Application of namoscated in the prevention and treatment of pulmonary arterial hypertension

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of namodenoson in prevention and treatment of pulmonary arterial hypertension. The application discloses that, as a specific small molecule inhibitor, namodenoson can effectively block the physical interaction between SHMT2 and RhoB, restore the ubiquitination degradation of RhoB, and thus reverse the rearrangement of endothelial cell skeleton and the damage of barrier function caused by overexpression of SHMT2. In a pulmonary arterial hypertension animal model, treatment with namodenoson can significantly reduce the mean pulmonary arterial pressure and the right ventricular systolic pressure, reduce the right ventricular hypertrophy, and improve the pulmonary vascular remodeling. The above protective effect of namodenoson is achieved by specific intervention on the SHMT2-RhoB axis, and the use of namodenoson in the treatment of PH can greatly shorten the period of preclinical and clinical research, reduce the research and development risk, and has extremely high clinical conversion value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of namodinosin in the prevention and treatment of pulmonary hypertension. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pulmonary hypertension (PH) is a fatal pulmonary circulatory disease characterized by progressively increasing pulmonary vascular resistance and pulmonary vascular remodeling, which can ultimately lead to right heart failure and even death. Although existing targeted drugs (such as prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase-5 inhibitors) can dilate blood vessels and improve hemodynamics and clinical symptoms to some extent, their effects on reversing or inhibiting pulmonary vascular remodeling are very limited. Therefore, elucidating the molecular mechanisms of pulmonary vascular remodeling and developing novel drugs that can intervene in this pathological process are key scientific issues that urgently need to be addressed in this field.

[0004] Endothelial dysfunction is the initiating factor in pulmonary endothelial remodeling (PH). Under pathological stimuli such as hypoxia, pulmonary artery endothelial cell permeability increases, leading to functional disorders and subsequently inducing angiogenesis and an inflammatory cascade. Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon metabolism, and recent studies have revealed that it also possesses non-metabolic "part-time functions" in addition to its metabolic functions. Existing research has reported that SHMT2 expression is significantly upregulated in the lung tissues of PH patients and animal models, and that endothelial cell-specific SHMT2 knockout can alleviate PH disease progression, suggesting that SHMT2 could be a potential target for PH treatment. However, existing inhibitors targeting SHMT2 (such as SHIN1 and SHIN2) mainly target its metabolic enzyme activity, resulting in poor specificity, significant toxicity, and no reported applications in PH. More importantly, the specific mechanism by which SHMT2 participates in endothelial dysfunction in PH through its non-classical function remains unclear, making the development of novel intervention strategies targeting this mechanism of significant clinical translational value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the inventors, through long-term technical and practical exploration, have developed the application of namodenoson in the prevention and treatment of pulmonary hypertension. This invention, through research, first discovered and confirmed that namodenoson (CAS: 163042-96-4), as a specific small molecule inhibitor, can effectively block the physical interaction between SHMT2 and RhoB, restore RhoB ubiquitination and degradation, thereby reversing endothelial cytoskeleton rearrangement and barrier function impairment caused by SHMT2 overexpression. In animal models of pulmonary hypertension, treatment with namodenoson significantly reduced mean pulmonary artery pressure and right ventricular systolic pressure, alleviated right ventricular hypertrophy, and improved pulmonary vascular remodeling. The protective effects of namodenoson are achieved through specific intervention of the SHMT2-RhoB axis. Based on these research findings, this invention was completed.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: A first aspect of the invention provides the use of namodinosin in the preparation of a medicament for the prevention and / or treatment of pulmonary hypertension.

[0007] The prevention and / or treatment of pulmonary hypertension includes at least one or more of the following effects: (a) Inhibit the interaction between SHMT2 and RhoB protein, and restore the ubiquitination and degradation of RhoB protein; (b) Improve hemodynamic parameters; (c) Protect the right heart structure; (d) Improve pulmonary vascular remodeling; (e) Protect the pulmonary vascular endothelial barrier.

[0008] Specifically, in (b), the improvement of hemodynamic parameters is manifested in reducing mean pulmonary artery pressure and reducing right ventricular systolic pressure. In (c), protecting the right ventricular structure specifically means reducing right ventricular hypertrophy and improving right ventricular systolic function; further, improving right ventricular systolic function specifically means increasing the tricuspid annulus systolic displacement (TAPSE) and increasing the pulmonary artery blood flow acceleration time to ejection time ratio (PAT / ET). In (d), the improvement of pulmonary vascular remodeling is specifically manifested in: reducing the thickness of the pulmonary arteriole wall and reducing the degree of pulmonary vascular muscularization.

[0009] In (e), protecting the pulmonary vascular endothelial barrier specifically manifests as reducing pulmonary vascular leakage and maintaining the integrity of intercellular connections.

[0010] In other words, Namodinosin effectively inhibited the development of pulmonary hypertension by intervening in the Shmt2-RhoB axis.

[0011] The product may be a drug or an experimental reagent, which is intended for use in basic research and can be used to construct relevant cell or animal models.

[0012] A second aspect of the present invention provides a pharmaceutical composition comprising namodinosin with at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0013] The other active pharmaceutical ingredients include substances that prevent and / or treat pulmonary hypertension.

[0014] The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0015] A third aspect of the present invention provides a method for preventing and / or treating pulmonary hypertension and related diseases, the method comprising administering the above-described namodinosine or the above-described pharmaceutical composition to a subject.

[0016] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects: The aforementioned technical solution reveals for the first time a novel pathological mechanism by which SHMT2 directly interacts with RhoB through its non-metabolic function (non-one-carbon metabolic activity), stabilizing RhoB protein through deubiquitination, and thereby promoting endothelial dysfunction in pulmonary hypertension, providing a new molecular target for PH intervention. Furthermore, this technical solution also discloses for the first time the novel application of the known drug namodinosin in the preparation of drugs for treating pulmonary hypertension. Namodinosin exerts its anti-PH effect by specifically blocking the SHMT2-RhoB interaction, a mechanism completely different from existing PH-targeting drugs and traditional inhibitors targeting SHMT2 metabolic activity, providing a novel treatment option for PH patients, especially those insensitive to existing treatments or who have developed resistance. Simultaneously, namodinosin has received orphan drug and fast track designations from the FDA and has demonstrated good safety and tolerability in phase II / III clinical trials for advanced liver cancer and non-alcoholic steatohepatitis. Its application in the treatment of PH can significantly shorten the preclinical and clinical research cycle, reduce development risks, and has extremely high clinical translational value and market potential. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1In this embodiment of the invention, Shmt2 in endothelial cells of pulmonary hypertension (PH) is significantly elevated; wherein, A: schematic diagram of proteomics detection process for hypoxic endothelial cells; B: heatmap analysis of differentially expressed genes in proteomics; C: correlation analysis of staining positivity intensity of Shmt2 in lung tissue of PH patients with mean pulmonary artery pressure; DE: expression level of Shmt2 in hypoxic hPAEC; F: expression level of Shmt1 in hypoxic hPAEC; GI: expression level of Shmt2 in the lungs of rat and mouse PH models; JK: staining level of Shmt2 in lung regions of PH patients and mouse models; LN: expression level of Shmt2 in sorted cell populations of the lungs of PH patients and mouse models; OP: changes in Shmt2 expression at the transcriptional and translational levels after HIF1a interference; Q: CHIP experiment showing the binding of HIF1a to the Shmt2 promoter.

[0019] Figure 2 In this embodiment of the invention, SHMT2 knockout in endothelial cells can alleviate the hypoxia-induced pulmonary hypertension phenotype in mice, while SHMT2 overexpression exacerbates the phenotype. A: Schematic diagram of the endothelial cell Shmt2 knockout mouse model; B: Cell sorting to verify the knockout specificity of Shmt2 in mouse lung tissue; CD: Right ventricular systolic pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) of Shmt2 ECKO mice and control mice; E: EM staining and α-SMA staining of mouse lung tissue; F: Statistical analysis of vascular myomorphization ratio in mouse lung tissue; N: No α-SMA signal; P: α-SMA covering a portion of the vascular circumference; F: α-SMA continuously covering the entire vascular circumference. G: Schematic diagram of the AAV9 vector for Shmt2 overexpression in endothelial cells; H: Cell sorting verification of the expression specificity of Shmt2 in endothelial cells in mouse lung tissue; IJ: Right ventricular systolic blood pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) in Shmt2 overexpressing mice and control mice; K: EM staining and α-SMA staining of mouse lung tissue; L: Statistical analysis of vascular myomorphization ratio in mouse lung tissue. p<0.05; p<0.01; p<0.001.

[0020] Figure 3This invention illustrates the effectiveness of targeted endothelial cell Shmt2 gene therapy in alleviating pulmonary hypertension. A: Schematic diagram of establishing a rat MCT pulmonary hypertension model and administering AAV9-Shmt2-shRNA intervention; B: Western blotting analysis of Shmt2 knockout efficiency in mouse lung tissue; C: Mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) in the AAV9-Shmt2-shRNA intervention group and control group; D: EM staining and α-SMA staining of rat lung tissue; E: Statistical analysis of vascular myositis ratio in mouse lung tissue; F: Statistical analysis of pulmonary vessel wall thickness in each group of rats; G: RV / LV+S ratio in each group of rats; H: Schematic diagram of establishing a rat Su5416 pulmonary hypertension model and administering AAV9-Shmt2-shRNA intervention; I: Mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) in the intervention group and control group of rats; J: Statistical analysis of pulmonary vessel wall thickness in each group of rats; K: EM staining and α-SMA staining of rat lung tissue; L: Statistical analysis of RV / LV+S ratio in each group of rats. p<0.05; p<0.01; p<0.001.

[0021] Figure 4 In this embodiment of the invention, SHMT2 promotes pulmonary artery endothelial cell dysfunction by targeting the non-metabolic function of RhoB. The data includes: AC: Proteomics analysis of differentially expressed genes in endothelial cells with Shmt2 interference and the control group, including heatmaps of differentially expressed genes, GO pathway enrichment analysis, and differential gene distribution analysis; D: Western blotting detection of Shmt2 expression levels in the cytoplasm and mitochondria; E: TEET detection of endothelial cell permeability resistivity; F: Phalloidin staining; G: FITC-dextran permeability detection; H: Schematic diagram of Shmt2 and Shmt2a sequences; I: Live cell mitochondrial staining and GFP fluorescence; J: Serine labeling and tracing experiments; KM: Endothelial cell permeability detection; N: Proteomics analysis of differentially expressed genes in endothelial cells with Shmt2 interference and the control group, including a volcano plot; OP: Changes in RhoB expression levels after Shmt2 interference; QR: RhoB expression levels after overexpression of Shmt2 and Shmt2a; SU: Endothelial permeability assay. p<0.05; p<0.01; p<0.001.

[0022] Figure 5 In this embodiment of the invention, RhoB is a key molecule that influences endothelial cell permeability and promotes pulmonary hypertension in Shmt2; wherein, A: schematic diagram of the RhoB overexpression experiment in Shmt2ECKO mice; B: Western blotting detection of RhoB expression levels in different cells of mouse lung tissue; CD: right ventricular systolic blood pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) of AAV9-ICAM2-RhoB and control-infected Shmt2ECKO mice; E: EM staining and α-SMA staining of mouse lung tissue; F: statistical analysis of vascular myomorphization ratio in mouse lung tissue, N: no α-SMA signal, P: α-SMA covering part of the vascular circumference; F: α-SMA continuously covering the entire vascular circumference. G: Electron microscopy of mouse lung vascular permeability; H: Evans blue assay of mouse lung vascular permeability; I: Schematic diagram of RhoB knockdown experiment in endothelial cells of SHMT2α-overexpressing mice; JK: Right ventricular systolic pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) in mice; L: EM staining and α-SMA staining of mouse lung tissue; M: Statistical analysis of vascular myomorphization ratio in mouse lung tissue; N: Evans blue assay of mouse lung vascular permeability. p<0.05; p<0.01; p<0.001.

[0023] Figure 6 In this embodiment of the invention, Shmt2 induces RhoB deubiquitination, leading to RhoB accumulation. The assays are as follows: AB: CHX assay to detect RhoB protein degradation rate; C: Protein degradation pathway inhibitor assay to detect the pathway by which Shmt2 promotes RhoB degradation; DE: CoIP assay to detect the interaction between Shmt2 and RhoB; F: Immunofluorescence staining; GH: Schematic diagram of Shmt2 and RhoB truncated mutants; IJ: CoIP assay of protein truncated mutants; KL: Ubiquitin assay to detect the ubiquitin type of RhoB; M: Site-directed mutagenesis assay to detect the K63 ubiquitination modification site of RhoB affected by Shmt2. p<0.01; p<0.001.

[0024] Figure 7This invention demonstrates how namodinosin effectively blocks the effects of Shmt2 and RhoB and delays pulmonary hypertension. The diagrams show: AC: Flowchart of small molecule screening and structural diagram of namodinosin; D: CoIP assay to detect the inhibitory effect of namodinosin on the binding of Shmt2 and RhoB; E: Ubiquitination assay; F: Western blotting to detect RhoB expression levels; G: Phalloidin staining; H: Permeability assay; I: H2-labeled serine tracing assay; J: Cytoplasmic and mitochondrial separation assay; KP: Treatment effect of namodinosin on Su5416-induced pulmonary hypertension in rats; QW: Treatment effect of namodinosin on MCT-induced pulmonary hypertension in rats. p<0.01; p<0.001.

[0025] Figure 8 In the embodiments of the present invention, A: Schematic diagram of namodinosin intervention in the Shmt2a overexpressing rat MCT pulmonary hypertension model; BC: α-SMA and EM staining of the pulmonary artery wall, and statistical analysis of vessel wall thickness and myomorphization ratio; DF: Treatment of Shmt2a overexpressing MCT-induced pulmonary hypertension rats with namodinosin; G: Schematic diagram of hypoxia modeling and namodinosin intervention in mice with endothelial-specific RhoB overexpression; HI: Right ventricular function indicators; JK: Vascular myomorphization ratio; G: Evans Blue staining of mouse lung tissue; LN: Electron microscopy detection of mouse pulmonary vascular permeability. p<0.01; p<0.001. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] In a typical embodiment of the present invention, the use of namodinosin in the preparation of a medicament for the prevention and / or treatment of pulmonary hypertension is provided.

[0029] It should be noted that this application is being disclosed for the first time and is different from its known clinical uses. The structural formula of namodinosin is as follows:

[0030] The prevention and / or treatment of pulmonary hypertension includes at least one or more of the following effects: (a) Inhibit the interaction between SHMT2 and RhoB protein, and restore the ubiquitination and degradation of RhoB protein; (b) Improve hemodynamic parameters; (c) Protect the right heart structure; (d) Improve pulmonary vascular remodeling; (e) Protect the pulmonary vascular endothelial barrier.

[0031] Specifically, in (b), the improvement of hemodynamic parameters is manifested in reducing mean pulmonary artery pressure and reducing right ventricular systolic pressure. In (c), protecting the right ventricular structure specifically means reducing right ventricular hypertrophy and improving right ventricular systolic function; further, improving right ventricular systolic function specifically means increasing the tricuspid annulus systolic displacement (TAPSE) and increasing the pulmonary artery blood flow acceleration time to ejection time ratio (PAT / ET). In (d), the improvement of pulmonary vascular remodeling is specifically manifested in: reducing the thickness of the pulmonary arteriole wall and reducing the degree of pulmonary vascular muscularization.

[0032] In (e), protecting the pulmonary vascular endothelial barrier specifically manifests as reducing pulmonary vascular leakage and maintaining the integrity of intercellular connections.

[0033] In other words, Namodinosin effectively inhibited the development of pulmonary hypertension by intervening in the Shmt2-RhoB axis.

[0034] According to the present invention, not only is the use of namodinosin in the preparation of a formulation for the prevention and / or treatment of pulmonary arterial hypertension disclosed, but also that the effect can be enhanced when namodinosin is administered in combination with at least one other pharmaceutically active ingredient. Namodinosin can also be used in combination with other non-pharmaceutical active ingredients as a substitute or supplement to other pharmaceutically active ingredients.

[0035] In another specific embodiment of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising namodinosin and at least one other pharmaceutical active ingredient and / or at least one other non-pharmaceutical active ingredient.

[0036] The other active pharmaceutical ingredients include substances that prevent and / or treat pulmonary hypertension or assist in the prevention and / or treatment of pulmonary hypertension.

[0037] The non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0038] In another specific embodiment of the present invention, the non-pharmaceutical active ingredient includes: Pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids; The ingredients include pharmacologically harmless salts (preferably sodium chloride), flavoring agents, vitamins (preferably vitamin A or vitamin E, tocopherol or provitamins), antioxidants (preferably ascorbic acid), and stabilizers and / or preservatives.

[0039] The dosage forms of the pharmaceutical preparations include: liquid dosage forms, solid dosage forms, external preparations, and sprays; In another specific embodiment of the present invention, the following dosage forms are included: true solutions, colloids, microparticles, emulsions, vortexes, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, inclusion complexes, fillers, patches, and liniments.

[0040] In another specific embodiment of the present invention, a method for preventing and / or treating pulmonary hypertension and related diseases is provided, the method comprising administering the above-mentioned namodinosine or the above-mentioned pharmaceutical composition to a subject.

[0041] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0042] Example 1. Research Subjects 1.1 In vivo experiments (1) Eight-week-old male C57BL / 6J mice, Shmt2flox / flox mice, Cdh5-CreERT2 mice and shmt2+ / - mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (2) Sprague–Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0043] 1.2 In vitro experiments (1) Human pulmonary artery endothelial cells were cultured in an incubator at 37°C and 5% CO2 using endothelial cell culture medium (#1001, ScienCell); (2) HEK-293T cells were cultured in DMEM medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin in a constant temperature incubator at 37°C and 5% CO2.

[0044] 2. Experimental Methods 2.1 Establishment of mouse and rat models of pulmonary hypertension In vivo experiments used 8-10 week old mice and rats. All animals were housed under 12-hour circadian conditions with free access to adequate food and water. Three rodent models of pulmonary hypertension (PH) were employed: a hypoxia-induced PH mouse model, a monoclotaline (MCT)-induced PH rat model, and a Sugen 5416 / hypoxia (SuHx)-induced PH rat model. In the chronic hypoxia-induced PH model, mice were randomly assigned to two groups, exposed to either a chronic hypoxic environment (10% O2) or a normoxic environment (21% O2) for 4 weeks in a ventilated incubator (BioSpherix, Lacona, NY, USA). In the MCT-induced PH model, Sprague–Dawley (SD) rats were subcutaneously injected with MCT (60 mg / kg, Sigma-Aldrich, St. Louis, Missouri, USA). In the SuHx-induced PH model, rats were first subcutaneously injected with the VEGF receptor inhibitor Sugen 5416 (20 mg / kg, Sigma-Aldrich) and then placed in a hypoxic chamber for 3 weeks.

[0045] 2.2 Construction and Identification of Endothelial Cell Shmt2-Specific Gene Knockout Mice (1) Construction of endothelial cell Shmt2-specific gene knockout mice: Shmt2 - / - Since mice are lethal during the embryonic stage, this study used Shmt2 mice. + / - Mice. Shmt2 + / -Both the shmt2flox / flox mice and the Cdh5-CreERT2 mice were of the C57BL / 6 strain. To obtain endothelial cell-specific SHMT2-deficient mice (Shmt2ECKO), Shmt2flox / flox mice were crossed with Cdh5-CreERT2 mice. After reaching adulthood, the mice were intraperitoneally injected for 5 consecutive days with tamoxifen (1 mg / day, Sigma-Aldrich, St. Louis, Missouri, USA) to induce gene knockout. Cdh5-Cre... - / - Mice were used as controls. Genomic DNA was extracted from mouse tails and genotyped using PCR.

[0046] (2) AAV9-mediated Shmt2 gene overexpression and shRNA-mediated Shmt2 knockdown: The coding sequences for Shmt2, Shmt2α, and RhoB were cloned into an AAV9 vector carrying the ICAM2 promoter (Vigene Biosciences, Rockville, Maryland, USA). Mice were randomly assigned to receive a single tail vein injection of AAV9-ICAM2-Shmt2 (or AAV9-ICAM2-Shmt2α, AAV9-ICAM2-RhoB), or AAV9-ICAM2-Ctrl, at a dose of 5 × 10¹¹ vg / mouse. Four weeks after administration, the expression levels of SHMT2 and RhoB were detected by flow cytometry sorting (FACS) and Western blot analysis. Endothelial cell-specific knockdown of Shmt2 or RhoB in rodents was achieved via AAV9-mediated shRNA delivery (Vezhen Biotechnology, Shmt2 shRNA: 5'-GGCAAAGAGATCCCTTATA-3', SEQ ID NO.1; RhoB shRNA: 5'-CGTGTTCAGTAAAGAC GAA-3', SEQ ID NO.2). AAV9-scrambled shRNA served as a control. SD rats or mice were randomly assigned to two groups, receiving either shRNA targeting Shmt2 or RhoB or an AAV9 vector carrying scrambled shRNA via tail vein injection. Lung tissue was collected 4 weeks after AAV administration, and gene silencing efficiency was assessed by Western blot analysis.

[0047] 2.3 Other methods: paraffin sectioning, HE staining, Masson staining, cell transfection, Western blot, immunoprecipitation, ubiquitination assay, RT-qPCR, etc.

[0048] 3. Results 3.1 Shmt2 levels in endothelial cells were significantly elevated in patients and animal models of pulmonary hypertension. To further investigate the target molecules of endothelial cell dysfunction during pulmonary hypertension, the team analyzed the proteomic sequencing results of dermal cells after 24 hours of hypoxia. Figure 1 AB), combined with previous experiments, found that Shmt2 is highly expressed in hypoxic endothelial cells and that the expression level of Shmt2 is correlated with the pulmonary artery pressure of patients. Figure 1 C). In vitro experiments showed that in hypoxic endothelial cells, Shmt2 was significantly elevated at both transcriptional and translational levels. Figure 1 DE), while the expression level of its cytoplasmic isoenzyme Shmt2 did not change significantly. Figure 1 F). Animal experiments showed that in both MCT and SuHx-stimulated rat pulmonary hypertension models and mouse hypoxic pulmonary hypertension models, the expression level of Shmt2 in the lungs was upregulated. Figure 1 GI), and lung tissue staining showed that shmt2 was highly expressed in the lung tissues of patients with pulmonary hypertension and a murine model of pulmonary hypertension, and was mainly expressed in the vascular endothelium and perivascular interstitial area. Figure 1 JK). Through tissue cell sorting, we found that in PH patients and a mouse PH model, Shmt2 was mainly highly expressed in CD31+ endothelial cells. Figure 1 LN). Furthermore, we found that hypoxia-induced upregulation of the transcription factor HIF1a led to increased expression of Shmt2 ( Figure 1 OP), HIF1a binds to the promoter region of Shmt2 ( Figure 1 Q).

[0049] 3.2 Endothelial cell overexpression of Shmt2 promotes the progression of pulmonary hypertension In mice with endothelial cell-specific knockout of Shmt2 ECKO, we first verified the specificity of Shmt2 knockdown in endothelial cells by sorting lung tissue cells. Figure 2 AB). After establishing a pulmonary hypertension model using Shmt2ECKO mice, it was found that reducing Shmt2 significantly alleviated the development of pulmonary hypertension, as evidenced by a significant decrease in right ventricular systolic blood pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) in the model group of Shmt2ECKO mice compared to the control mice. Figure 2 CD), vessel wall thickness and vascular muscularization ratio were significantly reduced ( Figure 2 EF). Through endothelial cell overexpression of Shmt2 ( Figure 2 We found that pulmonary hypertension was significantly aggravated in mice with endothelial cell Shmt2-OE (GH). Figure 2 IL).

[0050] 3.3 Shmt2 gene therapy targeting endothelial cells has preventive and therapeutic effects on pulmonary hypertension. We administered AAV9-mediated Shmt2 knockdown intervention to rat endothelial cells before establishing the MCT pulmonary hypertension model, and the pulmonary hypertension model was established two weeks later. Figure 3 A). The results showed that the level of Shmt2 in the lung tissue of rats treated with AAV9-Shmt2-shRNA was significantly reduced ( Figure 3 B). Mean pulmonary artery pressure (mPAP) and RVSP in rats with decreased endothelial cell Shmt2 levels. Figure 3 C). EM staining and α-SMA staining of rat lung tissue showed that knockdown of Shmt2 in endothelial cells significantly reduced the myomorphization ratio of lung vessels, vessel wall thickness, and RV / LV+S ( Figure 3 DG). Furthermore, during the progression of the Su5416-induced rat pulmonary hypertension model, we intervened with AAV9-Shmt2-shRNA in the model animals to knock down the level of Shmt2 in endothelial cells (DG). Figure 3 H). It was found that knocking down endothelial cell Shmt2 levels during the pathogenesis of pulmonary hypertension significantly reduced mean pulmonary artery pressure (mPAP), RVSP, RV / LV+S, and vessel wall thickness in rats. Figure 3 IK).

[0051] 3.4 Shmt2 in endothelial cells upregulates RhoB expression through non-metabolic pathways, promoting increased endothelial cell permeability. By analyzing the differentially expressed proteomic genes and enriched pathways in Shmt2-interfering and control hPAECs under hypoxic stimulation, we found that Shmt2 mainly affects endothelial cell proliferation and permeability, and the differentially expressed genes affected are mostly located in the cytoplasm (Figure 4 AC). Through cytoplasmic and mitochondrial isolation, we found that hypoxia led to upregulation of Shmt2 expression levels in both the cytoplasm and mitochondria. Figure 4 D). Through TEER permeability experiments, we found that hypoxia led to increased permeability of the endothelial cell monolayer membrane, and that interfering with Shmt2 expression could reverse the hypoxia-induced increase in endothelial cell permeability. Figure 4 E). Phalloidin staining showed a significant increase in F-actin in endothelial cells due to hypoxia, suggesting that endothelial cells were under stress and tended towards a contractile phenotype. Intervention with Shmt2 effectively reduced the increase in F-actin caused by hypoxia, while the one-carbon metabolic intermediates formic acid and glycine could not reverse this phenotype, indicating that Shmt2 affects the increase in stress fibers in endothelial cells caused by hypoxia, and is unrelated to its role in one-carbon metabolism. Figure 4 FG). Next, we constructed a lentiviral expression vector for Shmt2a without mitochondrial localization sequences (FG). Figure 4 H), by detecting GFP expression, it was confirmed that Shmt2a is located in the cytoplasm of endothelial cells and has no co-localization with mitochondria. Figure 4 I). Further, we use H... 2 Labeled serine tracing experiments confirmed that Shmt2a does not affect the one-carbon metabolism level of endothelial cells. Figure 4 J). Through endothelial cell permeability assays and phalloidin staining, we found that the permeability of endothelial cells overexpressing Shmt2 and Shmt2a was significantly increased. Figure 4 KM). By analyzing differentially expressed genes in endothelial cells with Shmt2 interference, we found that the gene RhoB, which is related to endothelial cell permeability, was significantly decreased after Shmt2 interference. Figure 4 N). In vitro experiments showed that hypoxia led to increased RhoB expression levels in endothelial cells. Both normoxic and hypoxic conditions caused significant downregulation of RhoB protein levels, but RhoB RNA levels did not decrease with Shmt2 knockdown in hypoxic-stimulated endothelial cells. Figure 4 The OP suggests that Shmt2 may affect RhoB expression through post-transcriptional modification. Overexpression of both Shmt2 and Shmt2a can upregulate RhoB expression levels. Figure 4 Further experiments showed that phalloidin staining and permeability assays demonstrated that interfering with RhoB could reverse the increased endothelial permeability caused by overexpression of Shmt2a, indicating that RhoB is a key target molecule of Shmt2 influencing endothelial cell permeability. Figure 4 SU).

[0052] 3.5 In vivo validation that RhoB is a key molecule of Shmt2 influencing endothelial cell permeability and promoting the progression of pulmonary hypertension. RhoB was specifically overexpressed in the lung endothelial cells of Shmt2ECKO mice using AAV9, and the specificity of RhoB overexpression in endothelial cells was verified by sorting lung tissue cells. Figure 5 AB). After establishing a pulmonary hypertension model using endothelial RhoB overexpression mice, it was found that RhoB overexpression reversed the pulmonary hypertension phenotype improved by Shmt2 knockout. Specifically, the right ventricular systolic blood pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) were significantly higher in the RhoB overexpression group compared to the Shmt2 knockout mice. Figure 5 CD), vessel wall thickness and vascular muscularization ratio were also significantly increased ( Figure 5 EF). Simultaneously, electron microscopy revealed that RhoB overexpression led to the opening of cell junctions in pulmonary small vessel endothelial cells, and Evans blue staining showed increased lung permeability in mice. Figure 5 GH).

[0053] Next, we used AAV9 to specifically knock down RhoB in the endothelium of mice that overexpressed Shmt2a. Figure 5 I). After establishing a pulmonary hypertension model, RhoB knockdown significantly reversed the aggravated pulmonary hypertension phenotype induced by Shmt2a overexpression, as evidenced by significantly higher right ventricular systolic blood pressure (RVSP) and right ventricular / left ventricular volume and fraction (RV / LV+S) in the RhoB knockdown group compared to Shmt2 knockout mice. Figure 5 JK), vessel wall thickness and vascular muscularization ratio were also significantly increased ( Figure 5 LM). Meanwhile, Evans blue staining showed increased lung permeability in mice ( ). Figure 5 N).

[0054] 3.6 Shmt2 leads to RhoB accumulation by inducing RhoB deubiquitination. To investigate the mechanism by which Shmt2 regulates RhoB, we used CHX experiments and found that knocking down Shmt2 significantly accelerated the degradation rate of RhoB, while overexpression of both Shmt2 and Shmt2a prolonged the degradation rate of RhoB, suggesting that Shmt2 plays a role in stabilizing RhoB. Figure 6 Treatment with inhibitors of multiple protein degradation pathways, including lysosomes, proteasomes, and autophagy, revealed that only chloroquine and ammonium chloride could inhibit RhoB degradation induced by Shmt2 interference, indicating that Shmt2 degrades RhoB through autophagy and lysosomal proteolysis. Figure 6 C). Further CoIP experiments revealed a direct binding between endogenous and exogenous Shmt2 and RhoB. Figure 6 DE). Immunofluorescence staining showed that the two co-localized ( Figure 6 F). Next, we constructed several truncated mutants of Shmt2 and RhoB and explored their binding regions using CoIP experiments. Figure 6 GH). Experimental results show that the deletion of amino acids 300-504 in Shmt2 prevents it from binding to RhoB, while the deletion of amino acids 141-196 in RhoB prevents it from binding to Shmt2. Figure 6 IJ). Further experiments showed that overexpression of Shmt2 and Shmt2a significantly reduced the ubiquitination of RhoB. The ubiquitination experiment further demonstrated that Shmt2 significantly reduced K63 ubiquitination of RhoB, but had no significant effect on K48 ubiquitination. Figure 6 KL). Through point mutation experiments, we found that Shmt2 mainly modifies the K63 ubiquitination of lysine at position 181. The K181R mutation leads to the disappearance of the K63 ubiquitination modification of RhoB, and it is not regulated by Shmt2. Figure 6 M).

[0055] 3.7 The small molecule drug Namodenoson effectively blocks the effects of Shmt2 and RhoB and delays pulmonary hypertension. Based on molecular docking and small molecule library screening, we first screened for the binding sites of Shmt2 and BRISC, and then screened for the binding sites of RhoB to obtain an inhibitor that inhibits the binding of both—namodinosin. Figure 7 AC). In vitro experiments showed that namodinosine can effectively inhibit the binding of the two (AC). Figure 7 D), while increasing the ubiquitination modification of RhoB ( Figure 7 E). Further in vitro experiments showed that Namodenoson effectively inhibited RhoB accumulation induced by Shmt2 overexpression (E). Figure 7 F) also reduced the increase in F-actin and endothelial cell permeability caused by Shmt2a overexpression. Figure 7 GH). Using H2-labeled serine tracing experiments, we found that namodinosin did not affect the one-carbon metabolism level of endothelial cells, nor did it affect the expression level of Shmt2 in the cytoplasm and mitochondria. Figure 7 IJ). Treatment of Su5416-induced pulmonary hypertension rats with namodinosine for 4 weeks showed significant improvements in the ratio of pulmonary artery acceleration time to ejection time (PAT / ET) and tricuspid annular systolic amplitude of motion (TAPSE). Figure 7 KL), mPAP, RVSP, and RV / (LV+S) were significantly reduced ( Figure 7 MO. Simultaneously, the thickness of the walls of small blood vessels in the lungs was significantly reduced ( Figure 7 P). Treatment with namodinosin in MCT-induced pulmonary hypertension rats effectively controlled the onset of pulmonary hypertension, as evidenced by significant reductions in mPAP, RVSP, and RV / (LV+S), improved PAT / ET and TAPSE, and decreased vessel wall thickness and myomorphization ratio. Figure 7 QW).

[0056] The above results indicate that namodinosine can significantly inhibit the binding of Shmt2 and RhoB, and effectively control the occurrence of pulmonary hypertension in vivo.

[0057] 3.8 In vivo validation of namodinosin's ability to delay pulmonary hypertension by intervening in the interaction between Shmt2 and RhoB We established a pulmonary hypertension model using rats overexpressing Shmt2a via MCT, and then treated the rats with namodinosin ( Figure 8A). Experiments showed that namodinosine significantly alleviated indicators of pulmonary hypertension exacerbated by Shmt2a overexpression, including decreased pulmonary vessel wall thickness and small vessel muscularization ratio. Figure 8 BC), PAT / ET and TAPSE improvements ( Figure 8 D), and mPAP, RVSP and RV / (LV+S) were significantly reduced ( Figure 8 EF). Furthermore, endothelial RhoB overexpression was performed on hypoxia-induced pulmonary hypertension mice treated with namodinosin (EF). Figure 8 G), the results showed that increased RhoB significantly reversed the protective effect of namodinosin against pulmonary hypertension, as evidenced by the significant increase in RVSP and RV / (LV+S) in namodinosin-treated mice due to RhoB overexpression. Figure 8 HI), the pulmonary artery vascular muscularization ratio was significantly increased ( Figure 8 JK). Evans Blue and electron microscopy results showed that namodinosin effectively improved hypoxia-induced disruption of intercellular junctions and increased vascular permeability in vascular endothelial cells, while RohB overexpression exacerbated these pathological phenotypes. Figure 8 The above results indicate that Namodinosin effectively inhibits the development of pulmonary hypertension by intervening in the Shmt2-RhoB axis.

[0058] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of namodinosin in the preparation of drugs for the prevention and / or treatment of pulmonary hypertension.

2. The application as described in claim 1, characterized in that, The prevention and / or treatment of pulmonary hypertension includes one or more of the following effects: (a) Inhibit the interaction between SHMT2 and RhoB protein, and restore the ubiquitination and degradation of RhoB protein; (b) Improve hemodynamic parameters; (c) Protect the right heart structure; (d) Improve pulmonary vascular remodeling; (e) Protect the pulmonary vascular endothelial barrier.

3. The application as described in claim 2, characterized in that, In (b), the improvement of hemodynamic parameters is specifically manifested in reducing mean pulmonary artery pressure and reducing right ventricular systolic pressure.

4. The application as described in claim 2, characterized in that, In (c), protecting the right ventricular structure specifically means reducing right ventricular hypertrophy and improving right ventricular systolic function.

5. The application as described in claim 4, characterized in that, The improvement in right ventricular systolic function is specifically manifested in increasing the systolic displacement of the tricuspid annulus and increasing the ratio of pulmonary artery blood flow acceleration time to ejection time.

6. The application as described in claim 2, characterized in that, In (d), the improvement of pulmonary vascular remodeling is specifically manifested in: reducing the thickness of the pulmonary arteriole wall and reducing the degree of pulmonary vascular muscularization.

7. The application as described in claim 2, characterized in that, In (e), protecting the pulmonary vascular endothelial barrier specifically manifests as reducing pulmonary vascular leakage and maintaining the integrity of intercellular connections.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises namodinosin with at least one other pharmaceutical active ingredient and / or at least one other non-pharmaceutical active ingredient.

9. The pharmaceutical composition according to claim 8, characterized in that, The other active pharmaceutical ingredients include substances that prevent and / or treat pulmonary hypertension or assist in the prevention and / or treatment of pulmonary hypertension; further, the non-active pharmaceutical ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

10. A method for preventing and / or treating pulmonary hypertension and related diseases, characterized in that, The method includes administering namodinosine or the pharmaceutical composition of claim 8 or 9 to a subject.

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