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

Abstract

The present invention relates to a reagent for detecting eosinophils, the application of eosinophils and their cytoplasmic contents and lipid metabolite hydroxydocosahexaenoic acid, belonging to the fields of medical treatment and pharmaceutical technology. The present invention provides the application of a reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary hypertension and / or judging the prognosis risk of pulmonary hypertension. By detecting the proportion of EOS in peripheral blood, the diagnosis of pulmonary hypertension and the judgment of the prognosis risk of pulmonary hypertension can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medicine and pharmaceuticals, and specifically relates to the application of reagents for detecting eosinophils, eosinophils, their cytoplasmic contents, and the lipid metabolite hydroxy docosahexaenoic acid (HDHA). Background Art

[0002] Pulmonary hypertension (PH) is a group of pulmonary circulation diseases caused by various etiologies and different pathogenesis, characterized by a progressive increase in mean pulmonary artery pressure. Currently, the diagnostic criterion is defined as a mean pulmonary artery pressure greater than or equal to 25 mmHg measured by right heart catheterization at rest and at sea level. During the progression of pulmonary hypertension, the dysfunction of pulmonary vascular resident cells and the abnormal infiltration of various immune cells often cause pulmonary vasoconstriction and pulmonary vascular remodeling, leading to thickening and stenosis of the pulmonary vessels, increased pulmonary artery pressure, and ultimately developing into right heart failure and right heart insufficiency.

[0003] Currently, most of the targeted drugs used clinically reduce pulmonary vascular resistance by dilating blood vessels, thereby alleviating pulmonary hypertension, but they cannot fundamentally reverse the situation of vascular remodeling. Therefore, there is still an urgent need to screen drugs that can effectively improve vascular remodeling and can quickly enter clinical practice for the treatment of pulmonary hypertension. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of reagents for detecting eosinophils (EOS), eosinophils, their cytoplasmic contents, and the lipid metabolite hydroxy docosahexaenoic acid (HDHA). By using the reagents for detecting eosinophils, the present invention can achieve the diagnosis of pulmonary hypertension and / or the judgment of the prognosis risk of pulmonary hypertension.

[0005] The present invention provides the application of reagents for detecting eosinophils in the preparation of products for diagnosing pulmonary hypertension and / or judging the prognosis risk of pulmonary hypertension.

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

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

[0008] The present invention also provides the application of EOS and / or EOS cytoplasmic contents in the preparation of drugs for maintaining the 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 drugs for treating pulmonary hypertension.

[0010] The present invention also provides the use of hydroxydocosahexaenoic acid in the preparation of a medicament for treating pulmonary hypertension.

[0011] The present invention also provides the use of hydroxydocosahexaenoic acid in the preparation of a medicament for inhibiting the proliferation of pulmonary vascular smooth muscle.

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

[0013] The present invention provides the use of a reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary hypertension and / or judging the prognosis risk of pulmonary hypertension. The present invention discovers that EOS is closely related to the occurrence of pulmonary hypertension. Therefore, it is proposed to use EOS as a biomarker for indicating the condition of PH and apply it to the preparation of related products for diagnosing PH. By detecting the proportion of EOS in peripheral blood routine, if it is down-regulated, it indicates the risk of pulmonary hypertension and poor prognosis of pulmonary hypertension. The application of the present invention can realize the diagnosis of pulmonary hypertension disease and the judgment of the prognosis risk of pulmonary hypertension.

[0014] The test results show that with the development of pulmonary hypertension, the proportion of EOS in the peripheral blood of mice decreases, and the infiltration of EOS in the lung tissue increases. EOS has a protective effect on pulmonary hypertension; the content of EOS can inhibit the proliferation and / or migration of pulmonary artery smooth muscle cells, the most important resident cells in the process of pulmonary vascular remodeling. The cytoplasmic content of EOS has a protective effect on maintaining the homeostasis of pulmonary artery smooth muscle. EOS and its cytoplasmic content have the effect of treating pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0016] Figure 1 It is a schematic diagram of the experimental results of the analysis of the proportion of EOS in the peripheral blood of patients with pulmonary hypertension provided by the present invention and its correlation with the disease severity; wherein, A. The diagram of the proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and normal healthy people; B. The diagram of the relationship between the proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and the NYHA cardiac function classification; C. The diagram of the relationship between the proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and the BMPR2 mutation;

[0017] Figure 2Schematic diagram of the experimental results of the proportion of EOS in the animal model of pulmonary hypertension provided by the present invention; wherein, A. Typical diagram of the proportion of EOS in the peripheral blood and flow cytometry of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen; B. Typical diagram of the proportion of EOS in the lung tissue and flow cytometry of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen.

[0018] Figure 3 Schematic diagram of the experimental results of EOS knockout aggravating hypoxia combined with Sugen-induced pulmonary hypertension in mice provided by the present invention; wherein, A. Results diagram of the right ventricular systolic pressure of EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; B. Results diagram of the right heart hypertrophy index of EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; C. Typical schematic diagram of the immunofluorescence staining of pulmonary vascular anti-α-smooth muscle actin in EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; wild-type mice were used as the experimental control group.

[0019] Figure 4 Schematic diagram of the experimental results of EOS inhibiting the abnormal function of pulmonary artery smooth muscle cells provided by the present invention; wherein, A. Results diagram of the proliferation of pulmonary artery smooth muscle cells after induction by growth factor PDGFbb and administration of EOS conditioned medium; B. Results diagram of the migration of pulmonary artery smooth muscle cells after induction by growth factor PDGFbb and administration of EOS conditioned medium.

[0020] Figure 5 Schematic diagram of the experimental results of EOS knockout affecting fatty acid metabolism in the lung tissue of mice induced by hypoxia combined with Sugen provided by the present invention; wherein, A. Results diagram of the principal component analysis of targeted metabolomics of the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; B. Heat map of differential expression of lipid metabolites in the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; C. Quantitative analysis results diagram of lipid metabolites in the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; wild-type mice were used as the experimental control group.

[0021] Figure 6 Schematic diagram of 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 implementation mode

[0022] The present invention provides an application of a reagent for detecting eosinophils in the preparation of a product for diagnosing pulmonary hypertension and / or judging the prognosis risk of pulmonary hypertension.

[0023] In the present invention, eosinophils can be used as biomarkers for the preparation of products for identifying and / or assisting in the diagnosis of pulmonary hypertension. In the present invention, the detection preferably includes detecting the proportion of eosinophils in peripheral blood. In the present invention, the product preferably includes a kit.

[0024] In the present invention, the sample for the detection preferably includes peripheral blood. Detecting the proportion of EOS in the routine blood test of peripheral blood, if it is down-regulated compared with healthy controls, indicates the risk of pulmonary hypertension and / or poor prognosis of pulmonary hypertension. In the present invention, the down-regulated proportion is preferably more than 25%, more preferably more than 30%. As shown by the clinical trial data of the present invention, the average value of male normal controls is 2.668, the average value of male patients is 1.78, with a 33% down-regulation; the average value of female normal controls is 2.024, the average value of female patients is 1.404, with a 30% down-regulation.

[0025] In the present invention, the pulmonary hypertension preferably includes pulmonary hypertension induced by hypoxia combined with Sugen.

[0026] The results of the examples of the present invention show that the proportion of EOS in the peripheral blood of patients with pulmonary hypertension decreases, and the lower the proportion of EOS in the peripheral blood, the more severe the condition of the patients with pulmonary hypertension. EOS can assist in the diagnosis of pulmonary hypertension disease. The proportion of EOS in the peripheral blood can also indicate the risk of BMPR2 mutation. The lower the proportion of EOS in the peripheral blood, the greater the risk of BMPR2 mutation and the greater the risk of poor prognosis. Animal experiments show that as pulmonary hypertension develops, the proportion of EOS in the peripheral blood of mice decreases, and the infiltration of EOS in the lung tissue increases. EOS has a protective effect on pulmonary hypertension, can assist in the diagnosis or judgment of pulmonary hypertension, and the lower the proportion of EOS in the peripheral blood, the more severe the condition. After knocking out EOS, the right ventricular systolic pressure and right ventricular hypertrophy index of mice increase significantly, and the pulmonary artery smooth muscle layer thickens significantly, indicating that EOS plays a protective role in the progression of pulmonary hypertension, and EOS can be used as an auxiliary diagnostic index. The detection method of the proportion of eosinophils can be carried out by routine blood test.

[0027] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of drugs for inhibiting the proliferation and / or migration of pulmonary artery smooth muscle.

[0028] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of drugs for maintaining the homeostasis of pulmonary artery smooth muscle.

[0029] The present invention also provides the use of EOS and / or EOS cytoplasmic contents in the preparation of drugs for treating pulmonary hypertension.

[0030] Basic research on EOS believes that the lysate contains all the cytoplasmic contents of EOS. In the embodiments of the present invention, the addition of the lysate can prove the role of the cytoplasmic contents of EOS. The test results show that EOS plays a protective role in the progression of pulmonary hypertension; the contents of EOS can inhibit the proliferation and / or migration of pulmonary artery smooth muscle, and the cytoplasmic contents of EOS have a protective effect on maintaining the homeostasis of pulmonary artery smooth muscle, and thus can play a role in the treatment of pulmonary hypertension.

[0031] The present invention also provides the use of the lipid metabolite hydroxydocosahexaenoic acid (HDHA) in the preparation of a drug for treating pulmonary hypertension. HDHA can effectively inhibit the proliferation of smooth muscle cells, which are the most important resident cells in the process of pulmonary vascular remodeling in vitro, and inhibit the infiltration of inflammatory cells such as neutrophils; in vivo experiments also show that after knocking out eosinophils, pulmonary vascular remodeling and surrounding inflammation are aggravated; HDHA is a natural metabolite of DHA, with less side effects and higher safety compared with other drugs. At the same time, it is also found that it can maintain immune homeostasis and 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). The downstream metabolites 14-HDHA and 17-HDHA of EOS have a therapeutic effect on pulmonary hypertension.

[0032] The present invention also provides the use of the 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 the downstream lipid metabolites of EOS, and the levels of 14-HDHA and 17-HDHA decrease significantly after knocking out EOS. Stimulation with a relatively low concentration of 14-HDHA and / or 17-HDHA can effectively inhibit the proliferation of pulmonary artery smooth muscle cells. The relatively low concentration described in the present invention is preferably 10-20 nM, more preferably 10 nM or 20 nM. The downstream metabolites 14-HDHA and / or 17-HDHA of EOS can effectively inhibit the proliferation of pulmonary vascular smooth muscle, and thus can be used in the preparation of therapeutic drugs for PH.

[0033] To further illustrate the present invention, the following describes in detail the reagents for detecting eosinophils, eosinophils and their cytoplasmic contents, and the application of the lipid metabolite hydroxydocosahexaenoic acid provided by the present invention with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] The proportion of EOS in the peripheral blood of patients with pulmonary hypertension and animal models decreases, and is related to the disease severity of patients with pulmonary hypertension.

[0036] 1.1 Test subjects and sample collection

[0037] The test subjects came from a single-center clinical cohort consisting of 123 patients with idiopathic or hereditary pulmonary hypertension and 119 age- and sex-matched healthy controls included in Fuwai Hospital, Chinese Academy of Medical Sciences. Diagnostic criteria for patients with idiopathic or hereditary pulmonary hypertension: at rest at sea level, mean pulmonary artery pressure detected by right heart catheterization ≥ 25 mmHg, pulmonary artery wedge pressure ≤ 15 mmHg. Exclusions: ① patients with allergies, infections, and autoimmune diseases; ② pulmonary diseases (tumors, infections, fibrosis, etc.), autoimmune diseases; and ③ adolescents under 18 years old.

[0038] Fasting venous blood was collected from patients and healthy controls and anticoagulated with ethylenediaminetetraacetic acid. This invention was first approved by the Institutional Review Board of Peking Union Medical College (2018043) and Fuwai Hospital (approval number: 2017-877). All subjects signed an informed consent form before being included in the cohort.

[0039] 1.2 Collection of peripheral blood from mice with pulmonary hypertension induced by hypoxia combined with Sugen

[0040] Male C57 / B6J mice aged 8-10 weeks and weighing more than 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%. Subcutaneous injection of Sugen 5416 was performed once a week, with a dose of 20 mg / kg / time, for a total of 3 injections. The mice were continuously raised in the hypoxic chamber for 3 weeks. The mice were anesthetized for right ventricular pressure measurement. If the right ventricular pressure rose above 30 mmHg, the modeling was completed. Peripheral blood from healthy controls and mice with pulmonary hypertension was collected and anticoagulated with ethylenediaminetetraacetic acid.

[0041] 1.3 Preparation of single-cell suspension of mouse lung tissue and flow cytometry analysis

[0042] Lung tissue digestive fluid was prepared using neutral protease (5 U / mL, Worthington) + type I collagenase (200 U / mL, Vetec) + elastase (0.02 U / mL, Sigma) + DNase (0.3 U / mL, Progema). The first right lobe of the lung tissue was minced in the digestive fluid and shaken thoroughly in a 37°C shaker for 25 min, then digested was terminated with an equal volume of complete medium (basic medium supplemented with 10% FBS). After lysing red blood cells, it was centrifuged and resuspended, and 100 μL was taken and added to the fluorescently conjugated target antibodies (CD45-FITC, CD11b-APC, SiglecF-PE) and incubated at room temperature in the dark for 30 min. After incubation, PBS was added to wash away the excess antibodies and centrifuged. The sample was passed through a cell sieve and then loaded onto a BD Accuri C6.

[0043] 1.4 Result Analysis

[0044] The results are shown in Figure 1 and Figure 2 , Figure 1 which is a schematic diagram of the experimental results of the proportion of EOS in the peripheral blood of patients with pulmonary hypertension and its correlation with the disease severity; among them, A. The proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and normal healthy people; B. The relationship between the proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and the NYHA cardiac function classification; C. The relationship between the proportion of EOS in the peripheral blood samples of patients with pulmonary hypertension and the BMPR2 mutation. Figure 2 which is a schematic diagram of the experimental results of the proportion of EOS in the pulmonary hypertension animal model; among them, A. The proportion of EOS in the peripheral blood of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen and the typical flow cytometry diagram; B. The proportion of EOS in the lung tissue of the mouse pulmonary hypertension model induced by hypoxia combined with Sugen and the typical flow cytometry diagram.

[0045] By analyzing the peripheral samples of patients with pulmonary hypertension and their disease-related indicators, the present invention finds that the proportion of EOS in the peripheral blood of patients with pulmonary hypertension decreases ( Figure 1 A in Figure 1 ), and the lower the proportion of EOS in the peripheral blood, the more severe the condition of the patients with pulmonary hypertension ( Figure 1 B in Figure 2 ). It can be seen that EOS can assist in the diagnosis of pulmonary hypertension disease. The lower the proportion of EOS in the peripheral blood, the higher the risk of disease and the more severe the condition; while the proportion of EOS in the peripheral blood of patients carrying the BMPR2 mutation is even less (

[0046] Example 2

[0047] Construction and functional evaluation of a pulmonary hypertension model in EOS knockout mice

[0048] 2.1 Establishment of a mouse PH model induced by hypoxia combined with Sugen

[0049] Male mice at 8 - 10 weeks of age with a body weight of over 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%. Subcutaneous injection of Sugen5416 was performed once a week, with a dose of 20 mg / kg / time, for a total of 3 injections. The mice were continuously raised in the hypoxic chamber for 3 weeks. The mice were anesthetized for right ventricular pressure measurement. When the right ventricular pressure rose above 30 mmHg, the modeling was completed.

[0050] (1) Control group 1: Normoxic wild - type: 4 mice, raised in a normoxic environment for 3 weeks; from the first day, subcutaneous injection of DMSO was performed once every 7 days, for a total of 3 injections.

[0051] (2) Control group 2: Normoxic EOS - knockout: 4 mice, raised in a normoxic environment for 3 weeks; from the first day, subcutaneous injection of DMSO was performed once every 7 days, for a total of 3 injections.

[0052] (3) Modeling group 1: Hypoxia combined with Sugen wild - type: 7 mice, raised in a hypoxic environment for 3 weeks; from the first day, subcutaneous injection of Sugen5416 (with DMSO as the solvent) was performed once every 7 days, for a total of 3 injections.

[0053] (4) Modeling group 2: Hypoxia combined with Sugen EOS - knockout: 9 mice, raised in a hypoxic environment for 3 weeks; from the first day, subcutaneous injection of Sugen5416 (with DMSO as the solvent) was performed once every 7 days, for a total of 3 injections.

[0054] 2.2 Measurement of right ventricular systolic pressure in mice

[0055] Under the condition of not exposing the mouse thorax, a 22 - gauge needle connected to the pressure sensor of the PowerLab instrument was inserted into the right ventricle of a mouse anesthetized intraperitoneally with 2% tribromoethanol through the xiphoid costal angle, and the position of the needle was determined by the waveform shown by the pressure sensor. After the waveform was stable, the right ventricular systolic pressure was recorded.

[0056] 2.3 Measurement of right ventricular hypertrophy index

[0057] After hemodynamic measurement, the chest was opened. After perfusion with pre - cooled saline, the whole heart and lungs were removed. The atria and the roots of the large blood vessels of the heart were removed along the atrioventricular groove, and the free wall of the right ventricle was separated along the posterior interventricular groove. After sucking off the excess moisture, the weight of the free wall of the right ventricle (RV) and the left ventricle + interventricular septum (LV + S) was weighed, and the right ventricular hypertrophy index = RV / LV + S was calculated.

[0058] 2.4 Immunofluorescence staining of lung tissue

[0059] Collect the left lung tissue, fix it with 10% neutral formaldehyde at 4°C, then dehydrate it with sucrose for 72 h, and embed it in OCT. All lung tissues are embedded in the same direction (transverse section). Cut the lung tissues into sections with a thickness of 5 μm. Incubate the sections with the primary antibody against α-smooth muscle actin overnight at 4°C. After washing the sections, add the corresponding HRP-conjugated secondary antibody and incubate in a wet box at room temperature for 30 min. After washing, add DAPI for mounting and take pictures.

[0060] 2.5 Result analysis

[0061] The results are shown in Figure 3 , Figure 3 which is a schematic diagram of the experimental results showing that EOS knockout exacerbates hypoxia combined with Sugen-induced pulmonary hypertension in mice. Among them, A. Right ventricular systolic pressure of EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; B. Right ventricular hypertrophy index of EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; C. Typical schematic diagram of immunofluorescence staining of pulmonary vascular α-smooth muscle actin in EOS knockout / wild-type mice under normal control and pulmonary hypertension conditions; Wild-type mice are the experimental control group.

[0062] Under normal conditions, there were no differences in right ventricular systolic pressure ( Figure 3 A in Figure 3 ), right ventricular hypertrophy index ( Figure 3 B in Figure 3 ), and pulmonary artery smooth muscle layer thickness ( Figure 3 C in Figure 3 ) between wild-type (control group 1) and EOS knockout group (control group 2) mice. Under the condition of hypoxia combined with Sugen-induced pulmonary hypertension modeling, after knocking out EOS, compared with modeling group 1, the right ventricular systolic pressure and right ventricular hypertrophy index of mice in modeling group 2 were significantly increased ( Figure 3 A and B in Figure 3 ). At the same time, the present invention found that compared with modeling group 1, the pulmonary artery smooth muscle layer of mice in modeling group 2 was significantly thickened ( Figure 3 C in Figure 3 ). According to Figure 3 , it can be seen that after lacking EOS, pulmonary hypertension in mice is aggravated, suggesting that EOS plays a protective role in the progression of pulmonary hypertension, and EOS can be used as an auxiliary diagnostic index.

[0063] Example 3

[0064] Determination of the proliferation and migration functions of pulmonary vascular smooth muscle cells

[0065] 3.1 Stimulation of pulmonary artery smooth muscle cells

[0066] After serum starvation of pulmonary artery smooth muscle cells for 24 h, add growth factor PDGFbb and gradient EOS cell lysates for stimulation, and divide them into: untreated control group, PDGFbb stimulation group, PDGFbb + 10 3 EOS lysate group (labeled as 10 3EOS), PDGFbb + 10 4 EOS lysis group (marked as 10 in the figure 4 EOS), PDGFbb + 10 5 EOS lysis group (marked as 10 in the figure 5 EOS) and PDGFbb + 10 6 EOS lysis group (marked as 10 in the figure 6 EOS). The culture media corresponding to the above groups are collectively referred to as EOS conditioned medium. The untreated control group only contains smooth muscle cell culture medium. The PDGFbb stimulation group is the smooth muscle cell culture medium containing the growth factor PDGFbb. The other four groups are the smooth muscle cell culture media containing the growth factor PDGFbb and EOS lysis solution).

[0067] 3.2 CCK8 proliferation assay of pulmonary artery smooth muscle cells

[0068] Pulmonary artery smooth muscle cells were seeded into 96 - well plates at 5000 cells / well. After serum starvation for 24 h, according to the experimental design, the corresponding conditioned medium was added, 100 μL of medium per well. After stimulation for 24 h, 10 μL of CCK8 stock solution was added to each well, incubated for 2 h, and the absorbance at 450 nm and 570 nm was read with an enzyme - linked immunosorbent assay (ELISA) reader to plot the standard curve and calculate the percentage of cell proliferation.

[0069] 3.3 Scratch and Transwell migration assays of pulmonary artery smooth muscle cells

[0070] Scratch assay: Pulmonary artery smooth muscle cells were seeded into 6 - well plates and serum - starved for 24 h. When the cell density was approximately 100% under the microscope, a scratch was made perpendicular to the well plate with a P200 pipette tip. After rinsing with PBS to remove floating cells, according to the experimental design, the corresponding conditioned medium was added, 1 mL of medium per well. Photos were taken at 0 h and 24 h of stimulation, and the scratch healing area was calculated.

[0071] Transwell assay: Pulmonary artery smooth muscle cells were seeded into the upper chamber of the Transwell insert, 200 μL of serum - free basal medium was added to each well. In the lower chamber, according to the experimental design, the corresponding conditioned medium was added, 600 μL of medium per well. After stimulation for 24 h, methanol fixation, crystal violet staining were performed, and the number of migrated cells was calculated by taking photos.

[0072] 3.4 Result analysis

[0073] The results are shown in Figure 4 , Figure 4Schematic diagram of experimental results showing the inhibitory effect of EOS on abnormal functions of pulmonary artery smooth muscle cells; among them, A. Proliferation of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and treatment with EOS conditioned medium. As the concentration of EOS lysate increased, the inhibitory effect on the proliferation of pulmonary artery smooth muscle cells became stronger; B. Migration of pulmonary artery smooth muscle cells after induction with growth factor PDGFbb and treatment with EOS conditioned medium. As the concentration of EOS lysate increased, the degree of scratch healing decreased and the number of Transwell transmembrane cells decreased.

[0074] After PDGFbb induced abnormal proliferation and migration of pulmonary artery smooth muscle cells, gradient EOS lysate was given, which could significantly inhibit cell proliferation and migration. At the same time, the present invention found that the inhibitory effect enhanced with the concentration gradient of EOS lysate, that is, the higher the concentration of EOS lysate, the stronger the inhibitory effect ( Figure 4 ), indicating that the content of EOS can inhibit the proliferation and migration of pulmonary artery smooth muscle, and the cytoplasmic content of EOS has a protective effect on maintaining the homeostasis of pulmonary artery smooth muscle.

[0075] Example 4

[0076] Detection of differential expression and function of downstream lipid metabolites of EOS

[0077] 4.1 Collection of lung tissues from a mouse model of pulmonary hypertension induced by hypoxia combined with Sugen

[0078] Male mice aged 8 - 10 weeks and weighing more than 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%. Subcutaneous injection of Sugen5416 was performed once a week, with a dose of 20 mg / kg / time, for a total of 3 injections. The mice were continuously raised in the hypoxic chamber for 3 weeks. The mice were anesthetized for right ventricular pressure measurement. If the right ventricular pressure rose above 30 mmHg, the modeling was completed. After right ventricular perfusion, the lung tissues of the mice were collected and frozen in liquid nitrogen.

[0079] (1) Wild - type modeling group: Hypoxia combined with Sugen wild - type: 9 mice, raised in a hypoxic environment for 3 weeks; starting from the first day, subcutaneous injection of Sugen5416 was performed once every 7 days, for a total of 3 injections.

[0080] (2) EOS knockout modeling group: Hypoxia combined with Sugen EOS knockout: 9 mice, raised in a hypoxic environment for 3 weeks; starting from the first day, subcutaneous injection of Sugen5416 was performed once every 7 days, for a total of 3 injections.

[0081] 4.2 Targeted metabolomics detection of mouse lung tissues

[0082] After taking the frozen tissue samples, weighing them and preparing the quality control samples, they were detected and analyzed using an Agilent ultra-high performance liquid chromatograph and a triple quadrupole mass spectrometer. The original data was processed using MassHunter software with default parameters and assisted by manual inspection to obtain the integrated area data of each compound and the internal standard. The concentration of each compound was regressed using the peak area ratio of the compound standard to the internal standard to obtain a quantitative standard curve of 10 points. The content of the test samples of each target substance was calculated and output using the standard curve, and the content of the original samples (per gram of lung tissue sample) was calculated. An integrated data analysis method was used for principal component analysis, heat map drawing, and quantitative analysis of lipid small molecule metabolites.

[0083] 4.3 Determination of the inhibitory effect of HDHA on proliferation

[0084] Pulmonary artery smooth muscle cells were seeded into 96-well plates at 5000 cells / well. After serum starvation for 24 h, an untreated control group (smooth muscle cell medium), a PDGFbb stimulation group (smooth muscle cell medium containing the growth factor PDGFbb), a PDGFbb + 14-HDHA group (smooth muscle cell medium containing the growth factor PDGFbb and 14-HDHA), and a PDGFbb + 17-HDHA group (smooth muscle cell medium containing the growth factor PDGFbb and 17-HDHA) were set up respectively. Two concentration gradients of 10 nM and 20 nM of the two types of lipid small molecules were set, with 100 μL of medium per well. After stimulation for 24 h, 10 μL of CCK8 stock solution was added to each well, incubated for 2 h, and the absorbance at 450 nm and 570 nm was read using an enzyme-linked immunosorbent assay reader to draw a standard curve and calculate the percentage of cell proliferation.

[0085] 4.4 Result analysis

[0086] 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 the lung tissue of mice induced by hypoxia combined with Sugen; among them, A. Principal component analysis of targeted metabolomics of the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; B. Heat map of differential expression of lipid metabolites in the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; C. Quantitative analysis of lipid metabolites in the lung tissue of EOS knockout / wild-type mice under pulmonary hypertension conditions; wild-type mice were used as 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 artery smooth muscle cells.

[0087] In mice with pulmonary arteries induced by hypoxia combined with Sugen, after knocking out EOS, obvious changes in fatty acid metabolism such as arachidonic acid, DHA, and EPA were shown compared with the wild-type control group (Figure 5 A and B in). At the same time, the present invention found that the levels of small molecule lipid metabolites 14-HDHA and 17-HDHA decreased significantly after EOS knockout ( Figure 5 C in). After stimulating pulmonary artery smooth muscle cells with 14-HDHA and 17-HDHA in vitro, proliferation of pulmonary artery smooth muscle cells could be effectively inhibited at relatively low concentrations ( Figure 6 ), and the downstream metabolites 14-HDHA and 17-HDHA of EOS have a therapeutic effect on pulmonary hypertension.

[0088] Example 5

[0089] Therapeutic effect of downstream metabolites 14-HDHA and 17-HDHA of EOS on pulmonary hypertension

[0090] 5.1 Establishment of a mouse PH model induced by hypoxia combined with Sugen and evaluation of the therapeutic effects of 14-HDHA and 17-HDHA

[0091] Male mice aged 8-10 weeks and weighing more than 25 g were placed in a hypoxic chamber with an oxygen concentration of 10%. Sugen5416 was subcutaneously injected once a week, with a dose of 20 mg / kg / time, for a total of 3 injections. The mice were continuously raised in the hypoxic chamber for 3 weeks. The mice were anesthetized for right ventricular pressure measurement. If the right ventricular pressure rose above 30 mmHg, the modeling was completed. From the second week of hypoxia, 14-HDHA and 17-HDHA were intraperitoneally injected every 3 days 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.

[0092] 5.2 Establishment of a rat PH model induced by hypoxia combined with Sugen and evaluation of the therapeutic effects of 14-HDHA and 17-HDHA

[0093] After subcutaneous injection of SD rats weighing about 200 g with Sugen5416 (dose: 20 mg / kg) once, they were quickly placed in a hypoxic chamber with an oxygen concentration of 10% and continuously raised for 3 weeks. After 3 weeks, they were placed in normoxia for 2 weeks to complete the modeling. During the normoxic stage 3 weeks after hypoxia, 14-HDHA and 17-HDHA were intraperitoneally injected every 3 days 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.

[0094] 5.3 Result analysis

[0095] Under normal conditions, there were no significant differences in the right ventricular systolic pressure, right heart hypertrophy index, and pulmonary artery smooth muscle layer thickness between the solvent control group mice (rats) and the mice (rats) administered 14-HDHA or 17-HDHA. Under the condition of hypoxic combined with Sugen-induced pulmonary hypertension modeling, the right ventricular systolic pressure and right heart hypertrophy index of the mice (rats) administered 14-HDHA or 17-HDHA were significantly lower than those of the solvent control group mice (rats), and the pulmonary artery smooth muscle layer was significantly thinner. It can be seen that the pulmonary hypertension in mice (rats) was improved after supplementing 14-HDHA or 17-HDHA, suggesting the therapeutic effect of 14-HDHA or 17-HDHA in pulmonary hypertension, and 14-HDHA or 17-HDHA can be used to prepare therapeutic drugs for pulmonary hypertension.

[0096] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of hydroxy docosahexaenoic acid in the preparation of a medicament for treating pulmonary hypertension; the hydroxy docosahexaenoic acid is 14-hydroxy docosahexaenoic acid and / or 17-hydroxy docosahexaenoic acid.

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