CN115466776A - Reagent for detecting eosinophils, eosinophils and application of eosinophils, cytoplasmic content of eosinophils and hydroxydocosahexaenoic acid - Google Patents
Reagent for detecting eosinophils, eosinophils and application of eosinophils, cytoplasmic content of eosinophils and hydroxydocosahexaenoic acid Download PDFInfo
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Abstract
The invention relates to a reagent for detecting eosinophils, eosinophils and the application of the eosinophils, the cytoplasmic content of the eosinophils and a lipid metabolite hydroxydocosahexaenoic acid, belonging to the technical field of medical treatment and medicine. The invention provides application of a reagent for detecting eosinophil in 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, diagnosis of pulmonary hypertension and judgment of pulmonary hypertension prognosis risk can be realized.
Description
Technical Field
The invention belongs to the technical field of medical treatment and medicine, and particularly relates to a reagent for detecting eosinophils, eosinophils and cytoplasm contents thereof and application of a lipid metabolite hydroxydocosahexaenoic acid.
Background
Pulmonary arterial Hypertension (PH) is a group of Pulmonary circulation diseases with progressively increased mean Pulmonary arterial pressure caused by various causes and different pathogenesis, and the mean Pulmonary arterial pressure measured by the right heart catheter in the resting and sea level state is defined as the diagnostic standard of the Pulmonary circulation diseases. In the process of pulmonary hypertension development, the dysfunction of the inherent cells of pulmonary blood vessels and the abnormal infiltration of various immune cells often cause pulmonary vasoconstriction and pulmonary vascular remodeling, which leads to the thickening and the stenosis of the pulmonary blood vessels of patients and the increase of the pulmonary artery pressure, and finally the pulmonary hypertension develops into right heart failure and right heart insufficiency.
At present, most of clinically used targeted drugs reduce pulmonary vascular resistance by expanding blood vessels to further relieve pulmonary hypertension, but cannot fundamentally reverse the situation of vascular remodeling, so that screening of drugs which can effectively improve vascular remodeling and can enter clinical medicine as soon as possible is still urgently needed for treating pulmonary hypertension.
Disclosure of Invention
The invention aims to provide a reagent for detecting Eosinophil (EOS), eosinophil and its cytoplasm content and application of lipid metabolite hydroxydocosahexaenoic acid (HDHA). The reagent for detecting eosinophilic granulocyte can realize the diagnosis of pulmonary hypertension disease and/or the judgment of pulmonary hypertension prognosis risk.
The invention provides application of a reagent for detecting eosinophil in preparation of a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension.
Preferably, the detection comprises detecting the proportion of eosinophils in the peripheral blood.
The invention also provides the use of EOS and/or EOS cytosol in the manufacture of a medicament for inhibiting proliferation and/or migration of pulmonary artery smooth muscle.
The invention also provides the use of EOS and/or EOS cytosolic content in the manufacture of a medicament for maintaining pulmonary artery smooth muscle homeostasis.
The invention also provides the use of EOS and/or EOS cytosolic content in the manufacture of a medicament for the treatment of pulmonary hypertension.
The invention also provides the application of the hydroxydocosahexaenoic acid in preparing a medicament for treating pulmonary hypertension.
The invention also provides the application of the hydroxydocosahexaenoic acid in preparing the medicine for inhibiting the proliferation of the smooth muscle of the pulmonary vessel.
Preferably, the hydroxydocosahexaenoic acid comprises 14-hydroxydocosahexaenoic acid and/or 17-hydroxydocosahexaenoic acid.
The invention provides application of a reagent for detecting eosinophil in preparation of a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension. The invention finds that EOS is closely related to the occurrence of pulmonary hypertension, and therefore proposes that EOS is used as a biomarker for prompting PH condition and is applied to the preparation of related products for diagnosing PH. Peripheral blood routinely measures EOS fraction, and if down-regulated, suggests risk of pulmonary hypertension and a poor prognosis of pulmonary hypertension. The application of the invention can realize the diagnosis of the pulmonary hypertension disease and the judgment of the pulmonary hypertension prognosis risk.
Test results show that with the development of pulmonary hypertension, the proportion of peripheral blood EOS of a mouse is reduced, the infiltration of pulmonary EOS is increased, and the EOS has the function of protecting the pulmonary hypertension; the content of EOS can inhibit the proliferation and/or migration of pulmonary artery smooth muscle cells which are the most important inherent cells in the process of pulmonary vascular remodeling, and the EOS cytoplasmic content has a protective effect of maintaining the pulmonary artery smooth muscle homeostasis. EOS and its cytosolic content have therapeutic effects on pulmonary hypertension.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic diagram of the EOS ratio in peripheral blood of a patient with pulmonary hypertension and the correlation analysis experiment result between the ratio and the severity of the disease; a, an EOS proportion graph in peripheral blood samples of pulmonary hypertension patients and normal healthy people; B. a relational graph of EOS proportion and NYHA cardiac function classification in peripheral blood samples of patients with pulmonary hypertension; C. a plot of EOS proportion versus BMPR2 mutation in peripheral blood samples from patients with pulmonary hypertension;
FIG. 2 is a schematic diagram of the results of an EOS ratio experiment in a pulmonary arterial hypertension animal model according to the present invention; a, EOS proportion in peripheral blood of a mouse pulmonary arterial hypertension model induced by hypoxia combined with Sugen and a typical flow cytometry graph; B. EOS proportion in mouse pulmonary arterial hypertension model lung tissue induced by hypoxia in combination with Sugen and typical graph of flow cytometry;
FIG. 3 is a schematic diagram of the experimental results of mouse pulmonary hypertension induced by EOS knockout/hypoxia combined with Sugen provided by the present invention; a, a result graph of right ventricular systolic pressure of an EOS knockout/wild type mouse under normal control and pulmonary arterial hypertension conditions; B. results of EOS knockout/wild type mice right heart hypertrophy index under normal control and pulmonary hypertension conditions; C. typical schematic diagram of immunofluorescence staining of anti-alpha-smooth muscle actin in lung blood vessel of EOS knockout/wild type mouse under normal control and pulmonary artery high pressure condition; wild type mice are experimental control group;
FIG. 4 is a schematic diagram of the experimental results of the inhibition of abnormal function of pulmonary artery smooth muscle cells by EOS provided by the present invention; a, a result graph of pulmonary artery smooth muscle cell proliferation after growth factor PDGFbb induction and EOS conditioned medium administration; B. a graph of the results of migration of pulmonary artery smooth muscle cells induced by growth factor PDGFbb and after administration of EOS conditioned medium;
FIG. 5 is a schematic diagram of the experimental results of the effect of EOS knockout on hypoxia combined with Sugen induced fatty acid metabolism in lung tissues of mice provided by the invention; a, an EOS knockout/wild type mouse lung tissue targeted metabonomics principal component analysis result graph under the condition of pulmonary hypertension; B. differential expression heatmap of EOS knock-out/wild type mouse lung tissue lipid metabolites under pulmonary arterial hypertension conditions; C. a result graph of quantitative analysis of lipid metabolites of mouse lung tissues of EOS knockout/wild type under pulmonary arterial hypertension conditions; wild type mice are experimental control group;
FIG. 6 is a schematic diagram showing the results of experiments on the inhibition of proliferation of pulmonary artery smooth muscle cells by 14-HDHA and 17-HDHA provided by the present invention.
Detailed Description
The invention provides an application of a reagent for detecting eosinophil in preparing a product for diagnosing pulmonary hypertension and/or judging the prognosis risk of pulmonary hypertension.
In the present invention, eosinophils can be used as a biomarker for the preparation of products for identifying and/or aiding in the diagnosis of pulmonary hypertension. In the present invention, the detection preferably comprises detecting the proportion of eosinophils in peripheral blood. In the present invention, the product preferably comprises a kit.
In the present invention, the sample to be tested preferably includes peripheral blood. Peripheral blood routinely measures EOS proportion, relative to healthy controls, if down-regulated, suggesting a risk of pulmonary hypertension and/or a poor prognosis of pulmonary hypertension. In the present invention, the proportion of the down-regulation is preferably 25% or more, more preferably 30% or more. As shown by the clinical test data of the invention, the average value of the male normal control is 2.668, the average value of the male patient is 1.78, and the down-regulation is 33%; the average for female normal controls was 2.024, the average for female patients was 1.404, with 30% downregulation.
In the present invention, the pulmonary arterial hypertension preferably comprises hypoxia in combination with Sugen-induced pulmonary arterial hypertension.
The results of the embodiment of the invention show that the proportion of EOS in peripheral blood of a patient with pulmonary hypertension is reduced, and the lower the proportion of EOS peripheral blood is, the more serious the condition of the patient with pulmonary hypertension is, and the EOS can assist in diagnosing the pulmonary hypertension disease. The proportion of peripheral blood EOS can also indicate the mutation risk of BMPR2, and the lower the proportion of peripheral blood EOS is, the higher the mutation risk of BMPR2 and the higher the risk of poor prognosis are. Animal experiments show that with the development of pulmonary hypertension, the proportion of peripheral blood EOS of a mouse is reduced, the infiltration of pulmonary EOS is increased, the EOS has the function of protecting the pulmonary hypertension, the pulmonary hypertension can be diagnosed or judged in an auxiliary manner, and the condition of the patient is more serious when the proportion of peripheral blood EOS is lower. After EOS knockout, the right ventricular systolic pressure and right heart hypertrophy index of the mouse are obviously increased, the pulmonary artery smooth muscle layer is obviously thickened, the EOS is prompted to have a protective effect in the process of pulmonary artery high pressure development, and the EOS can be used as an auxiliary diagnosis index. The method for detecting the proportion of eosinophilic granulocyte can be realized by conventional blood detection.
The invention also provides the use of EOS and/or EOS cytosolic content in the manufacture of a medicament for inhibiting proliferation and/or migration of pulmonary artery smooth muscle.
The invention also provides the use of EOS and/or EOS cytosolic content in the manufacture of a medicament for maintaining pulmonary artery smooth muscle homeostasis.
The invention also provides the use of EOS and/or EOS cytosolic content in the manufacture of a medicament for the treatment of pulmonary hypertension.
The EOS basal study suggests that lysates contain the entire cytosolic content of EOS. The embodiment of the invention can prove the effect of the EOS cytoplasmic content by adding the lysis solution. Test results show that EOS has a protective effect in the process of pulmonary hypertension development; the content of EOS can inhibit the proliferation and/or migration of pulmonary artery smooth muscle, and the content of EOS cytoplasm has a protective effect of maintaining the steady state of the pulmonary artery smooth muscle, so that the pulmonary artery hypertension treatment effect can be realized.
The invention also provides application of a lipid metabolite, namely hydroxydocosahexaenoic acid (HDHA) in preparing a medicament for treating pulmonary hypertension. HDHA can effectively inhibit the proliferation of smooth muscle cells which are the most important inherent 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 lung vessel remodeling and peripheral inflammation aggravate after eosinophil knockout; HDHA is a natural metabolite of DHA that is less and safer than other drugs with fewer side effects, while also being found to simultaneously 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 metabolites 14-HDHA and 17-HDHA downstream of EOS have therapeutic effect on pulmonary hypertension.
The invention also provides application of a lipid metabolite, namely hydroxydocosahexaenoic acid (HDHA), in preparing a medicament for inhibiting 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 lipid metabolites downstream of EOS, and the levels of 14-HDHA and 17-HDHA are significantly reduced after EOS knockout. Stimulation with a smaller concentration of 14-HDHA and/or 17-HDHA is effective in inhibiting pulmonary artery smooth muscle cell proliferation, preferably 10 to 20nM, more preferably 10nM or 20nM. The metabolite 14-HDHA and/or 17-HDHA downstream of EOS can effectively inhibit the proliferation of pulmonary vascular smooth muscle, thus being used for preparing PH therapeutic drugs.
To further illustrate the present invention, the reagent for detecting eosinophils, eosinophils and their cytosolic content, and the use of hydroxydocosahexaenoic acid provided in the present invention will be described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
The peripheral blood EOS ratio of the pulmonary hypertension patients and animal models is reduced, and is related to the disease severity of the pulmonary hypertension patients.
1.1 test population and sample Collection
Subjects were from a single-center clinical cohort consisting of 123 idiopathic or hereditary pulmonary hypertension patients enrolled in the hospital outside the hospital of the chinese medical academy of sciences and 119 age and sex matched healthy controls. Diagnostic criteria for patients with idiopathic or hereditary pulmonary hypertension: under the resting state of sea level, the average pulmonary artery pressure detected by the right heart catheter is more than or equal to 25mmHg, and the pulmonary arteriole wedge pressure is less than or equal to 15mmHg. And (3) excluding: (1) allergy, infection and autoimmune disease patients; (2) pulmonary diseases (tumors, infections, fibrosis, etc.), autoimmune diseases and (3) & lt 18 & gt youngsters.
Collecting venous blood of fasting patient and healthy control, and anticoagulating with EDTA. The invention firstly obtains the approval of the ethics committee of the institution of Beijing cooperative and medical college (2018043) and the Fufangyou hospital (approval number: 2017-877). All subjects signed informed consent prior to enrollment into the cohort.
1.2 Collection of peripheral blood of mice with pulmonary hypertension induced by hypoxia in combination with Sugen
Placing a C57/B6J male mouse with the age of 8-10 weeks and the weight of more than 25g into a low oxygen chamber with the oxygen concentration of 10%, subcutaneously injecting Sugen5416 once a week with the dose of 20 mg/kg/time for 3 times, continuously raising the male mouse in the low oxygen chamber for 3 weeks, and measuring the pressure of the right ventricle of an anesthetized mouse, wherein the modeling is completed if the pressure of the right ventricle is increased to more than 30 mmHg. Collecting peripheral blood of healthy control and pulmonary hypertension mice, and adopting ethylenediamine tetraacetic acid for anticoagulation.
1.3 mouse Lung tissue Single cell suspension preparation and flow cytometry analysis
Preparing lung tissue digestive juice by using neutral protease (5U/mL, worthington) + collagenase type I (200U/mL, vetec) + elastase (0.02U/mL, sigma) + DNase (0.3U/mL, progema), taking the first leaf on the right side of the lung tissue, shearing the first leaf in the digestive juice, shaking the shaker at 37 ℃ for 25min, stopping digestion by using an equal volume of complete culture medium (adding basal medium of 10 FBS), centrifuging after red cracking, resuspending, taking 100 mu L of the mixture, adding fluorescein-conjugated target antibody (CD 45-FITC, CD11b-APC, siglecF-PE), and incubating the mixture at room temperature for 30min in a dark place. After incubation, PBS was added to wash off excess antibody and centrifuged, and the sample was screened through a cell sieve and loaded onto BDAccuri C6.
1.4 analysis of results
The results are shown in fig. 1 and fig. 2, and fig. 1 is a schematic diagram of the results of the analysis experiment of the EOS ratio in the peripheral blood of a patient with pulmonary hypertension and the correlation between the EOS ratio and the severity of the disease; the EOS ratio of the peripheral blood samples of the pulmonary hypertension patients and the normal healthy people is A; B. the relation between EOS ratio in peripheral blood sample of pulmonary hypertension patient and NYHA heart function classification; C. correlation of EOS ratio in peripheral blood sample of pulmonary hypertension patients and BMPR2 mutation. FIG. 2 is a schematic diagram of the results of an EOS ratio experiment in a pulmonary hypertension animal model; a, EOS proportion in peripheral blood of a mouse pulmonary arterial hypertension model induced by hypoxia combined with Sugen and a typical flow cytometry graph; B. hypoxia was combined with Sugen-induced EOS proportion in lung tissue of mouse pulmonary arterial hypertension model and typical flow cytometry plots.
By analyzing peripheral samples of patients with pulmonary hypertension and indexes related to the disease conditions of the patients, the invention discovers that the proportion of EOS in peripheral blood of the patients with pulmonary hypertension is reduced (A in figure 1), and the lower the proportion of EOS in the peripheral blood is, the heavier the disease conditions of the patients with pulmonary hypertension are (B in figure 1), so that EOS can assist in diagnosing the pulmonary hypertension diseases, and the lower the proportion of EOS in the peripheral blood is, the higher the risk of the diseases is, the more serious the disease conditions are; whereas patients carrying BMPR2 mutations had a lower EOS peripheral blood fraction (C in fig. 1), indicating that lower EOS peripheral blood fraction indicates a greater risk of BMPR2 mutations and a greater risk of poor prognosis. Meanwhile, by detecting peripheral blood and lung tissue samples of a mouse pulmonary arterial hypertension disease model induced by hypoxia combined with Sugen, the invention discovers that with the development of pulmonary arterial hypertension, the EOS proportion of peripheral blood of a mouse is reduced, the EOS infiltration of lung tissue is increased (figure 2), EOS has the function of protecting the pulmonary arterial hypertension, and the diagnosis or judgment of the pulmonary arterial hypertension can be assisted, wherein the lower the EOS proportion of the peripheral blood is, the more serious the disease condition is.
Example 2
Pulmonary artery hypertension model construction and function evaluation of EOS knockout mouse
2.1 establishment of mouse PH model induced by hypoxia in combination with Sugen
Placing a male mouse with the age of 8-10 weeks and the weight of more than 25g in a low oxygen chamber with the oxygen concentration of 10%, subcutaneously injecting Sugen5416 once a week with the dosage of 20 mg/kg/time for 3 times, continuously feeding the male mouse in the low oxygen chamber for 3 weeks, and measuring the pressure of the right ventricle of an anesthetized mouse, wherein the modeling is completed if the pressure of the right ventricle is increased to more than 30 mmHg.
(1) Control group 1: normoxic wild type: 4 mice were raised in normoxic environment for 3 weeks; DMSO was injected subcutaneously every 7 days from day 1 for a total of 3 injections.
(2) Control group 2: normoxic EOS knockout: 4 mice were raised in an normoxic environment for 3 weeks; DMSO was injected subcutaneously every 7 days from day 1 for a total of 3 injections.
(3) Building a module 1: hypoxia in combination with Sugen wild type: 7 mice were raised in a hypoxic environment for 3 weeks; sugen5416 (DMSO as solvent) was injected subcutaneously every 7 days from day 1 for 3 total injections.
(4) Building a module 2: hypoxia combined sugeneeos knockdown: 9 mice were raised in a hypoxic environment for 3 weeks; sugen5416 (DMSO as solvent) was injected subcutaneously every 7 days from day 1 for 3 total injections.
2.2 measurement of the Right ventricular systolic pressure in mice
Under the condition of not exposing the thoracic cavity of the mouse, a 22-gauge needle connected with a pressure sensor of a PowerLab instrument is inserted into the right ventricle of the mouse which is subjected to abdominal cavity anesthesia by 2% tribromoethanol through a sword rib angle needle insertion, and the position of the needle is determined by a waveform displayed by the pressure sensor. Right ventricular systolic pressure was recorded after the waveform stabilized.
2.3 right ventricular hypertrophy index determination
After the hemodynamic measurement is completed, the chest is opened, and the whole heart and lungs are taken out after the perfusion with precooled physiological saline. The heart was isolated from the atrioventricular groove by removing the atria and the root of the great vessel along the atrioventricular groove, the right ventricular free wall was separated along the posterior interventricular groove, the weight of the right ventricular free wall (RV) and the left ventricular + ventricular septum (LV + S) was measured after absorbing the excess water, and the right ventricular hypertrophy index = RV/LV + S was calculated.
2.4 immunofluorescent staining of Lung tissue
Collecting left lung tissue, fixing with 10% neutral formaldehyde at 4 deg.C, precipitating sugar 72h, and embedding with OCT. All lung tissue is embedded in the same direction (cross section). Lung tissue was sectioned at 5 μm thickness. And (3) incubating overnight at 4 ℃ with a primary antibody for resisting alpha-smooth muscle actin, washing the film, dripping corresponding HRP coupled secondary antibody, incubating for 30min at a room temperature in a wet box, washing, dripping DAPI sealing film, and taking a picture.
2.5 analysis of results
The result is shown in FIG. 3, and FIG. 3 is a schematic diagram of the experimental result of inducing pulmonary hypertension of mice by EOS knockout-emphasized hypoxia combined with Sugen; wherein, A, right ventricular systolic pressure of EOS knockout/wild type mice under normal control and pulmonary hypertension conditions; B. right heart hypertrophy index of EOS knockout/wild type mice under normal control and pulmonary hypertension conditions; C. typical schematic diagram of immunofluorescence staining of anti-alpha-smooth muscle actin in lung blood vessel of EOS knockout/wild type mouse under normal control and pulmonary artery high pressure condition; wild type mice were experimental controls.
Under normal conditions, wild type (control 1) and EOS knockout (control 2) mice had no difference in right ventricular systolic pressure (a in fig. 3), right heart hypertrophy index (B in fig. 3), and pulmonary artery smooth muscle layer thickness (C in fig. 3). Under the condition of modeling the pulmonary hypertension induced by hypoxia combined with Sugen, after EOS is knocked out, the right ventricular systolic pressure and the right heart hypertrophy index of the mice in the modeling group 2 are obviously increased compared with the modeling group 1 (A and B in figure 3). Meanwhile, the invention finds that the pulmonary artery smooth muscle layer of the mouse in the modeling group 2 is obviously thickened compared with the pulmonary artery smooth muscle layer in the modeling group 1 (C in figure 3). As can be seen from FIG. 3, in the absence of EOS, pulmonary hypertension of the mice is aggravated, which suggests that EOS is protective in the process of pulmonary hypertension progression, and EOS can be used as an auxiliary diagnostic index.
Example 3
Determination of proliferation and migration function of pulmonary vascular smooth muscle cells
3.1 pulmonary artery smooth muscle cell stimulation
After 24h of serum starvation of pulmonary artery smooth muscle cells, adding growth factor PDGFbb and gradient EOS cell lysate for stimulation, and dividing into: untreated control group, PDGFbb stimulated group, PDGFbb +10 3 EOS lysate group (marked 10 in the figure) 3 EOS)、PDGFbb+10 4 EOS lysate group (10 in the figure) 4 EOS)、PDGFbb+10 5 EOS lysate group (10 in the figure) 5 EOS) and PDGFbb +10 6 EOS lysate group (10 in the figure) 6 EOS), the media corresponding to each group above are collectively referred to as EOS conditioned media, the untreated control group contains only smooth muscle cell media, the PDGFbb stimulation group is smooth muscle cell media containing growth factor PDGFbb, and the other four groups are smooth muscle cell media containing growth factor PDGFbb and EOS lysate).
3.2 pulmonary artery smooth muscle cell CCK8 proliferation assay
And (3) paving the pulmonary artery smooth muscle cells into a 96-well plate according to 5000 cells per well, after serum starvation is removed for 24 hours, adding a corresponding conditioned medium according to experimental design, adding 100 mu L of the medium into each well, adding 10 mu L of CCK8 stock solution into each well after 24 hours of stimulation, incubating for 2 hours, reading the absorbance at 450nm and 570nm by using an enzyme labeling instrument, drawing a standard curve, and calculating the cell proliferation percentage.
3.3 pulmonary artery smooth muscle cell scratch and Transwell migration experiments
Scratch test: pulmonary artery smooth muscle cells were plated in 6-well plates and serum starvation was removed for 24h. And when the cell density is observed to be about 100% under a microscope, scratching is performed by using a P200 gun head vertical to the pore plate, after the floating cells are removed by PBS (phosphate buffer solution) washing, adding a corresponding conditioned medium according to the experimental design, stimulating the culture medium for 0h and 24h for photographing, and calculating the healing area of the scratch, wherein each well contains 1mL of the culture medium.
Transwell experiment: pulmonary artery smooth muscle cells are paved into an upper chamber of a Transwell chamber, 200 mu L of serum-free basal medium is added into each hole, corresponding conditioned medium, 600 mu L of medium is added into a lower chamber according to experimental design, after 24h of stimulation, methanol fixation, crystal violet staining and photographing are carried out to calculate the number of migrated cells.
3.4 analysis of results
The results are shown in FIG. 4, and FIG. 4 is a diagram illustrating the results of experiments on the inhibition of abnormal function of the pulmonary artery smooth muscle cells by EOS; the proliferation condition of the pulmonary artery smooth muscle cells after induction and administration of the growth factor PDGFbb to the EOS conditioned medium is stronger in the inhibition effect of the pulmonary artery smooth muscle cell proliferation along with the increase of the concentration of EOS lysate; B. the migration condition of pulmonary artery smooth muscle cells after induction and administration of growth factor PDGFbb to EOS conditioned medium is reduced in the healing degree of scratches and the number of Transwell transmembrane cells as the concentration of EOS lysate is increased.
After PDGFbb induces abnormal proliferation and migration of pulmonary artery smooth muscle cells, gradient EOS lysate is given to obviously inhibit the proliferation and migration of the cells, and meanwhile, the invention finds that the inhibition effect is enhanced along with the concentration gradient of the EOS lysate, namely the higher the concentration of the EOS lysate is, the stronger the inhibition effect is (figure 4), which indicates that the content of the EOS can inhibit the proliferation and migration of the pulmonary artery smooth muscle, and the EOS cytoplasmic content has the protective effect of maintaining the steady state of the pulmonary artery smooth muscle.
Example 4
Differential expression and effect detection of EOS downstream lipid metabolite
4.1 Lung tissue drawing of mouse pulmonary arterial hypertension model induced by hypoxia in combination with Sugen
Placing a male mouse with the age of 8-10 weeks and the weight of more than 25g in a low oxygen chamber with the oxygen concentration of 10%, subcutaneously injecting Sugen5416 once a week with the dosage of 20 mg/kg/time for 3 times, continuously feeding the male mouse in the low oxygen chamber for 3 weeks, and measuring the pressure of the right ventricle of an anesthetized mouse, wherein the modeling is completed if the pressure of the right ventricle is increased to more than 30 mmHg. After perfusion in the right ventricle, the lung tissues of the mice are collected and frozen by liquid nitrogen.
(1) Wild type modeling group: hypoxia in combination with Sugen wild type: 9 mice were raised in a hypoxic environment for 3 weeks; sugen5416 was injected subcutaneously every 7 days from day 1 for a total of 3 injections.
(2) An EOS knockout building module: hypoxia combined Sugen EOS knockdown: 9 mice were raised in a hypoxic environment for 3 weeks; sugen5416 was injected subcutaneously every 7 days from day 1 for a total of 3 injections.
4.2 mouse Lung tissue Targeted Metabonomics detection
Taking a frozen tissue sample, weighing and preparing a quality control sample, detecting and analyzing by adopting an Agilent ultra-high performance liquid chromatograph and a triple quadrupole mass spectrometer, processing original data by MassHunter software, using default parameters and assisting manual inspection to obtain integral area data of each compound and an internal standard. And performing regression analysis on the concentration of the compound by adopting the peak area ratio of each compound standard substance to the internal standard substance to obtain a quantitative standard curve with 10 points, calculating and outputting the content of the on-machine test sample of each target substance by adopting the standard curve, and calculating the content of the original sample (each gram of lung tissue sample). And performing principal component analysis and heat map drawing by adopting an integrated data analysis method, and performing quantitative analysis on the lipid small molecule metabolite.
4.3 measurement of proliferation inhibitory Effect of HDHA
Pulmonary artery smooth muscle cells are plated into 96-well plates at 5000/well, after serum starvation is removed for 24h, an untreated control group (smooth muscle cell culture medium), a PDGFbb stimulation group (smooth muscle cell culture medium containing growth factor PDGFbb), a PDGFbb +14-HDHA group (smooth muscle cell culture medium containing growth factors PDGFbb and 14-HDHA) and a PDGFbb +17-HDHA group (smooth muscle cell culture medium containing growth factors PDGFbb and 17-HDHA) are respectively set, concentration gradients of 10nM and 20nM of the two lipid small molecules are respectively set, 100 uL of the culture medium is added into each well after 24h stimulation, 10 uL of CCK8 stock solution is added into each well, 2h incubation is carried out, absorbance at 450nM and 570nM is read by a microplate reader, a standard curve is drawn, and the cell proliferation percentage is calculated.
4.4 analysis of results
The results are shown in fig. 5 and fig. 6, and fig. 5 is a schematic diagram of the experimental results of the effect of EOS knockout on hypoxia combined with Sugen induced fatty acid metabolism in lung tissues of mice; a, analyzing main components of EOS knockout/wild mouse lung tissue targeted metabonomics under pulmonary arterial hypertension; B. differential expression heatmap of lipid metabolites in lung tissue of EOS knockout/wild type mice under pulmonary arterial hypertension conditions; C. quantitative analysis of EOS knockout/wild type mouse lung tissue lipid metabolites under pulmonary arterial hypertension conditions; wild type mice were experimental controls. FIG. 6 is a graph showing the results of experiments on the inhibition of proliferation of pulmonary artery smooth muscle cells by 14-HDHA and 17-HDHA.
In mice with hypoxia in combination with Sugen induced pulmonary artery, after EOS knock-out, significant changes in fatty acid metabolism such as arachidonic acid, DHA, and EPA were shown compared to wild-type controls (a and B in fig. 5). Meanwhile, the invention discovers that the levels of the small molecule lipid metabolites 14-HDHA and 17-HDHA are obviously reduced after EOS knockout (C in figure 5). After being stimulated by 14-HDHA and 17-HDHA of pulmonary artery smooth muscle cells in vitro, the lung artery smooth muscle cell proliferation can be effectively inhibited at a smaller concentration (figure 6), and the 14-HDHA and 17-HDHA of EOS downstream metabolites have a therapeutic effect on pulmonary artery hypertension.
Example 5
Therapeutic effect of downstream metabolites of EOS, 14-HDHA and 17-HDHA, on pulmonary hypertension
5.1 establishment of mouse PH model induced by hypoxia in combination with Sugen and evaluation of therapeutic effects of 14-HDHA and 17-HDHA
Placing a male mouse with the age of 8-10 weeks and the weight of more than 25g in a low oxygen chamber with the oxygen concentration of 10%, subcutaneously injecting Sugen5416 once a week with the dosage of 20 mg/kg/time for 3 times, continuously feeding the male mouse in the low oxygen chamber for 3 weeks, and measuring the pressure of the right ventricle of an anesthetized mouse, wherein the modeling is completed if the pressure of the right ventricle is increased to more than 30 mmHg. From the second week of hypoxia, 14-HDHA and 17-HDHA were intraperitoneally injected every 3 days until the end of modeling, at which time the right ventricular systolic pressure, right heart hypertrophy index and pulmonary artery smooth muscle layer thickness were evaluated.
5.2 establishment of hypoxia-Sugen-induced rat PH model and evaluation of therapeutic Effect of 14-HDHA and 17-HDHA
Approximately 200g of SD rats were injected once with Sugen5416 (20 mg/kg) subcutaneously and then immediately placed in a 10% oxygen-enriched hypoxic chamber for 3 weeks. After 3 weeks, the cells were kept in constant oxygen for 2 weeks to complete the modeling. In the normoxic stage after 3 weeks of hypoxia, 14-HDHA and 17-HDHA are respectively injected into the abdominal cavity every 3 days until the modeling is finished, and the right ventricular systolic pressure, the right heart hypertrophy index and the thickness of the pulmonary artery smooth muscle layer are evaluated at the modeling end.
5.3 analysis of results
Under normal conditions, there was no significant difference in the right ventricular systolic pressure, right heart hypertrophy index and pulmonary artery smooth muscle layer thickness between mice (rats) given solvent control and mice (rats) given 14-HDHA or 17-HDHA. Under the modeling condition of hypoxia combined with Sugen induced pulmonary hypertension, the right ventricular systolic pressure and the right heart hypertrophy index of a mouse (rat) given with 14-HDHA or 17-HDHA are obviously reduced and the pulmonary artery smooth muscle layer is obviously thinned compared with a solvent control group mouse (rat). It is known that the pulmonary hypertension of mice (rats) is improved after the supplementation of 14-HDHA or 17-HDHA, which suggests the therapeutic effect of 14-HDHA or 17-HDHA in the pulmonary hypertension, and 14-HDHA or 17-HDHA can be used for preparing the medicaments for treating the pulmonary hypertension.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (8)
1. Application of the reagent for detecting eosinophil in preparing a product for diagnosing pulmonary hypertension and/or judging the risk of prognosis of pulmonary hypertension.
2. The use of claim 1, wherein said detecting comprises detecting the proportion of eosinophils in peripheral blood.
Use of EOS and/or EOS cytosolic content in the manufacture of a medicament for inhibiting proliferation and/or migration of pulmonary artery smooth muscle.
Use of EOS and/or EOS cytosolic content in the manufacture of a medicament for maintaining pulmonary artery smooth muscle homeostasis.
Use of EOS and/or EOS cytosolic content in the manufacture of a medicament for the treatment of pulmonary hypertension.
6. Application of hydroxydocosahexaenoic acid in preparing medicine for inhibiting proliferation of pulmonary vascular smooth muscle is provided.
7. Application of hydroxydocosahexaenoic acid in preparing medicine for treating pulmonary hypertension is provided.
8. Use according to claim 6 or 7, wherein the hydroxydocosahexaenoic acid comprises 14-hydroxydocosahexaenoic acid and/or 17-hydroxydocosahexaenoic acid.
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| PCT/CN2022/125577 WO2024065892A1 (en) | 2022-09-26 | 2022-10-17 | Use of reagent for detecting eosinophil, eosinophil and cytoplasm content thereof, and hydroxy docosahexaenoic acid |
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