A pulmonary vascular endothelial barrier injury model and its construction method and application

Abstract

The present invention discloses a pulmonary vascular endothelial barrier damage model, a construction method and an application thereof, and relates to the field of biotechnology. The construction method of the pulmonary vascular endothelial barrier damage model comprises the following steps: after adaptive feeding healthy mice for one week, anesthetizing them with drugs, pre-treating oncostatin M recombinant protein and instilling it into the mouse lungs through the airway to obtain a pulmonary vascular endothelial barrier damage model; the present invention also discloses a pulmonary vascular endothelial barrier damage model and an application of the model in preparing a pulmonary vascular disease research model and an application of the model in preparing drugs for treating pulmonary vascular diseases; the pulmonary vascular endothelial barrier damage model provided by the present invention, a construction method and an application thereof, oncostatin M protein binds to type II oncostatin M receptors on endothelial cells, activates downstream signaling pathways, and causes endothelial barrier damage, thereby providing a clear target for endothelial barrier damage and facilitating in-depth exploration of the mechanism of lung injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a pulmonary vascular endothelial barrier damage model, a construction method, and an application thereof. Background Art

[0002] Vascular endothelial cells (ECs) are widely found lining arteries, veins, microvessels, and lymphatic vessels throughout various tissues and organs in the human body. As the first interface between blood and vascular tissue, they play a critical role in maintaining vascular permeability, transmitting vascular information, regulating vascular tone, preventing hemorrhage, secreting vasoactive substances, and maintaining oxygen and nutrient supply to all tissues in the body. When stimulated by infection, endotoxins, hypoxia, oxidized low-density lipoproteins, and inflammatory cytokines, ECs can transition from a quiescent state to an activated state. Activated ECs secrete a range of proinflammatory cytokines, chemokines, enzymes, and adhesion molecules. Inflammatory cytokines increase the production of inflammatory proteins and activate multiple signaling pathways, shifting ECs into a proinflammatory state. This inflammatory phenotype of ECs can trigger inflammation in the vascular wall, impairing its normal function, manifesting as weakened barrier function, increased permeability, and exacerbated inflammation. Pulmonary endothelial cells are at the center of the inflammatory storm associated with acute respiratory distress syndrome (ARDS). Loss of the endothelial barrier can lead to pulmonary edema (a hallmark of acute lung injury) and high-altitude pulmonary edema. The pathogenesis of these two diseases remains unclear, and to date, no drugs have been approved to treat these life-threatening conditions. Endothelial cell barrier damage plays a crucial role in the pathological processes of acute lung injury, ARDS, atherosclerosis, hypertension, diabetes, and cerebrovascular disease. Therefore, establishing a model of endothelial inflammatory damage is crucial for studying pulmonary vascular diseases and accelerating the development of effective therapeutic drugs.

[0003] Establishing a model of pulmonary endothelial injury is an important approach for studying pulmonary vascular disease. In recent years, with the development of various methods, including chemical induction, gene editing, physical induction, and combined induction, significant progress has been made in establishing models of vascular endothelial injury. Lipopolysaccharide (LPS) has been widely used to establish models of pathogen-associated sepsis. However, the purity of LPS extracted by different manufacturers varies, and it may contain bacterial lipoproteins or other bacterial substances. Furthermore, LPS primarily triggers an inflammatory response by activating the TLR4 signaling pathway, indirectly damaging endothelial cells. Its direct damage to the endothelial barrier is relatively limited, and its pathogenesis is relatively simple, which may not fully simulate the complex pathophysiological process. TNF-α is primarily used to establish models of endothelial injury in preeclampsia. Ox-LDL-induced endothelial injury is primarily oxidative and is used to establish models of endothelial barrier damage in atherosclerotic diseases. AGEs are important models for studying endothelial injury associated with diseases such as diabetes. Hcy-induced endothelial barrier injury models are used to study vascular diseases associated with hyperhomocysteinemia. Ang II-induced endothelial barrier injury models are important for studying cardiovascular diseases such as hypertension. High cholesterol diets primarily cause atherosclerosis in rats. None of the aforementioned chemical methods for inducing endothelial injury are fully applicable to establishing models of pulmonary endothelial barrier injury. H2O2, MCT, and rotenone are highly toxic to endothelial cells. While readily available, inexpensive, and exhibit significant damaging effects, H2O2 is unstable and easily decomposes. Different endothelial cells vary greatly in their sensitivity to MCT and rotenone. Therefore, H2O2, MCT, and rotenone can only be used as tools for in vitro studies of endothelial injury and cannot be guaranteed to be safe, effective, or reproducible in vivo. Although hypoxia and mechanical ventilation can induce endothelial barrier damage, these methods require specific experimental conditions and equipment and are not suitable for most laboratory applications. Although gene editing technology can precisely regulate gene expression, its operation is complex and the cost is high, making it unsuitable for early promotion.

[0004] Although some progress has been made in establishing pulmonary vascular endothelial injury models and they have been used to some extent, their respective shortcomings provide opportunities for exploring new methods and new research. Summary of the Invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a pulmonary vascular endothelial barrier injury model, construction method and application. OSM recombinant protein binds to type II OSM recombinant protein receptors on endothelial cells, activating downstream signaling pathways, leading to damage to endothelial cell tight junctions. OSM recombinant protein can mainly damage the pulmonary vascular endothelial barrier, providing a clear target for endothelial barrier injury; it has low toxicity to tissue cells and tissue compatibility; it has a clear mechanism of action and repeatability; the protein preparation process is simple and cost-controlled, making it easier for more laboratories to carry out research and improving resource utilization efficiency.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for constructing a pulmonary vascular endothelial barrier injury model comprises the following steps: after adaptive feeding of healthy mice for one week, the mice are anesthetized with drugs, and oncostatin M recombinant protein is pretreated and then instilled into the mouse lungs via the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0008] Furthermore, ketamine and xylazine were used for drug anesthesia, with the mass ratio of ketamine being 80 mg / kg and the mass ratio of xylazine being 10 mg / kg.

[0009] Furthermore, the pretreatment of the oncostatin M recombinant protein is to dilute the oncostatin M recombinant protein to 10-50 ng / μL with PBS, and the oncostatin M recombinant protein is used to treat the mice for 12-24 hours.

[0010] Furthermore, the pretreatment of the oncostatin M recombinant protein was performed by diluting the oncostatin M recombinant protein to 20 ng / μL with PBS, and the mice were treated with the oncostatin M recombinant protein for 24 hours.

[0011] A pulmonary vascular endothelial barrier injury model is used to prepare a research model for pulmonary vascular diseases. The preparation steps are as follows: healthy mice are adaptively fed for one week and then anesthetized with drugs. The oncostatin M recombinant protein is pretreated and then instilled into the mouse lungs through the airway to obtain a pulmonary vascular endothelial barrier injury model.

[0012] A pulmonary vascular endothelial barrier injury model is used in the preparation of drugs for treating pulmonary vascular diseases. The preparation is carried out using the following steps: healthy mice are adaptively fed for one week and then anesthetized with drugs. Oncostatin M recombinant protein is pretreated and then instilled into the mouse lungs through the airways to obtain a pulmonary vascular endothelial barrier injury model.

[0013] The technical solution of the present invention achieves the following beneficial technical effects:

[0014] 1. The present invention provides a pulmonary vascular endothelial barrier injury model, construction method, and application. OSM recombinant protein is used as a key factor in constructing the pulmonary vascular endothelial barrier injury model. OSM recombinant protein can primarily damage the pulmonary vascular endothelial barrier, rather than other functions of endothelial cells in other systemic diseases. It also has low toxicity to tissue cells and tissue compatibility. It has a clear mechanism of action and is reproducible. The protein preparation process is simple and cost-effective, making it easier for more laboratories to conduct research and improving resource utilization efficiency.

[0015] 2. The present invention provides a pulmonary vascular endothelial barrier injury model, construction method, and application. The feasibility of establishing C166 and HULEC-5a endothelial barrier injury models using OSM recombinant protein as a stimulus and different concentrations of OSM recombinant protein and different exposure times can induce endothelial barrier injury without causing massive cell death, thereby providing a reference for the establishment of subsequent inflammatory pulmonary vascular disease models and helping to explore related pathological processes and regulatory mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of a pulmonary vascular endothelial barrier injury model and its construction method, as well as the effect of the OSM recombinant protein on the vascular endothelium used in the present invention;

[0017] A shows immunofluorescence staining of OSM recombinant protein in lung tissue sections of mice in the PA and PBS groups observed by confocal microscopy. OSM recombinant protein is marked in red, and DAPI-stained cell nuclei are marked in blue. Scale bar: 20 μm.

[0018] B is a bar graph showing the OSM recombinant protein in the lung tissues of control mice and PAO1-infected mice detected by ELISA (**: P < 0.01, n = 4);

[0019] C is the confocal microscopy observation of VE-cadherin immunofluorescence staining of lung tissue sections of mice in the Ctrl and OSM groups, VE-cadherin is marked in red, and DAPI-stained cell nuclei are marked in blue;

[0020] The bar graph in D shows the results of FITC-dextran permeability analysis of vascular endothelial cells in mice in the Ctrl and OSM groups (***: P < 0.01, n = 4);

[0021] E is immunofluorescence staining of lung tissue sections of mice in each group observed under a confocal microscope. VE-cadherin is stained red, and cell nuclei are labeled with DAPI in blue. Scale bar: 10 μm.

[0022] The bar graph in F shows the results of FITC-dextran permeability analysis of vascular endothelial cells in mice with different treatments (**: P < 0.01, n = 4);

[0023] Figure 2 Schematic diagram of the effect of the OSM recombinant protein of the present invention on the expression of endothelial cadherin in mice and humans;

[0024] The bar graph in A shows the expression of Cdh5 mRNA in C166 cells after treatment with different concentrations of mOSM for different time periods detected by RT-qPCR;

[0025] B is the expression of VE-cadherin protein observed by fluorescence confocal microscopy after C166 cells were treated with 20 ng / mL mOSM for 12 hours. VE-cadherin was labeled with red fluorescence, and the cell nucleus was stained blue with DAPI. Scale bar: 5 μm.

[0026] The bar graph in C shows the expression of CDH5 mRNA in HULEC-5A after treatment with different concentrations of hOSM for different time periods as detected by RT-qPCR;

[0027] D is the expression of VE-cadherin protein observed by fluorescence confocal microscopy after HULEC-5A cells were treated with 20 ng / mL hOSM for 12 hours. VE-cadherin was labeled with green fluorescence, and the cell nucleus was stained blue with DAPI. Scale bar: 5 μm.

[0028] Figure 3 Schematic diagram of OSM promoting endothelial barrier damage;

[0029] A is an immunoblot showing the expression of OSMR, VE-cadherin and internal control β-actin proteins in HULEC-5A treated with different methods;

[0030] The bar graph in B shows the quantitative analysis of immunoblot bands (*: P < 0.05, **: P < 0.01, n = 3);

[0031] C is the expression of VE-cadherin protein in HULEC-5A cells treated with different methods observed by laser confocal microscopy. VE-cadherin is labeled in green, and the cell nucleus is labeled in blue by DAPI. Scale bar: 5 μm.

[0032] The bar graph in D shows the results of FITC-dextran relative permeability analysis of HULEC-5A cells treated with different methods (**: P < 0.01; ***: P < 0.001, n = 3);

[0033] The line graph in E shows the changing trend of transmembrane electrical impedance values ​​of different endothelial cells over time (**: P < 0.01, n = 3). DETAILED DESCRIPTION

[0034] A method for constructing a pulmonary vascular endothelial barrier injury model comprises the following steps: diluting OSM recombinant protein to 20 ng / μL with PBS, and instilling 50 μL of the diluted OSM recombinant protein into mice via the airway for 24 hours.

[0035] Mice were killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for frozen sections for use in later experiments.

[0036] Oncostatin-M (OSM) is a member of the IL-6 family of cytokines. It was first isolated from tissue lymphoma cells in 1986 and was named because of its ability to inhibit the proliferation of melanoma cells. OSM is mainly synthesized and secreted by multiple cells such as activated macrophages, neutrophils, T cells and mast cells. It has multiple biological activities and plays a wide range of roles in hematopoiesis, cell growth and differentiation, inflammatory response, metabolic regulation, tumor formation and immune regulation. OSM interacts with two independent complexes for cell signal transduction, namely the type I LIFRβ / gp130 receptor complex and the type II OSMRβ / gp130 receptor complex. After OSM binds to the type II OSMR complex, multiple signaling pathways are activated, including Janus-activated kinase / signal transducer and promoter of transcription 3 (JAK / STAT3), mitogen-activated protein kinase / extracellular-regulated kinase (MAPK / ERK), c-Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase / protein kinase B (PI3K / AKT) pathways. The OSM receptor (OSMR) is mainly expressed by mesenchymal cells such as fibroblasts, endothelial cells, osteoblasts and epithelial cells, as well as some cancer cells.

[0037] The present invention uses OSM recombinant protein as a key factor in constructing a pulmonary vascular endothelial barrier injury model. OSM can mainly damage the pulmonary vascular endothelium rather than the endothelial cells of other systemic diseases. It has low toxicity to tissue cells, tissue compatibility, a clear mechanism of action and repeatability. The protein preparation process is simple and cost-controlled, making it easier for more laboratories to carry out research and improving resource utilization efficiency.

[0038] Example 1 Verification of the effect of OSM recombinant protein on vascular endothelium in a mouse lung infection disease model

[0039] 1. Experimental materials:

[0040] Mouse OSM recombinant protein (495-MO-025, R&D Systems, USA), OSM antibody (NBP3-16686, Novus, USA), vascular endothelial cadherin antibody (VE-cadherin, Cell Signaling, USA), mouse OSM neutralizing antibody (AF-495-NA, R&D Systems, USA), rat (G3A1) IgG antibody (5415, CST, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen, USA), DAPI (4′6-diamidino-2-phenylindole, Thermo Fisher Scientific, USA), dextran fluorescein, 10,000 MW (D1821, Invitrogen, USA), FV3000 laser confocal microscope (OLYMPUS, Japan), Agilent Cary 60 UV spectrophotometer (Agilent, USA), Innova® 40 / 40R benchtop orbital shaker (Newton, USA) Brunswick), a Leica CM1860 cryostat (Leica Biosystems, Germany). PAO1 strains were provided by the Chengdu Institute of Respiratory Health. 6- to 8-week-old C57BL / 6J male mice were purchased from Chongqing Tengxin Biotechnology Co., Ltd. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Southwest Jiaotong University.

[0041] 2. Mouse Lung Infection Model

[0042] Healthy C57BJ / 6J mice were randomly divided into the following groups and treated accordingly.

[0043] In the PA group, mice were fed adaptively for 1 week and then anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). Each mouse was given 30 μL PAO1 (3 × 10 7 CFU) to induce lung infection. After 24 hours, the mice were killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for subsequent experiments.

[0044] In the PBS group, mice were fed adaptively for 1 week and then anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). Each mouse was given 30 μL PBS intranasally. 24 hours later, the mice were killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for subsequent experiments.

[0045] In the OSM group, mice were fed adaptively for one week and then anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). OSM recombinant protein was diluted to 20 ng / μL with PBS. 50 μL of the diluted OSM recombinant protein was injected into the mice via the airway using a microinjector for 24 hours. After 24 hours, the mice were killed by cervical dislocation under CO2 anesthesia, and lung tissue was collected for subsequent experiments.

[0046] In the Ctrl group, mice were fed adaptively for 1 week and then anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg). 50 μL of PBS was instilled into the airway of the mice for 24 hours. The mice were then killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for subsequent experiments.

[0047] In the anti-OSM+PA group, after one week of adaptive feeding, the mice were intraperitoneally injected with 100 μL of 2 μg / μL (200 μg / mouse) of OSM neutralizing antibody, anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg), and then each mouse was given 30 μL of PAO1 (3×10 7 CFU) for 24 hours to induce lung infection. After 24 hours, the mice were killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for subsequent experiments.

[0048] In the IgG+PA group, after one week of adaptive feeding, mice were intraperitoneally injected with 100 μL of 2 μg / μL (200 μg / mouse) IgG control antibody, anesthetized with ketamine (80 mg / kg) and xylazine (10 mg / kg), and then each mouse was given 30 μL of PAO1 (3×10 7 CFU) for 24 hours to induce lung infection. After 24 hours, the mice were killed by cervical dislocation under CO2 anesthesia, and lung tissues were collected for subsequent experiments.

[0049] In the IgG group, after one week of adaptive feeding, the mice were intraperitoneally injected with 100 μL, 2 μg / μL (200 μg / mouse) of IgG control antibody. 24 hours later, the mice were killed by cervical dislocation under CO2 anesthesia, and the lung tissues were collected for subsequent experiments.

[0050] Compared to some chemicals (such as H2O2, MCT, and rotenone), OSM recombinant protein, as a natural cytokine, has low cytotoxicity and good biocompatibility. Using OSM recombinant protein in experiments can reduce direct toxic damage to cells, improve experimental safety, and better simulate the in vivo physiological environment, reducing nonspecific reactions caused by exogenous chemicals. The OSM recombinant protein-induced endothelial barrier injury model is highly reproducible across laboratories, helping to ensure the reliability and consistency of experimental results and enhance research accuracy. OSM recombinant protein induces endothelial barrier injury by binding to OSMR and activating specific signaling pathways (such as JAK / STAT3 and MAPK / ERK). This well-defined mechanism of action makes experimental results more interpretable and more accurately reflects the pathophysiological process of endothelial barrier injury. At the same time, OSM recombinant protein can synergize with other cytokines (such as IL-6, TNF-α, etc.) to enhance the effect of endothelial barrier damage. This synergistic effect provides a new perspective for studying endothelial barrier damage under the combined action of multiple factors. Finally, the preparation of OSM recombinant protein is relatively simple and low-cost. The use of OSM recombinant protein does not require complex equipment or technology, which reduces the resource requirements of the experiment. Compared with some complex gene editing technologies or expensive chemical reagents, using OSM recombinant protein to construct models is more cost-effective.

[0051] 3. Experimental Process and Results

[0052] (1) Enzyme-linked immunosorbent assay (ELISA) detection: The left lung tissue was rinsed with cold PBS, minced with scissors, placed in a grinding tube for grinding, and then ultrasonically disrupted with an ultrasonic cell disruptor. The supernatant was collected as the lung tissue sample after centrifugation at 4°C and 12,000 g for 15 minutes. The OSM concentration in the lung tissue was determined using an ELISA kit provided by a commercial supplier according to the manufacturer's instructions.

[0053] (2) Multiple immunofluorescence staining of lung tissue: Mouse lung tissue was immersed in 4% paraformaldehyde for 48 hours, then immersed in 30% sucrose solution for dehydration for 24 hours, embedded in OCT and frozen for 24 hours, and then frozen sectioned with a thickness of 5µm. Subsequently, the tissue sections were permeabilized with 0.05% Tween20 for 10 minutes, incubated with 10% goat serum at room temperature for 1 hour to block nonspecific antibodies, and then incubated with antibodies (VE-cadherin rabbit monoclonal antibody, 1:400 dilution; OSM mouse monoclonal antibody, 1:200 dilution) at 4˚C overnight. Goat anti-rabbit Alexa Fluor™ 594 antibody (1:1000 dilution) was combined with the primary antibody at room temperature and incubated for 2 hours. The cell nuclei were stained with DAPI, and then the sections were sealed with anti-fluorescence quencher. The sections were observed under a laser confocal microscope and photographed.

[0054] (3) In vivo vascular permeation experiment: The dosage of FITC-dextran for each mouse was calculated based on 10 mg / kg body weight. After anesthesia, the mice in the IgG group, IgG+PA group, anti-OSM+PA group, Ctrl group, and OSM group were allowed to slowly inhale 25 μL of FITC-dextran (10,000 MW) through each nostril and kept in a vertical position for 2 minutes until their breathing stabilized. The mice were killed 1 hour later. The mice were disinfected with 70% ethanol, and the chest cavity was opened with sterile forceps and surgical scissors to expose the apex of the heart. A slight negative pressure was first applied to the piston of the 1 mL syringe, and a 22-gauge needle was inserted into the apex of the heart. When blood appeared in the syringe, the piston was gradually withdrawn to collect the blood. The collected blood was injected into a syringe pre-filled with 10 μL of EDTA (60 The cells were placed in an EP tube moistened with 5% paraformaldehyde (500 mg / mL) and centrifuged at 550 g for 10 min. The supernatant was collected and the fluorescence OD value of plasma FITC-dextran was detected using a multi-function microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0055] Lung infections caused by Pseudomonas aeruginosa (PA) infection can progress to ARDS without effective treatment. The important pathological changes of ARDS are pulmonary edema and the formation of hyaline membranes. Both of these pathological changes are related to endothelial barrier damage and increased cell permeability. After PA infects the lungs, immune cells secrete a large amount of cytokine OSM. After OSM binds to OSMR on endothelial cells, it destroys the tight junctions of endothelial cells, promotes cell permeability, and leads to damage to the pulmonary vascular endothelial barrier. After PA infection, OSM recombinant protein binds to type II OSM receptors on endothelial cells, activates downstream signaling pathways, and causes endothelial damage. This specific mechanism of action provides a clear target for studying endothelial barrier damage.

[0056] Test results such as Figure 1 As shown, Figure 1 In A and 1B, the red fluorescence of OSM recombinant protein in the lung tissue of the PA group was significantly increased compared with that in the PBS group, and the OSM recombinant protein in the lung tissue homogenate of the PA group was significantly increased compared with that in the PBS group (P<0.01).

[0057] Mouse OSM recombinant protein was instilled into the lungs of healthy mice. After 24 hours of treatment, the expression of adhesion protein (VE-cadherin) was detected to reflect the continuity of the endothelium and the permeability of the endothelium, thereby detecting the damage of the endothelial barrier function and reflecting the success of the model. The results are as follows Figure 1 C shows that compared with the Ctrl group, the expression of VE-cadherin in the lung tissue of mice in the OSM group was reduced and the endothelial integrity was destroyed.

[0058] Detection of FITC-dextran labeled FITC in plasma to reflect the permeability changes of vascular endothelium, the results are as follows Figure 1 As shown in D, compared with the Ctrl group, the FITC fluorescence in the plasma of mice in the OSM group increased (P < 0.001), indicating that the vascular endothelial permeability was increased.

[0059] OSM antibodies were used to neutralize the endogenous OSM produced after PA infection, and then the integrity of the vascular endothelium was detected. The results were as follows Figure 1 As shown in E and 1F, compared with the PA group, the expression of VE-cadherin in the lung tissue of mice in the anti-OSM+PA group was increased, the endothelium remained intact, the FITC fluorescence in the mouse plasma was reduced (P<0.01), and the vascular endothelial permeability was reduced.

[0060] In summary, after PA infection, OSM in lung tissue increased. After nasal inhalation of 1 μg of OSM recombinant protein into the mouse lungs for 24 hours, it led to increased pulmonary vascular permeability and damaged endothelial barrier in mice.

[0061] Example 2 OSM recombinant protein reduces the expression of VE-cadherin in mouse and human endothelial cells

[0062] 1. Experimental materials:

[0063] Recombinant human oncostatin M protein (295-OM-050, R&D Systems, USA), recombinant mouse OSM protein (495-MO-025, R&D Systems, USA), mouse vascular endothelial cells C166 and human pulmonary microvascular endothelial cells (HULEC-5A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCDB131 medium (10372019, Gibco, USA), epidermal growth factor (AF-100-15, PeproTech, USA), L-glutamine (A2916801, Gibco, USA), hydrocortisone (HY-N0583R, MCE, USA), Trizol reagent (Invitrogen, USA), reverse transcription kit (Takara Bio, Japan), real-time fluorescence quantitative polymerase chain reaction kit (RT-qPCR, Applied Biosystems, USA), vascular endothelial cadherin (VE-cadherin, cell line 1000, USA) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Signaling Company, USA), goat anti-rabbit Alexa Fluor™ 594 antibody (Invitrogen) and goat anti-rabbit Alexa Fluor™ 488 antibody (Invitrogen, USA), DAPI (4′6-diamidino-2-phenylindole, Thermo Fisher Scientific), laser confocal microscopy (OLYMPUS, Japan), and CFX Connect™ fluorescence quantitative PCR detection system (Bio-Rad, USA).

[0064] 2. Experimental Process and Results

[0065] (1) Cell culture: C166 cells were grown adherently in high-glucose DMEM containing 10% FBS and 1% streptomycin / penicillin antibiotics and stably subcultured in a 5% CO2, 37°C constant temperature and humidity cell culture incubator. 2.0×10 cells were cultured per well. 5 Cells were evenly seeded in 6-well plates and treated with different concentrations of mOSM (mouse OSM recombinant protein) (10 ng / mL, 20 ng / mL), and then the cells were collected for subsequent experiments.

[0066] HULEC-5A cells were grown adherently in MCDB131 medium containing 10% FBS, 10 ng / mL EGF, 1 μg / mL hydrocortisone, 10 mM L-glutamine, and 1% streptomycin / penicillin antibiotics and stably subcultured in a 5% CO2, 37°C constant temperature and humidity cell culture incubator. 2.5 × 10 cells were cultured per well. 5Cells were evenly seeded in 6-well plates and treated with different concentrations of hOSM (human OSM recombinant protein) (10 ng / mL, 20 ng / mL, 30 ng / mL), and then the cells were collected for subsequent experiments.

[0067] The cells in the Ctrl group were treated with an equal volume of PBS for the same time, and the cells were collected for subsequent experiments.

[0068] (2) RT-qPCR experiment: Cells were collected, total RNA was extracted with Trizol, and complementary DNA (cDNA) was synthesized according to the instructions of the TAKARA reverse transcription kit. RT-qPCR was performed using cDNA as a template. The reaction conditions were: 95°C for 30 s, followed by denaturation at 95°C for 5 s, and annealing at 60°C for 34 s, for a total of 40 cycles. β-actin was used as an internal reference. The relative expression of CDH5 was calculated using the 2–ΔΔCt formula. The primer sequences are as follows:

[0069] Mouse β-actin upstream 5′-GGCTGTATTCCCCTCCATCG-3′, mouse downstream 5′-CCAGTTGGTAACAATGCCATGT-3′;

[0070] Human β-actin upstream 5′-AAATCTGGCACCACACCTTC-3′, downstream 5′-GGGGTGTTGAAGGTCTCAAA-3′;

[0071] Human CDH5 upstream 5'-TTGGAACCAGATGCACATTGAT-3', downstream 5'-TCTTGCGACTCACGCTTGAC-3'.

[0072] (3) Cell immunofluorescence staining: 10×10 4 HULEC-5A / C166 cells were evenly seeded in 24-well plates at 4 °C / well. After infection, the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.05% tween 20 for 10 minutes, and blocked with 10% goat serum at room temperature for 1 hour. The cells were then incubated with antibody (rabbit monoclonal antibody to VE-cadherin, 1:300 dilution) overnight at 4 °C. Subsequently, they were incubated with goat anti-rabbit Alexa Fluor™ 594 antibody or goat anti-rabbit Alexa Fluor™ 488 antibody (1:1000 dilution) at room temperature for 2 hours. Cell nuclei were stained with DAPI, and the sections were mounted with anti-fluorescence quencher. The sections were observed using a laser confocal microscope and photographed.

[0073] Endothelial cells of different species, including mouse vascular endothelial cells C166 and human pulmonary microvascular endothelial cells (HULEC-5A), were selected and treated with different OSM concentrations for different times. VE-cadherin (protein encoded by the CDH5 gene) is the core of controlling the opening, closing and permeability changes of the endothelial barrier. RT-qPCR and immunofluorescence were used to detect the expression of CDH5 mRNA and VE-cadherin protein on the two cell lines. C166 cells were treated with different concentrations of mOSM (10 ng / mL, 20 ng / mL) for 12 h and 24 h.

[0074] Combine Figure 2 RT-qPCR was used to detect the expression of Cdh5 mRNA. Figure 2 As shown in A, compared with the Ctrl group, there was no significant change in Cdh5 mRNA expression after 12 and 24 h of treatment with 10 ng / mL mOSM; after 12 h of treatment with 20 ng / mL mOSM, Cdh5 mRNA expression was significantly decreased (P<0.05), while after 24 h of treatment, Cdh5 mRNA expression was not statistically significant.

[0075] Immunofluorescence detection of VE-cadherin protein expression Figure 2 As shown in B, compared with the Ctrl group, after C166 cells were treated with 20 ng / mL mOSM for 12 hours, the VE-cadherin fluorescence was significantly weakened and its integrity was destroyed.

[0076] After HULEC-5A cells were treated with different concentrations of hOSM (10 ng / mL, 20 ng / mL, 30 ng / mL) for 12 h and 24 h, the expression of CDH5 mRNA was detected by RT-qPCR. Figure 2 As shown in C, compared with the Ctrl group, there was no significant change in CDH5 mRNA expression after 12 h of treatment with 10 ng / mL hOSM, but the expression of CDH5 mRNA was significantly decreased after 12 h of treatment with 20 ng / mL hOSM (P<0.05), and the expression of CDH5 mRNA was significantly decreased after 12 h of treatment with 30 ng / mL hOSM (P<0.05). Compared with the Ctrl group, there was no statistically significant change in CDH5 mRNA expression after 24 h of treatment of HULEC-5A cells with three different concentrations of hOSM.

[0077] Immunofluorescence was used to detect VE-cadherin protein expression. Figure 2 As shown in D, compared with the Ctrl group, VE-cadherin fluorescence was significantly weakened after HULEC-5A cells were treated with 20 ng / mL hOSM for 12 h, and endothelial integrity was destroyed.

[0078] In summary, treatment of C166 cells with mouse OSM recombinant protein at a concentration of 20 ng / mL for 12 hours can lead to a decrease in the expression of cellular VE-cadherin; treatment of HULEC-5A cells with human OSM recombinant protein at a concentration of 20 ng / mL for 12 hours can lead to a decrease in the expression of cellular VE-cadherin. OSM recombinant protein reduces the expression of VE-cadherin in mouse and human endothelial cells.

[0079] Example 3 OSM promotes vascular endothelial barrier damage via OSMR

[0080] 1. Experimental materials:

[0081] Recombinant human oncostatin M protein (295-OM-050, R&D Systems, USA) and human pulmonary microvascular endothelial cells (HULEC-5A) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCDB131 medium (10372019, Gibco, USA) was purchased from Gibco, USA. Epidermal growth factor (AF-100-15, PeproTech, USA) was purchased from Gibco, USA. L-glutamine (A2916801, Gibco, USA) was purchased from Gibco, USA. Hydrocortisone (HY-N0583R, MCE, USA) was purchased from MCE, USA. Lipofectamine RNAiMAX transfection reagent was purchased from Invitrogen, USA. Opti-MEM (31985070, Gibco, USA) was purchased from Gibco, USA. VE-cadherin rabbit polyclonal antibody was purchased from Invitrogen, USA. OSMRβ mouse monoclonal antibody was purchased from Santa Clara, CA, USA. Cruz Company, USA), β-actin mouse polyclonal antibody (sc-47778, Santa Cruz Company, USA), HRP-conjugated goat anti-rabbit antibody (7074, Cell Signaling, USA), HRP-conjugated goat anti-mouse antibody (7076, Cell Signaling, USA), goat anti-rabbit Alexa Fluor™ 488 antibody (Invitrogen, USA), DAPI (4′6-diamidino-2-phenylindole, Thermo Fisher, USA), RIPA lysis buffer, protease phosphatase inhibitors, Triton X 100, 4% paraformaldehyde, sodium dodecylbenzenesulfonate, N,N-methylenebisacrylamide, skim milk powder, etc., are all products of Solebol; polyvinylidene fluoride (PVDF) membrane is a product of Merck; ECL luminescent solution is purchased from Millipore, USA; PageRuler™ prestained protein molecular weight marker (26617, Thermo Fisher, USA), Pierce™ BCA protein detection kit (23227, Thermo Fisher, USA) Fisher Company), dextran fluorescein, 3000MW (D3305, Invitrogen Company, USA), laser confocal microscope (OLYMPUS Company, Japan), Bio-Rad vertical electrophoresis apparatus (Bio-Rad Company, USA), electronic pressing imaging system, products of e-blot Company, CellZscope fully automatic cell transmembrane resistance measuring instrument (NanoAnalytics Company, Germany).

[0082] 2. Experimental Process and Results

[0083] (1) Cell culture and transfection: The HULEC-5A cell culture method and the reagents used were the same as in Example 2, with 2.5×10 cells per well. 5 HULEC-5A cells were evenly seeded in 6-well plates. After 12 hours, according to the instructions of the RNAiMAX transfection reagent, 30 pmol of siRNA was mixed and diluted with 150 μL of opti-MEM reduced serum medium and incubated at room temperature for 5 minutes. The RNAiMAX transfection reagent was diluted with 150 μL of opti-MEM reduced serum medium and incubated at room temperature for 3 minutes. Subsequently, the two opti-MEM containing OSMR siRNA and transfection reagent were gently mixed and incubated at room temperature for 15 minutes. Then, the two were carefully added to the cell culture medium. After culturing the cells for 6 hours, the culture medium was replaced with normal culture medium. Subsequent experiments were performed 24 hours after transfection.

[0084] The siRNA target sequence of OSMR is: GAAGAGAAGUGUCAAUAUCUU. The negative control group was transfected with Scrambled siRNA and was recorded as the Scramble group.

[0085] Cell immunofluorescence staining: 1.0×10 5 HULEC-5A cells were evenly seeded on 24-well laminin-coated slides. After OSM treatment, the cells were rinsed with pre-chilled PBS to remove excess culture medium, fixed with 4% paraformaldehyde for 15 minutes, rinsed three times with PBS, permeabilized with 0.05% tween20 for 10 minutes, rinsed twice with PBS, and incubated with 10% goat serum at 37°C for 1 hour. VE-cadherin rabbit polyclonal antibody was diluted 1:200 and incubated with cells overnight at 4°C. Alexa Fluor™ 488-labeled goat anti-rabbit antibody was diluted 1:1000 and incubated with cells at room temperature for 2 hours. Cell nuclei were stained with DAPI and incubated in the dark at room temperature for 10 minutes. The cells were then rinsed three times with PBS and 10 Add 1 μL of anti-fluorescence quencher to the center of the front of the adhesive slide, carefully peel the cell slide from the 24-well plate with ophthalmic tweezers, and stick the cell side to the anti-fade agent on the adhesive slide, trying to avoid bubbles. Then apply nail polish along the slide to prevent sliding, observe under a laser confocal microscope, and take pictures for record.

[0086] Immunoblotting: After treatment, cells were fully lysed in RIPA lysis buffer containing protein phosphatase inhibitors for 30 minutes. After measuring the protein concentration using a BCA protein assay kit, the same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 hours and then incubated with VE-cadherin (1:1000 dilution), OSMRβ (1:1000 dilution), and β-actin (1:10000 dilution) at 4°C overnight. After washing three times with TBST, the membrane was incubated with the corresponding HRP-labeled goat anti-mouse / rabbit antibodies at room temperature for 2 hours and scanned using an eBlot Touch imager. The immune bands were analyzed using Image J software.

[0087] In vitro cell permeability assay: 2×10 5 HULEC-5A cells were evenly seeded in the upper transwell chamber of a 24-well plate (3.0 μm pore size). Twelve wells were randomly divided into four groups: Scramble, OSM, siOSMR, and siOSMR+OSM, with three wells in each group. Cells in the Scramble and OSM groups were transfected with negative control siRNA, while cells in the siOSMR and siOSMR+OSM groups were transfected with OSMR siRNA. The cells were cultured at 37°C in a constant temperature and humidity until the cells became confluent monolayers. The monolayer endothelial cells in the OSM and siOSMR+OSM groups were treated with 30 ng / mL hOSM for 12 h. Subsequently, 100 μL of PBS containing FITC-dextran (1 ug / mL) was added to each upper chamber, and 500 μL of PBS without FITC-dextran (3000 ng / mL) was added to each lower chamber. After incubation at 37°C for 5 minutes, the fluorescence intensity of FITC-dextran transferred to the lower chamber was measured using a multifunctional microplate reader. The excitation wavelength and emission wavelength were 494 nm and 521 nm, respectively.

[0088] Cell transmembrane electrical impedance detection: 12×10 4HULEC-5A cells were evenly seeded in the upper transwell chamber (3.0 μm pore size) of a 24-well plate. The 15 wells were randomly divided into three groups: scramble group, OSM group, and siOSMR+OSM group, with 3 wells in each group. Cells in the scramble and OSM groups were transfected with negative control siRNA; cells in the siOSMR+OSM group were transfected with OSMR siRNA. After transfection, the upper chamber was transferred to a cell transmembrane electrical resistance measuring instrument and incubated at 37°C in a constant temperature and humidity until the cells were confluent and formed a monolayer. The monolayer endothelial cells in the OSM and siOSMR+OSM groups were treated with 30 ng / mL hOSM for 12 h, and the transcellular membrane electrical resistance of the cells in each well was measured in real time.

[0089] The results are as follows Figure 3 As shown, Figure 3 The immunoblotting results in A showed that OSMR expression increased significantly after OSM treatment of HULEC-5A. To explore whether OSM damages endothelial cells through OSMR signaling, OSMR in HULEC-5A was knocked down by siRNA. The immunoblotting results ( Figure 3 A, 3B) showed that compared with the Scramble group, the VE-cadherin protein expression in the OSM group was decreased, and the quantitative analysis results showed P=0.0098, which was a statistically significant difference. Compared with the OSM group, the VE-cadherin protein expression in the siOSMR+OSM group was increased, and the protein quantitative results showed P=0.0428, which was a statistically significant difference.

[0090] Immunofluorescence results Figure 3 As shown in C, compared with the scramble group, the VE-cadherin fluorescence of HULEC-5A cells in the OSM group was weakened and the continuity was destroyed. Compared with the OSM group, the VE-cadherin fluorescence of cells in the siOSMR+OSM group was enhanced and the continuity was restored. The in vitro cell FITC-dextran permeability test can reflect the permeability of the endothelium through the permeability of FITC-dextran in monolayer endothelial cells.

[0091] The cell permeability results are as follows Figure 3 As shown in D, compared with the Scramble group, the relative permeability of the cells in the OSM group was increased (P=0.0005), and compared with the OSM group, the permeability of the cells in the siOSMR+OSM group was decreased (P=0.0057).

[0092] The results of the endothelial cell transmembrane electrical impedance test are as follows Figure 3As shown in E, compared with the Scramble group, the transcellular membrane resistance of the OSM group was significantly decreased (P<0.01). Compared with the OSM group, the transcellular membrane resistance of the cells in the siOSMR+OSM group was increased (P<0.01), indicating that the integrity of the endothelial barrier was restored.

[0093] Therefore, the above results indicate that OSM promotes the reduction of intercellular adhesion protein expression and the increase of endothelial permeability through OSMR on endothelial cells, leading to endothelial barrier destruction.

[0094] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the claims of this patent application.

Claims

1. A method for constructing a pulmonary vascular endothelial barrier injury model, characterized in that: Healthy mice were fed adaptively for one week and then anesthetized with drugs. The oncostatin M recombinant protein was pretreated and then instilled into the mouse lungs through the airway to obtain a pulmonary vascular endothelial barrier injury model.

2. The construction method according to claim 1, characterized in that Ketamine and xylazine were used for anesthesia, with the mass ratio of ketamine being 80 mg / kg and the mass ratio of xylazine being 10 mg / kg.

3. The construction method according to claim 1, characterized in that The pretreatment of the oncostatin M recombinant protein is to dilute the oncostatin M recombinant protein to 10-50 ng / μL with PBS, and the mice are treated with the oncostatin M recombinant protein for 12-24 hours.

4. The construction method according to claim 3, characterized in that The pretreatment of oncostatin M recombinant protein was performed by diluting the oncostatin M recombinant protein to 20 ng / μL with PBS, and the mice were treated with the oncostatin M recombinant protein for 24 hours.

5. Use of a pulmonary vascular endothelial barrier injury model in preparing a pulmonary vascular disease research model, characterized in that: The preparation steps are as follows: healthy mice are adaptively fed for one week and then anesthetized with drugs, and the oncostatin M recombinant protein is pretreated and then instilled into the mouse lungs through the airway to obtain a pulmonary vascular endothelial barrier damage model.

6. Use of a pulmonary vascular endothelial barrier injury model in the preparation of a drug for treating pulmonary vascular diseases, characterized in that: The preparation steps are as follows: healthy mice are adaptively fed for one week and then anesthetized with drugs, and the oncostatin M recombinant protein is pretreated and then instilled into the mouse lungs through the airway to obtain a pulmonary vascular endothelial barrier damage model.

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