrier to perceive changes in vascular pressure and various stimuli. Endothelial cells are essential for maintaining vascular homeostasis and effective gas exchange. In the development of HPH, the hypoxic compensatory stage is mainly manifested by persistent pulmonary vascular contraction causing increased pulmonary artery pressure, and gradually transitions to irreversible remodeling of pulmonary vascular in the decompensated stage. Endothelial dysfunction is considered to be the triggering factor of vascular remodeling in pulmonary hypertension and the key core of disease progression. Endothelial dysfunction includes the multiple roles of endothelium in regulating vascular function. Improving endothelial dysfunction is also the cornerstone of current targeted treatment of pulmonary hypertension. The mechanism of endothelial dysfunction is complex and has not been fully elucidated. At present, clinical PH targeted drugs often only target a certain link of endothelial dysfunction, and single-drug treatment often has very limited effects. However, the combination of different drugs may affect the blood concentration of the drug. For example, when bosentan is used in combination with sildenafil or tadalafil, the concentration of sildenafil or tadalafil will be reduced. In addition, combined treatment also brings stronger adverse reactions and higher economic burden. Therefore, it is of great clinical significance to further clarify the mechanism of endothelial dysfunction and to find targets that can simultaneously affect multiple links of endothelial dysfunction.

[0004] WD repeat and SOCS box-containing protein 1 (WSB1) is a member of the SOCS box family. It was first discovered by researchers searching for proteins with a C-terminal SOCS box structure in a DNA database. It is located on chromosome 17p11.1. It has been found that WSB1 plays an important role in growth and development, immunity, tumorigenesis, and metastasis. However, there are no reports on the role of WSB1 in lung diseases. Summary of the invention

[0005] In order to solve the defects and shortcomings in the above-mentioned prior art, the purpose of the present invention is to determine the relationship between the expression of WSB1 and HPH disease, to provide an application of WSB1 as a drug target in screening drugs for alleviating or / and treating HPH disease and endothelial dysfunction induced by hypoxia in HPH disease, and further to provide an application of a WSB1 inhibitor in the preparation of drugs for alleviating or / and treating HPH disease and endothelial dysfunction induced by hypoxia in HPH disease.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In the present invention, WSB1 knockout and overexpression mice were constructed and HPH models were constructed to clarify the role of WSB1 in HPH. The results showed that systemic knockout of WSB1 can improve the HPH phenotype of mice, and overexpression of WSB1 can aggravate the HPH phenotype, confirming that elevated WSB1 is a damaging factor. IF double staining (immunofluorescence double staining) co-localized WSB1 and found that in the HPH model, WSB1 was enriched in endothelial cells that remodeled blood vessels.

[0008] The mechanism of action of WSB1 in HPH was studied: WSB1 was confirmed to be involved in multiple aspects of endothelial dysfunction, including early mitochondrial damage and dysfunction. In vivo experiments also confirmed that targeting endothelial cells with WSB1 can not only alleviate endothelial dysfunction, but also effectively prevent the occurrence and development of HPH. The data also showed that WSB1 aggravated hypoxia-induced endothelial dysfunction by promoting the accumulation of HIF-2α (hypoxia-inducible factor-2α).

[0009] The study also observed the effect of WSB1 on endothelial function and found that intervention with WSB1 can effectively improve the hypoxia-induced mitochondrial structure and function damage of endothelial cells in HPH mice. The study confirmed that endothelial cell WSB1 knockout improved PH phenotype and endothelial dysfunction; endothelial cell WSB1 knockout had similar PH improvement effects compared with WSB1 full knockout, suggesting that WSB1 is an important target for endothelial dysfunction.

[0010] Therefore, the WSB1 gene can be used as a drug target to construct an in vitro cell model or animal model with overexpression of the WSB1 gene, which can be used to screen drugs that alleviate or / and treat HPH diseases. The WSB1 gene or endothelial cell WSB1 gene can also be used as a target in gene therapy to design and prepare drugs and / or biological reagents that alleviate or / and treat HPH diseases, thereby achieving the purpose of alleviating or / and treating HPH diseases through genetic engineering technology.

[0011] For example, with WSB1 as the target gene, double-stranded siRNA (small interfering RNA) that can interfere with WSB1 expression is designed. After being synthesized by chemical methods, it is injected into the human body to silence the WSB1 gene through RNA interference to treat HPH. It is also possible to design and construct a mutant of WSB1, which enters the cell after injection to compete for the substrate of the original WSB1, thereby inhibiting the function of WSB1 and achieving the purpose of treatment. In addition, it is also possible to design small molecule compound inhibitors or antibodies with WSB1 as the target, or use in vitro cell models or animal models with overexpression of the WSB1 gene to screen and find molecules that can specifically inhibit WSB1. By constructing an endothelial cell targeted delivery system to deliver WSB1 antibodies, small molecule inhibitors, etc. to endothelial cells, an efficient and low-side effect treatment plan is achieved, providing a new option for HPH treatment.

[0012] The WSB1 gene sequence is a published gene sequence (NCBI Reference Sequence: NM_015626.10).

[0013] For the above functions of WSB1,

[0014] In a first aspect, the present invention provides the use of WSB1 as a drug target in screening drugs for alleviating and / or treating HPH diseases.

[0015] In a second aspect, the present invention provides the use of WSB1 as a drug target in screening drugs for alleviating and / or treating endothelial dysfunction induced by hypoxia in HPH diseases.

[0016] Preferably, the lower the expression level of WSB1, the better the therapeutic effect of the drug.

[0017] In a third aspect, the present invention provides use of a WSB1 inhibitor in the preparation of a medicament for alleviating or / and treating endothelial dysfunction induced by hypoxia in HPH diseases.

[0018] In a fourth aspect, the present invention provides a targeted drug for alleviating and / or treating hypoxia-induced endothelial dysfunction, comprising an inhibitor of WSB1.

[0019] In a fifth aspect, the present invention provides use of a WSB1 inhibitor in the preparation of a medicament for alleviating or / and treating HPH disease.

[0020] In a sixth aspect, the present invention provides a targeted drug for alleviating and / or treating HPH disease, comprising an inhibitor of WSB1.

[0021] Preferably, the WSB1 inhibitor is one of siRNA of the WSB1 gene, RNA interference vector of the WSB1 gene, antibody of WSB1, and other inhibitors capable of inhibiting the expression of WSB1.

[0022] Preferably, the targeted drug further comprises a pharmaceutically acceptable carrier, and the carrier is used for targeted delivery of the WSB1 inhibitor to endothelial cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] In the first aspect of the present invention, the expression of WSB1 in pulmonary hypertension was confirmed: WSB1 is highly expressed in pulmonary hypertension in humans and animals, and the role of WSB1 in pulmonary hypertension was confirmed: high expression of WSB1 can aggravate pulmonary hypertension. In addition, a mouse model of induced knockout of WSB1 was constructed, which confirmed that systemic knockout of WSB1 can improve the phenotype of hypoxic pulmonary hypertension in mice, and a WSB1 overexpression mouse model was constructed, which confirmed that overexpression of WSB1 can aggravate the phenotype of hypoxic pulmonary hypertension in mice. In the second aspect, the study of the mechanism of action of WSB1 in pulmonary hypertension was confirmed: WSB1 was confirmed to be involved in multiple aspects of endothelial dysfunction, including early mitochondrial damage and dysfunction. In vivo experiments also confirmed that targeting endothelial cells WSB1 can not only alleviate endothelial dysfunction, but also effectively prevent the occurrence and development of HPH. And the data showed that WSB1 aggravated hypoxia-induced endothelial dysfunction by promoting HIF-2α accumulation. Thirdly, it provides a new option for the treatment of hypoxic pulmonary hypertension by targeting endothelial cell WSB1, confirms that endothelial cell WSB1-specific (or targeted) knockout can improve the hypoxic pulmonary hypertension phenotype and endothelial dysfunction, and confirms that endothelial cell WSB1-specific (or targeted) knockout has a similar effect on improving hypoxic pulmonary hypertension as WSB1-complete knockout.

[0025] The present invention discovers a new use of WSB1. WSB1 is a damaging factor that promotes the progression of HPH disease. WSB1 participates in hypoxia-induced endothelial dysfunction by promoting the accumulation of HIF-2α in endothelial cells. Based on the research on the role and molecular mechanism of WSB1 in HPH disease, it provides a new target and new choice for the treatment of hypoxic pulmonary hypertension, and provides a target for the development of drugs for alleviating or / and treating HPH disease. Inhibitors of WSB1 can be used to prepare drugs for alleviating or / and treating HPH disease, and targeting inhibitors of WSB1 to endothelial cells is a new choice for the treatment of HPH. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Figure 1 Schematic diagram of gene identification for WSB1 knockout mice; A.Ubc CreERT2 Genotype identification diagram; B.Wsb1 flox / flox Genotype identification diagram; C.Wsb1 flox / flox ;Ubc CreERT2 Genotype mouse genetic identification diagram.

[0028] Figure 2 Schematic diagram of the breeding strategy for KO mice.

[0029] Figure 3The test chart of WSB1 protein expression level in lung tissue of HPH patients; A. Representative images of WSB1 immunohistochemistry in lung tissue of non-pulmonary hypertension control patients (Non-PH) and hypoxic pulmonary hypertension patients (HPH); B. Average gray value of WSB1 expression in lung tissue immunohistochemistry of each patient; C. Statistical chart of average gray value of lung tissue immunohistochemistry of two groups of patients.

[0030] Figure 4 The test graph of WSB1 expression level in lung tissue of HPH model mice; A. WB images of lung tissue of HPH model mice in normoxic group (Nx) and hypoxia-induced (Hx) (top) and corresponding statistical graphs (bottom); B. WB images (protein blot images) of lung tissue of HPH model mice induced by normoxic group (Nx) and hypoxia combined with sugen5416 (3-substituted indolin-2-one compound) (SuHx) (top) and corresponding statistical graphs (bottom).

[0031] Figure 5 The test diagram of the effect of WSB1 knockout on pulmonary hemodynamics in HPH mice; A. Representative pictures of jugular vein cannulation in each group of mice; B. Representative pictures of PWDoppler (PW Doppler ultrasound) of pulmonary artery sections of cardiac ultrasound in each group of mice; C. RVSP (right ventricular systolic pressure) quantitative analysis diagram; D. PAAT / PAET (pulmonary hypertension) quantitative analysis diagram.

[0032] Figure 6 This is a test chart of the effect of WSB1 knockout on pulmonary artery wall thickening in HPH mice; schematic diagram of lung tissue pathological staining of each group of mice, the top is HE staining (hematoxylin-eosin staining) schematic diagram, the bottom is αSMA (α-smooth muscle actin) immunohistochemical staining schematic diagram; quantitative statistical graph of WT / TT (the ratio of blood vessel wall diameter to total blood vessel diameter) of each group of mice; quantitative analysis graph of WA / TA (the ratio of pulmonary artery wall area to total blood vessel area) of each group of mice.

[0033] Figure 7 The figure shows the effect of WSB1 knockout on the muscularization of microvessels in HPH mice; the representative schematic diagrams of HE staining (top) and αSMA immunohistochemical staining (bottom) of microvessels (<50μm) in each group of mice; the quantitative statistical diagram of the muscularization of blood vessels in each group, which counts the proportions of no muscularization (N), partial muscularization (P) and complete muscularization (F) in the microvessels of each group of mice to quantify the degree of vascular muscularization.

[0034] Figure 8 This is a test diagram of the effect of WSB1 knockout on vascular collagen deposition in HPH mice; representative pictures of Masson staining of mice in each group; quantitative statistical chart of collagen deposition around the pulmonary arteries of mice in each group, which is quantified by calculating the percentage of perivascular collagen area to the total vascular area.

[0035] Fig. 9 Figure 2 shows the effect of WSB1 knockout on right heart remodeling and right heart function indices in mouse echocardiography; A. Representative echocardiogram of right ventricular sections in each group of mice; B. Statistical graph of right ventricular diastolic free wall thickness (RVFWT) in each group of mice; C. Statistical graph of right ventricular inner diameter (RVEDD) in diastole in each group of mice; D. Statistical graph of right ventricular output (RVCO) in each group of mice.

[0036] Fig.10 Figure 2 shows the effect of WSB1 knockout on right heart remodeling in HPH mice; A. Schematic diagram of HE staining of the coronal plane of the heart in each group of mice (top) and HE staining of the right ventricular local enlarged myocardium (bottom); B. Statistical analysis of the cross-sectional area of ​​right ventricular cardiomyocytes in each group of mice; C. Statistical diagram of the right heart remodeling index RV / (LV+S) in each group of mice.

[0037] Fig.11 The test diagram of the effect of WSB1 overexpression on the hemodynamic changes of mice; A. Representative pictures of jugular vein cannulation in each group of mice; B. Representative pictures of PWDoppler (PW Doppler ultrasound) of the pulmonary artery section of the heart ultrasound in each group of mice; C. RVSP quantitative analysis diagram; D. PAAT / PAET quantitative analysis diagram.

[0038] Fig.12 A test diagram of the effect of WSB1 overexpression on the thickening of the pulmonary artery wall in HPH mice; A. Schematic diagram of lung tissue pathological staining of mice in each group, the upper diagram is HE staining, and the lower diagram is αSMA immunohistochemical staining; B. Quantitative statistical diagram of WA / TA of mice in each group; C. Quantitative analysis statistical diagram of WT / TT of mice in each group.

[0039] Fig.13 A test diagram of the effect of WSB1 overexpression on vascular collagen deposition in HPH mice; A. Representative schematic diagram of Masson staining of mice in each group; B. Quantitative statistical diagram of collagen deposition around the pulmonary artery of mice in each group, which was quantified by calculating the percentage of collagen area around blood vessels to the total blood vessel area.

[0040] Fig.14 Figure 2 is a test diagram of the effect of WSB1 overexpression on the degree of microvascular muscularization in HPH mice; A. Representative schematic diagrams of HE staining (upper) and αSMA immunohistochemical staining (lower) of microvascular (20-50μm) in each group of mice; B. Quantitative statistical diagram of the degree of vascular muscularization in each group, which counts the proportions of no muscularization (N), partial muscularization (P) and complete muscularization (F) in the microvascular muscularization of each group of mice to quantify the degree of vascular muscularization.

[0041] Fig.15Figure 2 is a test diagram of the effect of WSB1 overexpression on right heart remodeling in HPH mice; A. Schematic diagram of HE staining of the coronal plane of the heart of each group of mice (top) and HE staining of the local enlarged myocardium of the right ventricle (bottom); B. Statistical analysis of the cross-sectional area of ​​right ventricular cardiomyocytes in each group of mice; C. Statistical diagram of the right heart remodeling index RV / (LV+S) of each group of mice.

[0042] Fig.16 Figure 2 is a test chart of the effect of WSB1 overexpression on right heart remodeling and right heart function indices in mouse echocardiography; A. Representative echocardiogram of right ventricular section in each group of mice; B. Statistical graph of right ventricular diastolic free wall thickness (RVFWT) in each group of mice; C. Statistical graph of right ventricular inner diameter (RVEDD) in diastole in each group of mice; D. Statistical graph of right ventricular output (RVCO) in each group of mice.

[0043] Fig.17 Figure 2 is a test diagram of the enrichment degree of WSB1 in the pulmonary vascular endothelial cells of HPH mice; A. Representative images of immunofluorescence double staining of WSB1 (green) and CD31 (red) in the lung tissues of mice in the Ctrl group (normal control group) and the HPH group (HPH model group); B. + Statistical graph of the mean fluorescence intensity of WSB1 in vascular smooth muscle cells (A). C. Representative images of double immunofluorescence staining of WSB1 (green) and αSMA (red) in lung tissues of Ctrl and HPH mice. D. + Statistical graph of the mean fluorescence intensity of WSB1 in 424 cells.

[0044] Fig.18 A test diagram of the changes in WSB1 expression in rPMVECs (mouse pulmonary microvascular endothelial cells) induced by hypoxia; A. Schematic diagram of WSB1 immunofluorescence staining of rPMVECs (red: WSB1; blue: DAPI-4',6-diamidino-2-phenylindole); B. Statistical graph of the average fluorescence intensity of WSB1 expression in normoxic group (Normoxia) and hypoxic group (Hypoxia) cells.

[0045] Fig.19 The test diagram of the effect of WSB1 knockdown on hypoxia-induced mitochondrial damage; A. Representative pictures of mitochondrial morphology staining; B. Statistical graph of mitochondrial number; C. Statistical graph of average mitochondrial length; D. Statistical graph of average fluorescence intensity of mitochondrial outer membrane protein Tomm20; E. Statistical graph of cellular ATP (adenosine triphosphate) content.

[0046] Fig. 20 Figure 1 shows the effect of WSB1 knockdown on endothelial cell apoptosis. A. Representative image of Tunel staining of rPMVEC. B. + Cell percentage statistics graph.

[0047] Fig.21 A. A test diagram of the effect of WSB1 knockdown on hypoxia-induced HIF-2α accumulation; A. A representative WB schematic diagram of the effect of WSB1 knockdown on HIF-2α expression; B. A statistical graph of the relative grayscale of WSB1 expression in the SiNC-NOR group (negative control group of siRNA), SiNC-HYP group (HYP model group of siRNA), and SiWSB1-HYP group (HYP model group with WSB1 gene knockout); C. A statistical graph of the relative grayscale of HIF-2α expression in the three groups of cells.

[0048] Fig. 22 A. A representative WB diagram of the effect of WSB1 overexpression on HIF-2α accumulation; B. A statistical diagram of the relative grayscale of HIF-2α expression in the NC-OE group (normal control group) and the WSB1-OE group (WSB1 overexpression group); C. A statistical diagram of the relative grayscale of WSB1 expression in group 2 cells.

[0049] Fig.23 The test diagram shows the effect of HIF-2α overexpression on mitochondrial damage caused by WSB1 knockdown; A. Representative pictures of mitochondrial morphological staining of two groups of cells; B. Statistical graph of average mitochondrial length; C. Statistical graph of mitochondrial number; D. Statistical graph of average fluorescence intensity of mitochondrial outer membrane protein Tomm20.

[0050] Fig.24 The test diagram shows the effect of endothelial cell WSB1 knockout on right heart remodeling and right heart function in HPH mice; A. Schematic diagram of HE staining of the coronal plane of the heart of each group of mice (top) and HE staining of locally enlarged cardiomyocytes in the right ventricle (bottom); B. Statistical analysis of the cross-sectional area of ​​right ventricular cardiomyocytes in each group of mice; C. Statistical diagram of the right heart remodeling index RV / (LV+S) of each group of mice.

[0051] Fig.25 Figure 3 shows the effect of endothelial cell WSB1 knockout on right ventricular remodeling and right heart function in HPH mice by echocardiography; A. Representative echocardiograms of right ventricular sections of mice in each group; B. Statistical graphs of right ventricular diastolic free wall thickness (RVFWT) of mice in each group; C. Statistical graphs of right ventricular inner diameter (RVEDD) of mice in each group; D. Statistical graphs of right ventricular output (RVCO) of mice in each group.

[0052] Fig.26The test figures show the effect of endothelial cell WSB1 knockout on the baseline hemodynamics and right heart function of mice; A. Representative images of RVSP of EC-WT group (wild-type normal group) mice and EC-KO group (endothelial cell WSB1 gene knockout normal group) mice; B. RVSP quantitative analysis of the two groups of mice; C. Representative PWDoppler images of pulmonary artery sections of cardiac ultrasound of the two groups of mice; D. PAAT / PAET statistical graphs of the two groups of mice; E. Representative images of right ventricular sections of cardiac ultrasound of the two groups of mice; F. RVFWT statistical graph of the two groups of mice; G. Representative images of heart HE staining of the two groups of mice; H. RV / (LV+S) statistical graph of the two groups of mice; I: RVCO statistical graph of the two groups of mice.

[0053] Fig. 27 The test diagram of the improvement effect of endothelial WSB1 knockout on mitochondrial damage in HPH mouse endothelial cells; A. Schematic diagram of CD31 and Tomm20 immunofluorescence double staining of each group of mice (left) and CD31 of each group of mice + Quantitative statistics of cell Tomm20 fluorescence intensity (right).

[0054] Fig.28 The test diagram shows the effect of endothelial WSB1 knockout on HIF-2α accumulation in endothelial cells of HPH mice; A. Schematic diagram of HIF-2α (green) and CD31 (red) immunofluorescence double staining of lung tissues of mice in each group, with the upper part showing the fluorescence of the muscularized blood vessel area (50-100μm) and the lower part showing the microvascular area (20-50μm); B. Statistical graph of the average fluorescence intensity of HIF-2α in endothelial cells.

[0055] Fig.29 These are test graphs for the effects of endothelial cell-specific knockout and systemic knockout of WSB1 on PH relief efficiency; A. RVSP statistical graph; B. PAAT / PAET statistical graph; C. RVCO statistical graph.

[0056] Fig.30 A. siRNA sequence knockdown efficiency detection diagram; A. Representative WB schematic diagram of the effects of different siRNA sequences on WSB1 expression in endothelial cells; B. Statistical graph corresponding to the WB schematic diagram; C. PCR test result diagram of different siRNA sequences. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] 1. Experimental Materials

[0059] 1.1 Experimental animals

[0060] C57BL / 6J mice were purchased from Jiangsu Weitonglihua Experimental Animal Technology Co., Ltd. (Jiangsu, China). flox / flox (Wsb1 flox / flox ) Mice were introduced from Professor Zhang Jinsan's team at Wenzhou Medical University, UBC CreERT2 (Ubc CreERT2 ) Mice were purchased from Jicui Yaokang and have been successfully bred and passaged. Male mice aged 8 weeks were selected for animal experiments. All animals were housed in a specific pathogen-free (SPF) barrier environment at the Experimental Animal Center of Wenzhou Medical University. All procedures were carried out in strict accordance with the relevant regulations approved by the Animal Ethics Committee of Wenzhou Medical University (Animal Ethics Number: wydw2021-0239).

[0061] 1.2 Lung tissue specimens from patients with pulmonary hypertension

[0062] With the approval of the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics Number: KY2023-R255) and the Ethics Committee of Shanghai Pulmonary Hospital (Ethics Number: K22-373Z), a total of 6 lung tissue specimens from patients with pulmonary hypertension and 6 from patients without pulmonary hypertension were collected from the First Affiliated Hospital of Wenzhou Medical University and Shanghai Pulmonary Hospital for subsequent experiments after obtaining informed consent from the patients.

[0063] 2. Experimental Methods

[0064] 2.1 Gene Mouse Tail WSB1 flox / flox 、UBC CreERT2 Gene identification

[0065] (1) When the mice are 2 weeks old, cut the tail to a length of about 0.3-0.5 cm;

[0066] (2) Use a PCR kit to directly operate on the mouse tail and prepare a protease mixture with Buffer L and protease in a mass ratio of 50:1;

[0067] (3) Add 100 μL of protease mixture to each tissue tube and centrifuge briefly to place the mouse tail at the bottom of the centrifuge tube;

[0068] (4) Incubate at 55°C for 15 min to release genomic DNA, followed by incubation at 95°C for 5 min to inactivate the protease;

[0069] (5) Centrifuge at 12000 rpm for 5 min and collect the supernatant as a template for subsequent PCR;

[0070] (6) Amplification of WSB1 using PCR technology flox / flox 、UBC CreERT2 Sequences of the primers used in the experiment:

[0071] UBC CreERT2 The forward primer for the gene was 5′-GACCAGGTTCGTTCACTCA-3′, and the reverse primer was 5′-AAGTTAGGAGCAAACAGTAGC-3′;

[0072] PCR amplification reaction system:

[0073] ddH2O (deionized water) 9.5µL; 2x PremixTag (dyeplus) 12.5µL; forward primer (10pmol / µl) 0.5µL; forward primer (10pmol / µl) 0.5µL; genomic DNA 2µL;

[0074] PCR amplification program settings:

[0075] Pre-denaturation stage: 94°C for 2 min, 1 cycle; denaturation stage: 94°C for 30 s, 35 cycles; annealing stage: 60°C for 30 s, 35 cycles; extension stage: 72°C for 1 min, 35 cycles; final extension stage: 72°C for 5 min, 1 cycle, and then maintained at 4°C;

[0076] (7) Prepare 120 mL of 1% agarose gel (1.2 g agarose + 120 mL deionized water), mix well, and boil in a microwave oven for 2-3 times, 1-2 min each time, until the agarose is completely dissolved; cool at room temperature for 3 min, add GoldView nucleic acid dye at a volume ratio of 1:10,000 and shake well; insert the comb into the gel plate and pour the mixed agarose gel into it, cool for about 45 min, remove the comb after the gel solidifies, and load the amplified product in step (6) for agarose gel electrophoresis. The voltage used during the electrophoresis is 110 V and the electrophoresis time is 35 min.

[0077] (8) Interpretation of nucleic acid gel results: Figure 1 As shown in A, Ubc CreERT2 The positive band of mouse is located at 993bp (①-③), the wild type has no band (④), and ⑤ is water; Figure 1 B, Wsb1 flox / flox The homozygote has a band at 441bp (①-②), and the wild-type band is located at 325bp (④); the heterozygote has bands at 441bp and 325bp (③), and ⑤ is water, with no band. flox / flox;Ubc CreERT2 Genotype mice, bands appeared at 993bp and 441bp ( Figure 1 C).

[0078] 2.2 Construction of WSB1 knockout mice

[0079] Breeding: Select the identified Wsb1 flox / flox Homozygous mice with Ubc CreERT2 Mice are bred, and the F1 generation of mice can obtain Wsb1 flox / wt 、Wsb1 flox / wt ; UbcCreERT2 two genotype mice. flox / wt ;Ubc CreERT2 When female mice of the same genotype are bred with male mice of the same genotype, Wsb1 can be obtained in the F2 generation of mice. flox / wt 、Wsb1 flox / wt ;Ubc CreERT2 、Wsb1 flox / flox 、Wsb1 flox / flox ;Ubc CreERT2 and Wsb1 wt / wt ;Ubc CreERT2 Five genotypes of mice, including Wsb1 flox / flox ;Ubc CreERT2 For KO mice, select Wsb1 wt / wt ;Ubc CreERT2 Mice were used as wild type (WT) controls. Figure 2 .

[0080] 2.3 Tamoxifen-induced CreERT2 mice

[0081] (1) Prepare tamoxifen (Tam) solution: Weigh an appropriate amount of tamoxifen powder and use corn oil to prepare a 20 mg / mL tamoxifen solution. Place the tamoxifen solution on a 37°C constant temperature shaker and shake at 220 rpm overnight to fully dissolve it. Because tamoxifen is photosensitive, it needs to be protected from light and stored at 4°C.

[0082] (2) Induction strategy: Select healthy mice of 8 weeks old and weigh them to ensure that their weight is above 20g. Inject the corresponding volume of tamoxifen solution at a dose of 100μg / g mouse body weight intraperitoneally. The control group of mice was injected with a corresponding volume of corn oil. Induce every other day, and after 3 inductions in total, wait for 1 week to allow tamoxifen to be fully eliminated without affecting subsequent experiments. The weight and adverse reactions of the mice should be closely monitored during the entire induction period and the waiting period. During the administration period, the weight of the mice will decrease and recover after stopping the drug.

[0083] 2.4 Construction of WSB1 overexpressing mice

[0084] The WSB1 overexpression adeno-associated virus (AAV) was commissioned to Shanghai Gene Gene Co., Ltd. to construct. 8-week-old healthy SPF-grade C57BL / 6J male mice were selected and anesthetized by inhalation of isoflurane at a volume concentration of 1-2%, and fixed on a mouse board with the abdomen facing up. After local disinfection of the neck skin, a midline incision was made in the neck, the anterior neck muscle was bluntly separated, the trachea was exposed, and AAV (50µL, total titer of 1E+11vg) was slowly instilled into the trachea through a catheter. After the injection, the mouse body was kept vertical for 3 minutes to allow AAV to fully enter the lungs on both sides. The skin was sutured layer by layer, and the mouse was returned to the cage until it was fully awake. The overexpression efficiency was tested 2 weeks after the adenovirus airway instillation.

[0085] 2.5 Establishment of pulmonary hypertension model

[0086] (1) Establishment of hypoxia-induced mild to moderate HPH model in mice: All experimental mice were housed in an SPF-grade barrier. Before modeling, the mice were adaptively housed for at least 1 week. Mice in the normoxic control group (Nx group) were housed in a standard SPF environment (oxygen volume concentration was 21%), and mice in the hypoxic group (Hx group) were placed in a hypoxic oxygen chamber to construct a pulmonary hypertension model. The air and nitrogen intake was adjusted by the control system to form a stable hypoxic environment (ensuring that the volume concentration of oxygen in the oxygen chamber was 10%, the relative humidity was maintained at 50%-70%, and the temperature was maintained at 20-26°C). Mice in the normoxic and hypoxic groups received the same diet and lighting conditions. Hypoxia modeling was performed for 24 hours every day for a total of 21 days. Every 3 days, the mice were removed from the oxygen chamber to replenish drinking water and feed, and the oxygen chamber was cleaned. After 3 weeks, all mice were anesthetized and invasive hemodynamic measurements were performed to determine whether the modeling was successful, and then the mouse lung and heart tissues were collected for further study;

[0087] (2) Establishment of severe HPH model in mice with hypoxia combined with Sugen5416 (SuHx):

[0088] ① Preparation of Sugen5416 solution: weigh 20 mg of Sugen5416 powder and dissolve it in 1 mL of DMSO (dimethyl sulfoxide, mother solution concentration 20 mg / mL). After fully dissolved, add 4 mL of PEG300 (polyethylene glycol 300), mix well, add 500 μL of Tween 80, and finally add 4.5 mL of normal saline, mix well, the final concentration is 2 mg / mL, prepare before each use, and use it immediately;

[0089] ②Model construction: All experimental mice were raised in SPF-level barriers. After 1 week of adaptive feeding, they were grouped by random number method. Mice in the normoxic group were raised in a standard SPF environment (oxygen volume concentration was 21%), and mice in the SuHx group were placed in a hypoxic oxygen chamber for modeling. The intake of nitrogen and air was adjusted by the control system to create a stable hypoxic environment (ensuring that the volume concentration of oxygen in the oxygen chamber was 10%, the relative humidity was maintained at 50%-70%, and the temperature was maintained at 20-26°C). Hypoxia modeling was performed for 24 hours every day for 3 weeks, and Sugen5416 (20 mg / kg) was subcutaneously injected on the first modeling day of each week. Mice in the normoxic group were subcutaneously injected with the same dose of solvent. Mice were taken out of the oxygen chamber every 3 days to supplement drinking water and feed, and the oxygen chamber was cleaned. Index detection and sample collection were performed after the modeling was completed.

[0090] 2.6 Right ventricular systolic pressure (RVSP) detection:

[0091] (1) Homemade pressure measuring catheter: Cut a 30 cm long PE (polyethylene) catheter (outer diameter 0.9 mm, inner diameter 0.5 mm), heat one end of the catheter with boiling water to soften it, insert a 1 mL syringe needle into it (avoid puncture, the insertion depth should be greater than 0.5 cm), then slowly bring the other end of the catheter close to the outer flame of the alcohol lamp, and quickly withdraw it when the catheter is slightly curled. Repeat this operation until the front end forms an arc of nearly 90 degrees, trim the length of the curved part to 2-3 mm for measuring RVSP;

[0092] (2) Connect the catheter to the pressure sensor and then to the corresponding PowerLab physiological recorder. Use saline containing heparin to remove air from the catheter;

[0093] (3) After weighing the mouse (BW), anesthetize the mouse according to its weight, fix its limbs and head on a mouse board, make a midline incision in the neck, carefully separate the right external jugular vein, avoid damaging the blood vessel and the nerves around the blood vessel, and leave slip knots at the proximal and distal ends of the blood vessel for later use;

[0094] (4) Under direct vision of a dissecting microscope, cannulate the jugular vein, ligate the distal jugular vein, use ophthalmic scissors to make a small cut in the right external jugular vein, cannulate the jugular vein under direct vision of a dissecting microscope, insert the catheter into the right ventricle and tie a slipknot at the proximal end. Record the pressure data after the waveform stabilizes. During the operation, be careful to avoid blood backflow into the sensor. Pull out the arterial cannula and quickly ligate the proximal end to minimize bleeding.

[0095] (5) Calculate the RVSP using the recorder analysis software.

[0096] 2.7 Transthoracic echocardiography to assess right ventricular function

[0097] (1) The mice were pre-skinned and depilated on their chests. The mice were anesthetized by inhalation of isoflurane (volume concentration of 1-2%) using an anesthesia machine. The mice were fixed on a 37°C constant temperature heating plate, and then the right heart function of the mice was evaluated using a VisualSonics Vevo 2100 ultrasound system (the model of the mouse sensor probe was MS-550D, with a frequency range of 22-55 MHz).

[0098] (2) Pulse Doppler ultrasound was used to record the right ventricular pulmonary artery blood flow outflow in the parasternal short axis and to measure the pulmonary artery acceleration time (PAAT) and pulmonary artery ejection time (PAET);

[0099] (3) Measure the right ventricular free wall thickness at end diastole (RVFWT) and the right ventricular internal diameter at end diastole (RVEDD) at the parasternal long-axis right ventricular outflow tract M-mode level;

[0100] (4) The recorded data were measured and analyzed using VevoLAB3.1.1 software.

[0101] 2.8 Lung tissue sampling and lung tissue pretreatment

[0102] After intubation, the mouse was anesthetized with an overdose of isoflurane and killed. The mouse was placed on a dissecting board with its abdomen facing up and its limbs fixed with rubber bands. The mouse's abdomen was first cut open with sterile surgical scissors, and then the chest cavity was opened. The diaphragm was cut off from the chest wall, and the chest wall was fixed with a needle to remove the thymus. A No. 27 scalp needle connected to a 20mL syringe was inserted into the right ventricle, and the left atrial appendage was quickly cut off with scissors. 10-20mL of sterile phosphate buffered saline (PBS) without Ca ions and Mg ions was perfused into the pulmonary blood vessels until the lung tissue turned white. The trachea was cut off, and the lung tissue was slowly peeled off along the dorsal surface of the mouse. It was placed in PBS for washing three times, and the esophagus, glands, lymph, etc. were removed with sterile tweezers, and the lung tissue was removed. If the lung tissue is used for frozen sections, the appropriate size of lung tissue is quickly cut for OCT embedding. If the lung tissue is used for subsequent experiments such as WB, the washed lung tissue is directly placed in a -80℃ refrigerator for freezing. If the lung tissue is used for subsequent experiments such as paraffin sections, the washed lung tissue is placed in 4% paraformaldehyde and immersed overnight at room temperature.

[0103] 2.9 Cardiac tissue collection and processing

[0104] Right heart remodeling index: After the mouse was intubated and killed, the chest cavity was quickly exposed, the heart was removed, the soft tissue around the heart was cut off, the right ventricular wall was carefully separated, and the excess liquid was wiped off with filter paper. The weight of the right ventricle (RV) and the weight of the left ventricle + ventricular septum (LV+S) were weighed respectively. The whole process was performed by one person to maintain consistent techniques. Calculate the right ventricular remodeling index: RV / (LV+S);

[0105] Heart tissue embedding: Place the thoroughly cleaned heart in 4% paraformaldehyde and soak it overnight at room temperature before subsequent embedding and slicing.

[0106] 2.10Western Blotting

[0107] Protein extraction: Prepare RIPA lysis buffer (the volume ratio of PierceRIPABuffer to phosphatase inhibitor is 100:1). Weigh 20 mg of tissue and add 200 μL RIPA lysis buffer to ensure that the tissue is immersed in the lysis buffer, then use a homogenizer to homogenize the tissue. After homogenization, let it stand on ice for 30 minutes (vortex mixing every 10 minutes). Centrifuge at 4°C and 12000 rpm for 5 minutes, then transfer the supernatant to a 1.5 mL centrifuge tube. Centrifuge again at 4°C and 12000 rpm for 30 minutes, and the resulting supernatant is the protein stock solution.

[0108] Table 1. Formula of standard protein gradient concentration solution

[0109]

[0110] Concentration determination: The concentration was determined using the BCA protein assay kit. First, the standard was prepared according to the formula in Table 1: 3 replicate wells were required for standards of different concentrations and samples to be tested, and 200 μL of working solution was required for each well. The working solution was prepared according to the number of samples required for detection (the volume ratio of A solution to B solution was 50:1). Take 6 μL of the protein stock solution and add 84 μL of PBS to each well for protein concentration determination. Add the prepared protein dilution to be tested and standard proteins of different concentrations to a 96-well plate, add 25 μL to each well, and then quickly add the pre-prepared working solution, 200 μL per well, to avoid bubbles during the whole process. Cover the plate, seal it, and bathe it in a water bath at 37°C for 30 minutes. Open the plate cover and measure the absorbance value at a wavelength of 562 nm on the microplate reader. Draw a standard curve based on the absorbance value of the standard and its own concentration, substitute the absorbance of the sample to be tested into the standard curve, and calculate the concentration of the protein dilution.

[0111] Sample preparation: Set the protein loading volume to 80µg / 20µL. Use PBS and 5x loading buffer to balance each sample to the same concentration based on the loading volume and protein concentration. Place the mixed sample tube in a metal bath and boil at 100°C for 10 min. After the sample cools down, package it and store the packaged samples at -80°C.

[0112] Table 2. Separation gel composition table (4 plates of 1.5 mL gel glass plates)

[0113]

[0114] Table 3.5% concentrated gel composition table

[0115]

[0116] Gel preparation: Fix the 1.5mL glass plate for immunoblotting on the gel preparation rack. Prepare the separation gel solution according to the formula in Table 2, and evenly add the separation gel to the glass plate. The height of the gel surface should be 0.5-1.0cm below the green belt on the upper part of the gel preparation rack. Gently add anhydrous ethanol to the upper layer of the separation gel to remove bubbles on its surface. Let it stand for 15 minutes, pour out the anhydrous ethanol and gently absorb the residual anhydrous ethanol with filter paper. Prepare the concentrated gel according to the formula in Table 3, mix well and quickly pour it on top of the separation gel, insert the comb of the corresponding specifications into the concentrated gel (insert quickly to avoid solidification, push away excess bubbles from left to right), and then let it stand at room temperature for 15 minutes before solidification.

[0117] Table 4. WB related solution configuration

[0118]

[0119] Electrophoresis: Prepare WB-related solutions according to the formula in Table 4, and add an appropriate amount of sample to each well. Add 2µL / well markers on both sides of the sample loading well. Perform constant voltage electrophoresis at 70V. When the molecular weight indicated by the markers shows a clear boundary, switch to 110V and continue electrophoresis. Stop electrophoresis when the target band is clearly discernible.

[0120] Electrotransfer: Soak the PVDF (polyvinylidene fluoride) membrane in methanol for 15 seconds for membrane activation; soak the transfer sandwich in electrotransfer solution to moisten it; carefully transfer the gel to the filter paper and cover it with the PVDF membrane, and be careful to remove all bubbles; clamp the sandwich (from bottom to top: sponge, filter paper, gel, PVDF membrane, filter paper, sponge), and perform electrotransfer at 300mA. The electrotransfer time is determined according to the molecular weight, at least 45 minutes.

[0121] Blocking: After electroporation, the strips were rinsed once with TBST and then blocked in a 5% skim milk blocking buffer at room temperature for 1.5 h. After blocking, the strips were washed three times with TBST buffer, with the shaker speed at 90 r / min, and each wash lasted for 7 min.

[0122] Primary antibody incubation: Add the target protein primary antibody to the strips and incubate for 12-16 hours at 4°C and a shaker speed of 40 r / min. After the primary antibody incubation, recover the primary antibody and wash the strips 3 times with TBST buffer. During the washing process, the shaker speed is 90-100 r / min, and each washing lasts for 10 minutes.

[0123] Secondary antibody incubation: After absorbing all the TBST buffer, add the corresponding secondary antibody and incubate at room temperature for 60 minutes at a shaking speed of 40 r / min. After incubation with the secondary antibody, wash 3 times with TBST buffer. During the washing process, the shaking speed is 90-100 r / min, and each time lasts for 10 minutes.

[0124] Strip exposure: Prepare the ECL exposure solution according to the instructions (the volume ratio of A solution to B solution is 1:1), and expose it in the exposure machine with the protein side facing up.

[0125] Analysis: Bands were processed and analyzed using Imagelab.

[0126] Re-wash and re-incubate: After exposure, wash the exposed strips three times with TBST buffer, with the shaker speed at 90r / min for 10min. Then use the primary and secondary antibody removal solution, with the shaker speed at 90r / min for 10min. Then repeat the above blocking, primary antibody incubation (replace the target protein primary antibody), secondary antibody incubation, and strip exposure operations.

[0127] 2.11 Paraffin-embedded tissue sections

[0128] (1) Fixation: Place fresh tissue in 4% paraformaldehyde for 24 hours;

[0129] (2) Water washing: Use clean water to rinse the fixed lung tissue overnight;

[0130] (3) Dehydration treatment: 1. Soak in 30% ethanol for 12 h; 2. Soak in 50% ethanol for 2 h; 3. Soak in 70% ethanol for 2 h; 4. Soak in 80% ethanol for 1 h; 5. Soak in 90% ethanol for 1 h; 6. Soak in 95% ethanol (I) for 30 min; 7. Soak again in fresh 95% ethanol (II) for 30 min; 8. Soak in 100% ethanol (I) for 30 min; 9. Soak again in fresh 100% ethanol (II) for 30 min;

[0131] (4) Transparent treatment: Soak in xylene (I) for 20 min; soak again in fresh xylene (II) for 20 min;

[0132] (5) Wax treatment: Use soft wax (52-54°C) for 2 hours; Use hard wax (60-62°C) for 1.5 hours;

[0133] (6) Use a tissue embedding machine to embed lung tissue, with the hilum facing upwards;

[0134] (7) Use a tissue slicer to slice the lung tissue, select the largest section, and slice with a thickness of 4 μm;

[0135] (8) Use a brush and tweezers to carefully select a wax sheet that is uniform in thickness, flat, and free of cracks. Take the sheet out of cold water, transfer it to 42°C warm water, and spread it until the wax sheet is completely flat.

[0136] (9) Tilt the adhesive slide at about 60 degrees and place it in warm water. Slowly move it close to the wax sheet so that it adheres smoothly to the slide. Mark the wax sheet number with a pencil.

[0137] (10) Place the adhered slide slightly tilted into the slice holder to control the moisture;

[0138] (11) Bake the slices in an oven at 65°C for 10 min and place in a slicing box for later use.

[0139] 2.12 Preparation of frozen lung tissue sections

[0140] (1) After the surface moisture of fresh lung tissue is absorbed by filter paper, it is immediately placed flat in a tissue embedding box with OCT (cryogenic embedding medium) on the bottom. Another layer of OCT (cryogenic embedding medium) is added on the tissue to completely cover the tissue.

[0141] (2) Quickly place the embedding box into liquid nitrogen for quick freezing;

[0142] (3) Place the embedded tissue block into a freezing microtome for slicing;

[0143] (4) During the sectioning process, the temperature of the cryostat cabinet should be kept at -15 to -20°C, and the temperature of the specimen head should be set to -20°C;

[0144] (5) Set the slice thickness to 7 μm and maintain a constant speed during the slicing process to ensure that the slices are complete;

[0145] (6) After the frozen sections are adsorbed onto the adhesive slides, they are briefly placed at room temperature to dry out excess moisture on the surface of the sections and then moved to -80°C for storage.

[0146] 2.13HE staining

[0147] (1) Drying: Place the paraffin sections in a 65°C oven for 4-6 hours;

[0148] (2) Dewaxing: Soak the sections in xylene (I) for 15 min, then soak in fresh xylene (II) for another 15 min.

[0149] (3) Hydration: The sections were immersed in gradient ethanol with a volume concentration of 100%, 95%, 80%, and 70% for 5 min each for hydration, and then the sections were washed in distilled water for 3 min.

[0150] (4) Add an appropriate amount of hematoxylin to the slices to fully cover the tissue, stain for 2 minutes, and use distilled water to remove residual stain;

[0151] (5) Add an appropriate amount of differentiation solution to cover the tissue and differentiate for 5 seconds until the slices turn light blue, then rinse with running water for 5 minutes;

[0152] (6) Add bluing solution for 30 seconds, then rinse with running water for 5 minutes;

[0153] (6) Add an appropriate amount of eosin dye solution to the slice to cover the tissue and stain for 6 minutes;

[0154] (7) Move the slices into 95% ethanol (I), fresh 95% ethanol (II), 100% ethanol (I), and fresh 100% ethanol (II) in turn and soak for 2-3 seconds each;

[0155] (8) Soak the sections in xylene (I) and fresh xylene (II) for 5 min each to make them transparent;

[0156] (9) Take out the slices from the fume hood, drop neutral resin on them, and seal them with a clean coverslip to avoid bubbles.

[0157] (10) Dry the slide in a fume hood and then examine it under a microscope;

[0158] (11) ImageProPlus was used to perform statistics on indicators such as vascular wall thickness and vascular area.

[0159] 2.14 Masson staining

[0160] (1) The steps of drying, dewaxing and hydrating paraffin sections are the same as (1) to (3) in 2.13;

[0161] (2) Add Bouin's fixative to the tissue sections, incubate at 37°C for 2 h, and then wash with running water;

[0162] (3) Use lapis lazuli blue solution (mass concentration: 0.5%) to dye for 3 min, then rinse with running water;

[0163] (4) Stain with Mayer's hematoxylin for 3 minutes and then rinse with running water;

[0164] (5) Use acidic ethanol for differentiation for 10 seconds, and then rinse with running water for 10 minutes;

[0165] (6) Stain with Ponceau fuchsin for 8 min and then wash;

[0166] (7) Treat with phosphomolybdic acid for 10 min, pour off the dye solution and directly add aniline blue for 9 min, then wash;

[0167] (8) Treat with weak acid for 2 minutes;

[0168] (9) Dehydrate with 95% ethanol for 5 seconds and 100% ethanol for 10 seconds, three times each. Transparentize with xylene for 2 minutes, three times each. Observe under a microscope after sealing with resin (collagen appears blue and cytoplasm appears red).

[0169] (10) ImageProPlus was used to count the collagen area around blood vessels.

[0170] 2.15 Immunohistochemistry

[0171] (1) Dewaxing of paraffin sections: Soak the paraffin sections in xylene (I) for 15 min, soak them in fresh xylene (II) for 15 min, soak them in 100% ethanol for 5 min, soak them in 95% ethanol for 5 min, soak them in 85% ethanol for 5 min, soak them in 75% ethanol for 5 min, and finally rinse them with distilled water.

[0172] (2) Antigen repair: Prepare citric acid antigen repair buffer (diluted to 1 times with distilled water, pH 6.8), place the antigen repair solution in a microwave oven, preheat on medium heat for 5 minutes, place the dewaxed and hydrated tissue sections in a repair box filled with citric acid antigen repair buffer (pH 6.8), heat on medium heat for 8 minutes, stop heating for 8 minutes, and heat on medium-low heat for 7 minutes. After natural cooling, place the slides in PBS and wash them slowly on a shaker for 3 times, 5 minutes each time;

[0173] (3) Blocking endogenous peroxidase: Place tissue sections in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 30 min. Then place the slides in clean PBS and wash on a shaker three times for 5 min each time.

[0174] (4) Blocking: After wiping off the PBS around the tissue section, use a histochemical pen to draw a circle around the tissue and add 10% goat serum in the circle to evenly cover the tissue. Block at room temperature for 1 hour.

[0175] (5) Primary antibody incubation: Gently shake off the blocking solution on the tissue sections, add an appropriate amount of primary antibody (prepared with 1% BSA concentration) on the sections, place the sections flat in a humidified box, and incubate in a refrigerator at 4°C overnight (12-18 hours);

[0176] (6) Secondary antibody incubation: The next day, take out the slides and equilibrate them at room temperature for 30 minutes. Carefully place the sections in PBS and shake on a shaker to wash 5 times, 5 minutes each time. After wiping off the liquid around the tissue, add the reaction enhancement solution in the circle, incubate at room temperature for 20 minutes, and wash with PBS 5 times, 5 minutes each time. After the sections are slightly dried, add the enhanced enzyme-labeled IgG polymer again, incubate at room temperature for 20 minutes, and wash with PBS 5 times, 5 minutes each time.

[0177] (7) DAB color development: After wiping off the PBS around the slice, add freshly prepared DAB color development solution into the circle and control the color development time under a microscope. The positive color is brown-yellow. Rinse the slice with tap water to stop the color development.

[0178] (8) Staining cell nuclei: Re-stain with hematoxylin for about 80 seconds, rinse with tap water, differentiate with hematoxylin differentiation solution for 5 seconds, rinse with tap water, blue reversion solution for 30 seconds, and rinse with running water;

[0179] (9) Dehydration and sealing: Soak the sections in 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 min each, then place them in xylene (I) and fresh xylene (II) for 5 min each for dehydration and transparency. Take the sections out of xylene, let them dry slightly, and then seal them with neutral gum.

[0180] (10) Microscopic examination.

[0181] 2.16 Immunofluorescence analysis of paraffin sections of lung tissue

[0182] (1) Dewaxing of paraffin sections: Same as immunohistochemistry in 2.15 above;

[0183] (2) Antigen retrieval: Same as immunohistochemistry in 2.15 above;

[0184] (3) Blocking: Same as immunohistochemistry in 2.15 above;

[0185] (4) Incubation with primary antibody: Same as immunohistochemistry in 2.15 above;

[0186] (5) Incubation with secondary antibody: The next day, wash with PBST / PBS alternately for 5 times, 5 minutes each time. Prepare an appropriate amount of secondary antibody with a volume concentration of 1% BSA, select the corresponding secondary antibody according to the source of the primary antibody (the dilution ratio is 1:1000), and incubate at room temperature in the dark for 1 hour. Wash with PBST / PBS alternately for 5 times, 4 minutes each time;

[0187] (6) Nuclear staining and sealing: Add anti-fluorescence quenching sealing medium containing 4',6-diamidino-2-phenylindole (DAPI, nuclear staining solution) to the lung tissue on the slide, and carefully seal the slide with a coverslip to avoid the formation of bubbles;

[0188] (7) Microscopic examination: After air drying, observe and photograph the samples using an immunofluorescence microscope with a light source of appropriate excitation wavelength for different fluorescent dyes.

[0189] 2.17 Immunofluorescence analysis of frozen lung sections

[0190] (1) Fixation: Take fresh frozen tissue sections, air-dry them in a fume hood, add an appropriate amount of 4% paraformaldehyde to each section and fix them at room temperature for 20 minutes, then wash them three times with PBS, each time for 4 minutes;

[0191] (2) Punching: Add an appropriate amount of Triton X-100 (Triton X-100) with a volume concentration of 0.3% to each slice and treat at room temperature for 10 minutes;

[0192] (3) Antigen repair: discard the 0.3% TritonX-100, add an appropriate amount of 1x frozen section antigen repair solution to each slide, incubate at room temperature for 15 min, and then wash with PBS three times, each time for 4 min;

[0193] (4) Incubation with primary antibody, incubation with secondary antibody, cell nuclear staining, sealing and microscopic examination are the same as described in 2.16.

[0194] 2.18 Statistics of vascular muscularization

[0195] The degree of muscularization of distal pulmonary arterioles was evaluated by the coverage of immunohistochemical αSMA-positive vascular smooth muscle cells, and the stratification was as follows: 1) non-muscularization, no obvious αSMA-positive cells; 2) partial muscularization, αSMA-positive cells formed a crescent-shaped structure; 3) complete muscularization, αSMA-positive cells formed a circular medial muscle layer. In order to conduct objective analysis, at least 100 blood vessels with appropriate diameters were counted from the lung sections of each group of mice, and the final data were presented as the proportion of non-muscularization, partial muscularization, and complete muscularization.

[0196] 3. Experimental results

[0197] 3.1 Expression and role of WSB1 in pulmonary hypertension

[0198] Figure 3 The test results showed that the expression of WSB1 protein in the lung tissue of HPH patients was increased. Figure 4 The test results showed that WSB1 expression was upregulated in the lung tissue of both the hypoxia-induced (Hx) mouse HPH model and the hypoxia combined with sugen5416 (SuHx)-induced mouse HPH model.

[0199] The WT-Ctrl group was the wild-type blank control group; the KO-PH group was the WSB1 gene knockout-HPH model group; and the WT-PH group was the wild-type HPH model group.

[0200] Figure 5 The experimental results showed that compared with the WT-PH group mice, the KO-PH group mice had improved right ventricular systolic pressure (RVSP) and pulmonary arterial hypertension (PAAT / PAET), indicating that WSB1 knockout improved pulmonary hemodynamic changes in HPH mice.

[0201] Figure 6 The experimental results showed that compared with the WT-PH group mice, the ratio of vascular wall diameter to total vascular diameter (WT / TT) and the ratio of pulmonary artery vascular wall area to total vascular area (WA / TA) were both downregulated in the KO-PH group mice, indicating that WSB1 knockout improved the thickening of the pulmonary artery vascular wall in HPH mice.

[0202] Figure 7 The experimental results showed that compared with the WT-PH group mice, the proportion of non-muscularization in the microvessels of the KO-PH group mice increased, and the proportion of complete muscularization decreased, indicating that WSB1 knockout improved the muscularization of the microvessels of HPH mice.

[0203] Figure 8 The experimental results showed that compared with the WT-PH group, the collagen deposition around the pulmonary artery in the KO-PH group mice was significantly improved (left figure). After quantitative calculation, the proportion of collagen area around the pulmonary artery in the KO-PH group mice was significantly decreased compared with that in the WT-PH group mice, indicating that WSB1 knockout improved the vascular collagen deposition in HPH mice.

[0204] Fig. 9 The experimental results showed that the right ventricular diastolic free wall thickness (RVFWT) of KO-PH mice was significantly reduced compared with that of WT-PH mice ( Fig. 9 A, 9B), both right ventricular internal diameter and right ventricular output increased during diastole ( Fig. 9 C, 9D), indicating that WSB1 knockout has a positive effect on right heart remodeling and right heart function in HPH mice. Fig.10 The experimental results in this study demonstrated that WSB1 knockout improved right heart remodeling in HPH mice.

[0205] The NC-Ctrl group was the normal control group; the OE-Ctrl group was the WSB1 overexpression-normal control group; the NC-PH group (NC-HPH group) was the normal HPH model group; and the OE-PH group (OE-HPH group) was the WSB1 overexpression-HPH model group.

[0206] Fig.11The experimental results showed that compared with the NC-PH group, the overexpression of WSB1 in the OE-PH group mice led to an increase in cardiac systolic pressure (RVSP), a decrease in the PAAT / PAET ratio, and a more severe pulmonary hypertension. The OE-Ctrl group mice also showed the same situation compared with the NC-Ctrl group mice, indicating that the overexpression of WSB1 aggravated the hemodynamic changes in mice.

[0207] Fig.12 The experimental results showed that the ratio of the vessel wall diameter to the total vessel diameter (WT / TT) and the ratio of the pulmonary artery vessel wall area to the total vessel area (WA / TA) were both upregulated in the OE-HPH group compared with the NC-HPH group. The same situation was also observed in the OE-Ctrl group compared with the NC-Ctrl group, indicating that WSB1 overexpression leads to thickening of the pulmonary artery vessel wall in HPH mice.

[0208] Fig.13 The experimental results showed that compared with the NC-HPH group, the collagen deposition around the pulmonary artery of the OE-HPH group mice was significantly more serious. After quantitative calculation, the proportion of collagen area around the pulmonary artery of the OE-HPH group mice was significantly increased, indicating that WSB1 overexpression aggravated the vascular collagen deposition in HPH mice.

[0209] Fig.14 The experimental results showed that compared with the NC-HPH group mice, the proportion of non-muscularization in the microvessels of the OE-HPH group mice was reduced, and the proportion of complete muscularization was increased, indicating that WSB1 overexpression aggravated the muscularization of the microvessels of HPH mice.

[0210] Fig.16 The experimental results showed that the right ventricular diastolic free wall thickness (RVFWT) of OE-HPH mice was increased compared with that of NC-HPH mice ( Fig.16 A, 16B), right ventricular internal diameter and right ventricular output decreased during diastole ( Fig.16 C, 16D), indicating that WSB1 overexpression has a reverse effect on right heart remodeling and right heart function in mice. Fig.15 The experimental results showed that WSB1 overexpression aggravated right heart remodeling in HPH mice.

[0211] Through genetic engineering, WSB1 was knocked out and overexpressed to observe the effect of changes in WSB1 expression on the outcome of HPH. It was found that knocking out WSB1 improved the HPH phenotype while overexpressing WSB1 aggravated the HPH phenotype, confirming that elevated WSB1 is a damaging factor.

[0212] 3.2 Study on the mechanism of action of WSB1 in pulmonary hypertension

[0213] The Ctrl group was the normal control group, and the HPH group was the HPH model group. The SiNC-NOR group was the normoxic control of short interfering RNA (siRNA), the SiNC-HYP group was the HYP model of short interfering RNA (siRNA), and the SiWSB1-HYP group was the HYP model with WSB1 gene knockout. The NC-OE group was the normal control group, the WSB1-OE group was the WSB1 overexpression group, and the HIF-2α-OE group was the HIF-2α overexpression group.

[0214] Fig.17 The experimental results showed that through cellular localization of WSB1, elevated WSB1 was enriched in the pulmonary vascular endothelial cells of HPH group mice.

[0215] Fig.18 The experimental results showed that compared with the normoxic group mice and the hypoxic group mice, the average fluorescence intensity of WSB1 expression in the pulmonary microvascular endothelial cells of the hypoxic group mice was significantly higher, indicating that hypoxia induced an increase in WSB1 expression in the pulmonary microvascular endothelial cells of mice.

[0216] Fig.19 The experimental results showed that WSB1 knockdown improved hypoxia-induced mitochondrial damage. Fig. 20 The experimental results showed that WSB1 knockdown had no effect on endothelial cell apoptosis.

[0217] Figures 17 to 19 Our results suggest that WSB1 is involved in multiple aspects of endothelial dysfunction, including early mitochondrial damage and dysfunction.

[0218] Fig.21 The experimental results showed that WSB1 knockdown inhibited hypoxia-induced HIF-2α accumulation. Fig. 22 The experimental results showed that overexpression of WSB1 promoted the accumulation of HIF-2α. Fig.23 The experimental results showed that for HYP model mice with WSB1 gene knockout, HIF-2α overexpression reversed the protective effect of WSB1 knockdown on mitochondrial damage.

[0219] Figure 21 to Figure 23 Our results suggest that WSB1 exacerbates hypoxia-induced endothelial dysfunction by promoting HIF-2α accumulation.

[0220] rPMVECs (mouse pulmonary microvascular endothelial cells) were plated into the well plate and placed in a cell culture incubator for further culture. When the cells grew to 70-80% confluence, they were transfected. The transfection was performed at room temperature. Two EP tubes of appropriate size were prepared. 125μL OPTI-MEM medium (reduced serum medium) and 3.25μL siRNA (20µM) were added to one tube. The sequence of siRNA is shown in Table 5. 125μL OPTI-MEM medium and 7.5μL Lipofectamine3000 liposome transfection reagent were added to the other tube. The tubes were allowed to stand for 2 minutes, and then the two tubes were mixed to form the transfection complex. The cells were incubated at room temperature for 20 minutes. During the incubation at room temperature, the cell medium was replaced. 2mL of basal medium was added to each well. After the incubation was completed, the transfection complex was added to the cells. The cells were gently shaken and mixed using the inverted eight-character method. The cells were placed in a 37°C constant temperature incubator for incubation for 6 hours and then the medium was replaced (replaced with complete medium). The knockdown efficiency was tested at the PCR and WB levels 24h and 48h after transfection. The test results are shown in Fig.30 As shown, the WB test results (Figure A, B) and PCR test results (Figure C) both indicate that siRNA3 has better knockdown efficiency. Among them, the Control group is the normal control group, the NC group is the negative control group, the siRNA1 group is the siRNA1 treatment group, the siRNA2 group is the siRNA2 treatment group, and the siRNA3 group is the siRNA3 treatment group.

[0221] Table 5. siRNA sequences

[0222]

[0223] The PCR experimental steps are as follows:

[0224] (1) Total RNA extraction.

[0225] (2) For cell samples, remove the culture medium and wash twice with 1 mL of pre-cooled PBS. Make sure to remove all residual liquid for the last time. Add 1 mL of TRIzol (total RNA extraction reagent) to the culture dish and blow it around every corner. Collect the TRIzol containing cells and add it to the enzyme-free EP tube. Let it stand on ice for 15 minutes. Add 200 μL of bromotrichloropropane (used to replace chloroform) to the TRIzol containing the sample, continue to shake and mix for 15 seconds, let it stand on ice for 15 minutes, and then place it in a pre-cooled 4°C centrifuge at 12,000 rpm for 10 minutes. At this time, you can see that the liquid is divided into three layers, the top layer is a transparent aqueous phase (containing RNA), the middle white layer (containing protein and DNA), and the bottom layer is a red layer (containing protein, DNA, phenol and bromotrichloropropane). Pipette 400 μL of the upper aqueous phase into a new enzyme-free EP tube (be careful in this step to avoid absorbing the substances in the middle layer). Add 400 μL of isopropanol to each tube, mix gently up and down, and let stand for 10 minutes. Then centrifuge at 12,000 rpm for 10 minutes at 4°C. The white precipitate obtained is RNA. Gently pour off the supernatant, turn it upside down on a paper towel to drain it slightly, add 1 mL of anhydrous ethanol and wash it repeatedly twice. Finally, dry the obtained white precipitate in a fume hood for 5-10 minutes, add 20 μL of DEPC water (water treated with diethyl pyrophosphate), and mix it by blowing. Use an ultra-micro spectrophotometer to detect RNA concentration and purity. The gun tips, EP tubes, etc. used in the entire experiment need to be sterilized and de-enzymed to minimize RNA enzyme contamination during the operation. The surrounding environment of mRNA extraction needs to be kept clean to prevent contamination by proteins, especially saliva.

[0226] (3) Reverse transcription: After measuring the mRNA concentration using an ultra-micro spectrophotometer, reverse transcription was performed using a reverse transcription kit (TOROIVDqRT Master Mix). The required template volume was calculated based on the RNA concentration measured in (1). The volume of enzyme-free water was (10 times the RNA template volume) µL. The reverse transcription system was prepared on ice (as per Table 6).

[0227] Table 6. Preparation of reverse transcription system

[0228]

[0229] After the system is configured, place it in a PCR instrument for reverse transcription. Set the reverse transcription program according to Table 7. The complementary DNA (cDNA) after reverse transcription can be directly PCR or stored at -80℃.

[0230] Table 7. Reverse transcription program settings

[0231]

[0232] (4) PCR: The experiment was performed using the SYBR Green qPCR Master Mix kit. The reaction system is shown in Table 8.

[0233] Table 8. qPCR reaction system

[0234]

[0235] To reduce the error of sample addition, the total amount of other reagents in Table 8 except the DNA template was calculated according to the number of samples, and premixed in the enzyme-free EP tube prepared in advance. 18 μL of premix was added to each PCR reaction well, and then 2 μL of DNA template was added. The PCR plate was placed in the reverse transcription instrument for qPCR experiment. The reaction procedure is shown in Table 9.

[0236] Table 9. qPCR program settings

[0237]

[0238] Table 10. Primer sequences

[0239]

[0240] (5) Data analysis: The data were exported from Bio-Rad and the results were calculated according to the 2-△△Ct method. △Ct = (Ct value of target mRNA - Ct value of internal reference) mean ± standard deviation, △△Ct = (△Ct value of target mRNA in the test sample - △Ct value of target mRNA in the reference sample) mean ± standard deviation, relative sample initial template amount = (2-△△Ct) mean ± standard deviation.

[0241] The primer sequences corresponding to the WSB1 gene and the β-Actin gene are shown in Table 10.

[0242] 3.3 Targeting endothelial cell WSB1 is a new option for HPH treatment

[0243] The EC-WT-Ctrl group was the wild-type blank control group; the EC-WT-PH group was the wild-type HPH model group; the EC-KO-PH group was the endothelial cell WSB1 gene knockout-HPH model group; and the EC-WT-Ctrl group was the wild-type normal control group.

[0244] Fig.24 The results of the experiment showed that endothelial cell WSB1 knockout improved right heart remodeling and right heart function in HPH mice.

[0245] Fig.25 The experimental results showed that the right ventricular diastolic free wall thickness (RVFWT) of EC-KO-PH group mice was significantly reduced ( Fig.25A, 25B), both right ventricular internal diameter and right ventricular output increased during diastole ( Fig.25 C, 25D), indicating that endothelial cell WSB1 knockout has a positive effect on right heart remodeling and right heart function indicators in HPH mice.

[0246] Fig.26 The experimental results showed that endothelial cell WSB1 knockout had no effect on the baseline hemodynamics and right ventricular function of normal mice. The EC-WT group was the wild-type normal group; the EC-KO group was the endothelial cell WSB1 gene knockout normal group.

[0247] Fig. 27 The results of the experiment showed that endothelial cell WSB1 knockout improved mitochondrial damage in endothelial cells of HPH mice. Fig.28 The results of the experiment showed that knockout of endothelial cell WSB1 improved the accumulation of HIF-2α in endothelial cells of HPH mice. Fig.29 The results of the experiment showed that endothelial cell-specific knockout of WSB1 had similar effects on the remission efficiency of HPH mice as systemic knockout.

[0248] The present invention observed the effect of WSB1 on endothelial function from multiple dimensions and found that intervention with WSB1 can effectively improve the hypoxia-induced mitochondrial structure and function destruction of vascular endothelial cells, endothelial-mesenchymal transition, and the inhibitory effect of endothelial cells on abnormal proliferation of smooth muscle cells in HPH mice. The study confirmed that knockout of endothelial cell WSB1 improves PH phenotype and endothelial dysfunction ( Figure 24-28 WSB1 knockout in endothelial cells has similar PH improvement effect as WSB1 knockout ( Fig.29 The results show that WSB1 is an important target for endothelial dysfunction. Therefore, endothelial cell WSB1 can be used as a new target for HPH treatment. Based on the treatment concept of targeting endothelial cell WSB1 without disturbing other cells, an endothelial cell targeted delivery system can be constructed to deliver WSB1 antibodies, small molecule inhibitors, etc. to achieve efficient and low-side effect treatment plans, providing new options for HPH treatment.

[0249] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a WSB1 inhibitor in the preparation of a drug for alleviating or / and treating hypoxic pulmonary hypertension, characterized in that: The WSB1 inhibitor is siRNA, and the sequence of the siRNA is TCCTGATGGAGCATTACTA.

2. A WSB1 inhibitor, characterized in that The inhibitor is siRNA, and the sequence of the siRNA is TCCTGATGGAGCATTACTA.

3. The WSB1 inhibitor according to claim 2, characterized in that The inhibitor of WSB1 is used for preparing drugs for alleviating and / or treating hypoxic pulmonary hypertension.

4. A targeted drug for alleviating or / and treating hypoxic pulmonary hypertension, characterized in that: The invention comprises an inhibitor of WSB1, wherein the inhibitor is siRNA, and the sequence of the siRNA is TCCTGATGGAGCATTACTA.

5. The targeted drug according to claim 4, characterized in that: The targeted drug also includes a pharmaceutically acceptable carrier.

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