Method for rapidly constructing mouse model of high altitude polycythemia and application thereof

Abstract

The application discloses a method for rapidly constructing a mouse model of high altitude polycythemia and application thereof. Specifically disclosed is a method for constructing a mouse model of high altitude polycythemia, which comprises placing a mouse in a low-pressure and low-oxygen environment and performing erythropoietin intervention to obtain a mouse model of high altitude polycythemia. The method can efficiently and stably induce a mouse model conforming to the core characteristics of high altitude polycythemia, and the HAPC mouse model is constructed in only 14 days. The HAPC mouse model constructed by the method is reliable and efficient in HAPC mechanism analysis, can realize high-throughput analysis in multiple omics levels, identifies a large number of statistically significant differential molecules, can stably reproduce the core pathological characteristics of HAPC, and can be applied to screening of candidate drugs or therapeutic targets for preventing and treating HAPC, and has important significance in the research on the pathogenesis of HAPC, drug screening and development.

Description

Technical Field

[0001] This invention relates to the field of biomedical and animal model construction technology, specifically to a method for rapidly constructing a mouse model of high-altitude polycythemia and its application. Background Technology

[0002] High-altitude polycythemia (HAPC) is the most common chronic altitude sickness, posing a serious health threat to approximately 140 million people living at high altitudes worldwide. Characterized by excessive red blood cell proliferation and high blood viscosity, it can lead to serious complications such as thrombosis, cerebral ischemia, high-altitude heart disease, and kidney failure. The international diagnostic criteria (often referred to as the Qinghai criteria) established at the 6th World Congress of High Altitude Medicine and Physiology in 2004 define HAPC as a peripheral blood hemoglobin concentration ≥210 g / L for men and ≥190 g / L for women, usually accompanied by corresponding clinical manifestations of polycythemia. Although these diagnostic criteria are widely adopted clinically, providing clear boundaries for disease identification, the underlying molecular mechanisms driving the development and progression of HAPC remain poorly understood. This knowledge gap results in a lack of efficient and specific clinical interventions, highlighting the urgent need for basic research into its pathophysiology.

[0003] The development of acute hypoxic-ischemic purpura (HAPC) is the result of the interaction of multiple factors, including hypoxic environment, sex, and individual genetic susceptibility. Its core pathophysiological process begins with chronic hypoxia, primarily activating the hypoxia-inducible factor-2α-mediated signaling axis, leading to upregulation of erythropoietin (EPO) synthesis and secretion in tissues such as the kidneys. EPO is a key hormone regulating erythropoiesis; it binds to EPO receptors on the surface of hematopoietic stem / progenitor cells in the bone marrow, driving the proliferation, differentiation, and maturation of erythroid progenitor cells—a normal compensatory response to hypoxia. However, in HAPC patients, this feedback regulation is abnormal, characterized by persistently elevated EPO levels or an imbalance in the EPO / soluble EPO receptor ratio. This imbalance leads to excessive erythropoiesis, ultimately resulting in a pathological state characterized by excessive erythrocyte production and high blood viscosity. Besides hypoxia and genetic factors, sex (significantly higher incidence in males than females), duration of high-altitude residence, and accompanying chronic inflammatory states have also been confirmed as important influencing factors in the occurrence and development of this disease.

[0004] To deeply elucidate the pathological essence of the aforementioned complex mechanisms and advance the development of therapeutic drugs, the rapid construction of animal models capable of simulating the clinical characteristics of human high-altitude hypoxia-prone chronic HAPC (HAPC) is crucial. However, translational research aimed at elucidating these complex mechanisms has long been limited by the efficiency of animal model construction. Currently, the mainstream method for constructing HAPC animal models is to simulate the chronic hypobaric and hypoxic environment at high altitudes. In terms of model animal selection, SD or Wistar rats have long been the primary choice due to their moderate size, low cost, and relatively stable response to hypoxia. In recent years, with the deepening of mechanistic research, the application of mouse models, which have advantages such as clear genetic background and mature gene manipulation tools, has gradually increased. Regarding modeling methods, the simple chronic hypobaric hypoxia method is the most classic, which involves placing animals in a hypobaric chamber at a specific altitude (e.g., 4000-6000 meters) for several hours daily for several weeks to simulate long-term high-altitude exposure. To accelerate model formation, some studies employ a composite factor modeling method, such as adding low temperature or using chemical inducers like cobalt chloride to the low-altitude hypoxia model. A successful model must reproduce clinical characteristics in terms of core hematological indicators, changes in blood rheology, pathological damage to multiple organs, and molecular functional phenotypes. However, current mainstream models generally suffer from lengthy modeling cycles. For example, one study established a rat model of HAPC by simulating an environment at an altitude of 5000 meters and exposing the rat to hypoxia for 28 days, while another study simulating an environment at an altitude of 4500 meters required a total of 42 days. These lengthy modeling times not only increase research costs and animal welfare concerns but also severely restrict the development of high-throughput drug screening and timely mechanism studies that require rapid validation, becoming a major bottleneck in the translational medicine of HAPC. In addition, existing models also face challenges such as species differences, incomplete phenotypic matching, high operating costs, and insufficient standardization of mouse models. Therefore, developing novel animal models that can efficiently, stably, and comprehensively simulate clinicopathological features is crucial for accelerating the mechanistic research and drug development of HAPC. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to quickly construct a reliable, efficient, stable, and comprehensive mouse model of high altitude polycythemia vera that simulates the clinical pathological characteristics of HAPC.

[0006] To address the aforementioned technical problems, this invention first provides a method for constructing a mouse model of high-altitude polycythemia, the method comprising placing mice in a low-pressure, low-oxygen environment and intervening with erythropoietin to obtain a mouse model of high-altitude polycythemia.

[0007] Furthermore, the method may include the following steps: S1) Place the mice in a low-pressure, low-oxygen environment; S2) The mice were raised in a low-pressure, low-oxygen environment for 14 days, and erythropoietin was administered every 2 days to obtain a mouse model of high-altitude polycythemia.

[0008] In the above method, the erythropoietin intervention can be achieved by administering erythropoietin to mice.

[0009] Furthermore, the application may be by injection.

[0010] Furthermore, the injection may be an intraperitoneal injection.

[0011] In the above method, in step S2), the dose of erythropoietin intervention each time can be 100 IU / animal.

[0012] In the above method, the low-pressure, low-oxygen environment can be a simulated low-pressure, low-oxygen environment of a plateau.

[0013] In the above method, the low-pressure, low-oxygen environment can be a simulated low-pressure, low-oxygen environment of a plateau at an altitude of 6,000 meters.

[0014] Furthermore, a low-pressure, low-oxygen chamber can be used to simulate the low-pressure, low-oxygen environment of a plateau at an altitude of 6,000 meters.

[0015] The low-pressure hypoxic chamber, as is well known to those skilled in the art, is a ground-based experimental device that simulates high-altitude environments such as low pressure and hypoxia. It consists of a chamber body, an air extraction system, a control system, and an oxygen supply system. By adjusting the ratio of air extraction to air intake, it changes the pressure inside the chamber, simulating high-altitude environments from 5,000 meters to 16,000 meters.

[0016] Further, step S1) can be: placing the mouse in a low-pressure hypoxia chamber and setting the low-pressure hypoxia chamber to simulate an altitude of 6000 meters.

[0017] In some implementations, step S1) is: placing the mouse in a low-pressure hypoxic chamber and raising the chamber environment at a rate of 10 m / s to simulate the corresponding air pressure and oxygen partial pressure conditions at an altitude of 6000 meters (approximately equivalent to an oxygen concentration of 10%).

[0018] Furthermore, the feeding described in step S2) is normal feeding (maintaining normal diet and water).

[0019] In the above method, the weight of the mouse can be 18-22 g.

[0020] Further, step S2) can be to raise the mice in a low-pressure, low-oxygen environment for 14 days, and inject the mice with erythropoietin solution every 2 days during this period to obtain a mouse model of high-altitude polycythemia.

[0021] Furthermore, the number of injections can be 7.

[0022] This invention also provides the application of a mouse model of high-altitude polycythemia constructed by any of the methods described herein in any of the following: A1) Application in screening therapeutic targets for high-altitude polycythemia; A2) Its application in screening drugs for the prevention or treatment of high-altitude polycythemia; A3) Application in the study of the pathogenesis of high-altitude polycythemia.

[0023] The mouse model of high-altitude polycythemia vera constructed by this invention can be determined as a successful model if mice that meet the diagnostic criteria for HAPC in Qinghai can be detected by routine blood tests.

[0024] Furthermore, the high-altitude polycythemia mouse model constructed in this invention can be judged as having been successfully constructed by the following indicators: hemoglobin concentration and hematocrit.

[0025] Specifically, for male mice, the hemoglobin concentration was ≥210 g / L. For female mice, the hemoglobin concentration was ≥190 g / L.

[0026] Furthermore, the mouse model of high-altitude polycythemia constructed in this invention can also be identified by at least one of the following methods: B1) Changes in routine blood tests: Significantly increased red blood cell count and hematocrit; B2) Changes in blood rheology: Whole blood viscosity and plasma viscosity are significantly increased; B3) Pathological damage to multiple organs: including disordered arrangement of myocardial fibers, thickening of alveolar septa, glomerular congestion and / or abnormal ratio of hematopoietic tissue to adipose tissue in bone marrow; B4) Abnormal cardiac function: Thickening of the anterior wall of the right ventricle and other changes consistent with pulmonary hypertension and increased cardiac load.

[0027] Furthermore, the mice may be SPF-grade mice.

[0028] Furthermore, the mouse may be a male mouse.

[0029] The mice described in this article may be 7-9, 7, 8, or 9 weeks old.

[0030] In this article, the erythropoietin mentioned can be human erythropoietin.

[0031] This invention establishes a rapid method for constructing a mouse model of high-altitude polycythemia (HAPC). This method, through a combination of a hypobaric, hypoxic environment and erythropoietin intervention, can stably induce a phenotype consistent with the clinical and pathological characteristics of HAPC within two weeks. Compared to the conventional modeling time (approximately one month), the HAPC mouse model constructed according to this invention requires only 14 days, significantly reducing research costs and animal welfare concerns, and meeting the needs of high-throughput drug screening and time-sensitive mechanism studies requiring rapid validation. The method of this invention can efficiently and stably induce a mouse model consistent with the core characteristics of high-altitude polycythemia, exhibiting significant increases in erythroid markers, good survival rates, and controllable weight loss. The HAPC mouse model constructed using this method is reliable and efficient in HAPC mechanism analysis, enabling high-throughput analysis at multiple omics levels, identifying a large number of statistically significant differentially expressed molecules, and stably reproducing the core pathological characteristics of HAPC. It can be applied to screen candidate drugs or therapeutic targets for the prevention and treatment of HAPC, and has significant implications for the study of HAPC pathogenesis, drug screening, and development. Attached Figure Description

[0032] Figure 1 This document outlines the rapid establishment process and results of a mouse model of high-altitude polycythemia (HAPC). A: Schematic diagram of experimental design. KM and C57 mice were used, and HAPC mouse models were constructed using erythropoietin (EPO) intervention, chronic hypobaric hypoxia, or a combination of both. B: Effect of EPO intervention on body weight in C57 and KM mice under normobaric and normoxic conditions (n=8); C: Effect of EPO intervention on blood routine indicators in C57 and KM mice under normobaric and normoxic conditions (n=8); D: Effect of EPO intervention on survival rate in C57 and KM mice under chronic hypobaric hypoxia (n=7); E: Effect of EPO intervention on body weight changes in C57 and KM mice under chronic hypobaric hypoxia (n=7); F: Effect of different modeling methods and animal strains on achieving HAPC hematological criteria. Based on comparative analysis, subsequent experiments selected chronic hypobaric hypoxia combined with EPO intervention for HAPC model construction (n=7). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0033] Figure 2This study describes multi-organ injury in a HAPC mouse model. A: Hemorheological parameters of HAPC-KM mice, reflecting changes in blood viscosity and fluidity (n=7); B: HE staining results of multiple tissues (heart, lung, kidney, sternum) in HAPC-KM mice; C: Representative echocardiographic images of HAPC-KM mice; D: Quantitative analysis of echocardiographic parameters in HAPC-KM mice, including diastolic thickness of the right ventricular anterior wall, systolic diameter of the right ventricular interior, diastolic and systolic thickness of the left ventricular posterior wall, and ejection fraction (n=7); E: Changes in electrocardiographic parameters (P wave amplitude and QRS interval) of HAPC-KM mice (n=7); F: Behavioral assessment of HAPC-C57BL / 6 mice using the open field test (n=6); G: Analysis of erythroid differentiation in the bone marrow of HAPC-KM mice using flow cytometry, showing the distribution of cells at each differentiation stage; H: Statistical quantification of the proportion of erythroid differentiated cells at each stage in the bone marrow of HAPC-KM mice using flow cytometry (n=3). *P<0.05; **P<0.01; ***P<0.001.

[0034] Figure 3 Proteomics analysis of erythrocyte membranes in HAPC-KM mice. A: Venn diagram showing the number of proteins identified in the erythrocyte membranes of control and HAPC model KM mice; B: Principal component analysis plot showing the differences and separation of overall proteomic features between the two groups; C: Volcano plot screening differentially expressed proteins between the two groups, with significantly upregulated and downregulated proteins highlighted; DF: Gene ontology enrichment analysis of differentially expressed proteins, including biological processes, cellular components, and molecular functions; G: Hierarchical clustering heatmap of differentially expressed proteins.

[0035] Figure 4 Phosphorylated proteomics analysis of erythrocyte membranes in HAPC-KM mice. A: Venn diagram comparing the number of phosphorylated proteins identified in the control and model groups; B: Venn diagram comparing the number of phosphorylated peptides identified in the two groups; C: Volcano plot screening differentially phosphorylated peptides between the two groups, with significantly hyperphosphorylated and hypophosphorylated peptides highlighted; D: Principal component analysis of phosphorylated proteomics data, showing the overall differences and separation between the two groups; E, F: Gene ontology enrichment analysis of proteins containing differentially phosphorylated sites, showing the significantly enriched biological processes and molecular functions; G: Quantitative bar chart of differentially phosphorylated sites in important erythrocyte membrane proteins.

[0036] Figure 5Metabolomics analysis of erythrocytes from HAPC-KM mice. A: Principal component analysis plots of metabolomics data obtained in positive and negative ion modes, respectively; B: Volcano plots of significantly different metabolites between the model group and the control group, based on data from positive ion mode (left) and negative ion mode (right); C: Pathway enrichment analysis of significantly altered metabolites; D: Schematic diagram of the glycerophospholipid metabolic pathway, highlighting (in red) 11 key nodes (metabolites) that showed significant changes in the HAPC model group; E: Bar chart showing the relative quantitative levels of differentially metabolites enriched in the glycerophospholipid metabolic pathway identified in Figure D in the control and model groups.

[0037] Figure 6 Lipidome analysis of HAPC-KM mouse erythrocytes. A: Overview of lipidome identification results. A total of 376 lipid molecules in 28 classes were identified in all samples; B: Principal component analysis plot of the overall lipidome, showing the separation between the control and model groups; C: Volcano plot of differentially abundant lipids between the control and model groups; D: Top 10 most significantly altered lipid molecules ranked by statistical significance; E: Classification and distribution of all differentially abundant lipids. The bar chart shows the number of significantly differentially abundant lipids belonging to each major lipid class.

[0038] Figure 7 This is a KEGG pathway diagram for glycerophospholipid metabolism. KEGG, Kyoto Encyclopedia of Genetics and Genomes. Red circles represent differentially expressed metabolites. Light blue boxes indicate identified erythrocyte membrane proteins, with no significant differences in expression levels. Pink boxes indicate identified phosphorylated erythrocyte membrane proteins, with no significant differences in modification levels. Red boxes represent Lpin2 and Cds2, whose phosphorylation sites were significantly altered in the model group. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] In the following examples, RBC (Red Blood Cell) represents red blood cells; HCT (Hematocrit) represents hematocrit (also known as erythrocyte volume compression); HGB (Hemoglobin) represents hemoglobin; and EPO (Erythropoietin) represents erythropoietin.

[0042] In the following examples, GraphPad Prism 10 was used for statistical analysis of animal experimental data. Unless otherwise stated, error bars represent the mean ± standard deviation of at least three independent experiments. Differences between groups were assessed using Student's t-test, with a p-value <0.05 considered statistically significant. GO enrichment analysis was performed using the R package clusterProfiler, and hierarchical clustering heatmaps were generated using the R package pheatmap.

[0043] All animal procedures described below were performed in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Academy of Military Medical Sciences.

[0044] Example 1: A method for rapidly constructing a mouse model of high-altitude polycythemia. The inventors of this application, through extensive and in-depth research, have established a rapid method for constructing a mouse model of high-altitude polycythemia (HAPC). A schematic diagram of this method is shown below. Figure 1 As shown in Figure A.

[0045] 1. Screening of methods for constructing a mouse model of high-altitude polycythemia. a. Laboratory animals and grouping: Animal sources: Eight-week-old male SPF-grade C57BL / 6J mice weighing 20-22g and eight-week-old male SPF-grade Kunming (KM) mice weighing 18-20g were used in the experiment and were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0046] Husbandry environment: All mice were acclimatized for 3 days in a standard environment (12-hour light / dark cycle, temperature 22±2℃, relative humidity 50±10%), with free access to food and water.

[0047] Randomization: After acclimatization, mice of both strains were randomly grouped using a random number table as follows: Atmospheric pressure and normal oxygen EPO experiment (corresponding) Figure 1 B, C): ddH2O control group: intraperitoneal injection of sterile double-distilled water under normal pressure and oxygen conditions; EPO intervention group: intraperitoneal injection of erythropoietin under normal pressure and oxygen conditions.

[0048] Low-pressure, low-oxygen experiment (corresponding) Figure 1D, E, F): Normobaric normoxic-ddH2O group: sterile double-distilled water was injected intraperitoneally under normobaric normoxic conditions; Hypobaric hypoxic-ddH2O group: sterile double-distilled water was injected intraperitoneally under chronic hypobaric hypoxic conditions; Hypobaric hypoxic-EPO group: erythropoietin was injected intraperitoneally under chronic hypobaric hypoxic conditions. Each group consisted of at least 7 mice.

[0049] b. Model building intervention methods: Erythropoietin Intervention: Drug: Recombinant human erythropoietin (EPO, catalog number: kx30H-EP, Beijing Kexingyuan Biotechnology Co., Ltd.). Dosage and Administration: Mice in the EPO intervention group were intraperitoneally injected with 10 IU or 100 IU per injection. The control group was injected with an equal volume of sterile double-distilled water (ddH2O). The injection frequency was once every other day, for a total of 7 injections.

[0050] Low-pressure hypoxic environment exposure: Equipment: Low-pressure hypoxic chamber (Guizhou Fenglei Co., Ltd.); Conditions: Mice in the hypoxic group were placed inside the chamber, and the environment inside the chamber was raised at a rate of 10 m / s to simulate the atmospheric pressure and oxygen partial pressure conditions corresponding to an altitude of 6000 meters. The mice were continuously fed in this hypoxic environment until the end of the experiment.

[0051] c. Observation indicators and detection methods: Weight monitoring (corresponding) Figure 1 B, E): Methods: Using an electronic balance, the weight of each mouse was measured regularly (every 2 days) before the start of the experiment (day 0) and during the intervention period. The changes in the weight of each group of mice over time were recorded and calculated.

[0052] Survival rate records (corresponding) Figure 1 D): Methods: Throughout the intervention period, the survival of mice in each group was observed and recorded daily. Survival curves were calculated and plotted using the initial number of animals as the baseline.

[0053] Complete blood count (CBC) test (corresponding) Figure 1 C, F): Sampling: 24 hours after the last intervention, mice were deeply anesthetized with isoflurane, and whole blood samples were collected through the orbital venous plexus and placed in anticoagulant tubes containing heparin sodium. Testing: Samples were immediately tested using a fully automated blood analyzer (Sysmex). Key observational indicators included: hemoglobin concentration, red blood cell count, and hematocrit.

[0054] Data analysis and model selection (corresponding) Figure 1 F): Statistics: Data analysis was performed using GraphPad Prism software. Student's t-test or one-way ANOVA was used for comparisons between groups. Data are expressed as mean ± standard deviation.

[0055] Model determination and screening: A comprehensive comparison was made of the hematological indicators (whether they met the diagnostic criteria for HAPC, such as hemoglobin >210 g / L), weight change trends, and survival rates of mice in each group. Ultimately, chronic hypobaric hypoxia combined with EPO administration was determined to be the optimal modeling protocol for subsequent experiments.

[0056] In summary, this experiment, through systematic comparison, confirms that chronic low-pressure hypoxia (simulating an altitude of 6000 meters) exposure for approximately two weeks in C57BL / 6J or Kunming mice, with intraperitoneal injection of 100 IU EPO every other day (for a total of 7 times), can efficiently and stably induce an animal model consistent with the core characteristics of high-altitude polycythemia, characterized by significantly elevated erythroid markers, good survival rate, and controllable weight loss.

[0057] 2. Methods for constructing a mouse model of high-altitude polycythemia This invention claims protection for a method for efficiently constructing a mouse model of high-altitude polycythemia. This method, through the combined application of a chronic low-pressure, low-oxygen environment and erythropoietin intervention, can stably induce a phenotype consistent with the clinical and pathological characteristics of the disease within two weeks. The specific operational steps of this model construction method are as follows: Animal preparation and grouping: Eight-week-old male SPF-grade mice (C57BL / 6J or Kunming mice) weighing 18-22g were selected. After acclimatization for 3 days in a standard environment (12 / 12-hour light / dark cycle, 22±2℃, 50±10% humidity), the mice were randomly divided into a model group and a corresponding control group.

[0058] Chronic hypobaric hypoxia exposure and EPO intervention (core modeling steps): Mice in the model group were placed in a precisely adjustable hypobaric hypoxia chamber (DWCF50-IIA, Guizhou Fenglei Co., Ltd.). The chamber environment was raised at a rate of 10 m / s to simulate the atmospheric pressure and oxygen partial pressure at an altitude of 6000 meters (approximately equivalent to an oxygen concentration of 10%). Mice were continuously housed in this hypoxic environment while maintaining normal food and water intake. Simultaneously, during the hypoxia exposure period, the model group mice underwent erythropoietin intervention. Specifically, intraperitoneal injections were administered every 2 days, with each injection containing 100 IU of recombinant human erythropoietin (dissolved in an appropriate amount of sterile solvent), for a total of 7 injections. The entire combined intervention (hypobaric hypoxia exposure + EPO administration) lasted 14 days.

[0059] Criteria for successful model construction: Mice that meet the Qinghai diagnostic criteria for HAPC are considered to have successfully constructed the model.

[0060] Specifically, the success of model construction can be determined using the following indicators: hemoglobin concentration. For male mice, a hemoglobin concentration ≥210 g / L. For female mice, a hemoglobin concentration ≥190 g / L.

[0061] After the above combined intervention, the model group mice stably exhibited the following core characteristics, which helped to determine the successful establishment of the high-altitude polycythemia model: (1) Changes in routine blood indicators: The number of red blood cells and hematocrit are significantly increased.

[0062] (2) Changes in blood rheology: whole blood viscosity and plasma viscosity increased significantly.

[0063] (3) Pathological damage to multiple organs: Histological examination after dissection (such as H&E staining) reveals typical lesions, including disordered arrangement of myocardial fibers, thickening of alveolar septa, glomerular congestion, and abnormal ratio of hematopoietic tissue to adipose tissue in bone marrow.

[0064] (4) Abnormal cardiac function: Echocardiography can reveal changes such as thickening of the anterior wall of the right ventricle, which are consistent with pulmonary hypertension and increased cardiac load.

[0065] 3. Identification of a mouse model of high-altitude polycythemia Routine blood analysis: A 0.5 mL blood sample was collected from the orbital cavity and injected into an anticoagulant tube containing heparin sodium (HY-17567A, MCE, China). The sample was gently inverted several times to ensure thorough mixing. Blood cell counts, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT), were measured using a fully automated blood analyzer (XN-1000V, Sysmex).

[0066] Blood rheology assay: Whole blood samples were collected using the orbital venous plexus method with heparin-anticoagulated capillary blood collection tubes. The blood collection process required rapid and gentle handling to avoid hemolysis and coagulation activation. The collected whole blood samples were gently mixed and used immediately for testing. The analysis was performed using a fully automated blood rheology analyzer (Shanghai Langyi Medical Instruments Co., Ltd.). The anticoagulated whole blood sample was directly aspirated into the sample cell, and the instrument measured its viscosity at different set shear rates. The viscosity values ​​at the following shear rates are reported: High shear rate (200 s⁻¹) -1 ), reflecting the deformability of erythrocytes; low shear rate (1 s), reflecting the deformability of erythrocytes; -1 The viscosity of whole blood in each mouse was recorded at high and low shear rates (in mPa·s), reflecting erythrocyte aggregation. The hemorheological parameters of the model group and the control group were statistically compared to assess whether the HAPC model successfully replicated the core pathological feature of high blood viscosity.

[0067] Hematoxylin and eosin (H&E) staining: Tissue samples from the heart, lungs, kidneys, and sternum were subjected to H&E staining for histopathological evaluation. Tissue samples were fixed, embedded in paraffin, sectioned, and stained with H&E. Pathological morphology was observed under an optical microscope, and the extent of damage was assessed based on the stained sections.

[0068] Echocardiography and electrocardiogram monitoring: After hypoxia exposure, mice were anesthetized with isoflurane, their chest hair was shaved, and echocardiography and electrocardiogram (ECG) were performed. A 6LAB animal ultrasound system (Beijing Yiren Hengye Technology Co., Ltd.) equipped with a French VERMON high-frequency probe was used to record mouse echocardiography. M-mode and two-dimensional parasternal long-axis scans were performed at the papillary muscle level to assess changes in ejection fraction (EF) (%) and fractional shortening (FS) (%). Electrocardiogram (ECG) was performed using a multi-channel physiological signal acquisition and processing system. Throughout the experiment, the temperature of the operating table where the mice were placed was maintained at 37°C.

[0069] 4. Results This embodiment successfully constructed a stable and representative mouse model of high-altitude polycythemia. First, it evaluated whether EPO combined with hypoxia exposure could rapidly and stably construct a mouse model of high-altitude polycythemia. The experimental design is as follows: Figure 1 As shown in Figure A, two mouse strains, KM and C57BL / 6, were used. Under normobaric and normoxic conditions, it was observed that the body weight of KM mice gradually increased over time, while the body weight of C57BL / 6 mice remained relatively stable; EPO intervention had no significant effect on the body weight of either mouse strain. Figure 1 B). Blood routine tests showed that EPO significantly increased peripheral blood erythroid proliferation markers (RBC, HCT, HGB), while causing a significant decrease in platelet count in C57BL / 6 mice. Figure 1 C). However, during the two-week experimental period, the use of EPO alone failed to bring the blood routine indicators of mice to the Qinghai diagnostic criteria for HAPC (HGB ≥ 210 g / L).

[0070] Therefore, we further investigated under hypobaric and hypoxic conditions. Survival analysis showed that C57BL / 6 mice tolerated hypobaric and hypoxic conditions better than KM mice, and EPO intervention reduced the mortality rate of both mouse strains to some extent. Figure 1 D). Weight monitoring showed that the mice's body weight dropped to its lowest point on day 5 of low-pressure, low-oxygen exposure, and then gradually recovered. Figure 1 E). Importantly, under these conditions, peripheral blood HGB in both mouse strains reached the Qinghai diagnostic criteria for HAPC within 2 weeks. Figure 1 F). In summary, by combining chronic hypobaric hypoxia with EPO intervention, this invention successfully and rapidly constructed a mouse model meeting the hematological criteria of HAPC within 2 weeks, significantly shorter than the conventional modeling time (approximately 1 month). This model is stable and reliable, and subsequent experiments all used this combined approach.

[0071] Example 2: Phenotypic analysis of HAPC model mice 1. Experimental Methods Hematoxylin and eosin (H&E) staining: Tissue samples from the heart, lungs, kidneys, and sternum were subjected to H&E staining for histopathological evaluation. Tissue samples were fixed, embedded in paraffin, sectioned, and stained with H&E. Pathological morphology was observed under an optical microscope, and the extent of damage was assessed based on the stained sections.

[0072] Echocardiography and electrocardiogram monitoring: After hypoxia exposure, mice were anesthetized with isoflurane, their chest hair was shaved, and echocardiography and electrocardiogram (ECG) were performed. A 6LAB animal ultrasound system (Beijing Yiren Hengye Technology Co., Ltd.) equipped with a French VERMON high-frequency probe was used to record mouse echocardiography. M-mode and two-dimensional parasternal long-axis scans were performed at the papillary muscle level to assess changes in ejection fraction (EF) (%) and fractional shortening (FS) (%). Electrocardiogram (ECG) was performed using a multi-channel physiological signal acquisition and processing system. Throughout the experiment, the temperature of the operating table where the mice were placed was maintained at 37°C.

[0073] Open field test: After hypoxia exposure, mice were subjected to an open field test. Mice were allowed to move freely within a test enclosure (50 cm × 50 cm × 35 cm) for 5 minutes, and the number of steps taken by the mice was recorded using tracking software (Shanghai Xinruan Information Technology Co., Ltd.).

[0074] Bone marrow cell flow cytometry analysis: Based on Ter119 and CD44 surface markers, the proportion of erythroid precursor cells at different developmental stages in the bone marrow was quantitatively analyzed by flow cytometry. After filtering, the cell concentration of bone marrow cells was adjusted, and they were stained with FITC-labeled anti-mouse Ter119 antibody and APC-labeled anti-mouse CD44 antibody at 4°C in the dark for 30 minutes. After washing, the cells were analyzed by flow cytometry.

[0075] 2. Results The results are as follows Figure 2 As shown, HAPC model mice exhibited systemic multi-organ damage. Compared with the control group, the low-shear and high-shear whole blood viscosity of HAPC model mice was significantly increased ( Figure 2 A) indicates a high-viscosity state of the blood. Multiple histopathological examinations (H&E staining) showed significant tissue damage and structural abnormalities in the heart, lungs, liver, and sternum (hematopoietic tissue) of the model group mice. Figure 2B). In the model group mice, significant pathological changes were observed in multiple organs: the heart showed disordered arrangement of myocardial fibers, abnormal morphology of cardiomyocytes, and widening of the interstitium with scattered inflammatory cell infiltration; the lungs showed alveolar collapse and fusion, significant thickening of alveolar walls, interstitial edema, and inflammatory cell infiltration; the kidneys showed compression and crowding of glomerular capillary loops, diffuse swelling of renal tubular epithelial cells with vacuolar degeneration and luminal narrowing, and widening of the renal interstitium with inflammatory cell infiltration; the sternal bone marrow showed decreased hematopoietic cell density and a large number of fat vacuoles, resulting in a severe imbalance between hematopoietic tissue and adipose tissue. In contrast, the control group mice had normal morphology and structure of all organs: regular arrangement of myocardial fibers, intact alveolar structure, no abnormalities in the morphology of glomeruli and renal tubules, and dense hematopoietic tissue in the bone marrow without obvious pathological changes. Echocardiography revealed significant changes in the structure and function of the heart in the model group mice. Figure 2 C), specifically manifested as a significant increase in right ventricular anterior wall diastolic thickness (RVAWd), right ventricular systolic diameter (RVIDs), left ventricular posterior wall diastolic thickness (LVPWd) and systolic thickness (LVPWs), and ejection fraction (EF). Figure 2 D). Simultaneous electrocardiogram analysis showed that the P wave amplitude and QRS interval were significantly prolonged in the model group mice ( Figure 2 E). Open field experiments showed that the activity time and frequency in the central region of the model group mice were significantly reduced, suggesting a decrease in their autonomous exploration behavior (E). Figure 2 F). Furthermore, flow cytometry analysis of bone marrow erythroid differentiation revealed an increasing proportion of Ter119+ cells in the model group mice, with significant changes in erythroid cell proportions at certain time points. Figure 2 G, Figure 2 H). Figure 2 The results shown further confirm the successful construction of the high-altitude polycythemia mouse model.

[0076] Example 3: Application of HAPC model mice In recent years, the rapid development and integration of multi-omics technologies have provided powerful tools for systematically elucidating the pathogenesis of various complex diseases, including cancer, metabolic diseases, and neurodegenerative diseases. Notably, current omics research on HAPC primarily focuses on changes in plasma components, while the core effector cells of the disease—red blood cells themselves—have received relatively little attention. Although mature red blood cells lack a nucleus, they are far from being functionally inert oxygen carriers. In addition to their basic gas transport functions, they are widely involved in immune regulation and play a crucial role in maintaining acid-base balance and regulating blood rheology. Recent research further challenges conventional wisdom, revealing the active role of red blood cells in immune regulation and even identifying specific red blood cell subsets with immunomodulatory functions (such as CD63-positive nucleated red blood cells). Furthermore, the metabolic state of red blood cells is crucial to their function. This suggests that under HAPC conditions, the molecular and functional remodeling of red blood cells themselves may play a key but not yet fully understood role in the pathogenesis of the disease.

[0077] Against this backdrop, multi-omics technologies offer a powerful approach for systematically decoding the complex mechanisms of HAPC. By integrating proteomic, phosphoproteomic, metabolomic, and lipidomic data, we can map causal networks from upstream signaling to downstream phenotypes, precisely locating key regulatory nodes and potential therapeutic targets.

[0078] This invention establishes an efficient HAPC mouse model and further utilizes this model to conduct comprehensive multi-omics analysis of erythrocytes, revealing the molecular map of erythrocyte remodeling and its dysregulation in HAPC, thereby providing new insights into the pathophysiology of the disease and laying the foundation for novel intervention measures. At the same time, it demonstrates the reliability, efficiency, and wide applicability of the HAPC mouse model.

[0079] 1. Proteomic analysis of erythrocyte membranes in HAPC-KM mice (1) Experimental methods Mouse erythrocyte isolation and membrane protein extraction: After centrifugation of whole blood, erythrocytes were mixed with hypotonic Tris-HCl buffer and incubated at 4°C for 1 hour to extract membrane proteins. The membrane precipitate was collected by centrifugation and washed until white. The precipitate was resuspended in pre-cooled SDC lysis buffer, denatured at 95°C for 5 minutes, and then sonicated. The supernatant was collected by centrifugation, and the protein concentration was determined by the BCA method.

[0080] Sample preparation: 100 μg of protein was subjected to reductive alkylation, followed by trypsin digestion at 37°C with shaking overnight. The reaction was terminated with TFA, the precipitate was removed by centrifugation, and the supernatants were washed and combined. The supernatants were then desalted using a C18 column. The peptides were concentrated and dried under vacuum, and quantified using Nanodrop. 5 μg of the peptides were reconstituted in a solution containing 1% acetonitrile / 1% formic acid for LC-MS / MS analysis. The remaining sample was used for enrichment of phosphorylated peptides.

[0081] LC-MS / MS analysis: Analysis was performed using a Vanquish Neo liquid chromatography system coupled with an Orbitrap Exploris 480 mass spectrometer. Peptides were loaded onto a pre-column and then subjected to nonlinear gradient separation on the analytical column for 150 minutes. Mass spectrometry parameters: MS1 scan range m / z 350-1200, resolution 60,000; MS / MS scan period 2 seconds, fragmentation of ions with charge states 2-6, resolution 15,000.

[0082] Data processing: Raw data were retrieved from the Swiss-Prot mouse database using Proteome Discoverer 3.0 software. Search parameters: complete trypsin digestion, allowing a maximum of 3 missed cleavage sites, and an FDR threshold of 1% for peptide and protein identification. Differentially expressed proteins were screened using the limma package in R, with a threshold of |log2(fold change)|>1 and a p-value <0.05.

[0083] (2) Results The results are as follows Figure 3 As shown, erythrocyte membrane proteomics revealed significant alterations in key pathways. Erythrocyte membrane proteomics analysis identified nearly 2000 proteins, of which 1666 were proteins shared by both groups (…). Figure 3 A). Principal component analysis showed that the overall proteomic characteristics of the control group and the model group did not exhibit a significant separation trend. Figure 3 B). Differential expression analysis identified 32 significantly differentially expressed proteins, of which 13 were upregulated and 19 were downregulated in the model group. Figure 3 C). GO enrichment analysis showed that ( Figure 3These differentially expressed proteins (DECs) are mainly involved in the following biological processes: regulation of protein stability, intracellular iron ion homeostasis, interferon-mediated signaling pathway, and glycosyl compound catabolic process, suggesting an imbalance of protein and iron ion homeostasis in HAPC mouse erythrocytes, which is related to abnormal erythroid proliferation and immune regulation. In terms of cellular components, the differentially expressed proteins were significantly enriched in the cell membrane and related structures, confirming the high purity of the extracted membrane proteins. Molecular functional analysis showed that the differentially expressed proteins mainly involve G protein activity, structural constituent of cytoskeleton, protein serine / threonine kinase activator activity, ubiquitin ligase inhibitor activity, and phosphatidylglycerol binding, indicating that post-translational modifications of proteins may have a significant impact on erythrocyte skeletal stability in model group mouse erythrocytes. Finally, we used a clustering heatmap to visually demonstrate the expression patterns of these 32 differentially expressed proteins between the two groups. Figure 3 G).

[0084] 2. Phosphorylated proteomics analysis of erythrocyte membranes in HAPC-KM mice (1) Experimental methods Sample preparation: The remaining peptides were enriched using the High-Select Fe-NTA phosphorylated peptide enrichment kit. The enriched phosphorylated peptides were desalted using a self-made C18 column, vacuum dried, and approximately one-third of the enriched product was analyzed by LC-MS / MS.

[0085] LC-MS / MS analysis: Liquid phase gradient and column conditions were the same as for membrane proteomics analysis. Mass spectrometry parameters were slightly adjusted: MS / MS acquisition trigger threshold was set to 4000, and maximum injection time was 120 ms.

[0086] Data processing: The Swiss-Prot mouse database was searched using Proteome Discoverer 3.0 software. Serine, threonine, and tyrosine phosphorylation were set as variable modifications. The IMP ptmRS node was enabled to calculate the phosphorylation site localization probability, and only sites with a probability > 0.8 were retained for downstream analysis. Differentially phosphorylated peptides were screened using the limma package, with a threshold set to |log2(fold change)| > 1 and P < 0.05.

[0087] (2) Results The results are as follows Figure 4 As shown, phosphorylated proteomics analysis revealed widespread hyperphosphorylation of the membrane cytoskeleton and hemoglobin. A total of 902 phosphorylated membrane proteins were identified in the erythrocyte membrane phosphorylated proteomics analysis, of which 782 (approximately 86.7%) were shared by both the control and model groups. Figure 4 A). Based on this, 2746 high-confidence phosphorylated peptides were further identified, of which 2311 (approximately 84.2%) were detected in both groups. Figure 4 B) indicates that phosphorylation modification is widespread and highly reproducible in erythrocyte membrane proteins. Differential analysis showed that, compared to the control group, a total of 84 phosphorylated peptides in the model group underwent significant changes (B). Figure 4 Of these, 58 were significantly upregulated and 26 were significantly downregulated. Notably, the number of differentially phosphorylated peptides was significantly higher than that of previously identified differentially expressed membrane proteins, suggesting that phosphorylation modification may act as a more sensitive functional regulatory layer in response to hypoxic stress when protein expression levels do not change significantly. Principal component analysis further confirmed that, based on the full spectrum of phosphorylated peptide expression data, there was a clear separation trend between the control group and the model group erythrocyte samples. Figure 4 (D) This indicates that chronic hypoxia can systematically alter the phosphorylation modification profile of erythrocyte membranes, which contrasts with the previously observed indistinguishable phenomenon between the two groups in membrane proteomics, highlighting the dynamic role of post-translational modifications in hypoxic adaptation.

[0088] GO functional enrichment analysis revealed that proteins corresponding to differentially phosphorylated peptides were significantly enriched in pathways such as blood coagulation, cell size regulation, erythrocyte homeostasis, lipid transport, and glucose homeostasis at the biological process level. Figure 4 E), these processes are highly consistent with the phenotypes commonly seen in HAPC disease states, such as hypercoagulability, altered erythrocyte morphology, and metabolic remodeling. At the molecular functional level, these proteins are mainly involved in transmembrane transport activities (including chloride and metal ion transport), spectroscopy binding, hemoglobin binding, and oxygen binding. Figure 4 (F) indicates that phosphorylation modification may directly participate in the functional regulation of erythrocytes in hypoxic environments by affecting ion homeostasis, cytoskeleton stability, and oxygen-binding capacity. To further elucidate the role of phosphorylation modification in erythrocyte physiological and pathological processes, we focused on demonstrating changes in phosphorylation levels of key proteins closely related to cytoskeleton integrity and oxygen transport function, providing modalomics evidence for further analysis of HAPC erythrocyte dysfunction.

[0089] 3. Metabolomics analysis of erythrocytes from HAPC-KM mice (1) Experimental methods Sample extraction: Perform on ice. Add 200 μL of cold methanol containing internal standard, vortex for 2 minutes, sonicate in an ice bath to homogenize the sample, and vortex again. Centrifuge at 4°C, collect the supernatant, freeze-dry, and store at -20°C. Before injection, reconstitute with 20% methanol aqueous solution, centrifuge, and collect the supernatant for positive and negative ion mode analysis.

[0090] Non-targeted metabolomics analysis: Data were acquired separately in positive and negative ion modes, including retention time, primary and secondary mass spectrometry information. Blank and QC samples were set up. After system equilibration, blank, two QC injections, and experimental samples in random order were injected sequentially, followed by one final QC injection.

[0091] Chromatographic conditions: A Waters BEH C8 column was used in positive ion mode, and a Waters HSS T3 column was used in negative ion mode. Other parameters remained the same: column temperature 50℃, injection volume 5 μL, flow rate 0.35 mL / min, mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution program: 0–1 min, 5% B; 1.1–11 min, 5% to 100% B; 11.1–13 min, 100% B; 13.1–15 min, 5% B.

[0092] Mass spectrometry conditions: Positive ion mode: Primary full scan combined with secondary DDA scan. Spray voltage 3.8 kV, capillary temperature 320℃, auxiliary gas heater temperature 350℃, sheath gas flow rate 35 Arb, auxiliary gas flow rate 8 Arb, S-lens RF level 50%, full scan range m / z 70-1050, full scan resolution 70,000, MS / MS resolution 17,500, TopN 5, normalized collision energy 20 / 40. Negative ion mode: Spray voltage 3.0 kV, other ion source parameters consistent with positive ion mode.

[0093] (2) Results The results are as follows Figure 5 As shown, metabolomics analysis highlights glycerophospholipid metabolism disorders. Using non-targeted metabolomics techniques, we identified 1576 metabolites in mouse erythrocytes. In unsupervised principal component analysis, both positive and negative ion modes showed a clear separation trend between the control and model group samples, indicating a systematic difference in the metabolic profiles of the two groups of erythrocytes. Figure 5 A). Further screening of differentially metabolites based on statistical significance (p-value) and fold change (fold change) Figure 5B). In positive ion mode, compared with the control group, 79 metabolites were significantly upregulated and 38 metabolites were significantly downregulated in the model group; in negative ion mode, 42 metabolites were significantly upregulated and 27 metabolites were significantly downregulated in the model group. A total of 196 differentially expressed metabolites were included in subsequent analyses. Pathway enrichment analysis was performed on these differentially expressed metabolites. Figure 5 The results showed that these metabolites were significantly enriched in glycerophospholipid metabolism, D-glutamine and D-glutamate metabolism, arginine and proline metabolism, and tryptophan metabolism. Among these, the glycerophospholipid metabolism pathway showed the most significant enrichment, suggesting that this pathway may play a central role in HAPC erythrocyte metabolic remodeling. To further reveal the altered patterns of this pathway, we mapped 27 differentially metabolites from 11 classes onto the glycerophospholipid metabolism map (marked in red). Figure 5 D). Quantitative analysis showed that the vast majority of these metabolites exhibited a significant downregulation trend in the model group ( Figure 5 E) indicates that the synthesis or turnover of glycerophospholipids in the erythrocytes of HAPC mice may be inhibited, which may in turn affect cell membrane integrity and signal transduction function.

[0094] 4. Lipidome analysis of HAPC-KM mouse erythrocytes (1) Experimental methods Sample Extraction: Total lipids were extracted from erythrocyte samples using a modified Bligh / Dyer method. The simplified procedure is as follows: 100 μL of erythrocyte sample was accurately transferred and added to a chloroform-methanol mixture (chloroform:methanol volume ratio 1:2) containing a known concentration of isotopic internal standard. The mixture was vortexed and sonicated on ice to fully lyse the cells and extract lipids. Subsequently, appropriate amounts of chloroform and water were added, vortexed, and centrifuged. The lower organic phase (chloroform layer) was collected, which is the total lipid extract. This extract was dried under a gentle nitrogen stream to remove the organic solvent, yielding the dried lipid fraction.

[0095] Separation and Detection: Lipid separation and detection were performed using a Jasper high-performance liquid chromatography system coupled with a SCIEX TRIPLE QUAD4500 MD triple quadrupole mass spectrometer, employing an electrospray ionization source. Key ion source parameters were optimized as follows: curtain gas pressure 20 psi, ion spray voltage 5500 V, source temperature 400℃, and nebulizer and auxiliary gas pressures both 35 psi. Chromatographic separation was performed on a Phenomenex Luna silica column (3 μm particle size, 150 mm × 2.0 mm id) maintained at 40℃. Mobile phase A was chloroform:methanol:ammonia (89.5:10:0.5, v / v / v), and mobile phase B was chloroform:methanol:ammonia:water (55:39:0.5:5.5, v / v / v / v). Elution was performed using a 36-minute nonlinear gradient: 0-2 minutes, 25% B; 2-25 minutes, linearly increasing from 25% to 100% B; 25-30 minutes, maintaining at 100% B; 30-30.1 minutes, rapidly decreasing from 100% to 25% B; 30.1-36 minutes, equilibrating at 25% B. The flow rate was 0.3 mL / min, and the injection volume was 5 μL.

[0096] Identification and Differential Screening: Lipid identification and absolute quantification employed a multiple reaction monitoring (MRM) mode based on targeted lipidomics. First, for various lipids to be detected (such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol esters), a pre-optimized MRM detection method incorporating specific parent-daughter ion pairs and collision energies was established by injecting standards. Raw mass spectrometry data were imported into software such as SCIEX OS or Analyst for processing. Lipid identification was performed by comparing retention times and characteristic MRM ion pairs. Quantification was achieved by calculating the ratio of the characteristic peak area of ​​each lipid to its corresponding internal standard peak area, and then substituting this ratio into a standard curve prepared from the internal standard to obtain the absolute or relative concentration of each lipid molecule. To screen for differentially expressed lipids between the HAPC model group and the control group, the quantified lipid abundance data were imported into statistical software. Due to potential variance differences between groups, the Games-Howell test was used to compare inter-group differences. Significance thresholds and fold change thresholds were set, and lipid molecules that simultaneously met the conditions of P value < 0.05 and fold change (FC) > 1.5 (upregulation) or FC < 1 / 1.5 (downregulation) were defined as significantly differentially expressed lipids.

[0097] (2) Results The results are as follows Figure 6 As shown, lipidomics analysis confirmed extensive remodeling of erythrocyte membrane lipids. Through targeted lipidomics analysis, we identified 376 lipid molecules across 28 classes in mouse erythrocytes. Among these, phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine and phosphatidylcholine containing free fatty acids were the most abundant in terms of variety. Figure 6 A). Principal component analysis showed that the lipid profiles of the control group and the model group exhibited a significant separation trend ( Figure 6 B) indicates that the HAPC model induces systemic changes in erythrocyte lipid composition. Further analysis identified 40 differentially expressed lipids, of which 39 were significantly downregulated in the model group, and only 1 was significantly upregulated (B). Figure 6 C). According to the statistical significance ranking, the top 10 most significantly different lipids were mainly lysophosphatidylcholine and sphingomyelin (C). Figure 6 D), which corroborates the aforementioned results of glycerophospholipid metabolism disorders in metabolomics. Further analysis of the differentially regulated lipid categories revealed that the only upregulated lipid belonged to the ceramide class; while the main downregulated lipids were enriched in ether-linked phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine. Figure 6 These lipids (E) are all directly involved in glycerophospholipid metabolism and membrane structure regulation. Specifically, PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the main glycerophospholipids constituting the cell membrane bilayer, jointly maintaining membrane integrity, fluidity, and signal transduction. LPC (lysophosphatidylcholine) is a hydrolysis product of PC under the action of phospholipase A2, and usually acts as a molecule regulating inflammation and membrane remodeling; its accumulation can affect membrane stability and cell function. PE-O (ether-linked phosphatidylethanolamine) is a special type of PE whose ether bond structure enhances membrane antioxidant capacity and plays a role in signal transduction. The downregulation of these lipids collectively suggests that HAPC erythrocytes may experience inhibition of membrane lipid metabolism, changes in membrane structure, and decreased antioxidant capacity. This is consistent with the conclusion of overall glycerophospholipid metabolic pathway disorder and supports the hypothesis that impaired membrane function participates in the development of HAPC disease.

[0098] 5. Multi-omics integrated analysis (1) Experimental methods Following the methods described above, metabolites, proteins, phosphorylated proteins, and lipids showing significant differences between the model group and the control group were screened. We integrated and mapped all molecules identified in the four omics studies that belong to the glycerophospholipid metabolic pathway (including information on their expression, modification, and abundance changes) into a standard glycerophospholipid metabolic pathway map (https: / / www.kegg.jp / pathway / mmu00564). In this pathway map, we used different shapes to distinguish molecule types: circles represent metabolites, and rectangles represent proteins or enzymes. Colors were used to indicate the direction and degree of change; light green indicated unidentified proteins; pink boxes indicated identified erythrocyte membrane phosphorylated proteins with no statistically significant difference in phosphorylation modification levels; light blue boxes marked identified erythrocyte membrane proteins with no significant change in expression levels; and red boxes marked phosphorylated proteins with significantly reduced phosphorylation sites. Through integrated analysis and network visualization, we identified key nodes (Lpin2, Cds2 and their related metabolites) that have undergone significant changes at multiple omics levels and are located at the core of the regulatory network, thus clearly presenting the core inhibitory axis from upstream enzyme activity regulation to downstream lipid composition changes.

[0099] (2) Results The results are as follows Figure 7 As shown, multi-omics integrated analysis points to glycerophospholipid metabolism disorder. Through integrated analysis of erythrocyte proteomics, phosphorylated proteomics, metabolomics, and lipidomics data, we mapped molecules from each omics onto the glycerophospholipid metabolism pathway map, systematically demonstrating the multidimensional regulatory changes of this pathway in the HAPC model. Figure 7In this integrated pathway diagram, red circles represent differentially expressed metabolites that show significant changes in the model group, primarily concentrated at key nodes in glycerophospholipid synthesis and conversion. Light blue boxes mark identified erythrocyte membrane proteins whose expression levels did not change significantly, indicating no significant alterations at the transcriptional or translational levels. Pink boxes represent identified phosphorylated erythrocyte membrane proteins, whose phosphorylation modification levels also showed no statistically significant differences. Notably, red boxes highlight Lpin2 and Cds2, proteins with significantly altered phosphorylation sites in the model group. In this metabolic pathway, the differentially expressed metabolites marked by red circles generally show a downregulated trend, while the phosphorylation levels of key enzymes involved in this pathway regulation, Lpin2 and Cds2, are also significantly reduced. This convergent change collectively suggests that in HAPC model erythrocytes, the glycerophospholipid metabolic pathway may involve multi-level inhibition, from upstream enzyme activity regulation to downstream product synthesis. This metabolic remodeling not only affects the lipid composition and structural integrity of the cell membrane, but also suggests that under chronic hypoxic stress, erythrocytes may precisely reprogram lipid metabolism pathways by finely regulating the phosphorylation state of key synthetic enzymes, thus becoming one of the core links in their membrane dysfunction and metabolic adaptation disorder.

[0100] In summary, the results indicate that the high-altitude polycythemia vera (HAPC) mouse model constructed in this invention is reliable and efficient in elucidating the HAPC mechanism. It can achieve high-throughput analysis at multiple omics levels, identify a large number of statistically significant differentially expressed molecules, and stably reproduce the core pathological features of HAPC. It can be applied to screen candidate drugs or therapeutic targets for the prevention and treatment of HAPC, and has important significance in the study of the pathogenesis of HAPC, drug screening, and development.

[0101] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing a mouse model of high-altitude polycythemia, characterized in that, The method involves placing mice in a low-pressure, low-oxygen environment and intervening with erythropoietin to obtain a mouse model of high-altitude polycythemia.

2. The method according to claim 1, characterized in that, The method includes the following steps: S1) Place the mice in a low-pressure, low-oxygen environment; S2) The mice were raised in a low-pressure, low-oxygen environment for 14 days, and erythropoietin was administered every 2 days to obtain a mouse model of high-altitude polycythemia.

3. The method according to claim 1 or 2, characterized in that, The erythropoietin intervention involved administering erythropoietin to mice.

4. The method according to claim 3, characterized in that, The application is by injection.

5. The method according to claim 4, characterized in that, The injection was administered intraperitoneally.

6. The method according to any one of claims 2-5, characterized in that, In step S2), the dose of erythropoietin intervention is 100 IU / animal per intervention.

7. The method according to any one of claims 1-6, characterized in that, The low-pressure, low-oxygen environment described is a simulation of the low-pressure, low-oxygen environment of a plateau.

8. The method according to any one of claims 1-7, characterized in that, The low-pressure, low-oxygen environment simulates the low-pressure, low-oxygen environment of a plateau at an altitude of 6,000 meters.

9. The method according to any one of claims 1-8, characterized in that, The mice weighed 18-22 g.

10. The use of the high-altitude polycythemia mouse model constructed by any one of claims 1-9 in any of the following: A1) Application in screening therapeutic targets for high-altitude polycythemia; A2) Its application in screening drugs for the prevention or treatment of high-altitude polycythemia; A3) Application in the study of the pathogenesis of high-altitude polycythemia.

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