Application of THBS4 inhibitor in preparation of medicine for preventing and / or treating pulmonary arterial hypertension

Abstract

The invention relates to an application of a THBS4 inhibitor in preparation of a medicine for preventing and / or treating pulmonary arterial hypertension. Three pulmonary arterial hypertension rat models (a hypoxia induction model, a hypoxia combined SUGEN induction model and a monocrotaline induction model) are constructed, the expression change of THBS4 is verified through qRT-PCR and western blot, and the correlation between the expression level of THBS4 and pulmonary arterial hypertension is verified in combination with in-vitro cell experiments and in-vivo intervention. Results show that THBS4 is remarkably up-regulated in pulmonary arteries of the three models, the expression level of THBS4 is positively correlated with the severity of diseases, and THBS4 is an important regulatory factor for promoting vascular remodeling in pulmonary arterial hypertension. In vivo, by knocking down THBS4, pulmonary vascular remodeling and right ventricular hypertrophy are remarkably relieved, and it is prompted that THBS4 inhibitors show good application prospects in the aspect of preventing and / or treating pulmonary arterial hypertension.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of THBS4 inhibitors in the preparation of drugs for the prevention and / or treatment of pulmonary hypertension. Background Technology

[0002] Pulmonary hypertension (PH) is a progressive and fatal cardiopulmonary disease characterized by structural remodeling of the distal pulmonary arterioles. This leads to a significant increase in pulmonary vascular resistance, a persistent increase in right ventricular load, and ultimately, right ventricular dysfunction and early death. The pathogenesis of PH is complex, driven by a combination of factors, including abnormal genetic and epigenetic regulation, oxidative stress, inflammation and immune dysfunction, ion channel dysregulation, and extracellular matrix (ECM) metabolic imbalance. These mechanisms work together to promote abnormal proliferation and migration of pulmonary vascular wall cells, thereby inducing irreversible vascular remodeling.

[0003] ECM (endovascular membrane) is a dynamic network composed of macromolecules such as collagen, glycoproteins, and proteoglycans. It not only provides structural support for blood vessels but also participates extensively in biological processes such as cell adhesion, migration, proliferation, and differentiation. In pulmonary artery disease (PH), abnormal ECM deposition or degradation promotes phenotypic transformation of structural cells such as pulmonary artery smooth muscle cells (PASMCs) and fibroblasts, driving vascular wall fibrosis and remodeling, thereby exacerbating vascular stiffness and right ventricular load. In recent years, multiple studies have indicated that specific ECM-related proteins, such as Periodin (POSTN), ADAMTS8, SPARC, and RAGE, play key regulatory roles in the development and progression of PH, suggesting that ECM-mediated pathological pathways may provide new therapeutic targets for intervention in vascular remodeling in PH.

[0004] The thrombospondins (TSPs) family is a highly conserved ECM-binding glycoprotein that plays a crucial role in vascular homeostasis, tissue repair, and cellular remodeling. Among them, THBS1 and THBS2 have been shown to be involved in the pathological process of pulmonary embolism (PH): THBS1 is significantly upregulated in the pulmonary artery, lung tissue, and plasma of patients and can directly promote vascular remodeling and inflammatory responses; THBS2 is also elevated in PH models and patients and is closely related to cardiac structural remodeling.

[0005] THBS4 is another important member of the TSP family, playing a key regulatory role in cardiovascular development, damage repair, and mechanosensing. Studies have shown that THBS4 is a key mediator in the mechanotransduction pathway of cardiomyocytes, regulating the transition of the ventricle from compensation to heart failure. In addition, THBS4 can also affect the activity of perivascular adipose tissue fibroblasts and PASMCs, participating in the regulation of vascular wall structure and function.

[0006] However, the expression level of THBS4 in patients with pulmonary hypertension, and its mechanism for use in the prevention or treatment of pulmonary hypertension, remains unclear. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of THBS4 inhibitors in the preparation of medicaments for the prevention and / or treatment of pulmonary hypertension.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the use of THBS4 inhibitors in the preparation of medicaments for the prevention and / or treatment of pulmonary hypertension.

[0010] This invention constructed three rat models of pulmonary hypertension (PH) (hypoxia-induced model, hypoxia combined with SUGEN-induced model, and lily alkaloid-induced model), and verified changes in THBS4 expression using qRT-PCR and Western blotting. Furthermore, it combined in vitro cell experiments and in vivo intervention to verify the correlation between THBS4 expression levels and PH. The results showed that THBS4 was significantly upregulated in the pulmonary arteries of all three models, and its expression level was positively correlated with disease severity, making it an important regulatory factor driving vascular remodeling in PH. In vivo, knockdown of THBS4 significantly reduced pulmonary vascular remodeling and right ventricular hypertrophy, suggesting that THBS4 inhibitors show great promise for the prevention and / or treatment of PH.

[0011] Preferably, the active ingredient in the THBS4 inhibitor is selected from any one or a combination of at least two of the following: shRNA molecules targeting the THBS4 gene or their pharmaceutically acceptable salts; signaling pathway inhibitors capable of inhibiting THBS4 gene expression; or growth factor inhibitors capable of inhibiting THBS4 gene expression.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0014] Preferably, the shRNA molecule targeting the THBS4 gene can downregulate the expression of the THBS4 gene, and the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.5 (for example, it can be the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5), preferably the sequence shown in SEQ ID NO.3.

[0015] Preferably, the signaling pathway inhibitors capable of inhibiting THBS4 gene expression include any one or a combination of at least two of the following: HIF-1α pathway inhibitors, SMAD2 pathway inhibitors, and MAPK pathway inhibitors.

[0016] Preferably, the active ingredient in the HIF-1α pathway inhibitor is any one or a combination of at least two of Oltipraz, its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0017] Preferably, the active ingredient in the SMAD2 pathway inhibitor is any one or a combination of at least two of AZ12601011, its pharmaceutically acceptable salt, isomer, solvate, and metabolite.

[0018] Preferably, the active ingredient in the MAPK pathway inhibitor is selected from any one or a combination of at least two of Adezmapimod, SCH772984, SP600125, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0019] Preferably, the growth factor inhibitor capable of inhibiting THBS4 gene expression includes a TGF-β receptor inhibitor.

[0020] Preferably, the active ingredient in the TGF-β receptor inhibitor is selected from any one or a combination of at least two of SB525334, SB431542, SIS3, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0021] Secondly, the present invention provides the use of THBS4 inhibitors in the preparation of medicaments for the prevention and / or treatment of pulmonary vascular remodeling caused by pulmonary hypertension.

[0022] Preferably, the active ingredient in the THBS4 inhibitor is selected from any one or a combination of at least two of the following: shRNA molecules targeting the THBS4 gene or their pharmaceutically acceptable salts; signaling pathway inhibitors capable of inhibiting THBS4 gene expression; or growth factor inhibitors capable of inhibiting THBS4 gene expression.

[0023] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0025] Preferably, the shRNA molecule targeting the THBS4 gene can downregulate the expression of the THBS4 gene, and the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.5 (for example, it can be the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5), preferably the sequence shown in SEQ ID NO.3.

[0026] Preferably, the signaling pathway inhibitors capable of inhibiting THBS4 gene expression include any one or a combination of at least two of the following: HIF-1α pathway inhibitors, SMAD2 pathway inhibitors, and MAPK pathway inhibitors.

[0027] Preferably, the active ingredient in the HIF-1α pathway inhibitor is any one or a combination of at least two of Oltipraz, its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0028] Preferably, the active ingredient in the SMAD2 pathway inhibitor is any one or a combination of at least two of AZ12601011, its pharmaceutically acceptable salt, isomer, solvate, and metabolite.

[0029] Preferably, the active ingredient in the MAPK pathway inhibitor is selected from any one or a combination of at least two of Adezmapimod, SCH772984, SP600125, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0030] Preferably, the growth factor inhibitor capable of inhibiting THBS4 gene expression includes a TGF-β receptor inhibitor.

[0031] Preferably, the active ingredient in the TGF-β receptor inhibitor is selected from any one or a combination of at least two of SB525334, SB431542, SIS3, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0032] Thirdly, the present invention provides the use of THBS4 inhibitors in the preparation of medicaments for the prevention and / or treatment of pulmonary hypertension-induced right ventricular hypertrophy and / or right ventricular failure.

[0033] Preferably, the active ingredient in the THBS4 inhibitor is selected from any one or a combination of at least two of the following: shRNA molecules targeting the THBS4 gene or their pharmaceutically acceptable salts; signaling pathway inhibitors capable of inhibiting THBS4 gene expression; or growth factor inhibitors capable of inhibiting THBS4 gene expression.

[0034] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0035] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0036] Preferably, the shRNA molecule targeting the THBS4 gene can downregulate the expression of the THBS4 gene, and the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.5 (for example, it can be the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5), preferably the sequence shown in SEQ ID NO.3.

[0037] Preferably, the signaling pathway inhibitors capable of inhibiting THBS4 gene expression include any one or a combination of at least two of the following: HIF-1α pathway inhibitors, SMAD2 pathway inhibitors, and MAPK pathway inhibitors.

[0038] Preferably, the active ingredient in the HIF-1α pathway inhibitor is any one or a combination of at least two of Oltipraz, its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0039] Preferably, the active ingredient in the SMAD2 pathway inhibitor is any one or a combination of at least two of AZ12601011, its pharmaceutically acceptable salt, isomer, solvate, and metabolite.

[0040] Preferably, the active ingredient in the MAPK pathway inhibitor is selected from any one or a combination of at least two of Adezmapimod, SCH772984, SP600125, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0041] Preferably, the growth factor inhibitor capable of inhibiting THBS4 gene expression includes a TGF-β receptor inhibitor.

[0042] Preferably, the active ingredient in the TGF-β receptor inhibitor is selected from any one or a combination of at least two of SB525334, SB431542, SIS3, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0043] Fourthly, the present invention provides the use of THBS4 inhibitors in the preparation of pulmonary artery smooth muscle cell proliferation inhibitors and / or pulmonary artery smooth muscle cell phenotypic transformation regulators.

[0044] The pulmonary artery smooth muscle cell phenotype transformation regulator is a regulator that promotes the transformation of pulmonary artery smooth muscle cells to a contractile phenotype.

[0045] Preferably, the active ingredient in the THBS4 inhibitor is selected from any one or a combination of at least two of the following: shRNA molecules targeting the THBS4 gene or their pharmaceutically acceptable salts; signaling pathway inhibitors capable of inhibiting THBS4 gene expression; or growth factor inhibitors capable of inhibiting THBS4 gene expression.

[0046] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0047] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0048] Preferably, the shRNA molecule targeting the THBS4 gene can downregulate the expression of the THBS4 gene, and the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.5 (for example, it can be the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5), preferably the sequence shown in SEQ ID NO.3.

[0049] Preferably, the signaling pathway inhibitors capable of inhibiting THBS4 gene expression include any one or a combination of at least two of the following: HIF-1α pathway inhibitors, SMAD2 pathway inhibitors, and MAPK pathway inhibitors.

[0050] Preferably, the active ingredient in the HIF-1α pathway inhibitor is any one or a combination of at least two of Oltipraz, its pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0051] Preferably, the active ingredient in the SMAD2 pathway inhibitor is any one or a combination of at least two of AZ12601011, its pharmaceutically acceptable salt, isomer, solvate, and metabolite.

[0052] Preferably, the active ingredient in the MAPK pathway inhibitor is selected from any one or a combination of at least two of Adezmapimod, SCH772984, SP600125, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0053] Preferably, the growth factor inhibitor capable of inhibiting THBS4 gene expression includes a TGF-β receptor inhibitor.

[0054] Preferably, the active ingredient in the TGF-β receptor inhibitor is selected from any one or a combination of at least two of SB525334, SB431542, SIS3, or their pharmaceutically acceptable salts, isomers, solvates, and metabolites.

[0055] Fifthly, the present invention provides an shRNA molecule that can downregulate the expression of the THBS4 gene.

[0056] Preferably, the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO.1-SEQ ID NO.5 (for example, it may be the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5), and preferably the sequence shown in SEQ ID NO.3.

[0057] In a sixth aspect, the present invention provides an expression vector comprising the shRNA molecule as described in the fifth aspect.

[0058] Preferably, the expression vector is an adeno-associated virus type 2 vector or an adeno-associated virus type 9 vector.

[0059] In a seventh aspect, the present invention provides a recombinant virus-like particle, the recombinant virus particle comprising the expression vector as described in the sixth aspect.

[0060] Eighthly, the present invention provides the use of the shRNA molecule described in the fifth aspect, the expression vector described in the sixth aspect, or the recombinant virus-like particle described in the seventh aspect in the preparation of a medicament for the prevention and / or treatment of pulmonary hypertension, pulmonary vascular remodeling caused by pulmonary hypertension, right ventricular hypertrophy caused by pulmonary hypertension, or right ventricular failure caused by pulmonary hypertension.

[0061] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0062] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, solubilizers, osmotic pressure regulators, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0063] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] This invention constructed three rat models of pulmonary hypertension (PH) (hypoxia-induced model, hypoxia combined with SUGEN-induced model, and lily alkaloid-induced model), and verified changes in THBS4 expression using qRT-PCR and Western blotting. Furthermore, it combined in vitro cell experiments and in vivo intervention to verify the correlation between THBS4 expression levels and PH. The results showed that THBS4 was significantly upregulated in the pulmonary arteries of all three models, and its expression level was positively correlated with disease severity, making it an important regulatory factor driving vascular remodeling in PH. In vivo, knockdown of THBS4 significantly reduced pulmonary vascular remodeling and right ventricular hypertrophy, suggesting that THBS4 inhibitors show great promise for the prevention and / or treatment of PH. Attached Figure Description

[0066] Figure 1 This is a comparison of the expression levels of THBS4 in an animal model of pulmonary hypertension and human patients with pulmonary hypertension, as shown in Example 1. In this figure, A is a comparison of the expression levels of THBS4 mRNA in rat pulmonary arteries, B is a comparison of the expression levels of THBS4 protein in rat pulmonary arteries, C is a comparison of the expression levels of THBS4 mRNA in rat lung tissue, and D is a comparison of the expression levels of THBS4 protein in the serum of IPAH patients and healthy controls.

[0067] Figure 2This is a comparison chart of the knockdown effects of different shRNAs on THBS4 in Example 2.

[0068] Figure 3 This is a graph showing the effect of knocking down THBS4 on right ventricular systolic pressure and right ventricular hypertrophy index in Example 2.

[0069] Figure 4 This is a graph showing the effect of knocking down THBS4 on the degree of endothelial thickening and vessel wall remodeling in rat pulmonary vessels in Example 2.

[0070] Figure 5 This is a graph showing the effect of knocking down THBS4 on the phenotypic transformation of PASMC cells in Example 3.

[0071] Figure 6 This is a graph showing the effect of knocking down THBS4 on the proliferation of PASMC cells in Example 3.

[0072] Figure 7 This is a graph showing the effects of different HIF-1α pathway inhibitors on THBS4 expression in Example 4.

[0073] Figure 8 This is a graph showing the effects of different TGF-β inhibitors on the expression of SMAD2 pathway inhibitors and MAPK pathway inhibitor THBS4 in Example 5.

[0074] Figure 9 This is a graph showing the effect of low concentration of the SMAD2 inhibitor AZ12601011 on THBS4 expression in Example 5. Detailed Implementation

[0075] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0076] The studies described below were conducted in strict accordance with relevant Chinese regulations on the protection and use of laboratory animals. All animal experimental protocols were approved by the Animal Ethics Committee of Shenzhen University (Approval No.: 2021003; A202300688), and the use of human samples was approved by the Ethics Committee of Shanghai Pulmonary Hospital (Approval No.: K23-206). All subjects signed written informed consent forms.

[0077] The experimental methods involved in the following implementation methods are as follows:

[0078] 1. Construction of a rat model of pulmonary hypertension (PH):

[0079] Three classic PH models were constructed using 8-week-old male SD rats:

[0080] (1) Hypoxia (HYP) induction model: Rats were kept in a continuous hypoxia (10% O2) environment for 4 weeks to obtain the hypoxia induction experimental model; rats were kept in a normoxic (21% O2) environment for 4 weeks to obtain the hypoxia induction control model.

[0081] (2) Hypoxia combined with Sugen (HySu) induction model: Rats were subcutaneously injected with SU5416 (20 mg / kg, MCE, #HY-10374), and then fed in a hypoxic environment for 3 weeks, and then fed in a normoxic environment for 2 weeks to obtain the HySu induction experimental model; The preparation method of SU5416 is as follows: first, SU5416 is dissolved in 1 volume of DMSO, which is a yellow transparent liquid, and then 4 volumes of CMC solution (CMC solution: 0.5% (w / v) sodium carboxymethyl cellulose, 0.9% (w / v) sodium chloride, 0.4% (v / v) Tween 80, 0.9% (v / v) benzyl alcohol and water are added to further dissolve it;

[0082] An equal volume of CMC solution was injected subcutaneously, and the animals were then kept in a normoxic environment for 5 weeks to obtain a HySu-induced control model.

[0083] (3) Monocrotaline (MCT) induction model: Rats were subcutaneously injected with MCT (60 mg / kg, SigmaAldrich, #C2401) and fed in a normoxic environment for 3 weeks to obtain the MCT induction experimental model; Rats were subcutaneously injected with an equal volume of physiological saline and fed in a normoxic environment for 3 weeks to obtain the MCT induction control model.

[0084] 2. Hemodynamic monitoring and histopathological analysis of rats:

[0085] After anesthetizing rats, a pressure catheter was inserted through the right jugular vein, and right ventricular systolic pressure (RVSP) was recorded in real time using a BIOPAC MP150 system for hemodynamic monitoring. After hemodynamic monitoring, the animals were sacrificed, and cardiopulmonary tissues were harvested. The distal pulmonary artery of the right lung was rapidly frozen in liquid nitrogen for RNA and protein analysis; the left lung lobe was fixed in neutral formalin, embedded in paraffin, and sectioned for histological analysis. Pulmonary vascular remodeling was assessed by measuring vessel wall thickness and calculating the vessel-media thickness ratio ((outer diameter - inner diameter) / outer diameter); the right ventricular hypertrophy index (RVHI) was calculated using the formula RV / (LV+S).

[0086] 3. Detection of THBS4 protein in human serum:

[0087] The THBS4 protein in serum samples from patients with pulmonary hypertension and healthy controls was quantitatively detected using the THBS4 enzyme-linked immunosorbent assay kit (J&L Biological, JL19965). The procedure was performed according to the instructions.

[0088] 4. Methods for preparing lentiviruses:

[0089] (1) Preparation of shRNA vector: Three shRNA primers (sequences shown in Table 1, SEQ ID NO.1-SEQ ID NO.5) were synthesized for each shRNA sequence target (sequences shown in Table 1, SEQ ID NO.6-SEQ ID NO.20), and after annealing, they were cloned into the pLVX-U6-EGFP-Puro vector with BsmBI restriction site.

[0090] (2) Preparation of overexpression vector: The CDS sequences of THBS4 and SMAD2 were amplified from RPASMC and cloned into the pLVX-CMV-FLAG-EGFP vector, with the empty vector as a control; all constructions were verified to be correct by sequencing.

[0091] (3) Preparation of lentivirus: 293T cells were transfected with polyethyleneimine (PEI) and high-titer lentivirus was produced by combining packaging plasmids.

[0092] 5. AV-9 virus packaging and in vivo delivery are as follows:

[0093] AAV9 vectors expressing shTHBS4 or shControl (empty vector) were constructed and expression was driven by the U6 promoter. After annealing three oligonucleotides, the vectors were cloned into AAV plasmids and co-transfected into 293T cells along with pRC and pHelper packaging plasmids. Virus was collected after 72 h, extracted using the freeze-thaw method, and titer determined by qPCR. 5 × 10⁵ AAV9 molecules were administered via intratracheal spray to each rat. 12 Viral particles are delivered one week before hypoxia or HySu induction.

[0094] 6. RNA extraction and qRT-PCR analysis:

[0095] Total RNA was extracted using RNAiso Plus, and cDNA synthesis was performed using M-MLV reverse transcriptase and random / oligo(dT)18 primers (600 ng RNA per reaction). Quantitative PCR was performed using SYBR Green reagent (Applied Biosystems) and specific primers (sequences shown as SEQ ID NO.21-SEQ ID NO.34 in Table 1) on a LightCycler96 system (Roche). Expression levels were expressed as 2... -ΔΔCt The calculation was performed with β-Actin as the internal reference.

[0096] 7. Western blot:

[0097] Proteins were extracted using RIPA lysis buffer (containing 1 mM PMSF and Na3VO4 inhibitor), and concentrations were determined using the BCA method. Equal volumes of protein (20 μg) were subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane (0.45 μm pore size), blocked with 5% BSA, and incubated overnight at 4°C with primary antibody. Antibodies included: THBS4 (1:1000, Abcam ab263898), SMAD2 / pSMAD2 (1:1000, CST #5339 / #18338), α-SMA, SM22α, calponin (1:1000), PCNA (1:5000), β-actin (1:3000), and β-Tubulin (1:6000); HRP secondary antibody (1:5000) was incubated at room temperature for 1 hour, developed using SuperSignal chemiluminescence reagent, imaged using a BLT GelView 6000Plus, and the band grayscale was quantitatively analyzed using ImageJ.

[0098] 8. Histological analysis:

[0099] Lung tissue sections (5 μm thick) were embedded in paraffin and then stained with H&E. Pulmonary arterioles with diameters of 51–150 μm were selected to measure media thickness, and the media / CSA ratio was calculated. At least 50 vessels were analyzed in each group, and measurements were performed using ImageJ.

[0100] 9. Statistical Analysis:

[0101] All data are expressed as mean ± standard error and were derived from at least three independent experiments. Normality was tested using the Shapiro-Wilk method.

[0102] The methods for comparing groups are as follows: For comparisons between two groups: for normally distributed groups, a two-tailed t-test is used; for non-normally distributed groups, a Mann-Whitney U test is used. For comparisons between multiple groups: one-way or two-way ANOVA is used, combined with appropriate post-hoc tests.

[0103] All statistical analyses were performed using GraphPad Prism 9.0 and R 4.2.0, and p < 0.05 was considered statistically significant.

[0104] The sequence information involved in the following embodiments is shown in Table 1:

[0105] Table 1

[0106]

[0107] Example 1

[0108] This embodiment verifies that THBS4 is significantly upregulated in both animal models of pulmonary hypertension and human patients with pulmonary hypertension.

[0109] (1) THBS4 expression was upregulated in an animal model of pulmonary hypertension:

[0110] (1.1) Test subjects: rats in hypoxia-induced experiment / control model, HySu-induced experiment / control model, and MCT-induced experiment / control model;

[0111] (1.2) Test methods: The expression level of THBS4 in rat pulmonary artery / lung tissue was detected by qRT-PCR, and the expression level of THBS4 protein in rat serum was detected by Western blot.

[0112] (2) THBS4 expression is upregulated in patients with pulmonary hypertension:

[0113] The expression level of THBS4 protein in the serum of patients with idiopathic pulmonary arterial hypertension (IPAH) and healthy controls was detected by ELISA.

[0114] Test results are as follows Figure 1 As shown, A represents the comparison of THBS4 mRNA expression levels in rat pulmonary arteries, B represents the comparison of THBS4 protein expression levels in rat pulmonary arteries, C represents the comparison of THBS4 mRNA expression levels in rat lung tissue, and D represents the comparison of THBS4 protein expression levels in the serum of IPAH patients and healthy controls. It is evident that THBS4 exhibits consistent and significant upregulation in various animal models and human PAH samples.

[0115] Example 2

[0116] This embodiment demonstrates that knocking down THBS4 can improve hypoxia and HySu-induced pulmonary hypertension.

[0117] (1) To evaluate the inhibitory effect of shRNAs targeting different sequences on THBS4 expression.

[0118] In this embodiment, five AAV9 viral vectors were constructed, each containing a different shRNA targeting THBS4 driven by the U6 promoter (the target sequences of shTHBS4-1 to shTHBS4-5 are shown in SEQ ID NO. 1 to SEQ ID NO. 5, respectively). An empty control viral vector (shControl) was also established. The AAV9 virus was delivered to the lungs of hypoxic and HySu model rats via tracheal spray. After animal modeling, the expression level of THBS4 protein in rat serum was analyzed by Western blot. The results are shown below. Figure 2As shown, the target sequences shTHBS4-1 to shTHBS4-5, as shown in SEQ ID NO.1 to SEQ ID NO.5, can effectively knock down THBS4, showing a significant downregulation compared to the control shRNA. Furthermore, shTHBS4-3, with the target sequence SEQ ID NO.3, exhibits a superior THBS4 knockdown effect.

[0119] (2) Evaluate the effect of shRNA on pulmonary hypertension.

[0120] After AAV9 viral vectors containing shTHBS4-3 and empty control viral vectors (shControl) were delivered to the lungs of rats via tracheal spray, the intervention effect of knocking down THBS4 was evaluated in hypoxia-induced and HySu-induced models, respectively.

[0121] (2.1) After modeling, hemodynamic monitoring was performed on each rat, and the results are as follows: Figure 3 As shown, the right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) in the THBS4 knockdown group (shTHBS4) were significantly lower than those in the control group (shControl), indicating a significant improvement in right ventricular load.

[0122] (2.2) After hemodynamic monitoring was completed, the animals were euthanized, and heart and lung tissues were collected for further histopathological analysis. The results are as follows: Figure 4 As shown in the figure. The results indicate that knockdown of THBS4 significantly inhibits pulmonary vascular media thickening and vessel wall remodeling in PH rats.

[0123] Example 3

[0124] This embodiment verifies the effect of THBS4 knockdown on PASMC cell phenotypic transformation and proliferation.

[0125] (1) The effect of knocking down THBS4 on the phenotypic transformation of PASMC cells.

[0126] Phenotypic transformation and abnormal proliferation of PASMCs are key pathological steps driving pulmonary vascular remodeling. The main characteristic is the transformation of cells from a contractile phenotype (characterized by high expression of α-SMA, SM22-α and calponin) to a synthetic phenotype. The latter is characterized by downregulation of contractile markers and significant upregulation of synthesis-related molecules such as Erg.

[0127] In this embodiment, AAV9 viral vectors containing shTHBS4-3 (shTHBS4) and empty control viral vectors (shControl) were delivered to the lungs of rats via tracheal spray. The effect of THBS4 knockdown on PASMC cell phenotypic transformation was then evaluated in a hypoxia-induced model. The results are as follows: Figure 5As shown, THBS4 knockdown significantly upregulates the expression levels of contractile marker proteins (SM22-α, calponin, α-SMA, and SMMHC) while downregulating the synthetic marker Erg, suggesting that THBS4 knockdown may facilitate the conversion of PASMC to a contractile phenotype.

[0128] (2) Effect of knockdown of THBS4 on the proliferation of PASMC cells.

[0129] The expression level of PCNA, a protein related to the proliferation of PASMC cells, in the pulmonary artery of HySu-induced PH rats was detected, and the results are as follows: Figure 6 As shown, THBS4 knockdown significantly inhibited PCNA expression in the pulmonary artery.

[0130] The above results indicate that THBS4 mainly participates in the vascular remodeling process in pulmonary hypertension by promoting PASMC proliferation and driving its conversion from contractile to anabolic forms. This suggests that THBS4 can be used to prepare PASMC proliferation inhibitors or PASMC phenotype modulators and can inhibit vascular remodeling.

[0131] Example 4

[0132] This embodiment verifies the effect of HIF-1α pathway inhibitors on THBS4 expression.

[0133] A hypoxia stimulation model was established in vitro, and RPASMCs were treated with hypoxia (3% O2) for 48 h. Simultaneously, different concentrations of the HIF-1α pathway inhibitor Oltipraz (0 μm, 1 μm, 5 μm, 10 μm, 15 μm) were added to the cells for intervention. After the hypoxia treatment, the expression level of THBS4 mRNA in each cell was measured. The results are as follows: Figure 7 As shown.

[0134] It is evident that the HIF-1α pathway inhibitor Oltipraz can significantly block hypoxia-induced THBS4 upregulation and has a dose-dependent inhibitory effect on hypoxia-induced THBS4 expression.

[0135] Example 5

[0136] This embodiment verifies that TGF-β affects THBS4 expression through multiple signaling pathways.

[0137] Previous studies have shown that significant upregulation of THBS4 was observed in hypoxia, HySu, and MCT-induced PH rat models, suggesting that it may be regulated by multiple cytokines. To systematically evaluate its upstream regulatory mechanisms, we screened six candidate growth factors (TGF-β1, TGF-β2, VEGF, IL-1β, EGF, and FGF) in vitro. The results showed that TGF-β1 was the most significant factor in inducing upregulation of THBS4 expression in RPASMCs.

[0138] This embodiment further explores the molecular mechanism by which TGF-β1 induces THBS4 expression and analyzes the signaling pathways it depends on. The experimental design covers the classical SMAD pathway and several non-classical alternative pathways, including the p38 MAPK, ERK, PI3K / Akt, JNK, STAT3, and NF-κB pathways. Pathway blockade was achieved by applying specific small molecule inhibitors.

[0139] (1) Experimental method:

[0140] RPASMCs were cultured for 72 h at 21% O2 or 3% O2. After 12 h of starvation (0.2% FBS), cells were stimulated with TGF-β1 (20 ng / mL) for 6 h. Simultaneously, one hour before stimulation, cells were pretreated with the following pathway inhibitors: TGF-β receptor inhibitors SB525334, SB431542, and SIS3 (1 μM); SMAD2 pathway inhibitor AZ12601011 (1 μM); and MAPK pathway inhibitors Adezmapimod (3 μM), SCH772984 (1 μM), and SP600125 (5 μM). THBS4 RNA and protein were extracted from the cells after treatment, and THBS4 expression was analyzed.

[0141] (2) Experimental results:

[0142] (2.1) Effects of different inhibitors on THBS4 expression, such as Figure 8 As shown, TGF-β1 receptor inhibitors SB525334 and SB431542 can completely block TGF-β1-induced upregulation of THBS4 expression; in addition, phosphorylation inhibitor AZ12601011 targeting SMAD2 and p38 MAPK inhibitor Adezmapimod can also significantly inhibit THBS4 expression upregulation.

[0143] (2.2) Effects of low concentrations of the SMAD2 inhibitor AZ12601011 (0 μm, 0.01 μm, 0.02 μm, 0.06 μm, 0.1 μm) on THBS4 expression as follows: Figure 9As shown, AZ12601011 inhibits THBS4 expression in a concentration-dependent manner, and can significantly interfere with its expression level even under low-dose conditions.

[0144] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0145] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0146] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Use of a THBS4 inhibitor in the preparation of a medicament for preventing and / or treating pulmonary arterial hypertension.

2. Use according to claim 1, characterized in that, The active ingredient in the THBS4 inhibitor is selected from any one or a combination of at least two of an shRNA molecule against the THBS4 gene or a pharmaceutically acceptable salt thereof, a signal pathway inhibitor capable of inhibiting the expression of the THBS4 gene, or a growth factor inhibitor capable of inhibiting the expression of the THBS4 gene; Preferably, the medicament further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, a solubilizer, an osmotic pressure regulator, a coating material, a colorant, a pH regulator, an antioxidant, or a bacteriostatic agent.

3. Use according to claim 2, characterized in that, The signal pathway inhibitor capable of inhibiting the expression of the THBS4 gene comprises any one or a combination of at least two of a HIF-1α pathway inhibitor, a SMAD2 pathway inhibitor, a MAPK pathway inhibitor; Preferably, the active ingredient in the HIF-1α pathway inhibitor is Oltipraz, any one or a combination of at least two of a pharmaceutically acceptable salt, an isomer, a solvate, a metabolite thereof; Preferably, the active ingredient in the SMAD2 pathway inhibitor is AZ12601011, any one or a combination of at least two of a pharmaceutically acceptable salt, an isomer, a solvate, a metabolite thereof; Preferably, the active ingredient in the MAPK pathway inhibitor is selected from Adezmapimod, SCH772984, SP600125, or any one or a combination of at least two of a pharmaceutically acceptable salt, an isomer, a solvate, a metabolite thereof; Preferably, the growth factor inhibitor capable of inhibiting the expression of the THBS4 gene comprises a TGF-β receptor inhibitor; Preferably, the active ingredient in the TGF-β receptor inhibitor is selected from SB525334, SB431542, SIS3, or any one or a combination of at least two of a pharmaceutically acceptable salt, an isomer, a solvate, a metabolite thereof.

4. Use of a THBS4 inhibitor in the preparation of a medicament for preventing and / or treating pulmonary vascular remodeling caused by pulmonary arterial hypertension.

5. Use of a THBS4 inhibitor in the preparation of a medicament for preventing and / or treating right ventricular hypertrophy and / or right ventricular failure caused by pulmonary arterial hypertension.

6. Use of a THBS4 inhibitor in the preparation of a pulmonary arterial smooth muscle cell proliferation inhibitor and / or a pulmonary arterial smooth muscle cell phenotype transformation regulator. The pulmonary arterial smooth muscle cell phenotype transformation regulator is a regulator for promoting the transformation of pulmonary arterial smooth muscle cells to a contractile phenotype.

7. An shRNA molecule, characterized in that, The shRNA molecule is capable of down-regulating the expression of the THBS4 gene; Preferably, the target nucleic acid sequence of the shRNA molecule is selected from any one of the sequences shown in SEQ ID NO. 1-SEQ ID NO. 5, preferably the sequence shown in SEQ ID NO.

3.

8. An expression vector, characterized by, The expression vector comprises the shRNA molecule as claimed in claim 7. Preferably, the expression vector is an adeno-associated virus type 2 or type 9 vector.

9. A recombinant virus-like particle, characterized in that, The recombinant virus particle comprises the expression vector of claim 8.

10. Use of the shRNA molecule of claim 7, the expression vector of claim 8 or the recombinant virus-like particle of claim 9 in the preparation of a medicament for preventing and / or treating pulmonary arterial hypertension, pulmonary vascular remodeling caused by pulmonary arterial hypertension, right ventricular hypertrophy or right ventricular failure caused by pulmonary arterial hypertension.

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