Reagents for detecting eosinophils, eosinophils and their cytoplasmic contents, and application of hydroxydocosahexaenoic acid

Abstract

The present invention relates to a reagent for detecting eosinophils, and the application of eosinophils and their cytoplasmic contents, as well as hydroxydocosahexaenoic acid, a lipid metabolic product, and belongs to the medical and pharmaceutical technical fields. The present invention provides an application of the reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary arterial hypertension and / or determining the prognostic risk of pulmonary arterial hypertension. By detecting the EOS ratio in peripheral blood, it is possible to diagnose pulmonary arterial hypertension and determine the prognostic risk of pulmonary arterial hypertension.

Description

[Technical field]

[0001] The present invention relates to the medical and pharmaceutical technical fields, and more particularly to a reagent for detecting eosinophils, and the application of eosinophils and their cytoplasmic contents and lipid metabolite hydroxydocosahexaenoic acid. [Background technology]

[0002] Pulmonary arterial hypertension (PH) is a group of pulmonary circulation diseases in which mean pulmonary artery pressure increases progressively due to various causes and onset mechanisms, and is currently defined as a mean pulmonary artery pressure of 25 mmHg or more measured by right heart catheterization at sea level while at rest. During the progression of pulmonary arterial hypertension, dysfunction of pulmonary vascular resident cells and abnormal infiltration of various immune cells cause pulmonary vascular constriction and pulmonary vascular remodeling, leading to pulmonary vascular thickening, stenosis, and increased pulmonary artery pressure, ultimately progressing to right heart failure and right heart dysfunction.

[0003] Currently, targeted drugs used in clinical practice mainly reduce pulmonary vascular resistance by dilating blood vessels, thus alleviating pulmonary arterial hypertension, but they cannot fundamentally reverse vascular remodeling. Therefore, it is still urgent to screen drugs that can effectively improve vascular remodeling and be put into clinical use as soon as possible for the treatment of pulmonary arterial hypertension. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a reagent for detecting eosinophils (EOS), and the application of eosinophils and their cytoplasmic contents, as well as hydroxydocosahexaenoic acid (HDHA), a lipid metabolic product. The present invention can utilize the reagent for detecting eosinophils to realize the diagnosis of pulmonary arterial hypertension and / or the assessment of the prognostic risk of pulmonary arterial hypertension.

[0005] The present invention provides an application of a reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary arterial hypertension and / or determining the prognostic risk of pulmonary arterial hypertension.

[0006] Preferably, the detecting comprises detecting the proportion of eosinophils in peripheral blood.

[0007] The present invention also provides an application of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for inhibiting proliferation and / or migration of pulmonary artery smooth muscle.

[0008] The present invention also provides the application of EOS and / or EOS cytoplasmic content in the preparation of a medicament for maintaining homeostasis of pulmonary artery smooth muscle.

[0009] The present invention also provides the application of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for treating pulmonary arterial hypertension.

[0010] The present invention also provides an application of hydroxydocosahexaenoic acid in preparing a medicament for treating pulmonary arterial hypertension.

[0011] The present invention also provides an application of hydroxydocosahexaenoic acid in preparing a medicament for inhibiting proliferation of pulmonary artery smooth muscle.

[0012] Preferably, the hydroxydocosahexaenoic acid includes 14-hydroxydocosahexaenoic acid and / or 17-hydroxydocosahexaenoic acid. Effect of the Invention

[0013] The present invention provides the application of eosinophil detection reagents in the preparation of products for the diagnosis of pulmonary arterial hypertension and / or the assessment of the prognosis risk of pulmonary arterial hypertension. The present invention has found that EOS is closely related to the occurrence of pulmonary arterial hypertension, and therefore proposes to use EOS as a biomarker to represent the status of PH pathology and to apply it to the preparation of related products for diagnosing PH. A decrease in the EOS rate in peripheral blood blood count indicates the risk of pulmonary arterial hypertension and poor prognosis of pulmonary arterial hypertension. The application of the present invention can realize the diagnosis of pulmonary arterial hypertension and the assessment of the prognosis risk of pulmonary arterial hypertension.

[0014] The study results showed that with the progression of pulmonary arterial hypertension, the proportion of EOS in the peripheral blood of mice decreased, the infiltration of EOS into lung tissue increased, and EOS had a protective effect against pulmonary arterial hypertension; the contents of EOS can inhibit the proliferation and / or migration of pulmonary arterial smooth muscle cells, which are the most important endogenous cells during the process of pulmonary vascular remodeling, and the cytoplasmic contents of EOS play a protective role in maintaining the homeostasis of pulmonary arterial smooth muscle. EOS and its cytoplasmic contents have a therapeutic effect on pulmonary arterial hypertension. [Brief description of the drawings]

[0015] In order to more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings from these drawings without creative efforts. [Figure 1] Schematic diagrams of experimental results of the EOS ratio in peripheral blood of patients with pulmonary arterial hypertension and its correlation with disease severity provided by the present invention; where A is a diagram showing the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and healthy subjects; B is a diagram showing the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and NYHA cardiac function class; C is a diagram showing the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and BMPR2 mutation; [Diagram 2]Schematic diagrams of experimental results of EOS percentage in an animal model of pulmonary arterial hypertension provided by the present invention; where A is a typical diagram of EOS percentage in peripheral blood and flow cytometry in a mouse model of pulmonary arterial hypertension induced by a combination of hypoxia and Sugen; B is a typical diagram of EOS percentage in lung tissue and flow cytometry in a mouse model of pulmonary arterial hypertension induced by a combination of hypoxia and Sugen; [Diagram 3] Schematic diagram of the experimental results that EOS knockout provided by the present invention aggravates pulmonary arterial hypertension induced by a combination of hypoxia and Sugen, where A is a result diagram of right ventricular systolic pressure of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; B is a result diagram of right heart hypertrophy index of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; C is a typical schematic diagram of pulmonary vascular anti-α-smooth muscle actin immunofluorescence staining of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; wild-type mice are the experimental control group; [Figure 4] FIG. 2 is a schematic diagram showing the experimental results of EOS provided by the present invention suppressing the abnormal function of pulmonary artery smooth muscle cells, in which A is a diagram showing the results of the proliferation of pulmonary artery smooth muscle cells after induction of growth factor PDGFbb and administration of conditioned medium to EOS; B is a diagram showing the results of the migration of pulmonary artery smooth muscle cells after induction of growth factor PDGFbb and administration of conditioned medium to EOS; [Diagram 5] Schematic diagram of the experimental results of the effect of EOS knockout provided by the present invention on fatty acid metabolism in mouse lung tissue induced by a combination of hypoxia and Sugen; where A is the principal component analysis result of the targeted metabolomics of EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension; B is the heat map of the differential expression of lipid metabolites in EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension; C is the quantitative analysis result of lipid metabolites in EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension; wild-type mice are the experimental control group; [Figure 6]FIG. 2 is a schematic diagram showing the experimental results of the inhibitory effect of 14-HDHA and 17-HDHA on the proliferation of pulmonary artery smooth muscle cells provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention provides the application of an eosinophil detection reagent in the preparation of a product for diagnosing pulmonary arterial hypertension and / or determining the prognostic risk of pulmonary arterial hypertension.

[0017] In the present invention, eosinophils can be used as a biomarker for preparing a product used for identification and / or auxiliary diagnosis of pulmonary arterial hypertension. In the present invention, said detection preferably comprises detecting the proportion of eosinophils in peripheral blood. In the present invention, said product preferably comprises a kit.

[0018] In the present invention, the detection sample preferably includes peripheral blood. When the EOS ratio in the peripheral blood count test is reduced compared to a healthy control, it indicates the risk of pulmonary arterial hypertension and / or the risk of poor prognosis of pulmonary arterial hypertension. In the present invention, the reduction ratio is preferably 25% or more, more preferably 30% or more. As shown in the clinical trial data of the present invention, the average value of the male normal control was 2.668, the average value of the male patient was 1.78, a decrease of 33%, and the average value of the female normal control was 2.024, the average value of the female patient was 1.404, a decrease of 30%.

[0019] In the present invention, the pulmonary arterial hypertension preferably includes pulmonary arterial hypertension induced by a combination of hypoxia and Sugen.

[0020] The results of the embodiment of the present invention show that the EOS ratio in peripheral blood is reduced in patients with pulmonary arterial hypertension, and the lower the EOS ratio in peripheral blood, the more serious the condition of patients with pulmonary arterial hypertension, and EOS can assist in the diagnosis of pulmonary arterial hypertension. The EOS ratio in peripheral blood can also indicate the risk of BMPR2 mutation, and the lower the EOS ratio in peripheral blood, the higher the risk of BMPR2 mutation and the higher the risk of poor prognosis. In animal experiments, with the progression of pulmonary arterial hypertension, the EOS ratio in the peripheral blood of mice decreases, and the infiltration of EOS into lung tissue increases, and EOS has the effect of protecting pulmonary arterial hypertension, and can assist in the diagnosis and judgment of pulmonary arterial hypertension, and the lower the EOS ratio in peripheral blood, the more serious the condition. After EOS knockout, the right ventricular systolic blood pressure and right ventricular hypertrophy index of the mice were significantly increased, and the pulmonary artery smooth muscle layer was significantly thickened, indicating that EOS plays a protective role in the progression of pulmonary arterial hypertension, and EOS can be used as an auxiliary diagnostic indicator. The detection method for the proportion of eosinophils can be performed by blood count test.

[0021] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for inhibiting proliferation and / or migration of pulmonary artery smooth muscle cells.

[0022] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for maintaining pulmonary artery smooth muscle homeostasis.

[0023] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for treating pulmonary arterial hypertension.

[0024] In basic research on EOS, it is believed that the lysate contains the entire cytoplasmic content of EOS. In the embodiment of the present invention, the effect of EOS cytoplasmic content can be demonstrated by adding lysate. The test results show that EOS plays a protective role in the progression of pulmonary arterial hypertension, the content of EOS can inhibit the proliferation and / or migration of pulmonary arterial smooth muscle, and the cytoplasmic content of EOS plays a protective role in maintaining the homeostasis of pulmonary arterial smooth muscle, which can play a role in the treatment of pulmonary arterial hypertension.

[0025] The present invention also provides the application of lipid metabolite hydroxydocosahexaenoic acid (HDHA) in the preparation of a drug for treating pulmonary arterial hypertension. HDHA effectively inhibits the proliferation of smooth muscle cells, which are the most important constituent cells in pulmonary vascular remodeling, in vitro, and also inhibits the infiltration of inflammatory cells such as neutrophils; in vivo experiments also showed that knocking out eosinophils aggravates pulmonary vascular remodeling and peripheral inflammation, and it was revealed that HDHA, a natural metabolic product of DHA, has fewer side effects and is safer than other drugs, and can maintain immune homeostasis and simultaneously inhibit smooth muscle proliferation. In the present invention, the hydroxydocosahexaenoic acid preferably includes 14-hydroxydocosahexaenoic acid (14-HDHA) and / or 17-hydroxydocosahexaenoic acid (17-HDHA). 14-HDHA and 17-HDHA, which are downstream metabolites of EOS, have a therapeutic effect on pulmonary arterial hypertension.

[0026] The present invention also provides the application of lipid metabolite hydroxydocosahexaenoic acid (HDHA) in the preparation of a drug for inhibiting the proliferation of pulmonary vascular smooth muscle. In the present invention, the hydroxydocosahexaenoic acid preferably includes 14-hydroxydocosahexaenoic acid and / or 17-hydroxydocosahexaenoic acid. 14-HDHA and 17-HDHA belong to downstream lipid metabolites of EOS, and the levels of 14-HDHA and 17-HDHA are significantly decreased after EOS is knocked out. Stimulation with low concentrations of 14-HDHA and / or 17-HDHA can effectively inhibit the proliferation of pulmonary artery smooth muscle cells, and the lower concentration in the present invention is preferably 10-20 nM, more preferably 10 nM or 20 nM. Administration of 14-HDHA and / or 17-HDHA, which are downstream metabolites of EOS, can effectively inhibit the proliferation of pulmonary vascular smooth muscle cells, and can be used in the preparation of a PH therapeutic drug.

[0027] To further illustrate the present invention, the application of eosinophil drugs, eosinophils and their cytoplasmic contents as well as the lipid metabolite hydroxydocosahexaenoic acid provided by the present invention will be described below with reference to the figures and examples, which should not be construed as limiting the scope of protection of the present invention. EXAMPLES

[0028] The percentage of EOS in peripheral blood is decreased in patients with pulmonary arterial hypertension and in animal models and is associated with disease severity in patients with pulmonary arterial hypertension.

[0029] 1.1 Subject population and sample collection The subjects were a single-center clinical cohort consisting of 123 patients with idiopathic or hereditary pulmonary arterial hypertension and 119 age- and sex-matched healthy controls enrolled at Fuwai Hospital, Chinese Academy of Medical Sciences. The diagnostic criteria for patients with idiopathic or hereditary pulmonary arterial hypertension were: mean pulmonary artery pressure ≥ 25mmHg and pulmonary artery wedge pressure ≤ 15mmHg at sea level, as detected by right heart catheterization. Exclusions: (1) patients with allergy, infection, or autoimmune disease; (2) patients with lung disease (tumor, infection, fibrosis, etc.), autoimmune disease, and (3) adolescents under 18 years of age.

[0030] Venous blood was collected from fasting patients and healthy controls and anticoagulated with ethylenediaminetetraacetic acid. All of the present invention was first approved by the Institutional Ethics Committee of Peking Union Medical College (No. 2018043) and Fuwai Hospital (Approval No.: 2017-877). All subjects signed informed consent forms before being included in the cohort.

[0031] 1.2 Collection of peripheral blood from mice with pulmonary arterial hypertension induced by a combination of hypoxia and Sugen Male C57 / B6J mice aged 8-10 weeks and weighing 25g or more were placed in a hypoxic chamber with an oxygen concentration of 10% and injected subcutaneously with Sugen5416 once a week, a total of three times at a dose of 20mg / kg / injection. The mice were then kept in the hypoxic chamber for three weeks. The right ventricular pressure of the anesthetized mice was measured, and modeling was completed when the right ventricular pressure reached 30mmHg or higher. Peripheral blood from healthy control and pulmonary arterial hypertension mice was collected and anticoagulated with ethylenediaminetetraacetic acid.

[0032] 1.3 Preparation of single cell suspension from mouse lung tissue and flow cytometry analysis Lung tissue digestion solution was prepared using neutral protease (5U / mL, Worthington) + type I collagenase (200U / mL, Vetec) + elastase (0.02U / mL, Sigma) + DNA enzyme (0.3U / mL, Progema). The first lobe of the right side of the lung tissue was minced with the digestion solution, thoroughly shaken at 37°C for 25 minutes, and then digestion was terminated with an equal volume of complete medium (basal medium containing 10% FBS). After red blood cell division, the cells were centrifuged, resuspended, and 100μL was taken and fluorescein-conjugated target antibodies (CD45-FITC, CD11b-APC, SiglecF-PE) were added and incubated for 30 minutes at room temperature in the dark. After incubation, PBS was added to wash out excess antibodies, centrifuged, and the samples were placed on a cell strainer and loaded onto a BD Accuri C6.

[0033] 1.4 Results analysis The results are shown in Figures 1 and 2. Figure 1 is a schematic diagram of the experimental results of the EOS ratio in peripheral blood of patients with pulmonary arterial hypertension and its correlation analysis with the severity of the disease; where A is the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and healthy subjects; B is the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and the NYHA cardiac function class; C is the relationship between the EOS ratio in peripheral blood samples of patients with pulmonary arterial hypertension and the BMPR2 mutation. Figure 2 is a schematic diagram of the experimental results of the EOS ratio in an animal model of pulmonary arterial hypertension; where A is a typical diagram of the EOS ratio in peripheral blood of a mouse pulmonary arterial hypertension model induced by a combination of hypoxia and Sugen, and flow cytometry; B is a typical diagram of the EOS ratio in lung tissue of a mouse pulmonary arterial hypertension model induced by a combination of hypoxia and Sugen, and flow cytometry.

[0034] By analyzing peripheral samples from patients with pulmonary arterial hypertension and their disease-related indicators, the present invention found that the proportion of EOS in peripheral blood was reduced in patients with pulmonary arterial hypertension (Figure 1A), and the lower the proportion of EOS in peripheral blood, the more serious the condition of patients with pulmonary arterial hypertension (Figure 1B), indicating that EOS can assist in the diagnosis of pulmonary arterial hypertension, and the lower the proportion of EOS in peripheral blood, the higher the risk of disease and the more serious the condition; in patients with BMPR2 mutation, the proportion of EOS in peripheral blood was even lower (Figure 1C), indicating that the lower the proportion of EOS in peripheral blood, the higher the risk of BMPR2 mutation and the higher the risk of poor prognosis. At the same time, by detecting peripheral blood and lung tissue samples of a mouse pulmonary arterial hypertension model induced by a combination of hypoxia and Sugen, the present invention showed that with the progression of pulmonary arterial hypertension, the proportion of EOS in the peripheral blood of mice decreased and the infiltration of EOS into the lung tissue increased (Figure 2), indicating that EOS has the effect of protecting pulmonary arterial hypertension and can assist in the diagnosis or judgment of pulmonary arterial hypertension, and the lower the proportion of EOS in peripheral blood, the more serious the condition. EXAMPLES

[0035] Construction and functional evaluation of pulmonary arterial hypertension model in EOS knockout mice 2.1 Establishment of a mouse PH model induced by the combination of hypoxia and Sugen Male mice aged 8 to 10 weeks and weighing 25g or more were placed in a hypoxic chamber with an oxygen concentration of 10% and Sugen5416 was injected subcutaneously once a week at a dose of 20mg / kg / injection for a total of three injections. The mice were then kept in the hypoxic chamber for three weeks, and the right ventricular pressure was measured. When the right ventricular pressure reached 30mmHg or higher, modeling was completed.

[0036] (1) Control group 1: normoxic wild type: 4 mice were kept under normoxic conditions for 3 weeks and injected subcutaneously with DMSO every 7 days starting from day 1, for a total of 3 injections.

[0037] (2) Control group 2: EOS knockout under normoxic conditions: 4 mice were kept under normoxic conditions for 3 weeks; DMSO was injected subcutaneously every 7 days from day 1, for a total of 3 injections.

[0038] (3) Modeling group 1: wild type combined with hypoxia and Sugen: 7 mice were kept in a hypoxic environment for 3 weeks; they were injected subcutaneously with Sugen5416 (DMSO as solvent) every 7 days from day 1, for a total of 3 injections.

[0039] (4) Modeling group 2: EOS knockout combined with hypoxia and Sugen: 9 mice were kept in a hypoxic environment for 3 weeks and injected subcutaneously with Sugen5416 (DMSO as solvent) every 7 days starting from day 1, for a total of 3 injections.

[0040] 2.2 Measurement of right ventricular systolic blood pressure in mice Without exposing the mouse's thoracic cavity, a 22-gauge needle connected to a pressure transducer in a PowerLab device was inserted from a xiphocostal angle into the right ventricle of a mouse anesthetized intraperitoneally with 2% tribromoethanol, and the position of the needle was determined using the waveform displayed by the pressure sensor. Once the waveform was stable, the right ventricular systolic pressure was recorded.

[0041] 2.3 Determination of right ventricular hypertrophy index After the hemodynamic measurements were completed, the chest was opened and washed with pre-cooled saline, and then the whole heart and lungs were removed. The heart was separated along the atrioventricular groove to remove the atria and the roots of the great vessels, and the right ventricular free wall was separated along the posterior interventricular groove. After absorbing excess water, the right ventricular free wall (RV) and the left ventricle + ventricular septum (LV+S) were weighed, and the right ventricular hypertrophy index = RV / LV+S was calculated.

[0042] 2.4 Immunofluorescence staining of lung tissue Left lung tissue was harvested and fixed in 10% neutral formaldehyde at 4°C, then precipitated with glucose for 72 hours and embedded in OCT. All lung tissues were embedded in the same orientation (transverse section). Lung tissues were sectioned at a thickness of 5 μm. After overnight incubation with anti-α-smooth muscle actin primary antibody at 4°C and washing of the slides, they were stained dropwise with the corresponding HRP-conjugated secondary antibody, incubated for 30 minutes in a humidified box at room temperature, washed, and then mounted with a drop of DAPI and photographed.

[0043] 2.5 Results analysis The results are shown in Figure 3, which is a schematic diagram of the experimental results showing that EOS knockout aggravates pulmonary arterial hypertension induced by a combination of hypoxia and Sugen, where A is the right ventricular systolic pressure of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; B is the right heart hypertrophy index of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; C is a typical schematic diagram of pulmonary vascular anti-α-smooth muscle actin immunofluorescence staining of EOS knockout / wild-type mice under normal control and pulmonary arterial hypertension; wild-type mice were the experimental control group.

[0044] Under normal conditions, wild-type (control group 1) and EOS knockout (control group 2) mice had no difference in right ventricular systolic blood pressure (A in Figure 3), right ventricular hypertrophy index (B in Figure 3), and pulmonary artery smooth muscle layer thickness (C in Figure 3). Under modeling conditions of pulmonary arterial hypertension induced by a combination of hypoxia and Sugen, after EOS knockout, right ventricular systolic blood pressure and right ventricular hypertrophy index were significantly increased in mice in modeling group 2 compared with modeling group 1 (A and B in Figure 3). At the same time, the present invention found that the pulmonary artery smooth muscle layer of mice in modeling group 2 was significantly thickened compared with modeling group 1 (C in Figure 3). According to Figure 3, after the lack of EOS, the pulmonary arterial hypertension of the mice was aggravated, suggesting that EOS plays a protective role in the progression of pulmonary arterial hypertension and EOS can be used as an auxiliary diagnostic indicator. EXAMPLES

[0045] Measurement of proliferation and migration function of pulmonary vascular smooth muscle cells 3.1 Stimulation of pulmonary artery smooth muscle cells Pulmonary artery smooth muscle cells were serum-starved for 24 hours, and then stimulated with the growth factor PDGFbb and gradient EOS cell lysate. The cells were divided into three groups: untreated control, PDGFbb-stimulated group, and PDGFbb+10 group. 3 EOS lysate group (10 3 EOS), PDGFbb+10 4 EOS lysate group (10 4 EOS), PDGFbb+10 5 EOS lysate group (10 5 EOS) and PDGFbb+10 6 EOS lysate group (10 6 The media corresponding to each of the above groups are collectively referred to as EOS conditioned media, the untreated control group contained only smooth muscle cell medium, the PDGFbb-stimulated group contained smooth muscle cell medium containing the growth factor PDGFbb, and the other four groups contained smooth muscle cell culture medium containing the growth factor PDGFbb and EOS lysate.

[0046] 3.2 Pulmonary artery smooth muscle cell CCK8 proliferation assay Pulmonary artery smooth muscle cells were seeded in 96-well plates at 5000 cells / well, and after serum starvation for 24 h, they were added into the corresponding conditioned medium according to the experimental design, 100 μL medium per well, and 24 h after stimulation, 10 μL of CCK8 stock solution was added to each well, incubated for 2 h, and the absorbance was read at 450 nm and 570 nm with a microplate reader, a standard curve was drawn, and the percentage of cell proliferation was calculated.

[0047] 3.3 Pulmonary artery smooth muscle cell scratch and Transwell migration experiments Scratch test: pulmonary artery smooth muscle cells were seeded in a 6-well plate and serum-starved for 24 hours. When the cell density was about 100% under the microscope, the well plate was scratched vertically with a P200 pipette tip, washed with PBS to remove floating cells, and then added with the corresponding conditioned medium according to the experimental design, 1 mL of medium per well. After stimulation for 0 hours and 24 hours, images were taken and the wound healing area was calculated.

[0048] Transwell experiment: pulmonary artery smooth muscle cells were seeded in the upper chamber of the transwell chamber, 200 μL of serum-free basal medium was added to each well, and the corresponding conditioned medium was added to the lower chamber according to the experimental design, 600 μL of medium per well. After 24 hours of stimulation, the cells were fixed with methanol, stained with crystal violet and photographed, and the number of migrated cells was calculated.

[0049] 3.4 Results analysis The results are shown in Figure 4, which is a schematic diagram of the experimental results showing that EOS suppresses the functional abnormality of pulmonary artery smooth muscle cells, where A shows the proliferation status of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and administration of conditioned medium to EOS. The higher the concentration of EOS lysate, the more the proliferation of pulmonary artery smooth muscle cells was suppressed; B shows the migration status of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and administration of conditioned medium to EOS. With an increase in the concentration of EOS lysate, the degree of wound healing decreased and the number of Transwell membrane-penetrating cells decreased.

[0050] After PDGFbb induces the abnormal proliferation and migration of pulmonary artery smooth muscle cells, administration of gradient EOS lysate can significantly inhibit the proliferation and migration of the cells. At the same time, the present invention discovered that the inhibitory effect increases with the concentration gradient of EOS lysate, that is, the higher the concentration of EOS lysate, the higher the inhibitory effect (Figure 4), indicating that the content of EOS can inhibit the proliferation and migration of pulmonary artery smooth muscle cells, and the cytoplasmic content of EOS has a protective effect on maintaining the homeostasis of pulmonary artery smooth muscle cells. EXAMPLES

[0051] Detection of differential expression and roles of lipid metabolites downstream of EOS 4.1 Lung tissue sampling of a mouse model of pulmonary arterial hypertension induced by a combination of hypoxia and Sugen Male mice aged 8 to 10 weeks and weighing more than 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%, and Sugen5416 was injected subcutaneously once a week at a dose of 20 mg / kg / injection, a total of three injections, and kept in the hypoxic chamber for three weeks. The mice were anesthetized and the right ventricular pressure was measured, and the modeling was terminated when the right ventricular pressure increased and exceeded 30 mmHg. After the right ventricle was perfused, mouse lung tissue was collected and frozen in liquid nitrogen.

[0052] (1) Wild-type modeling group: Wild-type combined with hypoxia and Sugen: 9 mice were kept in a hypoxic environment for 3 weeks and injected with Sugen5416 every 7 days starting from day 1, for a total of 3 injections.

[0053] (2) EOS knockout modeling group: EOS knockout was achieved by combining hypoxia and Sugen: 9 mice were kept in a hypoxic environment for 3 weeks and injected with Sugen5416 subcutaneously every 7 days from day 1, for a total of 3 injections.

[0054] 4.2 Targeted metabolomics measurements in mouse lung tissue Frozen tissue samples were collected, weighed, and prepared for quality control samples, then detected and analyzed by Agilent ultra-high performance liquid chromatography and triple quadrupole mass spectrometer. Raw data was processed by MassHunter software, using default parameters, and manually inspected to obtain integrated area data for each compound and internal standard. The peak area ratio of the standard and internal standard for each compound was used for regression analysis against the concentration of the compound to obtain a 10-point quantitative standard curve, and the standard curve was used to calculate and output the content of each target substance in the test sample loaded into the tester, and the content of the original sample (per gram of lung tissue sample) was calculated. Principal components were analyzed by integrated data analysis, heat maps were drawn, and quantitative analysis of lipid small molecule metabolites was performed.

[0055] 4.3 Measurement of the effect of HDHA on proliferation inhibition Pulmonary artery smooth muscle cells were seeded in a 96-well plate at 5000 cells / well, and after 24 hours of serum starvation, a non-treated control group (smooth muscle cell medium), a PDGFbb stimulation group (smooth muscle cell medium containing growth factor PDGFbb), a PDGFbb+14-HDHA group (smooth muscle cell medium containing growth factors PDGFbb and 14-HDHA), and a PDGFbb+17-HDHA group (smooth muscle cell medium containing growth factors PDGFbb and 17-HDHA) were set up, respectively, and a concentration gradient of 10nM and 20nM of two types of lipid small molecules was set up, respectively, and 100μL of medium was added to each well. After 24 hours of stimulation, 10μL of CCK8 stock solution was added to each well, and incubated for 2 hours. The absorbance at 450nm and 570nm was read with a microplate reader, a standard curve was drawn, and the cell proliferation rate was calculated.

[0056] 4.4 Analysis of results The results are shown in Figure 5 and Figure 6. Figure 5 is a schematic diagram of the experimental results of the effect of EOS knockout on fatty acid metabolism in mouse lung tissue induced by the combination of hypoxia and Sugen; where A is the analysis of the principal components of the target metabolomics of EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension; B is the heat map of the differential expression of lipid metabolites in EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension; C is the quantitative analysis of lipid metabolites in EOS knockout / wild-type mouse lung tissue under pulmonary arterial hypertension, and wild-type mice are the experimental control group. Figure 6 is a schematic diagram of the experimental results of the inhibitory effect of 14-HDHA and 17-HDHA on the proliferation of pulmonary arterial smooth muscle cells.

[0057] In mice with pulmonary artery induced by the combination of hypoxia and Sugen, after EOS was knocked out, the metabolism of fatty acids such as arachidonic acid, DHA, and EPA was significantly altered compared to the wild-type control group (Figure 5A and B). At the same time, the present invention discovered that the levels of small molecule lipid metabolites 14-HDHA and 17-HDHA were significantly decreased after EOS was knocked out (Figure 5C). After pulmonary artery smooth muscle cells were stimulated with 14-HDHA and 17-HDHA in vitro, low concentrations could effectively inhibit the proliferation of pulmonary artery smooth muscle cells (Figure 6), and 14-HDHA and 17-HDHA, downstream metabolites of EOS, had a therapeutic effect on pulmonary arterial hypertension. EXAMPLES

[0058] Therapeutic effects of 14-HDHA and 17-HDHA, downstream metabolites of EOS, on pulmonary arterial hypertension 5.1 Establishment of a mouse PH model induced by the combination of hypoxia and Sugen and evaluation of the therapeutic effects of 14-HDHA and 17-HDHA Male mice aged 8 to 10 weeks and weighing more than 25g were placed in a hypoxic chamber with an oxygen concentration of 10%, and Sugen5416 was injected subcutaneously once a week at a dose of 20mg / kg / injection, a total of three injections, and kept in the hypoxic chamber for three weeks. The mice were anesthetized and the right ventricular pressure was measured, and modeling was terminated when the right ventricular pressure was 30mmHg or higher. From the second week of hypoxia, 14-HDHA and 17-HDHA were injected intraperitoneally every three days, respectively, until the end of modeling. At the end of modeling, the right ventricular systolic pressure, right heart hypertrophy index, and pulmonary artery smooth muscle layer thickness were evaluated.

[0059] 5.2 Establishment of a rat PH model induced by the combination of hypoxia and Sugen and evaluation of the therapeutic effects of 14-HDHA and 17-HDHA SD rats weighing approximately 200 g were given a single subcutaneous injection of Sugen5416 (at a dose of 20 mg / kg), then quickly placed in a hypoxic chamber with an oxygen concentration of 10%, and maintained for 3 weeks. After 3 weeks, the rats were placed in normoxia for 2 weeks to complete modeling. During the normoxia phase after 3 weeks of hypoxia, 14-HDHA and 17-HDHA were injected intraperitoneally every 3 days until the end of modeling, and the right ventricular systolic pressure, right ventricular hypertrophy index, and pulmonary artery smooth muscle layer thickness were evaluated at the end of modeling.

[0060] 5.3 Analysis of results Under normal conditions, there was no significant difference in right ventricular systolic blood pressure, right ventricular hypertrophy index, and pulmonary artery smooth muscle layer thickness between the control group of mice (rats) administered with the solvent and the mice (rats) administered with 14-HDHA or 17-HDHA. Under modeling conditions in which pulmonary arterial hypertension was induced by a combination of hypoxia and Sugen, the mice (rats) administered with 14-HDHA or 17-HDHA had significantly lower right ventricular systolic blood pressure and right ventricular hypertrophy index, and the pulmonary artery smooth muscle layer was significantly thinner than the mice (rats) in the solvent control group. This shows that pulmonary arterial hypertension in mice (rats) was improved after supplementation with 14-HDHA or 17-HDHA, suggesting the therapeutic effect of 14-HDHA and / or 17-HDHA in pulmonary arterial hypertension, and suggesting that 14-HDHA or 17-HDHA can be used to prepare therapeutic drugs for pulmonary arterial hypertension.

[0061] Although the above embodiments have described the present invention in detail, they are only some embodiments of the present invention, not all embodiments, and other embodiments can be obtained according to the present embodiments without inventive step, and all of these embodiments belong to the protection scope of the present invention.

Claims

1. The use of the reagent for detecting eosinophils in the preparation of a product for the diagnosis of pulmonary arterial hypertension and / or for determining the prognostic risk of pulmonary arterial hypertension.

2. 2. The application of claim 1, wherein the detection comprises detecting a percentage of eosinophils in peripheral blood.

3. Use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for inhibiting proliferation and / or migration of pulmonary artery smooth muscle.

4. Use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for maintaining homeostasis of pulmonary artery smooth muscle.

5. Use of EOS and / or EOS cytoplasmic contents in the preparation of a medicament for treating pulmonary arterial hypertension.

6. Application of hydroxydocosahexaenoic acid in the preparation of medicine for inhibiting proliferation of pulmonary vascular smooth muscle.

7. Application of hydroxydocosahexaenoic acid in the preparation of medicine for treating pulmonary arterial hypertension.

8. The application according to claim 6 or 7, characterized in that the hydroxydocosahexaenoic acid includes 14-hydroxydocosahexaenoic acid and / or 17-hydroxydocosahexaenoic acid.

Images