Application of eosinophilic granulocyte cationic protein in preparation of medicine for preventing and treating pulmonary arterial hypertension

Abstract

The invention relates to application of eosinophilic granulocyte cationic protein in preparation of a medicine for preventing and treating pulmonary arterial hypertension, and belongs to the technical field of biological medicine. The invention finds that the ECP level in the plasma of a PAH patient and the ECP level combined with the endothelial surface of the lung far-end arteriole are obviously reduced for the first time, which prompts that the reduction of the ECP level is possibly closely related to the pulmonary vascular remodeling process, and further animal experiments prove that the ECP deficiency has a promoting effect on the generation and development of PAH. The invention also proves that the ECP can be specifically combined with the BMPR-1A / BMPR-2 receptor of the pulmonary artery endothelial cell. Besides, the invention also proves that the exogenous recombinant ECP can inhibit pulmonary vascular remodeling, relieve the occurrence and development of pulmonary arterial hypertension, including reducing vascular wall thickening and collagenous fiber deposition and relieving right heart function, and provides a new therapeutic target and a potential therapeutic strategy for PAH treatment.

Description

Technical Field

[0001] The invention relates to application of eosinophil cationic protein in preparing medicine for preventing and treating pulmonary hypertension, and belongs to the technical field of biomedicine. Background Art

[0002] Pulmonary hypertension ( P ulmonary A terial H Pulmonary arterial hypertension (PAH) is a chronic respiratory disease that mainly affects the pulmonary vascular system, has a high mortality rate and poor prognosis. The core pathological basis of PAH is pulmonary vascular remodeling, which mainly originates from the imbalance between proliferation and apoptosis of pulmonary vascular cells. Continuous proliferation of vascular cells leads to occlusive pulmonary vascular remodeling, which causes a gradual increase in pulmonary vascular resistance, and eventually leads to right heart failure or even death.

[0003] At present, first-line clinical drugs such as macitentan and tadalafil mainly relieve PAH symptoms by dilating occluded blood vessels. However, these drugs have limited effects in inhibiting abnormal proliferation of pulmonary vascular cells, reversing vascular remodeling, and preventing disease progression, and it is difficult to effectively avoid disease progression and the need for lung transplantation. Sotatercept, the latest drug developed by Merck, is a trap fusion protein targeting TGF-β superfamily members (such as ligands such as activin) and has significant anti-vascular remodeling effects. However, sotatercept is not effective in treating occlusive pulmonary vascular remodeling caused by obstruction of the bone morphogenetic protein receptor (BMPR) pathway. In addition, its high cost of treatment limits its widespread application and affects patient accessibility.

[0004] Although advances in diagnosis and treatment have improved the quality of life of patients and reduced hospitalization rates to a certain extent, the long-term survival rate of PAH patients has not been fully elucidated because the molecular mechanisms of occlusive pulmonary vascular remodeling have not been fully elucidated. At the same time, high treatment costs and prolonged hospitalization time further increase the socioeconomic burden. Therefore, in-depth research on the molecular mechanisms of pulmonary vascular remodeling and the development of effective interventions targeting these mechanisms are of great significance for improving patient prognosis and reducing socioeconomic burdens.

[0005] In recent years, an increasing number of studies have revealed that the interaction between immune cells and pulmonary artery structural cells plays a crucial role in the process of PAH vascular remodeling. As the barrier between the vascular wall and blood, pulmonary artery endothelial cells (PAECs) are responsible for the exchange of substances and gases, and also directly interact with immune cells and the cytokines and transmitters they secrete. Through direct interaction with PAECs, immune cells regulate processes such as their function, proliferation, migration, and apoptosis, thereby affecting the occurrence and development of vascular remodeling. Therefore, in-depth study of the cell communication mechanism and its regulatory network between PAECs and immune cells not only helps to clarify the molecular pathological mechanism of PAH, but also provides potential research directions for new treatment strategies targeting the interaction between immune cells and endothelial cells.

[0006] Eosinophils are a type of multifunctional immune cells that play important roles in the inflammatory processes triggered by parasitic infections and allergic reactions. A series of previous studies by the applicant have found that eosinophils are involved in the pathophysiological processes of various cardiovascular diseases, and it has been confirmed that eosinophils infiltrating into the lesion sites can improve the damage caused by lesions such as myocardial infarction, myocardial hypertrophy, and abdominal aortic aneurysm (Cardiovascular Research, 2023 a&b; Advanced Science, 2023), but at the same time, it will accelerate the progression of atherosclerosis and vascular calcification (European Heart Journal, 2023). Based on the analysis of the above studies and other relevant literature, the team summarized the diverse roles of eosinophils in cardiovascular diseases and published a review paper titled "Differential roles of eosinophils in cardiovascular disease" (Nature Reviews Cardiology, 2024). We proposed that the phenotypic and functional heterogeneity of eosinophils presented in the lesion tissues may be an important reason for their different manifestations in different pathological and physiological roles.

[0007] Eosinophils have been confirmed to be associated with the occurrence and development of pulmonary arterial hypertension (PAH). In clinical studies, it has been reported that the eosinophil count in patients' blood is positively correlated with the severity of PAH; however, other studies have found that the eosinophil count in PAH patients is significantly lower than that in the normal control group. In PAH animal models and their mechanism studies, Weng et al. confirmed that eosinophils induce the occurrence of PAH by releasing chemokines such as CCL11 and CCL24 to recruit macrophages; while Shu et al. found that eosinophils can prevent the progression of PAH by inhibiting perivascular inflammation and maintaining the homeostasis of pulmonary artery smooth muscle cells. The conclusions drawn from the above clinical and experimental studies are contradictory, suggesting that the mechanism of action of eosinophils in PAH is not clear and further in-depth exploration is urgently needed.

[0008] The most significant feature of eosinophils is that their cytoplasm is rich in secondary granules, which store a unique set of basic proteins, including eosinophil cationic protein ( E osinophil C ationic P rotein, ECP), etc. When stimulated by different stimuli, eosinophils will rapidly and selectively secrete the proteins in these granules, mediating cell-to-cell communication and interaction, and then regulating various inflammatory and immune responses. Therefore, focusing on the changes in granule proteins helps to more accurately reveal the role of eosinophils in diseases. Summary of the Invention

[0009] In view of the deficiencies of the prior art, the present invention provides the use of eosinophil cationic protein in the preparation of drugs for preventing and treating pulmonary arterial hypertension.

[0010] The technical solution of the present invention is as follows: Use of eosinophil cationic protein in the preparation of drugs for preventing and treating pulmonary arterial hypertension.

[0011] Preferably according to the present invention, the eosinophil cationic protein selectively activates the Smad1 / 5 / 8 signaling pathway by specifically binding to bone morphogenetic protein receptors BMPR-1A / BMPR-2 on the surface of pulmonary artery endothelial cells (PAECs), thereby inhibiting the abnormal apoptosis of PAECs and maintaining endothelial homeostasis.

[0012] Preferably according to the present invention, the eosinophil cationic protein is a recombinantly expressed eosinophil cationic protein.

[0013] Use of a substance overexpressing eosinophil cationic protein in the preparation of drugs for preventing and treating pulmonary arterial hypertension.

[0014] Preferably according to the present invention, the substance overexpressing eosinophil cationic protein includes: a recombinant vector containing the coding gene of eosinophil cationic protein, a recombinant virus containing the coding gene of eosinophil cationic protein, and a recombinant viral vector containing the coding gene of eosinophil cationic protein.

[0015] A drug for preventing and treating pulmonary arterial hypertension, comprising eosinophil cationic protein or a substance overexpressing eosinophil cationic protein.

[0016] Preferably according to the present invention, the drug for preventing and treating pulmonary arterial hypertension further comprises a pharmaceutically acceptable carrier or adjuvant.

[0017] Preferably according to the present invention, the dosage form of the drug for preventing and treating pulmonary arterial hypertension includes an oral preparation or an injection preparation.

[0018] Beneficial effects: As a sensitive marker of eosinophil activation, ECP is not only widely used as a biomarker for various human diseases to predict clinical events; but also has been proven to regulate the pathophysiological state of related cells, thereby affecting the disease process. The present invention first found that the ECP level in the plasma of PAH patients and the ECP level bound to the endothelial surface of distal pulmonary arteries were both significantly down-regulated, suggesting that the decrease in ECP level may be closely related to the process of pulmonary vascular remodeling. Further animal experiments confirmed the promoting effect of ECP deficiency on the occurrence and development of PAH. The present invention also confirmed that ECP can specifically bind to the BMPR-1A / BMPR-2 receptors of pulmonary artery endothelial cells. In addition, the present invention also confirmed that exogenous recombinant ECP can inhibit pulmonary vascular remodeling and relieve the occurrence and development of pulmonary arterial hypertension, including reducing vascular wall thickening, collagen fiber deposition and relieving right heart function, providing a new therapeutic target and potential therapeutic strategy for the treatment of PAH. Description of the drawings

[0019] Figure 1 Scatter plot of ECP levels detected by ELISA in the plasma of the PAH group and the Non-PAH group.

[0020] Figure 2 Results of immunohistochemical detection of ECP binding levels on the endothelial surface of distal pulmonary arteries in the PAH group and the Non-PAH group; among them, A is the immunohistochemical detection picture, and B is the scatter plot of ECP binding levels on the endothelial surface.

[0021] Figure 3 H&E staining results of distal pulmonary arteries in the PAH group and the Non-PAH group; among them, A is the H&E staining picture, and B is the bar chart of the proportion of the media in the total vascular thickness.

[0022] Figure 4Results of Masson staining of distal pulmonary arterioles in the PAH group and the Non-PAH group; among them, A is the picture of Masson staining, and B is the bar graph of the proportion of collagen fibers.

[0023] Figure 5 Results of immunofluorescence staining detection of the endothelial cell marker vWF in distal pulmonary arterioles in the PAH group and the Non-PAH group; among them, A is the picture of immunofluorescence staining detection, and B is the bar graph of optical density.

[0024] Figure 6 Results of immunofluorescence staining detection of the smooth muscle cell marker αSMA in distal pulmonary arterioles in the PAH group and the Non-PAH group; among them, A is the picture of immunofluorescence staining detection, and B is the bar graph of optical density.

[0025] Figure 7 Distribution of the main subsets of eosinophils in the lung tissue of PH model mice.

[0026] Figure 8 Gene expression distribution in each eosinophil subset in the hypoxic-induced PH model group (Hypoxia) and the normoxic control group (Normoxia) of mice.

[0027] Figure 9 Distribution and abundance of mEar1 gene expression in each eosinophil subset.

[0028] Figure 10 Results of flow cytometry detection of mEar1-positive eosinophils in the lung tissue of the hypoxic-induced PH model group and the normoxic control group of mice; among them, A is the picture of flow cytometry detection, and B is the bar graph of the proportion of mEar1-positive eosinophils.

[0029] Figure 11 Schematic diagram of the construction process of PH model mice.

[0030] Figure 12 For mEar1 - / - HE staining pictures, Masson staining pictures and immunofluorescence staining pictures of distal pulmonary arterioles in the PH models of mEar1 mice and wild-type mice.

[0031] Figure 13 For mEar1 - / - Results of right ventricular systolic pressure (RVSP) detection in the PH models of mEar1 mice and wild-type mice; among them, A is the curve of right ventricular pressure change, and B is the scatter plot of right ventricular systolic pressure.

[0032] Figure 14 For mEar1 - / - Scatter plot of Fulton index of right ventricular remodeling degree in the PH models of mEar1 mice and wild-type mice.

[0033] Figure 15 is mEar1 - / - Western blot detection graphs of key protein molecules in the lung tissues of PH models of mice and wild-type mice.

[0034] Figure 16 Schematic diagram of the construction process of PH model rats.

[0035] Figure 17 Bar graph of the right ventricular systolic pressure (RVSP) of rats in the Model group and the Control group.

[0036] Figure 18 Bar graph of the Fulton index of the degree of right ventricular remodeling of rats in the Model group and the Control group.

[0037] Figure 19 HE staining pictures of the lung tissues of rats in the Model group and the Control group, and immunofluorescence staining pictures of α-SMA and vWF.

[0038] Figure 20 Multicolor immunofluorescence detection pictures of lung tissue samples in the PAH group and the Non-PAH group.

[0039] Figure 21 Co-IP detection pictures of BMPR-1A, BMPR-1B, and BMPR-2 in ECP and PAECs.

[0040] Figure 22 Results of Scatchard plot evaluation of the binding ability between ECP and pulmonary artery endothelial cells; among them, A is the Scatchard plot curve under the condition of Control SiRNA, B is the Scatchard plot curve under the conditions of BMPR-1A SiRNA and BMPR-2 SiRNA, and C is the binding ability curve under different knockout conditions.

[0041] Figure 23 H&E staining pictures, Masson staining pictures, and immunofluorescence staining pictures of α-SMA and vWF of the lung tissues of mice in the Control group and the mEar1 group.

[0042] Figure 24 Scatter plot of the right ventricular systolic pressure (RVSP) of mice in the Control group and the mEar1 group.

[0043] Figure 25 Scatter plot of the Fulton index of the degree of right ventricular remodeling of mice in the Control group and the mEar1 group. Specific implementation manners

[0044] The technical solutions of the present invention will be further described below in conjunction with embodiments. However, the protection scope of the present invention is not limited thereto. Reagents and materials involved in the embodiments are all ordinary commercially available products without special instructions. Experimental steps involved in the embodiments are all conventional experimental operations in the art without special instructions.

[0045] Term Explanation: Pulmonary Arterial Hypertension: Generally expressed as PH in animal models and as PAH in humans or other conclusive statements.

[0046] Eosinophil Cationic Protein: Human eosinophil cationic protein is expressed as ECP, and murine eosinophil cationic protein is expressed as mEar1.

[0047] Material Source: All clinical samples of the present invention were taken from the Department of Cardiology, Guangdong Provincial People's Hospital. After obtaining the consent of the patients, 36 blood samples of PAH patients, 25 control blood samples of healthy people (Non-PAH), 8 lung tissue samples of PAH patients, and 7 control lung tissue samples of Non-PAH were included.

[0048] Example 1: The ECP level in PAH patients is negatively correlated with pulmonary vascular remodeling 1. Take the blood samples of the PAH group and the Non-PAH group. After separating the plasma, perform ELISA detection to analyze the ECP level in the plasma of PAH patients.

[0049] The ELISA detection results are as Figure 1 shown. Compared with the Non-PAH group, the ECP level in the plasma of the PAH group was significantly decreased.

[0050] 2. Take the lung tissue samples of the PAH group and the Non-PAH group, perform immunohistochemical analysis, and detect the ECP level bound to the surface of the vascular endothelium of the distal pulmonary arterioles.

[0051] The detection results by immunohistochemistry are as Figure 2 shown. Compared with the Non-PAH group, the ECP level bound to the surface of the vascular endothelium of the distal pulmonary arterioles in the PAH group was also significantly decreased.

[0052] 3. Take the lung tissue samples of the PAH group and the Non-PAH group, perform H&E staining and Masson staining respectively, and conduct histological morphology research on the distal pulmonary arterioles of PAH patients.

[0053] The H&E staining results of the distal pulmonary arterioles are asFigure 3 As shown, the number of vascular wall cells in the PAH group was significantly increased, suggesting that the vascular wall of the distal pulmonary arterioles in PAH patients was significantly thickened.

[0054] The Masson staining results of the distal pulmonary arterioles are as Figure 4 shown. The content of collagen fibers in the vascular wall of the PAH group was significantly increased, indicating that the collagen deposition in the distal pulmonary arterioles of PAH patients was significantly increased.

[0055] 4. Take lung tissue samples from the PAH group and the Non-PAH group, perform immunofluorescence staining of the endothelial cell marker vWF and the smooth muscle cell marker αSMA, and detect the proliferation levels of endothelial cells and smooth muscle cells in the distal pulmonary arterioles. Among them, the polyclonal antibody of vWF used for immunofluorescence staining was purchased from abcam (ab6994, 1:300), and the monoclonal antibody of α-SMA was purchased from Cell Signaling Technology (D4K9N, 1:300).

[0056] The detection results of immunofluorescence staining of the endothelial cell marker vWF are as Figure 5 shown, and the detection results of immunofluorescence staining of the smooth muscle cell marker αSMA are as Figure 6 shown. The results showed that compared with the Non-PAH group, the proliferation of endothelial cells and smooth muscle cells in the distal pulmonary arterioles of the PAH group was significantly increased.

[0057] The above results indicate that compared with the Non-PAH group, the plasma ECP level and the ECP level bound to the endothelial surface of the distal pulmonary arterioles in the PAH group were significantly down-regulated, accompanied by thickening of the distal pulmonary arteriole vascular wall, increased collagen deposition, and enhanced proliferation activity of endothelial cells and smooth muscle cells, suggesting that the decrease in ECP level may be closely related to the process of pulmonary vascular remodeling.

[0058] Example 2: Analysis of the characteristics and dynamic changes of eosinophil subsets in the lung tissue of PAH model mice IL-5 transgenic (IL-5 Tg, NJ1638) mice (purchased from Cyagen Biosciences Inc.) can significantly promote the differentiation, proliferation and survival of eosinophils by artificially overexpressing the interleukin-5 (IL-5) gene, resulting in a continuous increase in eosinophils in peripheral blood and tissues. The NJ1638 mice were continuously raised in a 10% O2 hypoxic environment for 4 weeks to induce a PH animal model; at the same time, a normoxic control group was set up.

[0059] Twenty-eight days after hypoxia exposure of the NJ1638 mice, eosinophils in the lung tissue were sorted and single-cell transcriptome sequencing was performed. The results are as Figure 7 shown. There was a mEar1-positive eosinophil subset in the lung tissue of the PH model mice (mEar1+ Eosinophils), and compared with the control group of mice, the results are as Figure 8 , the proportion of cells in this subset decreased significantly.

[0060] Furthermore, the distribution and abundance of mEar1 gene expression in each eosinophil subset were detected, and the results are as Figure 9 shown. In the PH model induced by hypoxia, mEar1 + The expression level of mEar1 in the Eosinophils subset was significantly lower than that in the control group.

[0061] In addition, this result was verified in the lung tissue of the PH model of wild-type mice induced by hypoxia. Specifically, C57 mice at 6 - 8 weeks old (purchased from Guangzhou Regene Biotechnology Co., Ltd.) were placed in a hypoxic environment with 10% O2 and continuously raised for 4 weeks to induce a PH animal model; meanwhile, a normoxic control group was set up.

[0062] mEar1-positive eosinophils in the lung tissue of PH model mice were detected by flow cytometry, and the results are as Figure 10 shown, which confirmed that the mEar1-positive eosinophil subset also decreased significantly during the occurrence of PH induced by hypoxia, suggesting that this cell subset may participate in the dynamic regulation of the pulmonary vascular microenvironment related to PAH.

[0063] Example 3: Deletion of mEar1 in mice accelerates the occurrence and progression of PH The construction process of the PH model mice is as Figure 11 shown. Specifically, mEar1 - / - mice (purchased from Cyagen Biosciences Inc.) and wild-type C57 mice (purchased from Guangzhou Regene Biotechnology Co., Ltd.) were placed in a hypoxic environment with 10% O2 and continuously raised for 4 weeks to induce a PH animal model.

[0064] 1. Take lung tissue samples from mEar1 - / - mice and wild-type mice with PH models, and perform H&E staining, Masson staining, and immunofluorescence staining of vWF, α-SMA, and mEar1 respectively to conduct histomorphological studies on the distal pulmonary arterioles of PH model mice. Among them, the vWF polyclonal antibody used for immunofluorescence staining was purchased from abcam (ab6994, 1:300), the α-SMA monoclonal antibody was purchased from Cell Signaling Technology (D4K9N, 1:300), and the mEar1 polyclonal antibody was purchased from biorbyt (orb13385, 1:300).

[0065] The staining results are as Figure 12 shown. Compared with the PH model of wild-type mice, mEar1 - / -In the PH model of mice, the vascular wall cells are significantly increased, the content of collagen fibers in the vascular wall is significantly increased, the proliferation of vascular endothelial cells and smooth muscle cells is significantly increased, but the binding level between the vascular endothelial surface and mEar1 is significantly down-regulated.

[0066] 2. Take mEar1 - / - PH models of mice and wild-type mice, and detect the right ventricular systolic pressure (RVSP) and Fulton index [RV / (LV+S)] of mice by right heart catheterization to evaluate the degree of right ventricular remodeling in mice.

[0067] The detection results of the right ventricular systolic pressure (RVSP) of mice are as Figure 13 shown, and the detection results of the Fulton index are as Figure 14 shown. The results show that compared with the PH model of wild-type mice, the right ventricular systolic pressure and Fulton index of the PH model of mEar1 - / - mice are significantly up-regulated.

[0068] The above experimental results show that in the PH model mice with mEar1 gene knockout (mEar1 – / – ), the distal pulmonary arterioles show significantly aggravated vascular remodeling characteristics, including obvious thickening of the vascular wall, increased collagen deposition, enhanced proliferation activity of endothelial cells and smooth muscle cells, and at the same time, the binding level of mEar1 on the surface of the distal pulmonary arterioles is basically disappeared. Functionally, the right ventricular function of the PH model of mEar1 – / – mice deteriorates further, manifested as a significant increase in the right ventricular systolic pressure (RVSP) and Fulton index [RV / (LV+S)].

[0069] 3. Take the lung tissue samples of mEar1 - / - mice and wild-type mice PH models, extract proteins and then perform WB detection to analyze the expression levels of key molecules of the Smad-dependent signaling pathway, phosphorylated Smad1 / 5 / 8, ID1 and the non-dependent signaling pathway phosphorylated p38MAPK, JNK and apoptosis-related protein Cleaved caspase-3. Among them, the polyclonal antibody against phosphorylated Smad1 / 5 / 8 used for WB detection was purchased from Sigma-Aldrich (AB3848-I, 1:1000), the polyclonal antibody against ID1 was purchased from abcam (ab168256, 1:1000), the monoclonal antibody against phosphorylated p38 MAPK was purchased from Cell Signaling Technology (4511T, 1:1000), the monoclonal antibody against phosphorylated JNK was purchased from Cell Signaling Technology (4668T, 1:1000), and the monoclonal antibody against Cleaved caspase-3 was purchased from Cell Signaling Technology (D 175, 1:1000).

[0070] The WB detection results are as Figure 15 shown. Molecular mechanism analysis shows that in the lung tissues of the mEar1 – / – mouse PH model, the transduction of the Smad-dependent signaling pathway is blocked, manifested as a significant decrease in the expression levels of phosphorylated Smad1 / 5 / 8 and its downstream target gene ID1; at the same time, there is abnormal activation of the Smad-independent signaling pathway, with significantly increased levels of phosphorylated p38 MAPK and JNK, and an increase in the expression of the key apoptosis protein Cleaved caspase-3, suggesting that the lack of ECP may promote PAH-related pulmonary vascular remodeling and right ventricular function impairment by inhibiting the Smad-dependent signal and over-activating the Smad-independent pathway.

[0071] Example 4: Deletion of mEar1 in rats accelerates the occurrence and progression of PH The construction process of the PH model rats is as Figure 16 shown. Specifically, male Sprague-Dawley (SD) rats aged 6 - 8 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected and divided into a Control group (igG control and Anti-ECP groups) and a Model group (igG control and Anti-ECP groups). The rats in the Control group were raised in a normoxic environment (21% O2); after the rats in the Model group were subcutaneously injected with Sugen5416 (20 mg / kg) in the neck, they were placed in a hypoxic chamber (oxygen concentration maintained at 10%) and continuously exposed for 3 weeks to induce a PH animal model. After 3 weeks, the rats in the Model group were moved to normoxic conditions and continued to be raised for 3 weeks. During the entire breeding period, the Anti-ECP rats in the Control group and the Model group were subcutaneously injected with an ECP neutralizing antibody (Anti-ECP) once every 5 days in the neck, and the igG control rats were subcutaneously injected with igG once every 5 days, 10 μg each time, for a total of 8 injections. After the breeding ended, hemodynamic parameters of the rats were measured and tissues were taken. The ECP neutralizing antibody (Anti-ECP) was purchased from Bojia, IPB2702.

[0072] 1. Take the rats in the Model group and the Control group, and detect the right ventricular systolic pressure (RVSP) and Fulton index [RV / (LV + S)] of the rats by right heart catheterization to evaluate the degree of right ventricular remodeling in the rats.

[0073] The detection results of the right ventricular systolic pressure (RVSP) of the rats are as Figure 17 shown, and the detection results of the Fulton index are as Figure 18As shown, the results showed that compared with the rats in the Control group, the right ventricular systolic pressure and Fulton index of the rats in the Model group were significantly up-regulated. Among the rats in the Model group, the right ventricular systolic pressure and Fulton index of the rats in the Anti-ECP group were significantly up-regulated compared with those in the IgG control group.

[0074] 2. Take the lung tissue samples of the rats in the Model group and the Control group, and perform H&E staining and immunofluorescence staining of vWF and α-SMA respectively to conduct a histomorphological study on the distal pulmonary arterioles of the PH model rats.

[0075] The staining results are as Figure 19 shown. Compared with the rats in the Control group, the vascular wall cells of the rats in the Model group were significantly increased, and the proliferation of vascular endothelial cells and smooth muscle cells was significantly increased. Among the rats in the Model group, the remodeling of the distal pulmonary arterioles of the rats in the Anti-ECP group was further aggravated.

[0076] The above experimental results indicate that in the PH model rats, the application of anti-ECP neutralizing antibody was observed to have a similar aggravation of the remodeling of the distal pulmonary arterioles as in mEar1 – / – mice, including thickening of the vascular wall, enhanced proliferation activity of endothelial and smooth muscle cells, accompanied by a significant increase in RVSP and Fulton index, further verifying the promoting effect of ECP deficiency on the occurrence and development of PAH.

[0077] Example 5: ECP can specifically bind to the BMPR-1A / 2 receptors of pulmonary artery endothelial cells 1. Take the lung tissue samples of the PAH group and the Non-PAH group, and use multi-color immunofluorescence detection to analyze the expression of BMPR-1A, BMPR-1B and BMPR-2 on the surface of the distal pulmonary arteriole endothelium in the PAH group and the Non-PAH group, and their co-localization with ECP on endothelial cells. Among them, the BMPR-1A antibody used for multi-color immunofluorescence detection was purchased from ThermoFisher Scientific Invitrogen (PA5-11856, 1:100), the BMPR-1B antibody was purchased from Proteintech (14312-1-AP, 1:300), the BMPR-2 antibody was purchased from Thermo Fisher Scientific Invitrogen (MA5-15826, 1:300), and the ECP antibody was purchased from biorbyt (orb 156688, 1:300).

[0078] The detection results are as Figure 20As shown, the expression level of BMPR-1B in the vascular endothelial cells of distal small pulmonary arteries in the PAH group was extremely low, while the expression levels of BMPR-1A and BMPR-2 were relatively high. However, compared with the Non-PAH group, the expressions of BMPR-1A, BMPR-1B, and BMPR-2 were all significantly downregulated in the lung tissues of PAH patients. In addition, there was an obvious co-localization between ECP and BMPR-1A and BMPR-2, and in the lung tissues of PAH patients, this co-localization signal was significantly weakened.

[0079] 2. After co-incubating the ECP recombinant protein (Aviscera Bioscience, human origin) with human pulmonary artery endothelial cells (PAECs, ATCC), Co-IP experiments were performed. The Co-IP detection results are as Figure 21 shown, confirming that ECP can bind to BMPR-1A and BMPR-2 in PAECs.

[0080] Furthermore, human pulmonary artery endothelial cells (PAECs, ATCC) were transfected with Control siRNA, BMPR-1A siRNA, BMPR-1B siRNA, BMPR-2 siRNA, or co-transfected with BMPR-1A siRNA and BMPR-2 siRNA to knockdown the expression levels of BMPR-1A or BMPR-1B or BMPR-2 or BMPR-1A and BMPR-2 in PAECs. After 48 hours, they were incubated with fluorescent / biotin-labeled ECP recombinant protein at gradient concentrations (0 - 100 nM) at 4°C for 2 hours. After washing, the binding signals were detected; the dissociation constant (Kd) and the maximum binding capacity (Bmax) were calculated by Scatchard plot (Bound / Free vs Bound) to verify the changes in the affinity between ECP and PAECs in the knockdown groups.

[0081] Among them, the nucleotide sequence of Control siRNA is as follows: 5′-UUCUCCGAACGUGUCACGU(dT)(dT)-3′, The nucleotide sequence of BMPR-1A siRNA is as follows: Sense strand: 5′-GAUCCGUCAUACGAAGAUA(dT)(dT)-3′, Antisense strand: 5′-UAUCUUCGUAUGACGGAUC(dT)(dT)-3′, The nucleotide sequence of BMPR-1B siRNA is as follows: Sense strand: 5′-GGACUAUAGCUAAGCAGAU(dT)(dT)-3′, Antisense strand: 5′-AUCUGCUUAGCUAUAGUCC(dT)(dT)-3′.

[0082] The nucleotide sequence of BMPR-2 siRNA is as follows: Sense strand: 5′-CCAAGAUGAAUACAAUCAA(dT)(dT)-3′, Antisense strand: 5′-UUGAUUGUAUUCAUCUUGG(dT)(dT)-3′.

[0083] The results of ligand-receptor binding experiments combined with Scatchard plot analysis are as Figure 22 shown. After knocking down BMPR-1A or BMPR-1B or BMPR-2, the affinity of ECP for PAECs was significantly reduced. Especially after knocking down BMPR-1A and BMPR-2 simultaneously, the affinity of ECP for PAECs was the lowest.

[0084] The above experimental results indicate that further immunoprecipitation, ligand-receptor binding experiments and Scatchard plot analysis results confirm that ECP can specifically bind to the BMPR-1A / BMPR-2 receptors of pulmonary artery endothelial cells.

[0085] Example 6: Therapeutic effect of murine mEar1 on PH C57 mice at 6-8 weeks of age (purchased from Guangzhou Regene Biotech Co., Ltd.) were selected and divided into a Control group and an mEar1 group. The mice in the Control group and the mEar1 group were placed in a 10% O2 hypoxic environment and continuously raised for 4 weeks to induce a PH animal model. During the feeding period, each mouse in the mEar1 group was subcutaneously injected with a total of 40 μg of mEar1 recombinant protein, injected 3 times a week, and each mouse in the Control group was subcutaneously injected with an equal amount of PBS buffer. Among them, the mEar1 recombinant protein was purchased from Aviscera Bioscience and was murine-derived.

[0086] 1. Take lung tissue samples from the mice in the Control group and the mEar1 group, and perform H&E staining, Masson staining, and immunofluorescence staining of α-SMA and vWF respectively to conduct histomorphological studies on the distal pulmonary arterioles of the PH model mice.

[0087] The staining results are as Figure 23 shown. Compared with the mice in the Control group, the content of vascular wall cells and vascular wall collagen fibers in the mice in the mEar1 group was significantly reduced, the proliferation of vascular endothelial cells and smooth muscle cells was significantly decreased, and vascular remodeling was effectively inhibited.

[0088] 2. Take the mice in the Control group and the mEar1 group, and detect the right ventricular systolic pressure (RVSP) and Fulton index [RV / (LV+S)] of the mice by right heart catheterization to evaluate the degree of right ventricular remodeling in the rats.

[0089] The detection results of the right ventricular systolic pressure (RVSP) of the mice are as Figure 24 shown, and the detection results of the Fulton index are as Figure 25 shown. The results show that compared with the mice in the Control group, the right ventricular systolic pressure and Fulton index of the mice in the mEar1 group are significantly down-regulated, and pulmonary hypertension is significantly alleviated.

[0090] In summary, eosinophil cationic protein (ECP) selectively activates the Smad1 / 5 / 8 signaling pathway by specifically binding to the bone morphogenetic protein receptors BMPR-1A / BMPR-2 on the surface of pulmonary artery endothelial cells (PAECs), thereby inhibiting the abnormal apoptosis of PAECs and maintaining endothelial homeostasis. At the same time, exogenous recombinant ECP can significantly improve the vascular remodeling of PAH, including reducing vascular wall thickening, collagen fiber deposition and alleviating right heart function, providing a new therapeutic target and potential therapeutic strategy for the treatment of PAH.

Claims

1. Use of eosinophil cationic protein in the preparation of a drug for preventing and treating pulmonary arterial hypertension.

2. The application according to claim 1, wherein The eosinophil cationic protein selectively activates the Smad1 / 5 / 8 signaling pathway by specifically binding to bone morphogenetic protein receptors BMPR-1A / BMPR-2 on the surface of PAECs, thereby inhibiting the abnormal apoptosis of PAECs and maintaining endothelial homeostasis.

3. The application according to claim 1, wherein The eosinophil cationic protein is a recombinantly expressed eosinophil cationic protein.

4. Use of a substance overexpressing eosinophil cationic protein in the preparation of a drug for preventing and treating pulmonary arterial hypertension.

5. The application according to claim 4, wherein The substance overexpressing eosinophil cationic protein includes: a recombinant vector containing the coding gene of eosinophil cationic protein, a recombinant virus containing the coding gene of eosinophil cationic protein, and a recombinant viral vector containing the coding gene of eosinophil cationic protein.

6. A drug for preventing and treating pulmonary hypertension, characterized in that, A substance including eosinophil cationic protein or a substance overexpressing eosinophil cationic protein.

7. The drug according to claim 6, characterized in that, The drug for preventing and treating pulmonary arterial hypertension further includes a pharmaceutically acceptable carrier or adjuvant.

8. The drug according to claim 6, characterized in that, The dosage form of the drug for preventing and treating pulmonary arterial hypertension includes an oral preparation or an injection preparation.

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