Application of Ero1α as a Biomarker and Therapeutic Target for Pulmonary Arterial Hypertension

Abstract

The present invention discloses the application of Ero1α as a biomarker and therapeutic target for pulmonary hypertension. Examples of the present invention show that Ero1α is highly expressed in hypoxic pulmonary artery smooth muscle cells and pulmonary tissue with pulmonary hypertension. Knocking down Ero1α reduces the proliferation and migration levels of pulmonary artery smooth muscle cells, while overexpressing Ero1α causes the opposite effect. At the animal level, knocking down the expression of Ero1α can significantly alleviate pulmonary hypertension caused by hypoxia and MCT. The present invention reveals that Ero1α can be used as a new target for the diagnosis and treatment of pulmonary hypertension, and therapeutic strategies targeting Ero1α can be developed to provide more effective treatment methods for patients with pulmonary hypertension. As a key biomarker and therapeutic target, Ero1α has important application prospects in the diagnosis and treatment of pulmonary hypertension.

Description

Technical Field

[0001] The present invention relates to the technical field of detection and treatment of pulmonary hypertension, and particularly relates to the application of Ero1α as a biomarker and therapeutic target for pulmonary hypertension. Background Art

[0002] Pulmonary hypertension (PH) is a disease characterized by pulmonary vasoconstriction and pulmonary vascular remodeling caused by various reasons. The global incidence of PH is about 1%, and in people over 65 years old, the incidence rate is as high as 10%. And since the day of right heart catheterization diagnosis, the 7-year survival rate is only 49%. Its high incidence and mortality make the research on the pathophysiological mechanism of PH extremely urgent. The current targeted therapeutic drugs for PH (such as prostaglandins, phosphodiesterase-5 inhibitors, endothelin receptor antagonists, and soluble guanylate cyclase stimulators) can improve the symptoms of patients and reduce the hospitalization rate. However, the main effect of these targeted therapeutic drugs is to dilate blood vessels, which can only temporarily relieve symptoms, cannot improve pulmonary vascular remodeling, and cannot reduce the mortality rate of PH patients. Therefore, studying the pathogenesis of pulmonary vascular remodeling is the top priority for improving the survival rate of PH patients.

[0003] Pulmonary vascular remodeling is the central link in the pathogenesis of PH. Among them, the "tumor cell-like" characteristics exhibited by pulmonary artery smooth muscle cells (PASMCs): abnormal biological behaviors such as excessive proliferation and migration are the key reasons leading to pulmonary vascular remodeling. Therefore, exploring the regulatory mechanism of the abnormal biological behaviors of PASMCs is of great significance for improving pulmonary vascular remodeling and finding new therapeutic targets for PH.

[0004] In PASMCs involved in the pathogenesis of PH, the expression levels of multiple proteins such as Drp1, Survivin, and Rhi Kinase have been found to be up-regulated, and they promote the development of PH, indicating that the changes in the protein expression levels in PASMCs have a crucial impact on the biological behaviors of PASMCs. However, there has been no scientific research team that has comprehensively studied the protein expression levels in PASMCs involved in the pathogenesis of PH. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides the application of Ero1α as a biomarker and therapeutic target for pulmonary hypertension, that is, a technology for comprehensively detecting and screening biomarkers for pulmonary hypertension through proteomic analysis, and further exploring its biological role in pulmonary hypertension through cell experiments and animal experiments. To achieve the above object, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention discloses a biomarker for pulmonary hypertension, and the biomarker is Ero1α gene or Ero1α protein;

[0007] In a second aspect, the present invention discloses the use of a biomarker for pulmonary hypertension, specifically:

[0008] The use includes at least one of (1)-(6):

[0009] (1) Use in the preparation of a product for screening pulmonary hypertension;

[0010] (2) Use in the preparation of a product for assessing the risk of pulmonary hypertension;

[0011] (3) Use in the preparation of a product for diagnosing pulmonary hypertension;

[0012] (4) Use in the preparation of a product for treating pulmonary hypertension;

[0013] (5) Use in the preparation of a product for monitoring the course of pulmonary hypertension;

[0014] (6) Use in the preparation of a product for analyzing the prognosis of pulmonary hypertension.

[0015] Preferably, the product for treating pulmonary hypertension includes: a product for reducing the expression level of Ero1α.

[0016] The reagent is a reagent for detecting the expression level of the biomarker.

[0017] It also includes a drug or a kit, including the biomarker described in claim 1, or its detection reagent, inhibitor; the drug or the kit includes at least one of the above-mentioned uses (1)-(6).

[0018] Preferably, the product for treating pulmonary hypertension includes a product for reducing the expression level of Ero1α.

[0019] It also includes a method for screening the above-mentioned drug or kit, which is screened by detecting the difference in the expression level of Ero1α in the subject and the control group.

[0020] It also includes the application of Ero1α gene as a target gene in screening drugs for preventing, alleviating and / or treating pulmonary hypertension, and the screening is to screen substances capable of inhibiting the expression level of Ero1α gene or its expression product.

[0021] It also includes the application of Ero1α protein as a target in screening drugs for preventing, alleviating and / or treating pulmonary hypertension, and the screening is to screen substances capable of reducing the activity of Ero1α protein.

[0022] Also included is the use of a substance that inhibits the expression of the Ero1α gene or reduces the expression level of its product in the preparation of a drug for preventing, alleviating, and / or treating pulmonary hypertension. The substance that inhibits the expression of the Ero1α gene includes a recombinant vector or recombinant virus capable of knocking down the Ero1α gene.

[0023] The present invention focuses on the Ero1α gene or the Ero1α protein expressed thereby as a biomarker for pulmonary hypertension (PH). Its applications cover the entire process from screening, risk assessment, diagnosis, treatment, disease course monitoring to prognosis analysis, demonstrating the broad potential of this biomarker in the development of pulmonary hypertension-related products.

[0024] In the above-mentioned scheme, the applications of the biomarker are elaborated as follows:

[0025] 1) Screening products: Used for early identification of pulmonary hypertension patients, in the form of blood test kits that can rapidly detect the Ero1α protein or its gene expression level.

[0026] 2) Risk assessment products: Help assess the risk of an individual developing pulmonary hypertension by analyzing the correlation between the Ero1α expression level and the risk.

[0027] 3) Diagnostic products: Used for the confirmation of pulmonary hypertension through highly specific Ero1α detection techniques.

[0028] 4) Treatment products: Focus on reducing the Ero1α expression level to treat pulmonary hypertension, including gene silencing therapy, small molecule inhibitors, etc.

[0029] 5) Disease course monitoring products: Track the changes in the condition of pulmonary hypertension patients by regularly detecting the Ero1α expression level.

[0030] 6) Prognosis analysis products: Evaluate the prognosis of pulmonary hypertension patients based on the relationship between the Ero1α expression level and the prognosis.

[0031] 7) Reagent and drug development: Reagents: Reagents for detecting the Ero1α expression level, including antibodies, probes, etc.; Drugs or test kits: Containing the Ero1α biomarker or its detection reagents, inhibitors, for the various applications mentioned above.

[0032] 8) Screening methods and applications: Screening methods, by detecting the difference in the Ero1α expression level in a subject, screening out effective drugs or test kits; Drug screening applications, using the Ero1α gene as a target gene to screen for drugs or substances that can inhibit its expression or reduce the activity of its product.

[0033] 9) Strategies for inhibiting Ero1α expression: Substances that inhibit the expression of the Ero1α gene, including recombinant vectors or recombinant viruses capable of knocking down the Ero1α gene, provide a new approach for the treatment of pulmonary hypertension.

[0034] The present invention not only reveals the importance of Ero1α in pulmonary hypertension but also provides a theoretical basis for the development of related diagnostic and therapeutic products, demonstrating the translational potential from basic research to clinical application.

[0035] The technical principle of the present invention is as follows:

[0036] To comprehensively explore the proteins in PASMCs that may affect their biological behavior, the present invention simultaneously submitted the data of rat PASMCs cultured under normoxia and hypoxia for data-independent analysis (DIA) of proteomics to screen for the proteins with upregulated expression in PASMCs cultured under hypoxia. The most significantly upregulated protein in PASMCs cultured under hypoxia was obtained through subsequent qRT-PCR and Western blot verification: endoplasmic reticulum oxidase 1α (Ero1α). Cell experiments confirmed that knocking down Ero1α could reduce the proliferation and migration levels of PASMCs cultured under normoxia and hypoxia, while overexpressing Ero1α had the opposite effect. Animal experiments confirmed that knocking down Ero1α could significantly alleviate pulmonary hypertension induced by hypoxia and MCT. This indicates that Ero1α is a key molecule that promotes the excessive proliferation and migration of PASMCs, thereby causing pulmonary vascular remodeling and ultimately leading to the occurrence and development of pulmonary hypertension. This reveals a key new target in the central environment of the pathogenesis of pulmonary hypertension and will provide an important basis for the prevention and treatment of pulmonary hypertension.

[0037] The advantages and beneficial effects of the present invention are as follows:

[0038] 1. Comprehensively explored the protein expression of pulmonary artery smooth muscle cells under hypoxia: Through proteomic analysis, the present invention comprehensively reveals the proteins with upregulated and downregulated expression in hypoxic pulmonary artery smooth muscle cells, which is of great significance for understanding the "tumor cell-like" characteristics of pulmonary artery smooth muscle cells under hypoxia - excessive proliferation and migration.

[0039] 2. Identified the molecule with the most significant increase in expression under hypoxia in hypoxic pulmonary artery smooth muscle cells: Through verification by experimental techniques such as qRT-PCR and Western blot, the molecule with the most obvious up-regulation of expression under hypoxia in hypoxic pulmonary artery smooth muscle cells was obtained: Ero1α. The Ero1α gene was discovered in 1998, and its research history is only over twenty years. There is still little research on the biological function of this molecule and it mainly focuses on neoplastic diseases. The present invention first confirmed that Ero1α is the molecule with the most significant up-regulation of expression in hypoxic pulmonary artery smooth muscle cells.

[0040] 3. Verified the harmful effect of Ero1α in pulmonary arterial hypertension: The present invention confirmed that Ero1α is highly expressed in hypoxic pulmonary artery smooth muscle cells. Pulmonary artery smooth muscle cells showed excessive proliferation and migration levels under hypoxic conditions, while knocking down Ero1α could reduce the proliferation and migration levels of pulmonary artery smooth muscle cells. At the same time, Ero1α was highly expressed in the lung tissues of hypoxic and MCT-induced pulmonary arterial hypertension, and was also highly expressed in the pulmonary arteries of human chronic obstructive pulmonary disease with pulmonary arterial hypertension. However, knocking down the expression of Ero1α in the lung tissue by transfecting adeno-associated virus significantly alleviated hypoxic and MCT-induced pulmonary arterial hypertension. That is, the present invention conducted double explorations from cell experiments and animal experiments, and verified that Ero1α promotes the development of pulmonary arterial hypertension, and knocking down the expression of Ero1α can significantly alleviate the development of pulmonary arterial hypertension.

[0041] In summary, the present invention comprehensively explored the protein expression levels of pulmonary artery smooth muscle cells under hypoxia through high-throughput sequencing technology, determined that Ero1α is the molecule with the most significant up-regulation of expression under hypoxia in hypoxic pulmonary artery smooth muscle cells through subsequent verification by qRT-PCR and Western blot. Further cell and animal experiments confirmed that the high expression of Ero1α promotes the occurrence and development of pulmonary arterial hypertension. Ero1α is expected to become a new potential target for the treatment of pulmonary arterial hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the schematic diagram of the present invention, in which: Hypoxia causes an increase in the expression level of Ero1α in pulmonary artery smooth muscle cells, thereby causing an enhancement in the proliferation and migration levels of pulmonary artery smooth muscle cells, and further causing the thickening of pulmonary arterioles, and ultimately leading to the occurrence and development of pulmonary arterial hypertension. Hypoxia: Hypoxia, PASMC: Pulmonary artery smooth muscle cell, ER: Endoplasmic reticulum, Nucleus: Nucleus, Proliferation: Proliferation, Migration: Migration, Pulmonary arteriole: Pulmonary arteriole, Vascular remodelling: Vascular remodelling, Vascular thickening: Vascular thickening.

[0043] Figure 2 For the screening process and expression verification of Ero1α of the present invention, wherein: (A) Flow chart of data-independent acquisition (DIA) detection and analysis of proteomics, with the culture time under normoxia (21% O2) and hypoxia (2% O2) both being 24 h; (B) Volcano plot of proteins detected by DIA: proteins up-regulated and down-regulated under hypoxia; (C) Heat map of 10 most significantly up-regulated proteins under hypoxia screened from the DIA identification results; (D) qRT-PCR detection of the mRNA expression levels of the proteins in the heat map in PASMCs, n = 3; (E) Western blot detection of the expression level of Ero1α in PASMCs cultured under hypoxia (2% O2), representative figure and (F) statistical chart, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001. Normoxia: normoxia, Hypoxia: hypoxia.

[0044] Figure 3 Knocking down Ero1α of the present invention reduces the proliferation and migration levels of PASMCs, wherein: (A) Representative figure and (B) statistical chart of Western blot verifying that si-Ero1α knocks down the expression levels of PASMCs under normoxia and hypoxia (2% O2, 24 h); (C) Representative figure and (D) statistical chart of EdU staining for detecting the proliferation level of PASMCs, magnification: 100 times; (E) Transwell for detecting the migration level of PASMCs, representative figure and (F) statistical chart, magnification: 200 times, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. Normoxia+si-NC: normoxia + si-NC, Normoxia+si-Ero1α: normoxia + si-Ero1α, Hypoxia+si-NC: hypoxia + si-NC, Hypoxia+si-Ero1α: hypoxia + si-Ero1α.

[0045] Figure 4Overexpression of Ero1α in the present invention increased the proliferation and migration levels of PASMCs. Among them: (A) Representative Western blot image and (B) statistical chart verifying that the Ero1α overexpression plasmid increased the expression levels of PASMCs under normoxia and hypoxia (2% O2, 24 h); (C) Representative EdU staining image and (D) statistical chart for detecting the proliferation level of PASMCs, magnification: 100×; (E) Representative Transwell image and (F) statistical chart for detecting the migration level of PASMCs, magnification: 200×, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. Normoxia+si-NC: normoxia + si-NC, Normoxia+si-Ero1α: normoxia + si-Ero1α, Hypoxia+si-NC: hypoxia + si-NC, Hypoxia+si-Ero1α: hypoxia + si-Ero1α.

[0046] Figure 5 The present invention shows the expression differences of Ero1α between the high-pressure group and the control group in human chronic obstructive pulmonary disease pulmonary hypertension specimens (Human COPD-PH sample), rat hypoxic pulmonary hypertension (Rat HPH sample), and rat monocrotaline-induced pulmonary hypertension (Rat MCT-PH sample) models. Among them: (A) Immunofluorescence detection of the expression level of Ero1α in the COPD-PH control and patient pulmonary arteries, magnification: 400×; (B) Immunofluorescence detection of the expression level of Ero1α in the pulmonary arteries of the normoxic control and hypoxic high-pressure groups in the rat HPH model, magnification: 400×; (C) Immunofluorescence detection of the expression level of Ero1α in the pulmonary arteries of the control and high-pressure groups in the rat MCT-PH model, magnification: 400×. Control: control, COPD-PH: chronic obstructive pulmonary disease pulmonary hypertension group, HPH: hypoxic pulmonary hypertension group, MCT-PH: monocrotaline pulmonary hypertension group.

[0047] Figure 6Knockdown of Ero1α expression in lung tissue of the present invention alleviated HPH, where: (A) Flow chart of HPH modeling; (B) Detection of eGFP-labeled AAV by upright fluorescence microscopy after frozen section of lung tissue, and the results showed that AAV infected pulmonary arteries as expected, magnification: 400 times; (C) Representative diagram of RVSP measurement; (D) Representative diagram of pulmonary arterioles stained with HE, magnification: 200 times; (E) Statistical chart of RVSP; (F) Statistical chart of pulmonary artery thickening index, pulmonary artery thickening index = (outer diameter - inner diameter) / outer diameter, and the unit of artery diameter is μm; (G) Statistical chart of right ventricular hypertrophy index; (H) Statistical chart of cardiac output; (I) Statistical chart of total pulmonary resistance, total pulmonary resistance = 80×RVSP (mmHg) / pulmonary blood flow (L / min), and the unit is dyn·s·cm -5 ; (J) Statistical chart of systemic blood pressure. HNO: Normoxia group, HHO: Hypoxia group, HNV: Hypoxia + AAV-CTL (control) group, HAVP: Hypoxia + AAV-Ero1α (prevention) group, HAVT: Hypoxia + AAV-Ero1α (treatment) group. n = 8-10, ns: No statistical difference, ***p < 0.001.

[0048] Figure 7 Knockdown of Ero1α expression in lung tissue of the present invention alleviated MCT-PH, where: (A) Flow chart of MCT-PH modeling; (B) Detection of eGFP-labeled AAV by upright fluorescence microscopy after frozen section of lung tissue, and the results showed that AAV infected pulmonary arteries as expected, magnification: 400 times; (C) Representative diagram of RVSP measurement; (D) Representative diagram of pulmonary arterioles stained with HE, magnification: 200 times; (E) Statistical chart of RVSP; (F) Statistical chart of pulmonary artery thickening index, pulmonary artery thickening index = (outer diameter - inner diameter) / outer diameter, and the unit of artery diameter is μm; (G) Statistical chart of right ventricular hypertrophy index; (H) Statistical chart of cardiac output; (I) Statistical chart of total pulmonary resistance, total pulmonary resistance = 80×RVSP (mmHg) / pulmonary blood flow (L / min), and the unit is dyn·s·cm -5 ; (J) Statistical chart of systemic blood pressure, MNC: Ethanol normal saline group, MCT: MCT group, MNV: MCT + AAV-CTL group, MAV: MCT + AAV-Ero1α group, n = 8-10, ns: No statistical difference, ***p < 0.001. Detailed implementation manners

[0049] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments.

[0050] Example 1: Cell experiment

[0051] (1) Screen for proteins with up-regulated expression under hypoxic culture of PASMCs.

[0052] 1) Protein mass spectrometry analysis: Perform data-independent acquisition (DIA) protein mass spectrometry analysis on rat PASMCs cultured under normoxia (21% O2, 24 h) and hypoxia (2% O2, 24 h) ( Figure 2 as shown in A below). Proteins with increased and decreased expression under hypoxia are shown in the volcano plot ( Figure 2 as shown in B below). The DIA process is based on the following three steps: First, construct a spectral library: The spectral library collects all detectable non-redundant high-quality peptide information (MS / MS spectra) of the sample as a peptide identification template for subsequent data analysis. It contains fragment ion intensities and retention times that describe the spectral peak characteristics of the peptides. The spectral library is constructed using data collected by performing data-dependent acquisition (DDA) on samples of interest. Second, obtain a large amount of sample data in DIA mode: The data-independent acquisition (DIA, also known as SWATH) mode uses the latest high-resolution mass spectrometry to simultaneously acquire peptide ion characteristics in terms of mass number and retention time. Third, data analysis: By deconvoluting the DIA data and combining it with the DDA spectral library, qualitative and quantitative information on peptides and proteins is obtained. The MSstats package is used to perform differential analysis on the data.

[0053] 2) Further screening by qRT-PCR: According to fold change > 5 and p < 0.05 under hypoxia, 10 proteins with the highest upregulated expression levels under hypoxia were initially screened out ( Figure 2 as shown in C below), and the mRNA expression levels of these 10 proteins were verified by qRT-PCR ( Figure 2 as shown in D below). The results showed that the expression levels of Ero1α, Slc2a1, and Fam162a were significantly increased under hypoxia, and the increase in the expression level of Ero1α was the most significant. The specific experimental procedure is as follows: Extract total RNA from rat PASMCs using TRIzol (TAKARA 9109). Use the TaKaRa RR036A kit for reverse transcription to synthesize cDNA, and then use the TaKaRa RR420A kit for quantitative real-time polymerase chain reaction (qRT-PCR). Apply 2 -ΔΔCtRelative quantification was performed using the following primer sequences: Ero1α-forward: 5’-TGG ACG AAT CTC TGA GTG AGG-3’, Ero1α-reverse: 5’-CAG CAT CGG GGG ACT GTA T-3’; Loxl2-forward: 5’-CCT ATA AGC CGG AGC AAC CC-3’, Loxl2-reverse: 5’-AGA CGG TAC CCC ATT CTC CA-3’; Slc2a1-forward: 5’-CTT ATT GCC CAG GTG TTC GG-3’, Slc2a1-reverse: 5’-GGC AGA AGG GCA ACA GGA TA-3’; Fam162a-forward: 5’-TGA GGG CAA GAA GCT TGA AC-3’, Fam162a-reverse: 5’-TAC GTC TAG TCC GCT TTG GC-3’; Slc30a6-forward: 5’-GCC TGA GAT ACA CAC GGG AAG-3’, Slc30a6-reverse: 5’-CTG CCA CAT GCT CTT GAA GC; Abcc10-forward: 5’-GGG GCC ACT TAC AGG TTT GA-3’, Abcc10-reverse: 5’-AAC CAC TGC ATC GTG GCA TA-3’; Cdca5-forward: 5’-AAG GTT CCT GAG ATC ACG TCT T-3’, Cdca5-reverse: 5’-ACG TCG GAG TCC TTA GGT TC-3’; Pros1-forward: 5’-CCC GAA ACG GAT TAT TTT TAT CCA-3’, Pros1-reverse: 5’-TGG TCT GGG ATG GCA TTG AC-3’; Tbc1d7-forward: 5’-AGA TGG TGG AAG ACA GCG TG-3’, Tbc1d7-reverse: 5’-AAA GCC TTG GGC AGC TGA G-3’; Rab29-forward: 5’-GGG ATA TTG CAG GGC AGG AG-3’, Rab29-reverse: 5’-TGA TGA CAC AGG CAG AAG CA-3’.

[0054] 3) Western blot verification: The expression of Ero1α in hypoxic cultured PASMCs was further verified by Western blot ( Figure 2 in E, Figure 2In Figure F), the results showed that Ero1α was the most significantly up-regulated protein in hypoxic cultured PASMCs, and its expression level was the highest at 24 h of hypoxia. Subsequently, 2% O2 and 24 h of hypoxia were used as the hypoxic culture conditions for subsequent hypoxic culture.

[0055]

[0056] The amino acid sequence of Ero1α protein is as follows: >NP_612537.1 ERO1-like protein alpha precursor [Rattus norvegicus] MGRAWGLLVGLLGVVWLLRLGHGEERRPETAAQRCFCQVSGYLDDCTCDVE TIDKFNNYRLFPRLQKLLESDYFRYYKVNLRKPCPFWNDINQCGRRDCAVKPCHSDEVPDGIKSASYKYSKEANLLEECEQAERLGAVDESLSEETQKAVLQWTKHDDSSDSFCEVDDIQSPDAEYVDLLLNPERYTGYKGPDAWRIWSVIYEENCFKPQTIQRPLASGQGKHKENTFYSWLEGLCVEKRAFYRLISGLHASINVHLSARYLLQDNWLEKKWGHNVTEFQQRFDGVLTEGEGPRRLKNLYFLYLIELRALSKVLPFFERPDFQLFTGNKVQDVENKELLLEILHEVKSFPLHFDENSFFAGDKHEAHKLKEDFRLHFRNISRIMDCVGCFKCRLWGKLQTQGLGTALKILFSEKLIANMPESGPSYEFQLTRQEIVSLFNAFGRISTSVRELENFRHLLQNVH (SEQ ID NO.2).

[0057] The Western blot detection steps are as follows: Total protein was extracted from PASMCs using a lysis buffer containing RIPA, protease inhibitor, and phosphatase inhibitor. Protein samples were electrophoresed on a 10% SDS-polyacrylamide gel, separated at a voltage of 60 mV until the marker was separated, and then separated at a voltage of 120 mV until the separation was complete. The gel containing the protein was transferred to a PVDF membrane at a current of 250 mA in transfer buffer for 2 hours. The PVDF membrane was blocked with 5% non-fat milk powder at room temperature for 1 hour, and then rinsed with TBST for 30 minutes (3 times, 10 minutes each). The membrane was incubated with the primary antibody overnight at 4°C on a shaker. The next day, the membrane was rinsed with TBST for 30 minutes (3 times, 10 minutes each), and then incubated with the secondary antibody diluted in 5% non-fat milk powder at room temperature for 1 hour. Subsequently, it was rinsed with TBST again for 15 minutes (3 times, 5 minutes each), and then the protein bands were developed and photographed. The antibodies used included Ero1α (Proteintech, catalog number 12007-1-AP) and actin (Proteintech, catalog number 23660-1-AP).

[0058] (2) Verify whether knocking down Ero1α reduces the proliferation and migration levels of PASMCs.

[0059] 1) Cell treatment:

[0060] The protein expression level of Ero1α was knocked down by transfecting small interfering RNA (siRNA). PASMCs were evenly seeded in six-well plates. After 24 hours, they were transfected with si-NC and si-Ero1α respectively. The medium was changed 24 hours after transfection. The culture plates of the hypoxic group were placed in a hypoxic (2% O2) incubator, and the culture plates of the normoxic group continued to be cultured in a normoxic environment. Cells were harvested 24 hours later. The siRNA sequence targeting Ero1α was 5'-GCAAGCATCAATGTACATT-3'. In each well of the six-well plate, the transfection mixture included 900 μL of complete medium, 50 μL of Opti-MEM containing 2.5 μL of Lipofectamine 3000 (Lipo3000), and 50 μL of Opti-MEM containing 2.5 μL of siRNA.

[0061] 2) Cell grouping:

[0062] Normoxia + si-NC, Normoxia + si-Ero1α, Hypoxia + si-NC, Hypoxia + si-Ero1α.

[0063] 3) Detection indicators and methods:

[0064] ①EdU staining was used to detect the cell proliferation level: The proliferation of PASMCs was detected using the EdU staining kit (Cell-LightTM EdU Apollo567 In Vitro Kit) from RiboBio. Reagent A of the EdU staining reagent was diluted in complete cell culture medium at a ratio of 1:1000, and 100 μL of the diluted reagent was added to the seeded PASMCs in a 96-well plate. After incubation for 2 hours, the cells were fixed with 4% paraformaldehyde and then treated with 0.5% Triton X-100 to increase cell membrane permeability. Subsequently, the cells were stained with Apollo for 30 minutes. After Apollo staining, the cells were treated with 0.5% Triton X-100 again to further increase permeability, and then stained with Hoechst 33342 for 30 minutes. To prevent fluorescence quenching, the stained images of PASMCs were immediately taken using a fluorescence microscope after staining.

[0065] ②Transwell was used to detect the cell migration level: PASMCs were seeded into a 12-well plate and transfected with siRNA or plasmid. After 24 hours of transfection, the cells were digested and prepared into a 200 μL culture medium suspension containing 10,000 cells. 600 μL of complete culture medium (without cells) was added to the lower chamber of Transwell (pore size 8 μm, Corning), and 200 μL of the cell suspension was added to the upper chamber. After 24 hours, the cells were transferred to normoxia (normoxia group) or hypoxia (hypoxia group) conditions for another 24 hours of culture, and then cell staining and photography were performed. The chambers were taken out of the incubator, rinsed with phosphate buffer (PBS), and then fixed with 4% paraformaldehyde (200 μL in the upper chamber and 800 μL in the lower chamber) for 20 minutes. Then the cells were rinsed with PBS again and stained with 0.1% crystal violet (200 μL in the upper chamber and 800 μL in the lower chamber) for 20 minutes. The chambers were washed in PBS buffer until the washing solution became clear. The non-migrated cells in the upper chamber were gently removed with a cotton swab. Images of PASMCs that had successfully migrated to the lower chamber were taken using an optical microscope.

[0066] 4) Experimental results: Transfection of si-Ero1α into PASMCs knocked down the expression of Ero1α under normoxia and hypoxia ( Figure 3 in A, Figure 3 in B). Then, the proliferation level of PASMCs was detected by EdU staining and the migration level of PASMCs was detected by Transwell. The results showed that PASMCs exhibited enhanced proliferation and enhanced migration under hypoxia, while knocking down Ero1α reduced the proliferation ( Figure 3 in C, Figure 3 in D) and migration level ( Figure 3 in E, Figure 3 in F) of PASMCs under normoxia and hypoxia.

[0067] (3) Verify whether overexpression of Ero1α enhances the proliferation and migration levels of PASMCs.

[0068] 1) Cell treatment:

[0069] Enhance the protein expression level of Ero1α by transfecting plasmids. PASMCs were evenly seeded in six-well plates. After 24 h, they were transfected with the control plasmid Ctrl and the Ero1α overexpression plasmid O-Ero1α respectively. The culture medium was changed 24 h after transfection. The culture plates of the hypoxic group were placed in a hypoxic (2% O2) incubator, and the culture plates of the normoxic group continued to be cultured in a normoxic environment. Cells were harvested 24 h later. Overexpression of Ero1α was achieved by transfecting the Ero1α overexpression plasmid. In each well of the six-well plate, the transfection mixture included 900 μL of complete medium, 50 μL of Opti-MEM containing 2.0 μL of Lipofectamine 3000, and 50 μL of Opti-MEM containing 800 ng of plasmid and 2.0 μL of P3000 reagent.

[0070] 2) Cell grouping:

[0071] Normoxia + Ctrl, Normoxia + O-Ero1α, Hypoxia + Ctrl, Hypoxia + O-Ero1α.

[0072] 3) Detection indexes and methods:

[0073] ① Proliferation level: Detect the cell proliferation level by EdU staining; the specific experimental method is the same as the part of transfecting si-Ero1α above.

[0074] ② Migration level: Detect the cell migration level by Transwell; the specific experimental method is the same as the part of transfecting si-Ero1α above.

[0075] 5) Experimental results: Transfection of O-Ero1α into PASMCs increased the expression of Ero1α under normoxia and hypoxia ( Figure 4 in A, Figure 4 in B). Then, the proliferation level of PASMCs was detected by EdU staining and the migration level of PASMCs was detected by Transwell. The results showed that PASMCs showed enhanced proliferation and migration under hypoxia, and overexpression of Ero1α further enhanced the proliferation ( Figure 4 in C, Figure 4 in D) and migration level ( Figure 4 in E, in F) of PASMCs under normoxia and hypoxia.

[0076] (4) Detect the expression levels of Ero1α in three specimens of human COPD-PH, rat HPH, and rat MCT-PH.

[0077] 1) Specimen selection:

[0078] COPD-PH specimens: Lung tissues of COPD-PH patients and age- and sex-matched control patients collected previously. The diagnosis of lung cancer was excluded in both the PH and control groups.

[0079] HPH specimens: Specimens of the high-pressure group and the control group of the previously established HPH animal model.

[0080] MCT-PH specimens: Specimens of the high-pressure group and the control group of the previously established MCT-PH animal model.

[0081] 2) Specimen treatment:

[0082] Detect the expression and localization of Ero1α in the pulmonary artery by immunofluorescence staining. The steps of immunofluorescence staining are as follows: The sections are baked in an oven at 60 °C for 30 - 60 minutes, and then dewaxed by soaking them in xylene I for 20 minutes, xylene II for 20 minutes, absolute ethanol I for 10 minutes, absolute ethanol II for 5 minutes, 85% alcohol for 5 minutes, and 75% alcohol for 5 minutes. Then the sections are rinsed with slowly flowing tap water, immersed in an antigen retrieval solution containing EDTA, heated to boiling by high-temperature microwave, cooled for 8 minutes, and repeated four times. After the sections are cooled to room temperature, they are rinsed three times with PBS, blocked with goat / donkey serum at room temperature for 40 minutes, and then incubated with the primary antibodies (α-SMA, BM0002, 1:200; Ero1α, Proteintech CatNo.12007-1-AP, 1:800) at 4 °C for 24 hours. The next day, the sections are washed with PBS for 15 minutes, incubated with the secondary antibody at room temperature for 50 minutes, and the subsequent steps are carried out in the dark to prevent photofluorescence quenching. The sections are washed with PBS for 15 minutes, stained with DAPI for 10 minutes, washed with PBS again, and sealed with neutral balsam. To prevent fluorescence quenching, images are taken immediately using a fluorescence microscope after staining.

[0083] 3) Experimental results: In the specimens of the three models, compared with the control, the pulmonary arterioles in the pulmonary hypertension group were significantly thickened, and the expression level of Ero1α in the pulmonary arterioles in the high-pressure group was significantly higher than that in the control group in human COPD-PH patients ( Figure 4 in A), the high-pressure group of the previously established rat HPH model ( Figure 5 in B), and the high-pressure group of the previously established rat MCT-PH ( Figure 5 in C). This indicates that Ero1α is very likely to be involved in the development of PH at the animal level.

[0084] Example 2: Animal model experiment.

[0085] (1)Verify whether knocking down Ero1α can alleviate HPH.

[0086] 1) Modeling method:

[0087] Before the start of the animal experiment, an adeno-associated virus AAV-Ero1α for knocking down Ero1α and an adeno-associated virus control AAV-CTL were constructed. Fifty male SD rats (body weight 90 - 140 g, 4 weeks old) were randomly divided into five groups. The rats were housed in a specific pathogen-free (SPF) animal breeding center throughout the experiment. First, 4-week-old male SD rats were placed in the SPF breeding room for 1 week for quarantine and environmental adaptation. Then, AAV-CTL and AAV-Ero1α (prevention) were respectively injected into the lung tissues of the corresponding groups of rats through the airway. After injection, the rats were continued to be raised for 2 weeks to wait for the injected AAV to express in the lung tissues. Then, the hypoxia process was initiated: Except for the rats in the normoxia group, all rats were exposed to a 10% O2 hypoxic environment for 4 weeks, that is, continuous hypoxia for 28 days, with 8 hours of hypoxia per day. Two weeks after hypoxia exposure (the 14th day of hypoxia), the treatment group of rats was given AAV-Ero1α (treatment) by airway injection, and then hypoxia continued until the 28th day, ending the modeling.

[0088] 2) Experimental grouping:

[0089] Normoxia group, hypoxia group, hypoxia + AAV-CTL (control) group,

[0090] Hypoxia + AAV-Ero1α (prevention) group, hypoxia + AAV-α (treatment) group.

[0091] 3) Detection indexes and methods:

[0092] ①Measure the right ventricular systolic pressure by right heart floating catheter; measure the systemic circulation pressure by left carotid artery intubation; measure the cardiac output by thermodilution method, and calculate the total pulmonary resistance (TPR), TPR = 80×RVSP (mmHg) / cardiac output (L / min), dyn·s·cm -5 .

[0093] ②Isolate the right ventricle (RV), left ventricle + interventricular septum (LV + Septum), weigh them after drying, and calculate the right ventricular hypertrophy index, calculation formula = RV / (LV + Septum).

[0094] ③Separate the lung tissue, embed it in paraffin, cut white sections, and the white sections are respectively used for: HE staining: Calculate the vascular thickening index of small pulmonary arteries (with a diameter of 50 - 200 μm). The formula for the thickening index = [outer diameter of the blood vessel (μm) - inner diameter of the blood vessel (μm)] / (outer diameter of the blood vessel (μm)). The steps of HE staining are as follows: 1. Deparaffinization: Immerse the paraffin sections in xylene I for 20 minutes, then xylene II for 20 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, 75% alcohol for 5 minutes, and finally rinse with slowly flowing tap water. 2. Hematoxylin staining: Stain the sections in hematoxylin solution for 3 - 5 minutes, rinse with tap water, differentiate with the differentiating solution, rinse with tap water again, blue with the blueing solution, and rinse with running water. 3. Eosin staining: Dehydrate the sections in 85% and 95% gradient alcohols for 5 minutes respectively, and then stain in eosin solution for 5 minutes. 4. Dehydration and mounting: Put the sections into absolute ethanol I, II, and III for 5 minutes each in sequence, then into xylene I and II for 5 minutes each until the tissue becomes transparent. Finally, mount with neutral gum. After completing these steps, take images with an optical microscope for subsequent analysis.

[0095] ④At the end of the modeling, place the lung tissue washed with normal saline in liquid nitrogen for quick freezing, then perform OCT embedding and frozen sectioning, and detect the expression and localization of AAV virus in the lung tissues of each group with an upright fluorescence microscope.

[0096] 4) Experimental results: The modeling process is shown in Figure ( Figure 5 A) as shown. With an upright fluorescence microscope, it can be observed that AAV carrying enhanced green fluorescence (eGFP) is transfected and expressed in the pulmonary artery as expected ( Figure 6 B) as shown. The analysis results after the modeling are shown in the figure. Hypoxia caused an increase in the right ventricular systolic pressure (RVSP) of rats ( Figure 6 C, E), thickening of small pulmonary arteries ( Figure 6 D, F), right ventricular hypertrophy ( Figure 6 G), decrease in cardiac output ( Figure 6 H), and increase in total pulmonary resistance ( Figure 6 I), while knocking down the expression of Ero1α in the prevention group and treatment group significantly alleviated the above phenomena. These results indicate that at the animal level, Ero1α promotes the occurrence and development of HPH, and knocking down Ero1α can significantly alleviate HPH. In addition, hypoxia did not cause an increase in systemic blood pressure, and there was no statistical difference in systemic blood pressure among each group ( Figure 6 J) as shown, indicating that pulmonary hypertension is not caused by an increase in the measured pressure of systemic hypertension.

[0097] (2) Verify whether knocking down Ero1α can alleviate MCT-PH.

[0098] 1) Modeling method:

[0099] Before the animal experiment, adeno-associated virus AAV-Ero1α with knocked-down Ero1α and adeno-associated virus control AAV-CTL were constructed. Forty male SD rats (weighing 140 - 190 g, 5 weeks old) were randomly divided into four groups. The rats were raised in a specific pathogen-free (SPF) breeding center throughout the experiment. After 5-week-old rats entered the SPF breeding center, they were first raised for 1 week for quarantine and environmental adaptation. At 6 weeks old, AAV-CTL and AAV-Ero1α were injected into the airway in the corresponding groups respectively. After the injection of AAV, the rats were raised for another 2 weeks to wait for the expression of AAV in the lung tissue of the injected rats. Two weeks later, the rats were 8 weeks old. At this time, ethanol normal saline and monocrotaline (MCT) solution (ethanol: normal saline = 2:8 as the solvent, the solute was MCT, the MCT concentration was 1%, and the dose was 60 mg / kg) were injected into the abdominal cavity in the corresponding groups. After the intraperitoneal injection of the MCT solution, the rats were raised for another 3 weeks to complete the model establishment.

[0100] 2) Experimental grouping:

[0101] Ethanol normal saline group, MCT group, MCT + AAV-CTL group, MCT + AAV-Ero1α group.

[0102] 3) Detection indexes and methods:

[0103] Same as the HPH model.

[0104] 4) Experimental results: The model establishment process is shown in Figure ( Figure 6 A). The AAV carrying eGFP was transfected and expressed in the pulmonary artery as expected under an upright fluorescence microscope ( Figure 7 B). The analysis results after the model establishment are shown in the figure. Intraperitoneal injection of the MCT solution caused severe PH in rats, specifically manifested as an increase in RVSP ( Figure 6 C, E), thickening of the small pulmonary arteries ( Figure 7 D, F), right ventricular hypertrophy ( Figure 7 G), decreased cardiac output ( Figure 7 H), and increased total pulmonary resistance ( Figure 7 I), while knocking down the expression of Ero1α significantly alleviated these phenomena. This indicates that Ero1α promoted the development of MCT-PH, and knocking down Ero1α could significantly relieve MCT-PH. In addition, the increase in pressure in the pulmonary hypertension group was not due to systemic hypertension, and there was no statistical difference in the systemic circulation pressure among the groups ( Figure 7 Figure 7 J).

Claims

1. Use of a reagent for detecting a biomarker in the preparation of a product for diagnosing pulmonary hypertension, characterized in that: The biomarker is the Ero1α gene or the Ero1α protein; The nucleotide sequence of the Ero1α gene is as shown in SEQ ID NO.1, and the amino acid sequence of the Ero1α protein is as shown in SEQ ID NO.2; The reagent is a reagent for detecting the expression level of the biomarker.

2. Use of a substance that inhibits the expression of Ero1α gene in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The substance for inhibiting the expression of the Ero1α gene is siRNA, and the sequence of the siRNA is 5'-GCAAGCATCAATGTACATT-3'.

Images