Application of 4-octyl itaconic acid in the preparation of drugs to improve pulmonary hypertension

Abstract

This invention discloses the application of 4-octyl itaconic acid (4-OI) in the preparation of drugs to improve pulmonary arterial hypertension (PAH). In vivo experiments showed that 4-OI significantly reduced pulmonary vascular wall thickening and collagen deposition in a rat model of PAH, effectively improving PAH and vascular remodeling. In vitro experiments further revealed that 4-OI reduces endoplasmic reticulum stress (ERS) by inhibiting the PERK-eIF2α pathway, thereby blocking SM22-dependent smooth muscle cell phenotypic transformation and endothelial-mesenchymal transition (EndMT) processes, ultimately inhibiting vascular remodeling and exerting a therapeutic effect on PAH. This invention closely links the three key pathological processes of ERS, EndMT, and vascular remodeling, providing a new perspective for a deeper understanding of the relationship between oxidative stress, endothelial dysfunction, and vascular structural changes in PAH. It also lays a theoretical foundation for the development of precision drugs for the treatment of PAH and vascular remodeling, and has clinical translational value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of 4-octylitaconic acid in the preparation of drugs for improving pulmonary hypertension. Background Technology

[0002] Pulmonary arterial hypertension (PAH) is a fatal disease characterized by progressively increasing pulmonary vascular resistance, ultimately leading to right heart failure and death; it is also known as "cardiovascular cancer." Epidemiological studies show that the 5-year survival rate for PAH is only 60-70%, and current treatments can only alleviate symptoms and cannot reverse the pathological progression. The pathological mechanism of PAH is extremely complex, resulting from the combined effects of multiple factors and processes. From an external perspective, physical, chemical, and biological factors such as hypoxia, tobacco, and dust, as well as interactive factors such as infection, inflammation and immunity, and drugs, can all induce the disease. From an internal perspective, individual differences in genetics, development, structure, and disease also affect the occurrence and development of PAH. Abnormal regulation of various vasoactive molecules, such as endothelin, angiotensin II, and prostacyclin, as well as multiple ion channels and signaling pathways, jointly participate in the pathogenesis of PAH. The core pathological mechanisms of PAH involve endothelial dysfunction, abnormal proliferation, and vascular remodeling. Endothelial dysfunction is considered an early event in PAH development, while vascular remodeling is the core link leading to its continued progression, specifically manifested as intimal thickening of pulmonary arterioles, smooth muscle layer hypertrophy, adventitia fibrosis, and luminal narrowing. Endothelial-mesenchymal transition (EndMT) is considered a key driver of vascular remodeling. Although targeted drugs such as endothelin receptor antagonists and phosphodiesterase-5 inhibitors have shown some efficacy in some patients, these drugs are only applicable to specific types of PAH. Systematic analysis of the pathogenesis of PAH, discovery of novel therapeutic targets, and design of innovative intervention strategies have become urgent needs in clinical treatment, and are of great significance for overcoming the PAH challenge and improving patient prognosis. 4-Octylitaconic acid (4-OI) is a derivative of itaconic acid with good membrane permeability, enabling it to cross the cell membrane and enter the cell interior. In recent years, 4-OI has shown significant therapeutic potential in lung disease research. It has made positive progress in chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and infectious diseases through multiple mechanisms such as anti-inflammatory and antioxidant effects and metabolic remodeling. Studies have shown that 4-OI can reduce endoplasmic reticulum stress (ERS) by activating the Nrf2 pathway, thereby protecting against renal ischemia-reperfusion injury. However, there are currently no existing technologies to investigate the effects of 4-OI on the ERS-EndMT axis in vascular endothelial cells, and there are no reports of its application in the treatment of PAH. Summary of the Invention

[0003] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of 4-octyl itaconic acid (4-OI) in the preparation of drugs to improve pulmonary arterial hypertension (PAH).

[0004] A second object of the present invention is to provide the use of 4-octylitaconic acid in the preparation of medicaments that inhibit endothelial-mesenchymal transition.

[0005] A third objective of this invention is to provide the use of 4-octylitaconic acid in the preparation of medicaments for inhibiting pulmonary vascular remodeling.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] This invention utilizes a polycystic urea heart disease (PAH) model induced by methyl lily alkaloid (MCT) and a human umbilical vein endothelial cell (HUVECs) model stimulated by transforming growth factor-β1 (TGF-β1, which can induce the expression of α-smooth muscle actin (α-SMA) and SM22, initiating not only mesenchymal transition but also potentially driving abnormal vascular wall thickening (a typical pathological feature of PAH)) to explore the mechanism of action of 4-OI in improving PAH. In vivo experiments assessed the degree of pulmonary vascular remodeling and fibrosis through lung tissue morphometry analysis (HE staining and Masson staining). The results showed that 4-OI significantly reduced pulmonary vascular wall thickening and collagen deposition in the PAH rat model, effectively improving PAH and vascular remodeling. In vitro experiments used CCK-8 and EdU to detect cell proliferation, Annexin V-FITC / PI double staining to analyze cell apoptosis, Transwell and cell scratch assays to assess migration ability, immunofluorescence to detect endoplasmic reticulum structure and cytoskeleton remodeling, and Western blot to detect endoplasmic reticulum stress. The expression of Stress (ERS) markers (p-PERK, p-eIF2α), Endothelial-mesenchymal transition (EndMT) markers (α-SMA), and SM22 was verified using the ERS agonist thapsigargin. In vitro experiments showed that 4-OI significantly inhibited TGF-β1-induced HUVEC proliferation, migration (exerting anti-fibrotic and anti-angiogenic effects by intervening in the TGF-β1-mediated cell motility signaling pathway), and cytoskeleton remodeling (reflecting its inhibitory effect on EndMT and pathological vascular remodeling). 4-OI also significantly reduced the expression of p-PERK, p-eIF2α, α-SMA, and SM22. 4-OI specifically inhibited ROS generation and ERS signaling activation in pulmonary artery endothelial cells, restoring the normal morphology of the endoplasmic reticulum and thus interrupting the EndMT process. In summary, this invention demonstrates that 4-OI alleviates ERS by inhibiting the PERK-eIF2α pathway in ERS, thereby blocking SM22-dependent smooth muscle cell phenotypic transformation and the EndMT process. In other words, 4-OI exerts its therapeutic effect on PAH by reducing vascular remodeling and EndMT.

[0008] Therefore, the present invention provides the use of 4-octylitaconic acid in the preparation of a drug for improving pulmonary hypertension.

[0009] This invention discovers that 4-OI has a positive effect in the treatment of PAH, especially in the intervention of EndMT and ERS: on the one hand, 4-OI can inhibit the initiation of ERS by inhibiting the PERK pathway, reducing ROS production, and alleviating oxidative stress; on the other hand, it can block the ERS-induced EndMT process, fundamentally slowing down vascular remodeling.

[0010] Furthermore, the drug achieves its therapeutic purpose by inhibiting one or more of the following: cell proliferation, migration, skeletal remodeling, pulmonary vascular wall thickening, collagen deposition, and reducing pulmonary artery fibrosis.

[0011] Furthermore, the drug achieves its therapeutic purpose by inhibiting endothelial-mesenchymal transition and pulmonary vascular remodeling.

[0012] This invention also provides the use of 4-octylitaconic acid in the preparation of drugs that inhibit endothelial-mesenchymal transition.

[0013] This invention also provides the use of 4-octylitaconic acid in the preparation of drugs that inhibit pulmonary vascular remodeling.

[0014] Furthermore, the drug achieves its therapeutic purpose by inhibiting endoplasmic reticulum stress.

[0015] The endoplasmic reticulum (ER) is the core site of intracellular protein synthesis, folding, and post-translational modification, and also participates in the regulation of lipid synthesis and calcium homeostasis. When cells encounter pathological stimuli such as hypoxia, oxidative stress, metabolic disorders, or pathogen infection, unfolded / misfolded proteins accumulate abnormally in the ER lumen, leading to end-stage renal disease (ERS). To cope with ERS, eukaryotes have evolved a highly conserved adaptive response mechanism—the unfolded protein response (UPR). UPR achieves bidirectional regulation from promoting survival to promoting apoptosis by dynamically balancing ER function and cell fate decisions. Its core signaling hubs include three transmembrane sensors: IRE1α (Inositol-Requiring Enzyme 1α), PERK (Protein Kinase RNA-like ER Kinase), and ATF6α (Activating Transcription Factor 6α), which synergistically regulate downstream gene expression to restore homeostasis or initiate programmed cell death. 4-OI can effectively regulate the activity of the unfolded protein response key pathway (PERK), alleviate ERS, and thus inhibit the EndMT process. This invention is the first to closely link these three key pathological processes—ERS, EndMT, and vascular remodeling—providing a novel perspective for understanding the complex relationship between oxidative stress, endothelial dysfunction, and structural changes in PAH.

[0016] Furthermore, the inhibition of endothelial-mesenchymal junction translates to inhibition of α-SMA and / or SM22 expression.

[0017] Furthermore, the inhibition of endoplasmic reticulum stress is the inhibition of PERK and / or eIF2α expression.

[0018] Furthermore, the concentration of 4-OI in the drug is 0.1–20 μM.

[0019] Furthermore, the concentration of 4-OI in the drug is 0.1–10 μM.

[0020] Preferably, the concentration of 4-OI in the drug is 10 μM.

[0021] This invention demonstrates that the addition of 4-OI significantly inhibits TGF-β1-induced HUVEC proliferation. Furthermore, this inhibitory effect exhibits a clear concentration-dependent characteristic, meaning the inhibitory effect becomes more pronounced with increasing 4-OI concentration. The inhibitory effect is most significant when the 4-OI concentration reaches 10 μM, indicating that 4-OI is a potential candidate drug for treating diseases caused by TGF-β1 overactivation and may also have therapeutic effects on PAH and vascular remodeling.

[0022] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides the application of 4-OI in the preparation of drugs to improve PAH. In vivo experiments show that 4-OI can significantly reduce pulmonary vascular wall thickening and collagen deposition in PAH rats, improving PAH and vascular remodeling. In vitro experiments further reveal that 4-OI alleviates ERS by inhibiting the PERK-eIF2α pathway, blocking SM22-dependent smooth muscle cell phenotypic transformation and EndMT processes, ultimately inhibiting vascular remodeling and exerting a therapeutic effect on PAH. This invention innovatively links the three key pathological processes of ERS, EndMT, and vascular remodeling, providing a new perspective for a deeper understanding of the complex relationship between oxidative stress, endothelial dysfunction, and vascular structural changes in PAH. It also provides a solid theoretical foundation for developing precision therapeutic drugs for PAH and vascular remodeling based on ERS regulation, possessing significant clinical translational value. Attached Figure Description

[0025] Figure 1 Quantitative morphological analysis of small arteries in the rat lungs using HE staining. Figure 1 In the figures, A represents representative HE-stained images of small pulmonary arteries in each group (scale bar = 50 μm); B represents WA (percentage of vessel wall area) = vessel wall area / (vessel wall area + lumen area) × 100%; C represents WT (percentage of vessel wall thickness) = 2 × vessel wall thickness / vessel outer diameter × 100%. Data are expressed as mean ± standard deviation, n = 4. One-way ANOVA was used to determine differences among multiple groups. P< 0.001, ** P<0.01.

[0026] Figure 2 Masson staining was performed on the small and medium pulmonary arteries of rats in each group. Figure 2 In the figure, A represents the Masson staining image of the small pulmonary arteries of rats in each group (scale bar = 50 μm), with blue representing collagen fibers and red representing muscle fibers; B represents the percentage of collagen fiber area (%) = area of ​​blue region / total tissue area × 100%. Data are expressed as mean ± standard deviation, n = 4, **** P< 0.0001, ** P< 0.01.

[0027] Figure 3 HUVECs cells were treated with different concentrations of 4-OI (0, 0.1, 1, 10 μM) for 48 h, followed by intervention with TGF-β1 10 ng / mL for 48 h. Cell proliferation was measured using the CCK-8 assay. Cell proliferation rate % = [(absorbance of treatment group - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%. Data are expressed as mean ± standard deviation, n=4; one-way ANOVA was used to determine differences among multiple groups. P< 0.0001, *** P< 0.001, * P< 0.05, ns ( P >0.05).

[0028] Figure 4 4-OI inhibited TGF-β1-induced cell proliferation in HUVECs. Figure 4 In the figure, A represents the cell proliferation results (scale bar = 100 μm) of HUVECs after pretreatment with 4-OI (10 μM, 48 h) and intervention with TGF-β1 (10 ng / mL) for 48 h, as detected by the EdU method; B represents the percentage of EdU-positive cells (%) = number of EdU-positive cells / total number of cells × 100%. Data are expressed as mean ± standard deviation, n=3. One-way ANOVA was used to determine differences among multiple groups. P< 0.001, * P< 0.05.

[0029] Figure 5 4-OI inhibited TGF-β1-induced cell proliferation in HUVECs in a manner independent of apoptosis. Figure 5In this context, A represents HUVECs cells treated with 10 μM 4-OI for 48 h followed by TGF-β1 intervention at 10 ng / mL for 48 h. Apoptosis was detected using Annexin V-FITC / PI double staining, with cells differentiated by four quadrants: the lower right quadrant represents early apoptotic cells, and the upper right quadrant represents late apoptotic cells. B represents the total apoptotic cells (%) = early apoptotic cells (%) + late apoptotic cells (%). Data are expressed as mean ± standard deviation, n=3. One-way ANOVA was used to determine differences among multiple groups, ns( P >0.05).

[0030] Figure 6 F-actin immunofluorescence staining was used to observe the cytoskeleton. Among other things, Figure 6 In the diagram, A represents a typical image of F-actin staining (scale bar = 20 μm); B represents SD (cytoskeleton density) % = cytoskeleton area / total cell area × 100%. Data are expressed as mean ± standard deviation, n = 3; one-way ANOVA was used to determine differences among multiple groups. P< 0.001, ** P< 0.01.

[0031] Figure 7 4-OI inhibits TGF-β1-induced cell migration in HUVECs. Figure 7 Figure A shows a comparison of cell migration at 0h and 12h after scratching (scale bar = 500 μm) and a Transwell migration result at 24h (scale bar = 100 μm); Figure B shows the statistical results of the scratching experiment, with wound closure rate (%) = (0h scratch area - 12h scratch area) / 0h scratch area × 100%; Figure C shows the Transwell experiment statistics, with the number of cells transferred per field of view. Data are expressed as mean ± standard deviation, n = 3; one-way ANOVA was used to determine differences between multiple groups. P< 0.0001, * P< 0.05.

[0032] Figure 8 4-OI inhibits TGF-β1-induced ROS generation in HUVECs. Among other things, Figure 8 In the table, A represents the ROS single-parameter fluorescence histogram, with the X-axis representing fluorescence intensity and the Y-axis representing cell count. The further to the right the histogram shifts, the higher the fluorescence intensity. B represents the MFI (mean fluorescence intensity), which can be directly calculated using the mean function in FlowJo software. Data are expressed as mean ± standard deviation, n=3. One-way ANOVA is used to determine differences between multiple groups. P< 0.001.

[0033] Figure 9TGF-β1 induced morphological changes in the endoplasmic reticulum of HUVECs by 4-OI repair. Figure 9 In this table, A represents the results of ER-Tracker Red fluorescence detection, revealing that TGF-β1 enhances the endoplasmic reticulum fluorescence signal in HUVECs, while 4-OI pretreatment significantly reduces this effect (scale bar = 100 μm); B represents the quantitative statistics of ER-Tracker Red fluorescence detection, where RFU (relative fluorescence unit) is calculated relative to other groups with the Control group as the baseline (set as 1), i.e., RFU = raw fluorescence intensity of the Control group / raw fluorescence intensity of the experimental group. Data are expressed as mean ± standard deviation, n = 3; one-way ANOVA was used to determine differences among multiple groups. P< 0.01.

[0034] Figure 10 To validate the three endoplasmic reticulum stress pathways (PERK, IRE1, ATF6) using qPCR. Figure 10 In the table, A represents TGF-β1 significantly activating PERK mRNA expression, a gene related to the PERK pathway, with the 4-OI+TGF-β1 group showing a significant inhibitory effect, leading to a decrease in PERK expression; B represents ATF4 mRNA expression in the PERK pathway; C represents ATF6 mRNA expression in the ATF6 pathway; and D represents XBP1 mRNA expression in the XBP1 pathway. Data are expressed as mean ± standard deviation, n=4; one-way ANOVA was used to determine differences among multiple groups. P< 0.0001, ** P< 0.01, ns (p>0.05).

[0035] Figure 11 4-OI improves pulmonary hypertension by inhibiting the endoplasmic reticulum stress-related pathway PERK. Among other things, Figure 11 In the table, A represents the effect of Western blot analysis on the expression of PERK pathway and EndMT marker proteins; B represents p-PERK protein expression; C represents p-eIF2a protein expression; D represents α-SMA protein expression; and E represents SM22 protein expression. Data are expressed as mean ± standard deviation, n=3; one-way ANOVA was used to determine differences among multiple groups. P< 0.0001, *** P< 0.001, ** P< 0.01, * P< 0.05.

[0036] Figure 12 4-OI improves pulmonary hypertension by inhibiting thapsidic reticulum stress induced by thapsidic acid. Figure 12In the table, A represents the effect of Western blot analysis on the expression of PERK pathway and EndMT marker proteins; B represents p-PERK protein expression; C represents p-eIF2a protein expression; D represents α-SMA protein expression; and E represents SM22 protein expression. Data are expressed as mean ± standard deviation, n=3; one-way ANOVA was used to determine differences among multiple groups. P< 0.0001, *** P< 0.001, ** P< 0.01, * P< 0.05.

[0037] Figure 13 Immunohistochemical analysis of 4-OI's effect on improving pulmonary vascular remodeling in pulmonary hypertension by inhibiting key biomarkers of ERS. Figure 13 In the figure, A represents immunohistochemical staining of small blood vessels in the rat lungs (scale bar = 50 μm), from left to right: control group (Control), PAH model group (MCT), and 4-OI treatment group (MCT+4-OI). From top to bottom, the expression localization of p-PERK, PERK, p-eIF2α, eIF2α, α-SMA, and SM22 proteins is shown (brown indicates positive signal, and hematoxylin counterstains the cell nuclei). BE represents semi-quantitative analysis of protein expression in each group: (B) p-PERK / PERK, (C) p-eIF2α / eIF2α, (D) α-SMA, and (E) SM22. H-score = ∑(percentage of positive cells × staining intensity score), where percentage of positive cells = positive cells in the field of view / total cells, and staining intensity score (0 = negative, 1 = weak, 2 = moderate, 3 = strong). Three non-overlapping fields of view were randomly selected from each group. Data are expressed as mean ± standard deviation, n = 4. One-way ANOVA was used to compare differences between groups. P< 0.0001, *** P< 0.001, ** P< 0.01, * P< 0.05. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] The structural formula of 4-OI is as follows (downloadable from PubChem https: / / pubchem.ncbi.nlm.nih.gov / ):

[0041] .

[0042] In this invention, GraphPad Prism 9.4.1 was used for statistical analysis of the data. Each experiment was repeated three times or more, and all data are expressed as mean ± standard deviation. Unpaired t-tests were used to compare data between two groups, and one-way ANOVA was used for data from three or more groups. A p-value < 0.05 was considered statistically significant.

[0043] Example 1: Effects of 4-octyl itaconic acid (4-OI) on the morphology of rat pulmonary arteries

[0044] I. Experimental Methods

[0045] 1. Animal culture and experimental grouping

[0046] (1) Animal selection and grouping: Healthy male SD rats with similar body weights were selected, aged 6-7 weeks and weighing approximately 220-260g. The SD rats were randomly divided into 4 groups, with 5 SD rats in each group.

[0047] Control group: treated with an equal volume of physiological saline.

[0048] MCT model group: a single subcutaneous injection of lily alkaloid (MCT, dose 60 mg / kg) was used to induce the PAH model.

[0049] MCT+4-OI: Starting on day 1 after MCT injection, administer 10 mg / kg of 4-OI daily.

[0050] (2) Model induction and drug treatment

[0051] MCT-induced PAH model: At the beginning of the experiment, animals in the MCT model group and the 4-OI treatment group were injected subcutaneously with a single dose of MCT (60 mg / kg), while the control group was injected with an equal volume of physiological saline. After injection, the animals were returned to their cages and fed normally, and their behavior and health status were observed.

[0052] Drug treatment: Starting from day 1 after MCT injection, animals in the 4-OI treatment group were intraperitoneally injected with 4-OI once daily for 3 consecutive weeks. Animals in the control group and MCT model group were given an equal volume of physiological saline.

[0053] 2. HE staining to observe pathological changes in lung tissue

[0054] Fixation: Quickly remove lung tissue and fix it in 4% paraformaldehyde for 24 hours;

[0055] Dehydration: The tissue was placed in 70%, 80%, 90%, 95% and 100% ethanol in sequence for 1-2 hours each time to dehydrate;

[0056] Transparency: Place the dehydrated tissue in xylene, usually twice, for 30 minutes each time, to make the tissue transparent;

[0057] Wax impregnation: The transparent tissue is placed in molten paraffin wax, usually twice, for 1-2 hours each time, to allow the paraffin wax to fully penetrate;

[0058] Embedding: The paraffin-impregnated tissue is placed into an embedding box, paraffin is added, and the tissue is cooled and solidified into a wax block;

[0059] Sectioning: Use a microtome to cut the wax block into sections 4-6 micrometers thick and place them on a glass slide;

[0060] Dewaxing and hydration: Dewax the sections in xylene twice, 10 minutes each time. Then, immerse the sections in 100%, 95%, 90%, 80%, and 70% ethanol sequentially, and finally rinse with distilled water for 2-3 minutes per rinse.

[0061] HE staining: Place the sections in hematoxylin staining solution for 5-10 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, blue with weak alkaline ammonia solution, rinse with tap water, place the sections in eosin staining solution for 1-3 minutes, and rinse with tap water.

[0062] Dehydration and clearing: Immerse the sections in 70%, 80%, 90%, 95%, and 100% ethanol sequentially, for 2-3 minutes at each stage. Clear the sections twice in xylene, 5 minutes each time.

[0063] Mounting: Add a drop of neutral resin to the slide, cover with a coverslip, and avoid air bubbles;

[0064] Microscopic examination: After the slide has dried, observe the pathological changes in the lung tissue using a microscope.

[0065] 3. Masson staining to observe pathological changes in lung tissue

[0066] Prepare paraffin sections using the same HE staining steps as described above.

[0067] Masson staining: Immerse the sections in Weigert iron hematoxylin staining solution (equal volumes of solution A and solution B) for 10 minutes. After rinsing with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds. After rinsing with tap water, blue the sections with a weakly alkaline ammonia solution. After rinsing with tap water, immerse the sections in Ponceau S-acid fuchsin solution for 10 minutes. After rinsing with tap water, differentiate the sections in 1% phosphomolybdic acid solution for 5-10 minutes. After rinsing with tap water, immerse the sections in Brilliant Green or aniline blue solution for 1-2 minutes.

[0068] The subsequent steps are the same as the HE staining steps above.

[0069] II. Experimental Results

[0070] A PAH animal model was established using SD rats via subcutaneous injection of MCT. HE staining results are shown below. Figure 1 As shown, the MCT group exhibited abnormal lung tissue structure and disordered arrangement, with widespread thickening of alveolar walls and focal enlargement and collapse of alveolar cavities; thickening of the walls of interstitial pulmonary arterioles, intimal hyperplasia, and luminal narrowing; increased lymphocyte infiltration in the alveolar septa and perivascular spaces; and increased collagen deposition and significant fibrosis in the pulmonary interstitium. Compared with the MCT group, the MCT+4-OI group showed improved lung tissue morphology, with thinning of the media and larger luminal diameters. These results indicate that 4-OI can inhibit pulmonary vascular remodeling in MCT (…). Figure 1 (A) Compared with the control group, the WA% and WT% of the MCT group were significantly increased ( P< 0.001); After 4-OI intervention, all of the above parameters were significantly improved ( P< 0.01) Figure 1 (B and C in the middle).

[0071] Masson staining results are as follows: Figure 2 As shown, the pulmonary arteriole in the MCT group showed significant thickening of the intima, with abundant collagen fiber deposition in the intima layer, appearing blue; the smooth muscle layer of the pulmonary arteriole media was significantly thickened, with increased collagen fibers, indicating structural remodeling of the vessel wall; increased collagen fibers in the membrane layer, overall thickening of the vessel wall, and narrowing of the vessel lumen; compared with the control group, the MCT group showed significant perivascular fibrosis of the pulmonary arteries (…). P< 0.001); After 4-OI intervention, the degree of perivascular fibrosis in the pulmonary artery decreased ( P < 0.01).

[0072] In summary, the results indicate that 4-OI significantly reduced pulmonary vascular wall thickening and collagen deposition in PAH rats, and improved pulmonary hypertension and vascular remodeling.

[0073] Example 2: 4-OI inhibits TGF-β1 (transforming growth factor-β1)-induced cell proliferation, cytoskeleton remodeling, and cell migration in human umbilical vein endothelial cells (HUVECs).

[0074] I. Experimental Methods

[0075] 1. Construction of HUVECs cell model of pulmonary hypertension

[0076] (1) Cell counting: Take 10 μL of cell suspension and mix it with trypan blue staining solution at a ratio of 1:1. Then take 10 μL of the mixture and slowly drip it into the cell counting plate from the edge to fill the gap between the counting plate and the coverslip.

[0077] (2) Cell seeding plate: Taking a 6-well plate as an example, the number of cells seeded per well is 300,000.

[0078] (3) TGF-β1 treatment: 10 ng / mL TGF-β1 was added to the cells for stimulation for 48 hours.

[0079] (4) Phenotypic detection: Western blotting was used to detect Smad2 / 3 phosphorylation.

[0080] 2. CCK-8 (Cell Counting Kit-8) assay for cell proliferation.

[0081] This is a reliable and widely used method in cell proliferation and cytotoxicity experiments. By measuring the optical density (OD) value produced by the cells, the number of cells can be accurately calculated, thus reflecting the cell proliferation status.

[0082] (1) Cell grouping:

[0083] 1) Control group: Cells were added to ordinary culture medium and cultured in an ordinary incubator (37℃, 5% CO2).

[0084] 2) TGF model group: After cell adhesion, cells were stimulated with 10 ng / mTGF-β1 for 48 hours;

[0085] 3) Drug group (TGF model group after 48 hours of drug incubation): After cell adhesion, cells were incubated with 4-OI culture medium prepared in ordinary medium for 48 hours, and then stimulated with 10 ng / mL TGF-β1 for 48 hours.

[0086] (2) Cell count: Same as above.

[0087] (3) Cell seeding plates: 96-well plates with 6000 cells / well and 100 culture medium per well. Each experimental group has 5 replicates. Sterile pure water is added to the surrounding blank wells. The plates are placed in a CO2 incubator and cultured normally for 24 hours.

[0088] (4) Intervention in cells: Different intervention methods were adopted according to different experimental groups.

[0089] (5) CCK-8 incubation: After cell intervention, the culture medium was aspirated, the cells were washed twice with PBS, and 100 μL of freshly prepared complete culture medium containing CCK-8 solution (CCK-8: 10% FBS F12 medium = 1:9) was added to each well. The cells were incubated in a cell culture incubator at 37°C in the dark for 2 hours.

[0090] (6) Measure the absorbance value: Use an ELISA reader to measure the absorbance value of each well at a wavelength of 450 nm, and calculate the relative cell viability. Cell viability = [(absorbance of drug group - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100%.

[0091] 3. EdU cell proliferation detection

[0092] EdU is a fluorescently labeled nucleotide analog that can be incorporated into newly synthesized DNA during cell division. By detecting EdU-labeled DNA, cell proliferation can be accurately assessed.

[0093] (1) Cell culture: Seed cells into culture plates and process them according to the experiment after they adhere to the plate.

[0094] (2) EDU labeling: Add EDU (50 μM) to the cell culture medium and incubate for 2 hours. EDU will be incorporated into the DNA of the proliferating cells. Discard the culture medium and wash the cells twice with PBS for 5 minutes each time.

[0095] (3) Cell fixation: Fix cells with 4% paraformaldehyde for 30 minutes, discard the fixative, add 2 mg / mL glycine, decolorize and incubate on a shaker for 5 minutes, discard the glycine solution, add PBS and wash for 5 minutes, then discard the PBS.

[0096] (4) Permeabilization treatment: Permeabilize cells with 0.5% Triton X-100 for 10 minutes to increase cell membrane permeability.

[0097] (5) Apollo staining: Add 1×Apollo staining reaction solution (prepared and used immediately), incubate at room temperature in the dark for 30 minutes to 1 hour, and EDU reacts with azide to generate fluorescent markers.

[0098] (6) Nuclear staining: Use 1×Hoechst33342 reaction solution and incubate at room temperature in the dark for 30 minutes. Then discard the reaction solution and wash 3 times with PBS.

[0099] (7) Fluorescence microscopy observation: Under a fluorescence microscope, EDU-labeled cells will show red fluorescence signals.

[0100] (8) Data analysis: The proportion of EDU positive cells was calculated using the image analysis software ImageJ to assess cell proliferation.

[0101] 4. Cell scratch test

[0102] (1) Cell seeding: Digest the cells and resuspend them in complete culture medium, and seed them evenly into 6-well plates.

[0103] (2) Place the culture plate in a 37℃, 5% CO2 incubator for 24 hours.

[0104] (3) Preparation before scratching: Take out the culture plate and gently wash the cells 1-2 times with PBS.

[0105] (4) Scratching procedure: Using a 200 μL sterile pipette tip, make straight scratches perpendicular to the culture plate on the cells. Two parallel scratches can be made on each well to increase the reproducibility of the experiment.

[0106] (5) Removal of scratched cells: Gently rinse the culture plate with PBS to remove cells that detached during scratching.

[0107] (6) Cell migration: Add serum-free culture medium (to reduce the influence of cell proliferation on migration), and put the culture plate back into a 37°C, 5% CO2 incubator for 12 hours.

[0108] (7) Observe and take pictures.

[0109] (8) Data analysis: Use image analysis software (such as ImageJ) to measure the width or area of ​​the scratch. Cell migration rate = (initial scratch area - current scratch area) / initial scratch area. Compare the migration ability of different experimental groups.

[0110] 5. Transwell invasion experiment

[0111] (1) Cell preparation: After the cells were treated according to the experimental plan, they were digested and resuspended in serum-free culture medium. The cell density of each group was adjusted to 1×10⁻⁶. 5 200μL per cell.

[0112] (2) Cell inoculation:

[0113] Add cells to the upper chamber: Add 200 μL of cell suspension to the upper chamber of the Transwell chamber, taking care to avoid generating air bubbles.

[0114] Add culture medium to the lower chamber: Add 400 μL of culture medium containing FBS to the lower chamber. Serum is used as a chemical inducer to attract cell migration.

[0115] 6. Annexin V-FITC / PI cell apoptosis assay

[0116] (1) Prepare an appropriate number of centrifuge tubes and label them. Collect the culture medium from the culture plate into the centrifuge tubes.

[0117] (2) Wash once with PBS, add EDTA-free trypsin for digestion, stop digestion with serum-containing culture medium, and collect cells into the corresponding centrifuge tubes. Centrifuge at 300×g for 5 minutes at 4°C.

[0118] (3) Carefully aspirate the supernatant, add 1 ml of PBS to wash, and centrifuge at 300×g for 5 minutes;

[0119] (4) Discard the supernatant, add 100 μL of 1×Annexin V binding buffer to resuspend, and transfer to a flow cytometer;

[0120] (5) Add 2.5 μL of Annexin V and PI dye in sequence, mix well, incubate at room temperature in the dark for 15 minutes, add 200 μL of 1× Annexin V binding buffer, mix the sample well, and load it onto the instrument immediately.

[0121] 7. Cytoskeleton staining experiment

[0122] (1) Cells were treated differently according to experimental requirements;

[0123] (2) Wash the cells twice with PBS, and then fix the cells with 4% paraformaldehyde for 15 minutes;

[0124] (3) Wash 2-4 times with PBS containing 0.1% Triton X-100, 5 minutes each time;

[0125] (4) Dilute Actin-Tracker Red 1:100 with antibody dilution buffer, add 200 μL to each confocal dish, and incubate at room temperature in the dark for 1 hour;

[0126] (5) Wash 2-4 times with PBS containing 0.1% Triton X-100, 5 minutes each time. After washing, allow the confocal dish to air dry and store in a refrigerator at 4°C protected from light;

[0127] (6) Use a laser confocal microscope to magnify 400 times, observe the cytoskeleton under oil immersion and take pictures.

[0128] II. Experimental Results

[0129] 1. 4-OI inhibits TGF-β1-induced cell proliferation in HUVECs

[0130] To investigate the effect of 4-OI on TGF-β1-induced HUVEC proliferation, a TGF-β1 concentration of 10 ng / mL (widely used in similar endothelial cell studies) was selected to treat HUVECs for 48 hours to observe a sufficiently significant cell proliferation response. Simultaneously, different concentrations of 4-OI (0, 0.1, 1, and 10 μM) were prepared in standard culture medium and incubated for 48 hours, followed by stimulation with 10 ng / mL TGF-β1 for another 48 hours. This step was to observe whether different concentrations of 4-OI inhibited TGF-β1-induced cell proliferation and to attempt to determine the relationship between this inhibition and the 4-OI concentration.

[0131] The CCK-8 assay was used to quantitatively assess cell proliferation, and the experimental results are as follows: Figure 3 As shown, the addition of 4-OI significantly inhibited TGF-β1-induced HUVEC proliferation. Furthermore, this inhibition exhibited a clear concentration-dependent effect, meaning the inhibitory effect became more pronounced with increasing 4-OI concentration. The inhibitory effect was most significant at a 4-OI concentration of 10 μM, suggesting that 4-OI is a potential candidate drug for treating diseases caused by TGF-β1 overactivation. Based on this finding, 4-OI (10 μM) was used as the concentration for subsequent studies to further explore its inhibitory mechanism on TGF-β1-induced HUVEC proliferation.

[0132] Next, the proliferation of HUVECs was assessed using EdU assay, and the results are as follows: Figure 4 As shown, the intensity of EdU-labeled red fluorescence in the TGF-β1 group was significantly increased compared to the Control group. Conversely, the intensity of EdU-labeled red fluorescence in the 4-OI + TGF-β1 group was significantly decreased compared to the TGF-β1 group, further suggesting that 4-OI may inhibit TGF-β1-induced cell proliferation in HUVECs.

[0133] 2. 4-OI inhibits TGF-β1-induced cell proliferation in HUVECs in apoptosis-independent manner.

[0134] When apoptosis was detected by Annexin V-FITC / PI double staining, the results were as follows: Figure 5 As shown, neither the TGF-β1 treatment group nor the 4-OI pretreatment group showed significant differences compared to the control group. p >0.05), indicating that the inhibitory effect of 4-OI on TGF-β1-induced HUVEC proliferation is independent of the apoptosis pathway.

[0135] 3,4-OI inhibits TGF-β1-induced cytoskeleton remodeling in HUVECs

[0136] EdU and CCK8 cell proliferation assays showed that 4-OI effectively inhibited TGF-β1-induced HUVEC proliferation. The cytoskeleton, mainly composed of microfilaments, microtubules, and intermediate filaments, is not only a scaffold structure maintaining cell morphology but also a key factor regulating cell proliferation. Immunofluorescence staining of the cytoskeleton (F-actin) was used to observe the effect of 4-OI on F-actin in TGF-β1-treated HUVECs. The experimental results are as follows: Figure 6 As shown, the TGF-β1 group significantly promoted cytoskeleton remodeling in HUVECs, specifically by enhancing pseudopodia formation and stress fiber assembly. Notably, 4-OI played a significant inhibitory role in this process. In the 4-OI+TGF-β1 group, stress fibers were reduced, F-actin distribution was closer to that of the control group, showing a uniform cortical distribution; pseudopodia formation was reduced, cell morphology returned to a cobblestone-like shape, polarization was weakened, and cell area decreased. This suggests that 4-OI can significantly inhibit TGF-β1-induced cytoskeleton remodeling in HUVECs, reduce stress fiber and pseudopodia formation, and thus may inhibit EndMT and pathological vascular remodeling.

[0137] 4. 4-OI inhibits TGF-β1-induced cell migration in HUVECs

[0138] To investigate the effect of 4-OI on TGF-β1-induced cell migration in HUVECs, the effect of 4-OI on the migration ability of TGF-β1-induced HUVECs was assessed using cell scratch assays and Transwell migration assays. Cell scratch results are shown below. Figure 7 As shown, in the Control group, 12 hours after scratching, HUVECs only partially filled the scratched area, with a healing rate of 40%. After TGF-β1 stimulation, cell migration accelerated, and the scratch healing rate was approximately 80% after 12 hours. In the group treated with 4-OI + TGF-β1, the scratch healing rate decreased to 60%. 4-OI treatment alone had no significant effect on scratch healing (healing rate approximately 40%), and there was no statistically significant difference compared to the control group.

[0139] Transwell migration 24h experiment results are as follows Figure 7As shown, the number of unstimulated HUVECs in the Control group was low, less than 200 per field of view. TGF-β1 significantly increased the migration ability of HUVECs, with the number of migrating cells increasing to approximately 600 per field of view, almost filling the entire field of view. The number of migrating cells decreased to 250 per field of view with 4-OI + TGF-β1, indicating that 4-OI can significantly antagonize the migration-promoting effect of TGF-β1, and the inhibitory effect is consistent with the cell scratch assay results. The scratch assay more directly reflects collective migration, while the Transwell assay quantifies single-cell migration. Both the scratch assay and the Transwell migration assay demonstrate that 4-OI can significantly inhibit TGF-β1-induced HUVEC migration, an effect consistent with its ability to regulate cytoskeleton remodeling, suggesting that 4-OI may exert its anti-fibrotic and anti-angiogenic effects by intervening in the TGF-β1-mediated cell motility signaling pathway.

[0140] In summary, 4-OI treatment significantly inhibited TGF-β1-induced abnormal proliferation of HUVECs (reducing EdU positivity rate). P< 0.001), skeletal remodeling and migration (reduced number of Transwell-migrating cells), P< The result (0.001) indicates that 4-OI may exert its anti-fibrotic and anti-angiogenic effects by intervening in the TGF-β1-mediated cell motility signaling pathway, thereby inhibiting endothelial-mesenchymal transition (EndMT) and pathological vascular remodeling.

[0141] Example 3: Study on the mechanism by which 4-OI improves EndMT and pulmonary hypertension by inhibiting ERS

[0142] I. Experimental Methods

[0143] 1. Intracellular reactive oxygen species detection

[0144] (1) After the cells were treated differently according to the experimental plan, the cell morphology was observed under a microscope;

[0145] (2) Dilute the stock solution DHE in advance with serum-free F12 medium at a dilution ratio of 1:1000 to make the final concentration 10 μM;

[0146] (3) Discard the supernatant, wash twice with PBS, add 1 mL of diluted DHE to each well of a 6-well plate, and incubate at 37°C for 30 minutes in a cell culture incubator;

[0147] (4) Observe and take pictures, remove DHE, wash the cells three times with serum-free culture medium, and observe the red fluorescence under a fluorescence microscope and take pictures.

[0148] (5) Add trypsin to digest the cells, add complete culture medium to collect the cells into a centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in serum-free culture medium, and transfer the cells to a flow cytometer for the experiment.

[0149] 2. qRT-PCR detection of PERK, ATF4, ATF6, and XBP1

[0150] (1) Primers required for qRT-PCR

[0151] Table 1 Primers required for qRT-PCR

[0152]

[0153] (2) After extracting total RNA from cells using the Trizol method, the RNA reverse transcription system was prepared according to Table 2 below:

[0154] Table 2 RNA reverse transcription system

[0155]

[0156] The sample was incubated in a PCR amplification instrument at 50°C for 15 minutes, then at 85°C for 5 seconds, and finally cooled at 4°C. The synthesized cDNA was then stored at -20°C.

[0157] (3) Real-time PCR

[0158] 1) Preparation of the reaction system

[0159] Table 3. Real-time PCR reaction system

[0160]

[0161] 2) Set up the reaction program

[0162] 3) Results Analysis: Statistical data was analyzed using... ΔΔ The relative expression levels of the target gene in each group were calculated using the Ct method.

[0163] 3. Endoplasmic reticulum fluorescence staining experiment

[0164] (1) Cells were treated differently according to experimental requirements;

[0165] (2) Wash the cells with PBS 1-2 times, add ER-Tracker dye to serum-free culture medium at a ratio of 1:1000 and mix well. Add 100 μL of diluted ER-Tracker dye to each dish and incubate for 1 hour in an incubator at 37°C and 5% CO2.

[0166] (3) Remove the staining solution and wash the cells 2-3 times with PBS to remove unbound dye;

[0167] (4) Add fresh culture medium, observe the endoplasmic reticulum using a fluorescence microscope, and take images.

[0168] 4. Western blot analysis of p-PERK, PERK, p-eIF2α, eIF2α, α-SMA, SM22, and GAPDH protein expression levels.

[0169] (1) Sample preparation: Lyse cells, centrifuge and collect supernatant; add 4× loading buffer, mix well, heat in a metal bath for 10 minutes, and store at -20℃.

[0170] (2) Western Blot: electrophoresis, transfer to membrane, block, incubate with primary antibody for the corresponding protein to be detected, incubate with secondary antibody, and develop.

[0171] 5. Immunohistochemical staining of tissues

[0172] (1) The preparation of paraffin sections and the dewaxing and hydration steps are the same as those in Example 1 for HE staining;

[0173] (2) Antigen retrieval: 1) Immerse the slides in EDTA (pH 9.0), heat to boiling in a pressure cooker (start timing 1 min 30 s from the start of pressure cooker steam), and allow to cool naturally to room temperature. Remove the slides from the retrieval solution and wash 3 times with TBS for 5 min each time; 2) Immerse the slides in citric acid (pH 6.0), heat to boiling in a pressure cooker (start timing 2 min from the start of pressure cooker steam), and allow to cool naturally to room temperature. Remove the slides from the retrieval solution and wash 3 times with TBS for 5 min each time;

[0174] (3) Draw circles with a hydrophobic pen, add 3% H2O2 and incubate at room temperature for 10-15 min to quench endogenous peroxidase activity, wash 3 times with PBS for 5 min each time;

[0175] (4) Blocking: Add 5% BSA dropwise and block at room temperature for 30 min;

[0176] (5) Primary antibody incubation: Add diluted primary antibody dropwise and incubate overnight at 4°C;

[0177] (6) Secondary antibody incubation: Place the slides at room temperature for 15 min to warm up, and wash 3 times with TBST buffer (5 min / time). Add HRP-labeled secondary antibody and incubate at room temperature for 30-60 min, then wash 3 times with TBST.

[0178] (7) Color development: Add DAB working solution, control the color development time under a microscope, and stop the color development with tap water;

[0179] (8) Counterstaining, dehydration, mounting, and microscopic examination: Same as HE staining steps in Example 1.

[0180] II. Experimental Results

[0181] 1. 4-OI inhibits TGF-β1-induced ROS generation in HUVECs

[0182] There is a close bidirectional regulatory relationship between reactive oxygen species (ROS) and endoplasmic reticulum stress. On the one hand, endoplasmic reticulum stress induces mitochondrial dysfunction through the PERK-eIF2α-ATF4 pathway, leading to ROS bursts. On the other hand, excessive ROS can oxidize protein disulfide isomerase (PDI) in the endoplasmic reticulum lumen, impairing protein folding ability and further aggravating endoplasmic reticulum stress.

[0183] This invention utilizes the specific reaction (oxidation reaction) between the redox-sensitive fluorescent probe DHE (dihydroethidine) and ROS. The oxidized product, ethidine oxide, can be incorporated into chromosomal DNA, producing red fluorescence. The content and changes of ROS are determined based on the red fluorescence in living cells. Experimental results are as follows: Figure 8 As shown, the intracellular ROS level in the TGF-β1 treatment group was higher than that in the control group. P< The ROS level was 0.001, while the 4-OI+TGF-β1 group reduced the ROS level to a level that was basically the same as the control group.

[0184] 2. Repair of TGF-β1-induced morphological changes in the endoplasmic reticulum of HUVECs by 4-OI

[0185] Based on the above research, it is hypothesized that TGF-β1 may promote abnormal activation of HUVECs by disrupting endoplasmic reticulum homeostasis, while 4-OI, as an anti-inflammatory metabolite, may exert a protective effect by maintaining endoplasmic reticulum morphology and function. To confirm this hypothesis, ER-Tracker Red fluorescence staining was used to systematically evaluate the regulatory role of 4-OI on TGF-β1-induced endoplasmic reticulum stress in HUVECs.

[0186] ER-Tracker Red is a live-cell red fluorescent probe that specifically labels the endoplasmic reticulum (ER). This probe uses fluorescently labeled glibenclamide, which specifically binds to sulfonylurea receptors on the ER membrane. These receptors are primarily located in the ATP-sensitive potassium channels (K+ATP channels) of the ER. The fluorescence intensity of this probe is closely related to the morphology, quantity, and stress state of the ER. Experimental results are as follows... Figure 9 As shown, the endoplasmic reticulum in the Control group exhibited uniformly distributed weak red fluorescence, indicating normal endoplasmic reticulum morphology and no significant stress. When cells are stimulated by TGF-β1, the endoplasmic reticulum expands, vesicles, or aggregates, leading to increased fluorescence intensity. P<The relative fluorescence intensity of the TGF-β1 group was 3 times that of the Control group (0.001), and the fluorescence distribution was uneven, with some areas showing aggregation or diffuse enhancement, reflecting an imbalance in endoplasmic reticulum homeostasis. The 4-OI+TGF-β1 group effectively alleviated this process, with a significantly weaker fluorescence intensity compared to the TGF-β1 group. P< The distribution was closer to the control (0.01), indicating that 4-OI effectively alleviated endoplasmic reticulum stress caused by TGF-β1 and partially restored the normal morphology of the endoplasmic reticulum.

[0187] Because the experiment used an inverted fluorescence microscope (rather than a higher resolution confocal microscope), although it was not possible to precisely observe subcellular structures (such as individual ER vesicles), it was still possible to qualitatively compare the changes in fluorescence intensity of different treatment groups, thereby assessing the degree of endoplasmic reticulum stress.

[0188] 3. qPCR validation of the three pathways of 4-OI in TGF-β1-induced endoplasmic reticulum stress in HUVECs

[0189] Previous studies have shown that 4-OI may exert a cytoprotective effect by regulating endoplasmic reticulum (ER) homeostasis. ER stress research should initially focus on three classic pathways: the PERK-eIF2α-ATF4 pathway; the IRE1α-XBP1 pathway; and the ATF6 pathway. To experimentally confirm whether 4-OI exerts its effect by regulating ER homeostasis, quantitative real-time PCR (qPCR) was used to systematically analyze the effects of 4-OI on TGF-β1-induced HUVECs. The qPCR results are as follows: Figure 10 As shown, TGF-β1 significantly activated the expression of PERK, a gene related to the PERK pathway, with PERK mRNA expression upregulated by approximately 1.5-fold. P< The 4-OI+TGF-β1 group showed a significant inhibitory effect, reducing PERK expression to 0.9-fold. After TGF-β1 stimulation, ATF4 expression in the PERK pathway did not reach the statistical significance threshold compared to the Control group. P< While the expression of 4-OI (n=0.05) showed an upregulation trend, the expression of PERK was significantly reduced in the TGF-β1 group, with a clear reversion trend observed in the 4-OI+TGF-β1 group. The other two pathways (XBP1 and ATF6) showed an overall upregulation trend in the mRNA expression of the key genes XBP1 and ATF6 in the TGF-β1 group, and 4-OI exhibited some inhibitory effect, but under the current experimental conditions (n=4), these changes did not reach statistical significance. These results preliminarily suggest that 4-OI may preferentially target the PERK pathway. 4-OI may affect TGF-β1-induced human umbilical vein endothelial cells (HUVECs) through multiple endoplasmic reticulum stress-related pathways, with the PERK pathway playing a crucial role.

[0190] 4. 4-OI improves pulmonary hypertension by inhibiting the endoplasmic reticulum stress-related pathway PERK.

[0191] To further verify the regulatory role of 4-OI on TGF-β1-induced endoplasmic reticulum stress in HUVECs, Western blot was used to detect endoplasmic reticulum stress marker proteins. The experimental results are as follows: Figure 11 As shown: After TGF-β1-induced HUVECs, α-SMA expression was significantly increased compared to the control group ( P< α-SMA (0.05%) is a classic EndMT marker, reflecting the early transformation of endothelial cells into a mesenchymal phenotype. Upregulation of α-SMA expression indicates that the cell has initiated the mesenchymal transformation process. Simultaneously, SM22 expression was also significantly increased compared to the Control group ( ). P< SM22 (0.0001) is a marker of smooth muscle cell (SMC) differentiation; upregulated expression suggests that cells may further differentiate into SMC-like cells. These results indicate that TGF-β1 not only initiates mesenchymal transition but may also drive abnormal thickening of the vascular wall (a typical pathological feature of pulmonary hypertension). In the 4-OI+TGF-β1 group, α-SMA expression was significantly decreased compared to the TGF-β1 group. P< 0.01). This indicates that 4-OI inhibits the EndMT process and maintains endothelial properties. Simultaneously, SM22 expression was also significantly reduced compared to the TGF-β1 group ( P< (0.01). This result suggests that 4-OI blockade of smooth muscle-like transdifferentiation may alleviate vascular remodeling.

[0192] Furthermore, protein expression in the PERK-eIF2α pathway was analyzed. TGF-β1 significantly activated the PERK signaling pathway, and p-PERK (phosphorylated PERK) expression was significantly increased compared to the control group. P< 0.05), downstream p-eIF2α (phosphorylated eIF2α) expression increased synchronously ( P< 0.01). Experimental results indicate that TGF-β1 can activate the PERK pathway, and the PERK pathway may be a key link between endoplasmic reticulum stress and cell phenotypic transformation. Inhibition of the PERK pathway by 4-OI resulted in simultaneous downregulation of α-SMA and SM22, suggesting that 4-OI exerts a pleiotropic protective effect by targeting PERK. Notably, the inhibitory effect of 4-OI even led to lower expression levels of p-PERK and α-SMA compared to the control group, suggesting that 4-OI may directly inhibit the basal activity of the PERK pathway or activate other protective pathways (such as antioxidant pathways).

[0193] 5. 4-OI improves pulmonary hypertension by inhibiting thapsidic gargin-induced endoplasmic reticulum stress.

[0194] Previous studies have shown that 4-OI improves pulmonary hypertension by inhibiting the endoplasmic reticulum stress-related pathway PERK. To further confirm this view, a further verification experiment was conducted by introducing Thapsigargin. Thapsigargin is a classic endoplasmic reticulum stress inducer, which can rule out the non-ER stress-dependent effects of TGF-β1. If 4-OI also alleviates TG-induced damage, it will strengthen the conclusion that "4-OI works by targeting endoplasmic reticulum stress." Western blotting was used for verification, and the experimental results are as follows: Figure 12 As shown, after Thapsigargin stimulation, the expression of p-PERK and p-eIF2α proteins was significantly increased compared to the Control group. P< (0.0001), which indicates that Thapsigargin successfully induced endoplasmic reticulum stress in HUVECs.

[0195] 6. Morphological evidence of how 4-OI reverses pulmonary vascular remodeling by inhibiting key markers of ERS.

[0196] To clarify the regulatory role of 4-OI on EndMT and ERS in PAH, immunohistochemistry was used to systematically analyze the expression distribution of ERS markers (p-PERK, p-eIF2α) and EndMT-related proteins (α-SMA, SM22) in pulmonary arterioles of the Control group, PAH model group (MCT), and 4-OI treatment group (MCT+4-OI), revealing the regulatory role of 4-OI on the ERS-EndMT axis. Immunohistochemistry used DAB as the chromogenic agent. Its basic principle is to utilize an enzymatic reaction, with specific antibodies recognizing target proteins and horseradish peroxidase catalyzing the formation of an insoluble brown precipitate, thereby localizing protein expression under a microscope.

[0197] Experimental results are as follows Figure 13 As shown, in the Control group, p-PERK was weakly expressed only in the cytoplasm of endothelial cells, while PERK protein was evenly distributed across all layers of the blood vessel wall. In the PAH model group, p-PERK exhibited significant nuclear-cytoplasmic colocalization characteristics, with dark brown granular deposits in the nuclei of endothelial cells and smooth muscle cells (SMCs) (p-PERK / PERK H-score ratio approximately 0.4, ***). P< The 0.001 vs Control group suggests persistent ERS activation. In the MCT+4-OI group, the p-PERK positivity rate decreased by approximately 25% (p-PERK / PERK H-score ratio approximately 0.3, * P< The 0.05 vs MCT model group indicates that 4-OI specifically inhibits PERK phosphorylation ( Figure 13(A and B in the original text). Additionally, strongly positive p-eIF2α granules (presumably detached endothelial cell fragments) were observed on the luminal surface of the pulmonary arterioles in the MCT model group, while only focal weak positivity was observed in the vessel wall SMCs. The positive signal in the luminal area was reduced in the MCT+4-OI group (*). P< 0.05 vs MCT model group), and punctate p-eIF2α expression was observed in intramural SMCs ( Figure 13 (A and C in the text).

[0198] Meanwhile, in the Control group, α-SMA was strictly confined to the endothelial stromal cells (SMCs) layer, with negative intimal endothelial cells. In the MCT model group, α-SMA invaded the intima and diffused into the adventitia, forming a "whole-wall positive" pattern. In the MCT+4-OI group, the α-SMA-positive area significantly shrank, and intimal infiltration and diffusion into the adventitia were significantly reduced (**). P< 0.01 vs MCT model group) Figure 13 (A and D in the text).

[0199] In the Control group, SM22 showed continuous strong positivity in membrane SMCs (H-score: 111.5±10.0), while in the MCT model group, SM22 expression extended to both intima and adventitia fibroblasts (H-score: 234.8±29.3, ****). P< 0.0001). The SM22 distribution in the 4-OI treatment group was close to normal (H-score: 149.5±30.5, *** P< 0.001), suggesting a reversal of the smooth muscle cell phenotype from a synthetic to a contractile phenotype ( Figure 13 (A and E in the text).

[0200] In summary, this study demonstrates that 4-OI can improve ERS-induced EndMT by inhibiting the PERK-eIF2α signaling pathway, thereby treating pulmonary vascular remodeling and pulmonary hypertension. This invention not only deepens our understanding of the pathogenesis of PAH but also lays an important theoretical foundation and experimental basis for developing novel PAH therapeutics targeting ERS. As a small molecule compound with both anti-inflammatory and metabolic regulatory properties, 4-OI shows promising clinical applications in the treatment of PAH.

Claims

1. Use of 4-octyl itaconic acid for the preparation of a medicament for improving pulmonary hypertension, characterized in that, The pulmonary arterial hypertension is caused by monocrotaline or transforming growth factor-β1.

2. Use according to claim 1, characterized in that, The drug achieves the therapeutic purpose by inhibiting one or more of cell proliferation, migration, skeleton reorganization, thickening of pulmonary vascular wall, collagen deposition, and reduction of pulmonary arterial vascular fibrosis.

3. Use according to claim 1, characterized in that, The drug achieves the therapeutic purpose by inhibiting endothelial-mesenchymal transition and inhibiting pulmonary vascular remodeling.

4. The use according to claim 1, characterized in that, The drug achieves the therapeutic purpose by inhibiting endoplasmic reticulum stress.

5. Use according to claim 3, characterized in that, The inhibition of endothelial-mesenchymal transition is inhibition of α-SMA and / or SM22 expression.

6. Use according to claim 4, characterized in that, The inhibition of endoplasmic reticulum stress is inhibition of PERK and / or eIF2α expression.

7. Use according to claim 1, characterized in that, The concentration of 4-octyl itaconic acid in the drug is 0.1-20 μM.

8. The use according to claim 1, characterized in that, The drug further comprises a pharmaceutically acceptable carrier.

Images