Application of TGF-β1 / Snai1 pathway mediated endothelial mesenchymal transition in treatment of choroidal neovascularization

Abstract

The application provides an application of TGF-beta 1 / Snai1 pathway-mediated endothelial mesenchymal transformation in treating choroidal neovascularization, and belongs to the technical field of biological medicine. The application first takes the inhibition of TGF-beta 1 / Snai1 pathway-mediated endothelial mesenchymal transformation as an effective strategy for intervening choroidal neovascularization fibrosis and vascular remodeling, provides corresponding drug use, a cell model and a screening method, the model established by the application can truly simulate the pathological microenvironment of choroidal neovascularization, provides a reliable tool for screening of anti-fibrosis and anti-vascular remodeling drugs, and further provides a drug screening method which is simple in operation, clear in result and can efficiently identify candidate substances with endothelial mesenchymal transformation inhibition activity. The application not only opens up a new direction for the treatment of choroidal neovascularization, but also provides a key experimental platform for related drug research and development, and has good clinical application prospect and industrial value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of TGF-β1 / Snai1 pathway-mediated endothelial-mesenchymal transition in the treatment of choroidal neovascularization. Background Technology

[0002] Choroidal neovascularization is a major cause of blindness in various eye diseases, including age-related macular degeneration and pathological myopia. Its pathological characteristics include abnormal vascular proliferation in the choroidal capillary layer, accompanied by vascular leakage and fibrous scarring. Currently, first-line clinical treatment involves anti-vascular endothelial growth factor (VEGF) drugs, which inhibit neovascularization and slow disease progression. However, this type of treatment has drawbacks, including poor response in some patients, the need for frequent injections, and a lack of effective interventions for existing fibrotic lesions.

[0003] In existing research models, cell culture systems used to simulate choroidal neovascularization are mostly under conventional conditions stimulated by a single factor, which cannot accurately reflect the complex microenvironment resulting from the interplay of multiple pathological factors such as hypoxia and oxidative stress in disease states. Meanwhile, existing drug screening systems primarily focus on the inhibitory effect on neovascularization, and the evaluation indicators for fibrosis and vascular remodeling are still incomplete. Therefore, establishing cell models capable of simulating the pathological microenvironment of choroidal neovascularization and developing drug screening methods targeting fibrosis and vascular remodeling are urgent technical problems to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide the application of TGF-β1 / Snai1 pathway-mediated endothelial-mesenchymal transition in the treatment of choroidal neovascularization, which solves the technical problems of the lack of effective intervention methods for choroidal neovascularization fibrosis and vascular remodeling in the prior art, and the lack of cell models that can realistically simulate the pathological microenvironment of choroidal neovascularization for drug screening.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of an inhibitor in the preparation of a drug for inhibiting choroidal neovascularization, fibrosis, or vascular remodeling; The inhibitor is a substance that inhibits the endothelial-mesenchymal transition mediated by the TGF-β1 / Snai1 pathway in endothelial cells.

[0007] In this invention, the TGF-β1 / Snai1 pathway refers to the signal transduction pathway composed of transforming growth factor-β1 (TGF-β1) and its downstream transcription factor Snai1.

[0008] Preferably, the above-mentioned inhibitor is a TGF-β1 inhibitor, a Snai1 inhibitor, or a TGF-β1 receptor inhibitor.

[0009] In this invention, the TGF-β1 inhibitor refers to a substance that can inhibit the expression or activity of TGF-β1, the Snai1 inhibitor refers to a substance that can inhibit the expression or activity of Snai1, and the TGF-β1 receptor inhibitor refers to a substance that can block the binding of TGF-β1 to its receptor or inhibit downstream signal transduction of the receptor.

[0010] Preferably, the above-mentioned inhibition of endothelial-mesenchymal conversion inhibits the transformation of endothelial cells into mesenchymal cell phenotypes.

[0011] In this invention, the endothelial-mesenchymal transition refers to the biological process by which endothelial cells gradually lose their endothelial cell phenotype and acquire a mesenchymal cell phenotype under specific microenvironmental stimulation.

[0012] Preferably, the above-mentioned inhibition of endothelial-mesenchymal transition manifests as upregulation of endothelial marker expression and downregulation of mesenchymal marker expression.

[0013] In this invention, the endothelial markers refer to molecular markers specifically expressed in endothelial cells and used to identify endothelial cell phenotypes, and the mesenchymal markers refer to molecular markers specifically expressed in mesenchymal cells and used to identify mesenchymal cell phenotypes.

[0014] Preferably, the endothelial markers are selected from CD31 or VE-cadherin, and the mesenchymal markers are selected from α-SMA, FSP1, or NG2.

[0015] In this invention, CD31 refers to platelet-endothelial cell adhesion molecule-1, VE-cadherin refers to vascular endothelial cadherin, α-SMA refers to α-smooth muscle actin, FSP1 refers to fibroblast-specific protein-1, and NG2 refers to glial antigen 2.

[0016] This invention also provides the application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization fibrosis or vascular remodeling. The hypoxia-induced RF / 6A model is obtained by a preparation method including the following steps: culturing RF / 6A cells under mixed gas conditions for 24-72 hours to obtain a hypoxia-induced RF / 6A cell model; the volume percentage of oxygen in the mixed gas is 0.5%-5%, the volume percentage of carbon dioxide is 3%-8%, and the balance is nitrogen, and the sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

[0017] In this invention, the hypoxia-induced RF / 6A model refers to a cell model that induces endothelial-mesenchymal transition in monkey choroidal retinal endothelial cells by simulating the local hypoxic microenvironment of choroidal neovascularization through reducing oxygen concentration.

[0018] The present invention also provides an application of an oxidative stress-induced human umbilical vein endothelial cell (Huvecs) model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization fibrosis or vascular remodeling. The oxidative stress-induced Huvecs model is obtained by a preparation method including the following steps: treating Huvecs cells in a culture medium containing tert-butyl hydroperoxide for 12-48 hours to obtain an oxidative stress-induced Huvecs cell model; the concentration of the tert-butyl hydroperoxide is 100-200 μM.

[0019] In this invention, the oxidative stress-induced Huvecs model refers to a cell model that induces endothelial-mesenchymal transition in human umbilical vein endothelial cells by oxidative damage induced by tert-butyl hydroperoxide, simulating the local oxidative stress microenvironment of choroidal neovascularization.

[0020] This invention also provides the application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of a model for screening substances that inhibit endothelial-mesenchymal transition. The hypoxia-induced RF / 6A model is obtained by a preparation method including the following steps: culturing RF / 6A cells under mixed gas conditions for 24-72 hours to obtain a hypoxia-induced RF / 6A cell model; the volume percentage of oxygen in the mixed gas is 0.5%-5%, the volume percentage of carbon dioxide is 3%-8%, and the balance is nitrogen, and the sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

[0021] In this invention, the hypoxia-induced RF / 6A model refers to a cell model that induces endothelial-mesenchymal transition in monkey choroidal retinal endothelial cells by simulating the local hypoxic microenvironment of choroidal neovascularization through reducing oxygen concentration.

[0022] The present invention also provides an application of an oxidative stress-induced human umbilical vein endothelial cell (Huvecs) model in the preparation of a model for screening substances that inhibit endothelial-mesenchymal transition. The oxidative stress-induced Huvecs model is obtained by a preparation method including the following steps: treating Huvecs cells in a culture medium containing tert-butyl hydroperoxide for 12-48 hours to obtain an oxidative stress-induced Huvecs cell model; the concentration of the tert-butyl hydroperoxide is 100-200 μM.

[0023] In this invention, the oxidative stress-induced Huvecs model refers to a cell model that induces endothelial-mesenchymal transition in human umbilical vein endothelial cells by oxidative damage induced by tert-butyl hydroperoxide, simulating the local oxidative stress microenvironment of choroidal neovascularization.

[0024] This invention also provides a method for screening candidate drugs for inhibiting choroidal neovascularization fibrosis or vascular remodeling, comprising the following steps: contacting the candidate drug with a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model or an oxidative stress-induced human umbilical vein endothelial cell Huvecs model, detecting the expression levels of endothelial markers and mesenchymal markers in the cells; if the candidate drug can upregulate endothelial markers and downregulate mesenchymal markers, then the candidate drug is determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling; the endothelial markers are selected from CD31 or VE-cadherin, and the mesenchymal markers are selected from α-SMA, FSP1, or NG2; the expression level of Snai1 is also detected in the above detection steps; if the candidate drug can downregulate the expression of Snai1, then the candidate drug is determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling.

[0025] In this invention, Snai1 refers to the zinc finger transcription factor Snai1.

[0026] The beneficial effects of this invention are: This invention, for the first time, utilizes inhibition of TGF-β1 / Snai1 pathway-mediated endothelial-mesenchymal transition (EMT) as an effective strategy for intervening in choroidal neovascularization fibrosis and vascular remodeling, providing corresponding drug applications, cell models, and screening methods. The established hypoxia-induced RF / 6A model and oxidative stress-induced Huvecs model can realistically simulate the pathological microenvironment of choroidal neovascularization, providing reliable tools for screening anti-fibrotic and anti-vascular remodeling drugs. The drug screening method based on these models is simple to operate, yields clear results, and can efficiently identify candidate substances with inhibitory activity against EMT. This invention not only opens up new directions for the treatment of choroidal neovascularization but also provides a crucial experimental platform for related drug development, demonstrating promising clinical application prospects and industrial value. Attached Figure Description

[0027] Figure 1The figures show the comparison of the relative expression levels of CD31, VE-cadherin, NG2, FSP1, and α-SMA mRNA and protein in different groups of the hypoxic RF / 6A cell model. The PCR results are shown in the figures for CD31 (A), VE-cadherin (B), NG2 (C), FSP1 (D), and α-SMA (E); the Western Blot results are shown in the figures for protein electrophoresis (G), CD31 (H), VE-cadherin (I), NG2 (J), FSP1 (K), and α-SMA (L). p<0.0001; Figure 2 The images show the dynamic changes of EndoMT-related molecular markers in TGF-β1-induced hypoxic RF / 6A cells. (AE) Morphological images of RF / 6A cells treated with different concentrations of TGF-β1 (phase contrast microscopy); (F) Results of CCK-8 assay for the optimal intervention concentration of TGF-β1; (G) Electrophoresis images of protein expression in each group; (H) Western blot results of CD31 protein expression; (I) Western blot results of VE-cadherin protein expression; (J) Western blot results of NG2 protein expression; (K) Western blot results of FSP1 protein expression; and (L) Western blot results of α-SMA protein expression. p<0.05, p<0.01, p<0.001; Figure 3The figures show the changes in Snail expression and EndoMT-related proteins in RF / 6A cells after hypoxia and TGF-β1 intervention. (A) Western blot results of CD31 protein expression after Snail overexpression; (B) Western blot results of VE-cadherin protein expression after Snail overexpression; (C) Western blot results of NG2 protein expression after Snail overexpression; (D) Western blot results of FSP1 protein expression after Snail overexpression; (E) Western blot results of α-SMA protein expression after Snail overexpression. (F) Electrophoresis results of Snail protein expression in the hypoxia group (48 h) and Snail overexpression group; (G) Electrophoresis results of Snail protein expression after intervention with different concentrations of TGF-β1; (H) Western blot results of Snail protein expression with TGF-β1 concentration; (I) Electrophoresis results of Snail protein expression in the interference control group, interference Snail group, overexpression Snail group, and overexpression control group; (J) Western blot results of Snail protein expression in the interference Snail group and overexpression Snail group; p< 0.05, p< 0.01, p< 0.001, p<0.0001; Figure 4 The results show the dynamic changes of TGF-β1 / Snail signaling pathway regulating EndoMT, including: (L) representative images of CD31, VE-cadherin, and NG2 protein expression in 7 groups of cells detected by immunofluorescence staining (×200, scale bar=50 μm); (M) quantitative analysis of CD31 average fluorescence intensity; (N) quantitative analysis of VE-cadherin average fluorescence intensity; (O) quantitative analysis of NG2 average fluorescence intensity; (P) DAPI staining results of each group of cells; p< 0.05, p<0.0001; Figure 5 The graph shows the results of the Transwell assay for the migration ability of RF / 6A cells in each group. AG: Transwell migration results of 7 groups of cells (×100, scale bar=100μm); H: Quantitative analysis of the number of migrating cells in each group. p<0.0001; Figure 6 The image shows the morphological observation results of normal Huvecs cells. In the image, A: ×40, scale bar = 250μm; B: ×100, scale bar = 100μm. Figure 7 Figure 1 shows the results of establishing a model of oxidative damage and the effect of TBHP on Huvecs cell viability. AD: Effect of 0-1000 μM TBHP treatment on cell viability at 12 h, 24 h, 48 h, and 72 h; EF: Dose-response curves of TBHP-induced oxidative damage in the 12 h and 24 h treatment groups; p <0.05, p <0.01, p <0.001; Figure 8 The figure shows the effect of TBHP on ROS levels in Huvecs. Left: ROS fluorescence staining in the Control group and TBHP group (×200, scale bar=50μm); Right: Quantitative analysis of ROS fluorescence intensity. p<0.0001; Figure 9 The image shows the results of qPCR detection of mRNA expression levels of relevant genes in Huvecs from each group. A: Endothelial markers CD31 and VE-cadherin; B: Mesenchymal markers α-SMA, FSP1, and NG2; C: TGF-β1 and Snai1; p <0.05, p <0.01, p <0.001; Figure 10 The image shows the results of immunofluorescence staining detection of CD31 and α-SMA expression in Huvecs from different groups. Top: CD31, α-SMA, DAPI, and Merge plots for the Control and TBHP groups; Bottom: Quantitative analysis of average fluorescence intensity of CD31 and α-SMA; p < 0.05. 0.01, p<0.001; Figure 11The graph shows the screening results of TGF-β1 treatment concentration versus time. A: Cell viability after 24 hours of treatment with different concentrations of TGF-β1; B: Cell viability after 48 hours of treatment with different concentrations of TGF-β1; ns: no statistically significant difference; p: no statistically significant difference. <0.05, p <0.01, p <0.001; Figure 12 Figure 1 shows the results of Snai1 interference target screening and interference efficiency verification. A: qPCR detection of relative Snai1 mRNA expression levels in each group; B: Western blot detection of Snai1 protein expression in each group (left: electrophoresis image, right: quantitative analysis); ns: no statistically significant difference, p <0.05, p <0.01, p <0.001; Figure 13 The image shows the expression results of EndoMT-related gene mRNAs in Huvecs from each group detected by qPCR. A: CD31, VE-cadherin; B: α-SMA, FSP1, NG2; C: TGF-β1, Snai1; ns: no statistically significant difference, p <0.05, p <0.01, p <0.001; Figure 14 The image shows the results of Western blot analysis of EndoMT-related protein expression in Huvecs from each group. Left: Electrophoresis images of proteins from each group; Right: Quantitative analysis of CD31, VE-cadherin, α-SMA, FSP1, and NG2 proteins; ns: no statistically significant difference, p: no statistically significant difference. <0.05, p<0.001; Figure 15 The image shows the results of TUNEL staining for apoptosis in Huvecs of each group. Left: TUNEL, DAPI, and Merge plots for each group; Right: Quantitative analysis of TUNEL positivity rate for each group; ns: No statistically significant difference. p<0.001. Detailed Implementation

[0028] This invention provides the application of a substance that inhibits the TGF-β1 / Snai1 pathway in endothelial cell-mediated endothelial-mesenchymal transition in the preparation of a drug for inhibiting choroidal neovascularization, fibrosis, or vascular remodeling.

[0029] In this invention, the TGF-β1 / Snai1 pathway refers to the signal transduction pathway composed of transforming growth factor-β1 (TGF-β1) and its downstream transcription factor Snai1. TGF-β1 belongs to the transforming growth factor β superfamily and is a multifunctional cytokine involved in various biological processes such as cell proliferation, differentiation, migration, apoptosis, and extracellular matrix synthesis. Snai1, also known as Snail, is a member of the zinc finger transcription factor family and plays an important role in embryonic development, tumor metastasis, and fibrotic diseases. The TGF-β1 / Snai1 pathway is one of the core signaling pathways mediating endothelial-mesenchymal transition; activation of this pathway can lead to endothelial cell phenotype loss and mesenchymal phenotype acquisition.

[0030] Preferably, the above-mentioned substance is a TGF-β1 inhibitor, a Snai1 inhibitor, or a TGF-β1 receptor inhibitor.

[0031] In this invention, the TGF-β1 inhibitor refers to a substance capable of inhibiting the expression or activity of TGF-β1. TGF-β1 inhibitors include, but are not limited to, small molecule compounds, neutralizing antibodies, antisense oligonucleotides, siRNA, shRNA, miRNA, peptides, proteins, or any substance capable of blocking the binding of TGF-β1 to its receptor. TGF-β1 inhibitors are commercially available, such as common TGF-β1 receptor kinase inhibitors like SB-431542, LY364947, and A83-01, and can also be prepared in-house using existing technologies. The Snai1 inhibitor refers to a substance capable of inhibiting the expression or activity of Snai1. Snai1 inhibitors include, but are not limited to, small molecule compounds, neutralizing antibodies, antisense oligonucleotides, siRNA, shRNA, miRNA, peptides, proteins, or any substance capable of blocking Snai1 transcription or translation. The TGF-β1 receptor inhibitor refers to a substance capable of blocking the binding of TGF-β1 to its receptor or inhibiting downstream signal transduction of the receptor. TGF-β1 receptors mainly include TGF-β1 type receptor (TβRI) and TGF-β1 type receptor (TβRII). TGF-β1 receptor inhibitors can exert their inhibitory effects by blocking receptor phosphorylation or interfering with the binding of the receptor to the ligand.

[0032] Preferably, the above-mentioned inhibition of endothelial-mesenchymal conversion inhibits the transformation of endothelial cells into mesenchymal cell phenotypes.

[0033] In this invention, endothelial-mesenchymal transition (EMT) refers to the biological process by which endothelial cells gradually lose their endothelial cell phenotype and acquire a mesenchymal cell phenotype under specific microenvironmental stimulation. EMT is an important mechanism of vascular remodeling and fibrosis, and has been reported in various diseases such as atherosclerosis, pulmonary hypertension, renal fibrosis, and tumor microenvironment remodeling. During EMT, endothelial cells lose their typical endothelial markers while acquiring mesenchymal markers. The cell morphology changes from cobblestone or pebble-like to a long spindle-shaped, fibroblast-like morphology, with looser intercellular connections and enhanced migration ability.

[0034] Preferably, the above-mentioned inhibition of endothelial-mesenchymal transition manifests as upregulation of endothelial marker expression and downregulation of mesenchymal marker expression.

[0035] In this invention, endothelial markers refer to molecular markers specifically expressed in endothelial cells and used to identify endothelial cell phenotypes. Endothelial markers typically include CD31, VE-cadherin, von Willebrand factor (vWF), endothelin-1 (ET-1), and angiotensin-converting enzyme (ACE). Mesenchymal markers refer to molecular markers specifically expressed in mesenchymal cells and used to identify mesenchymal cell phenotypes. Mesenchymal markers typically include α-SMA, FSP1, NG2, vimentin, fibronectin, and type I collagen. By detecting changes in the expression levels of these markers, it is possible to determine whether endothelial-mesenchymal transition has occurred in endothelial cells and the degree of this transition.

[0036] Preferably, the endothelial markers are selected from CD31 or VE-cadherin, and the mesenchymal markers are selected from α-SMA, FSP1, or NG2.

[0037] In this invention, CD31 refers to platelet-endothelial cell adhesion molecule-1, also known as PECAM-1, a transmembrane glycoprotein with a molecular weight of 130 kDa, belonging to the immunoglobulin superfamily. CD31 is mainly expressed on the surface of vascular endothelial cells, platelets, and certain immune cells, participating in processes such as intercellular adhesion, angiogenesis, and leukocyte migration. VE-cadherin refers to vascular endothelial cadherin, also known as CD144, a calcium-dependent transmembrane adhesion molecule specifically expressed on endothelial cells, participating in maintaining intercellular connections and vascular permeability. α-SMA refers to α-smooth muscle actin, a member of the actin family, mainly expressed on smooth muscle cells, pericytes, and myofibroblasts, serving as a marker for mesenchymal cells and myofibroblasts. FSP1 refers to fibroblast-specific protein-1, also known as S100A4, a calcium-binding protein mainly expressed on fibroblasts and myofibroblasts, participating in cell migration and invasion. NG2 refers to glial antigen 2, also known as chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan expressed in pericytes, smooth muscle cells, and certain immature cells, serving as a marker for pericytes and vascular smooth muscle cells. The detection of these markers can be achieved through methods such as real-time quantitative PCR, Western blotting, immunofluorescence staining, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). The relevant detection reagents are commercially available.

[0038] This invention also provides the application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization fibrosis or vascular remodeling. The hypoxia-induced RF / 6A model is obtained by a preparation method including the following steps: culturing RF / 6A cells under mixed gas conditions for 24-72 hours to obtain a hypoxia-induced RF / 6A cell model; the volume percentage of oxygen in the mixed gas is 0.5%-5%, the volume percentage of carbon dioxide is 3%-8%, and the balance is nitrogen, and the sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

[0039] In this invention, the hypoxia-induced RF / 6A model refers to a cell model that induces endothelial-mesenchymal transition (EMT) in monkey choroidal retinal endothelial cells by simulating the local hypoxic microenvironment of choroidal neovascularization through reduced oxygen concentration. RF / 6A cells are endothelial cell lines derived from the monkey choroidal retina and are commonly used in research on ocular vascular diseases. Under hypoxic conditions, the cells can simulate the local ischemic and hypoxic state of choroidal neovascularization. The volume percentage of oxygen in the mixed gas is preferably 0.8%~3%, more preferably 1%~3%, and most preferably 1%. The volume percentage of carbon dioxide is preferably 4%~6%, more preferably 5%. The culture time is preferably 36~60 hours, more preferably 48 hours. Hypoxic culture can be performed in a three-gas incubator, which can precisely control the ratio of oxygen, carbon dioxide, and nitrogen. This model can simulate the endothelial-mesenchymal transition induced by the hypoxic microenvironment during choroidal neovascularization, providing a reliable tool for screening candidate drugs that inhibit vascular remodeling and fibrosis.

[0040] The present invention also provides an application of an oxidative stress-induced human umbilical vein endothelial cell (Huvecs) model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization fibrosis or vascular remodeling. The oxidative stress-induced Huvecs model is obtained by a preparation method including the following steps: treating Huvecs cells in a culture medium containing tert-butyl hydroperoxide for 12-48 hours to obtain an oxidative stress-induced Huvecs cell model; the concentration of the tert-butyl hydroperoxide is 100-200 μM.

[0041] In this invention, the oxidative stress-induced Huvecs model refers to a cell model that induces oxidative damage through tert-butyl hydroperoxide, simulating the local oxidative stress microenvironment of choroidal neovascularization and inducing endothelial-mesenchymal transition in human umbilical vein endothelial cells. Huvecs cells are human umbilical vein endothelial cells and are a classic cell model for studying vascular endothelial function. Tert-butyl hydroperoxide is an organic peroxide that can induce an increase in intracellular reactive oxygen species (ROS) levels, simulating oxidative stress. The concentration of tert-butyl hydroperoxide is preferably 120-180 μM, more preferably 140-170 μM, and most preferably 161 μM. The treatment time is preferably 18-36 hours, more preferably 22-26 hours, and most preferably 24 hours. Oxidative stress is one of the important characteristics of the choroidal neovascularization microenvironment, and this model can simulate the oxidative stress-induced endothelial-mesenchymal transition process. The ROS level can be detected by the fluorescent probe DCFH-DA to verify whether the model has been successfully established. This model provides an effective experimental platform for screening drugs that combat oxidative stress-induced endothelial-mesenchymal transition.

[0042] This invention also provides the application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of a model for screening substances that inhibit endothelial-mesenchymal transition. The hypoxia-induced RF / 6A model is obtained by a preparation method including the following steps: culturing RF / 6A cells under mixed gas conditions for 24-72 hours to obtain a hypoxia-induced RF / 6A cell model; the volume percentage of oxygen in the mixed gas is 0.5%-5%, the volume percentage of carbon dioxide is 3%-8%, and the balance is nitrogen, and the sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

[0043] In this invention, the hypoxia-induced RF / 6A model refers to a cell model that induces endothelial-mesenchymal transition (EMT) in monkey choroidal retinal endothelial cells by simulating the local hypoxic microenvironment of choroidal neovascularization through reduced oxygen concentration. The specific construction method of this model is as described above. This model can be used to screen for substances that can inhibit hypoxia-induced EMT, including but not limited to small molecule compounds, natural product extracts, biomacromolecules, and nucleic acid drugs. During screening, the test substance is co-cultured with hypoxia-induced RF / 6A cells, and the expression changes of endothelial and mesenchymal markers are detected. If the test substance can upregulate endothelial markers and downregulate mesenchymal markers, it indicates that the substance has inhibitory activity against EMT. This model provides an efficient screening tool for discovering candidate drugs for anti-fibrosis and anti-vascular remodeling.

[0044] The present invention also provides an application of an oxidative stress-induced human umbilical vein endothelial cell (Huvecs) model in the preparation of a model for screening substances that inhibit endothelial-mesenchymal transition. The oxidative stress-induced Huvecs model is obtained by a preparation method including the following steps: treating Huvecs cells in a culture medium containing tert-butyl hydroperoxide for 12-48 hours to obtain an oxidative stress-induced Huvecs cell model; the concentration of the tert-butyl hydroperoxide is 100-200 μM.

[0045] In this invention, the oxidative stress-induced Huvecs model refers to a cell model that induces endothelial-mesenchymal transition (EMT) in human umbilical vein endothelial cells by oxidative damage induced by tert-butyl hydroperoxide, simulating the local oxidative stress microenvironment of choroidal neovascularization. The specific construction method of this model is as described above. This model can be used to screen for substances that can inhibit oxidative stress-induced EMT. The screening method is the same as described above. This model complements the hypoxia-induced RF / 6A model, respectively simulating two core characteristics of the choroidal neovascularization microenvironment, allowing for a comprehensive evaluation of the inhibitory effects of test substances under different pathological microenvironments. The combined use of the two models improves the reliability and universality of the screening results.

[0046] This invention also provides a method for screening candidate drugs for inhibiting choroidal neovascularization fibrosis or vascular remodeling, comprising the following steps: contacting the candidate drug with a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model or an oxidative stress-induced human umbilical vein endothelial cell Huvecs model, detecting the expression levels of endothelial markers and mesenchymal markers in the cells; if the candidate drug can upregulate endothelial markers and downregulate mesenchymal markers, then the candidate drug is determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling; the endothelial markers are selected from CD31 or VE-cadherin, and the mesenchymal markers are selected from α-SMA, FSP1, or NG2; the expression level of Snai1 is also detected in the above detection steps; if the candidate drug can downregulate the expression of Snai1, then the candidate drug is determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling.

[0047] In this invention, Snai1 refers to the zinc finger transcription factor Snai1. Snai1 is a member of the Snail family of zinc finger transcription factors, located downstream of the TGF-β1 signaling pathway, and plays a core regulatory role in endothelial-mesenchymal transition (EMT). Snai1 inhibits the transcription of endothelial markers such as CD31 and VE-cadherin by binding to the E-box sequence of the promoter region of target genes, while simultaneously activating the expression of mesenchymal markers such as α-SMA, FSP1, and NG2. Therefore, the expression level of Snai1 can serve as an important indicator of endothelial-mesenchymal transition activity. Methods for detecting Snai1 expression levels include real-time quantitative PCR, Western blotting, immunofluorescence staining, and immunohistochemistry. In the screening methods, the contact time between the candidate drug and the cell model can be adjusted according to the drug properties and experimental objectives, typically ranging from 24 to 72 hours. The detection time point can be selected at 24, 48, or 72 hours after drug treatment. The markers can be detected at the mRNA and / or protein levels. Candidate drugs screened using this method can be further validated in animal models to assess their therapeutic effects on choroidal neovascularization. This method provides an efficient and reliable screening strategy for drug development aimed at inhibiting choroidal neovascularization fibrosis and vascular remodeling.

[0048] In this invention, EndoMT refers to endothelial-mesenchymal transition, which is the biological process by which endothelial cells gradually lose their endothelial cell phenotype and acquire a mesenchymal cell phenotype under specific microenvironmental stimulation.

[0049] In this invention, TBHP refers to tert-butyl hydroperoxide, a commonly used oxidative stress inducer used to establish cellular oxidative damage models.

[0050] In this invention, DCFH-DA refers to 2',7'-dichlorodihydrofluorescein diacetate, a reactive oxygen species fluorescent probe. After entering the cell, it is hydrolyzed by esterase to DCFH, and then oxidized to fluorescent DCF in the presence of reactive oxygen species, which is used to detect intracellular reactive oxygen species levels.

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example This study focuses on the occurrence and regulation of endothelial-mesenchymal transition (EndoMT) in choroidal neovascularization (CNV). Using in vitro cell models, the regulatory role of the TGF-β1 / Snai1 signaling pathway on EndoMT under hypoxic and oxidative stress microenvironments was investigated, verifying the crucial role of EndoMT in CNV vascular remodeling and providing experimental evidence for the core hypothesis of this research. The study was divided into two parts, using monkey choroidal / retinal endothelial cells (RF / 6A) and human umbilical vein endothelial cells (Huvecs) as research subjects, respectively, simulating the local hypoxic and oxidative stress microenvironment of CNV. The TGF-β1 / Snai1 pathway-mediated EndoMT process was verified from multiple dimensions, including cell phenotype, molecular markers, signaling pathways, and cell function.

[0053] I. Monkey choroid / retinal endothelial cells Part One Experimental methods: 1. RF / 6A cell resuscitation, medium change, passage, and cryopreservation Preparation: Disinfect the laminar flow hood with alcohol swabs beforehand, prepare the necessary consumables, and irradiate the laminar flow hood with UV light for 30 minutes; prepare and preheat the necessary reagents. Thawing: Turn on the water bath beforehand, prepare a PE glove, turn on the centrifuge, remove RF / 6A cells from the liquid nitrogen tank and place them in the PE glove (to avoid contamination), thaw in a 37°C water bath, aspirate 1 mL of complete culture medium (containing 10% serum and 1% penicillin antibiotics), place it in a cryovial and mix well, transfer the cell suspension to a 5 mL centrifuge tube, balance, and centrifuge (1000 rpm / 5 min); aspirate 4 mL of complete culture medium into a T25 cell culture flask and label with the date, name, and cell name; remove the centrifuged cells, aspirate the supernatant with a pipette, add 1 mL of complete culture medium to resuspend the cells, add to the T25 culture flask, mix well, and shake in a figure-eight or cross pattern; observe cell morphology under an inverted microscope and transfer to a cell culture incubator. Medium Change: Remove the T25 cell culture flask from the cell culture incubator and observe the cell status under a microscope (if there are many floating cells or the culture medium color changes, the medium needs to be changed). After aspirating the old culture medium from the culture flask, wash twice with 3 mL of PBS, add 5 mL of complete culture medium, and place it in the cell culture incubator. Passaging: Remove the T25 culture flask from the incubator and observe the cell confluence under a microscope. When it reaches 80%-90%, aspirate the old culture medium, wash twice with 3 mL of PBS, add 1 mL of trypsin to the culture flask, shake the flask, and place it in the incubator for 2-3 minutes. During this time, observe the cell morphology under a microscope. Once the cells become round, immediately add 2 mL of complete culture medium to stop digestion and mix well. Transfer to a centrifuge tube and centrifuge (1000 rpm / 5 min). Then, add 4 mL of complete culture medium to a new T25 culture flask and label it. After centrifugation, discard the supernatant, add 2 mL of complete culture medium to resuspend the cells, and transfer them to two separate T25 culture flasks. Mix well and shake in a figure-eight or cross-shaped motion. Observe the cell morphology under a microscope and transfer them to the cell culture incubator. Cryopreservation: Prepare cell cryopreservation solution in advance (55% basal medium + 40% serum + 5% DMSO) and pre-cool it in a 4°C freezer; remove the T25 culture flask from the incubator and observe under a microscope that the cell confluence reaches 80%-90%. Aspirate the old culture medium and wash twice with 3mL PBS. Add 1mL trypsin to the culture flask, shake the flask, and place it in the incubator for 2-3 minutes. During this time, observe the cell morphology under a microscope. Once the cells become round, immediately add 2mL of complete culture medium to stop digestion and mix well. Transfer to a centrifuge tube and centrifuge (1000rpm / 5min). Label the cryopreservation tube (date, name, cell name, and passage number). Remove the centrifuge tube, discard the supernatant, add 1mL of pre-cooled cryopreservation solution, mix well, and transfer to a cryopreservation tube. Then place it in a -80°C freezer. After 2-3 days, transfer it to a liquid nitrogen tank.

[0054] 2. Establishment and grouping of RF / 6A cell hypoxia model Logarithmically growing RF / 6A cells were collected and divided into control and hypoxia groups (hypoxia for 6 h, 12 h, 24 h, and 48 h). Control group cells were cultured in a CO2 incubator (37℃, 5% CO2) for 24 h, while hypoxia group cells were cultured in a CO2 incubator for 24 h, followed by hypoxia culture in a tri-gas incubator (37℃, 3% O2, 5% CO2, 92% N2) for 6 h, 12 h, 24 h, and 48 h, respectively. The expression of CD31, VE-cadherin, NG2, FSP1, and α-SMAM RNA was detected by real-time quantitative PCR (qRT-PCR), and the expression of CD31, VE-cadherin, NG2, FSP1, and α-SMA proteins was detected by Western blotting.

[0055] 3. qPCR was used to detect the expression of CD31, VE-cadherin, NG2, FSP1, and α-SMAm RNA in cells of each group. Primer design: All primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the primer concentration was diluted to 10 μM before being dispensed.

[0056] Total RNA extraction: ① Lysis: P8 generation RF / 6A cells in logarithmic growth phase were used, and the cell density was adjusted to 2.5 × 10⁻⁶ cells. 6 / mL, seeded into six-well plates. After treatment with appropriate conditions, each group of cells was washed twice with PBS, and 350μL / well Buffer RLS + 50×DTT (50:1) cell lysis buffer was added. The cells were incubated at room temperature for 5 min to lyse the cells. The cells were repeatedly pipetted to detach them and transferred to 1.5mL centrifuge tubes. The cells were vortexed at high speed to mix. ② Purification: An equal volume of 70% ethanol was added to the cell lysis buffer and pipetted to mix. The mixture was transferred to a Universal RNA Minicolumn and centrifuged at 12,000 rpm for 1 min. The filtrate was discarded. 600μL Buffer RLS was added to the Universal RNA Minicolumn and centrifuged at 12,000 rpm for 1 min. The filtrate was discarded. 650μL of Buffer RLS was added to the Universal RNA Minicolumn and centrifuged at 12,000 rpm for 1 min. The filtrate was discarded. Centrifuge at 12,000 rpm for 1 min with Buffer RWB and discard the filtrate; place the Universal RNA Minicolumn adsorption column on a new 2.0 mL Collection Tube and centrifuge at 12,000 rpm for 2 min; place the Universal RNA Minicolumn adsorption column on a new RNase Free Tube, add 50 μL LEPC water to the center of the membrane, incubate at room temperature for 5 min, then centrifuge at 12,000 rpm for 2 min to elute RNA and collect the product; ③ Determine the total RNA concentration and purity: use Nanodrop2000 software to detect RNA concentration and purity; Genomic DNA removal and reverse transcription: prepare the reaction solution on ice, using a standard technical system; Quantitative PCR: prepare the reaction solution on ice, using a standard reaction system and procedure.

[0057] Data results use 2- CT methods are used to process data.

[0058] 4. Western blot analysis was performed to detect the expression of CD31, VE-cadherin, NG2, FSP1, and α-SMA proteins in cells of each group. Total protein extraction: ① Lysis: P7 generation RF / 6A cells in logarithmic growth phase were taken, and the cell density was adjusted to 2.5×106 / mL. They were seeded in six-well plates. After each group of cells was treated with the appropriate conditions, they were washed twice with PBS, and 150μL / well of RIPA+PMSF (100:1) cell lysis buffer was added. The cells were lysed on ice for 30 min. After lysis, the lysate was collected using a cell scraper. ② Centrifugation: The collected lysate was transferred to a 1.5mL centrifuge tube and centrifuged at 12000rpm for 15 min at 4℃. The supernatant was collected.

[0059] Protein Concentration Assay: ① Preparation of BCA Working Solution: Protein concentration was determined using a BCA protein concentration assay kit. The solution was prepared according to the sample quantity at a ratio of BCA:Cu = 50:1. After thorough mixing, it was placed on ice for later use. ② Dilution of Standards: 10 μL of BCA standard was diluted to 100 μL with PBS to achieve a final concentration of 0.5 mg / mL. Standards were added to the protein standard in 96-well plates at concentrations of 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, and 20 μL, respectively. Each well was then supplemented with PBS to a final volume of 20 μL. ③ Sample Dilution Gradient: Samples were diluted 4-fold, 8-fold, and 10-fold. For the 4-fold dilution, 5 μL of sample and 15 μL of PBS were added to each well, bringing the total volume to 20 μL. Three replicates were made per group. 200 μL of BCA working solution was added to each well. For the 8-fold dilution, the solution was diluted 8-fold. ④ For ELISA reader detection: Take 2.5 μL of sample and 17.5 μL of PBS respectively, with a total volume of 20 μL, and add them to the wells of a 96-well plate. Make 3 replicates per group. Add 200 μL of BCA working solution to each well. For ELISA reader detection: Place the 96-well plate in a 37℃ incubator and incubate for 40 min. Then use an ELISA reader to detect the OD value of each well at a wavelength of 562 nm and plot a standard curve to calculate the protein concentration of the sample. ⑤ Protein denaturation: Add SDS-PAGE Loading Buffer (5×) at a ratio of sample:loading buffer = 4:1, boil at 100℃ for 5 min, cool to room temperature, seal with sealing film, and store at -80℃.

[0060] Gel preparation and sample loading: ① Cleaning glass plates: After cleaning and drying the glass plates, place the 1mm thick glass plate and the thin glass plate together in the groove and place them on the gel casting rack. Clamp the gel casting rack to fix the glass plates. ② Separating gel: Prepare 10% separating gel according to the kit instructions. Mix quickly and gently add it to the glass plate interlayer, avoiding the formation of air bubbles. When it is about 2cm from the top of the glass plate, add deionized water to fill it. ③ Stacking gel: After the separating gel has solidified at room temperature for 30 minutes, gently tilt the gel casting rack to remove the deionized water. Blot up any remaining moisture on the glass plate with absorbent paper. Prepare 5% stacking gel according to the instructions. Mix quickly and add it to the glass plate interlayer. Immediately insert the comb and let it solidify at room temperature for 30 minutes.

[0061] Electrophoresis and Transfer: ① Electrophoresis Buffer Preparation: Prepare the electrophoresis buffer in advance according to the instructions; ② Sample Loading: Clamp the gel plate (with comb) to the electrophoresis tank clamp, fill with electrophoresis buffer, and check for leaks. After confirming there are no leaks, load samples according to the experimental group order. Add at least 30 μg of sample protein and 5 μL of pre-stained marker to each well, and transfer to the electrophoresis tank; ③ Electrophoresis: Set the stacking gel voltage to 80V. After the pre-stained markers separate and the stacking gel is fully emulsified, set the voltage to 120V and wait for the bromine... After the phenol blue reaches the bottom of the separating gel, the membrane transfer begins; ④ Transfer: Prepare the transfer solution in advance according to the instructions. Cut the PVDF membrane to the appropriate size and activate it with methanol for 2 minutes before use. Pour the prepared transfer solution into the tray and soak the filter paper and sponge pad in it. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp in the order from negative electrode to positive electrode. Place the transfer clamp vertically into the transfer tank according to the positive and negative electrodes. Transfer conditions: 300mA constant current, 30 minutes.

[0062] Immunological reaction: ① Blocking: Prepare 5% skim milk (50mL: 5g) in advance, and place the PVDF membrane in 5% skim milk for 1.5h; ② Washing: After blocking, discard the blocking solution and wash 3 times with 1×TBST for 10min each time; ③ Incubation with primary antibody: Prepare protein antibody in advance according to the antibody instructions, and add β-actin (1:1000), CD31 (1:1000), VE-cadherin (1:1000), NG2 (1:800), FSP1 (1:50) 0) α-SMA (1:2000), GAPDH (1:10000) primary antibody, incubate overnight on a shaker at 4℃; ③ Wash membrane: recover the primary antibody the next day, wash 3 times with 1×TBST, 10 min each time; ④ Incubate secondary antibody: prepare secondary antibody in advance according to the antibody instructions, after washing the membrane, add HRP-labeled goat anti-rabbit secondary antibody (1:20000) and HRP-labeled goat anti-mouse secondary antibody (1:10000) and incubate at room temperature in the dark for 1 h; ⑤ Wash membrane: after incubation, wash the membrane 3 times with 1×TBST, 10 min each time.

[0063] Exposure and image analysis: ECLA and solution B were mixed in a 1:1 ratio. The eluted PVDF membrane was taken out, dried with absorbent paper, and then the mixed ECL luminescent solution was added. After reacting for 1 minute, the excess liquid was dried with absorbent paper, and the membrane was placed in a chemiluminescence analyzer. The membrane was exposed and photographed according to the preset program. GAPDH or β-actin was used as an internal reference, and the grayscale value was analyzed using ImageG software.

[0064] 5. Establish a TGF-β1 concentration screening mechanism. RF / 6A cells in good logarithmic growth phase were selected for counting, digested, and prepared into a cell suspension. The cells were then cultured at 5 × 10⁻⁶ cells / mL. 3Cells were evenly seeded at a density of 100 μL per well in a 96-well plate, with 5 replicates per group. Working solutions of TGF-β1 were prepared according to concentration gradients of 1, 2.5, 5, and 10 ng / ml. The old culture medium was discarded, and the prepared media containing different concentrations of TGF-β1 were added to the corresponding wells. A control group was set up, containing complete culture medium without TGF-β1 (i.e., 0 ng / ml). After culturing for 48 h, the culture medium was discarded, and 100 μL of complete culture medium containing 10% CCK-8 was added to each well for 2 h. The absorbance (A) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as (Experimental group A - Blank group A) / (Control group A - Blank group A) × 100%.

[0065] 6. Snail's overexpression and silence One day before transfection, cells were pre-placed in DMEM medium containing 15% FBS and seeded in 6-well plates, aiming for a cell density of 50%–60% the following day. 12 μL of prepared siRNA and pC-Snai1 were added to 250 μL of serum-free medium and mixed thoroughly. 5 μL of lipofectamine 2000 was dissolved in 250 μL of serum-free DMEM, mixed thoroughly, and incubated at room temperature for 5 min. siRNA and pC-Snai1 were gently mixed with diluted lipofectamine 2000 and incubated at room temperature for 20 min. After washing the cells twice, the mixture was added to the plate and mixed thoroughly. After 6 h of incubation, the medium was replaced with fresh medium. The cells were then incubated in a cell culture incubator for another 48 h. Negative and positive control sequences for siRNA and pC-Snai1 were used as controls. After 48 h, Western blot was used to detect the expression of the snail protein. Specific sequences are shown in Table 1 below. Table 1. siRNA fragment sequences

[0066] 7. TGF-β1 was co-transfected with siR-snail and overexpressing snail, and grouped into different groups. RF / 6A cells in logarithmic growth phase were collected and divided into four groups: hypoxia for 48 h, hypoxia for 48 h + TGF-β1, hypoxia for 48 h + Snail overexpression, hypoxia for 48 h + Snail knockdown, hypoxia for 48 h + TGF-β1 + Snail knockdown, hypoxia for 48 h + TGF-β1 + Snail overexpression, and hypoxia for 48 h + TGF-β1 + empty vector. Following the Lipofectamine 2000 instructions, transfection and treatment were performed on each group. Cells were then incubated at 37°C in a 5% CO2 incubator for 24 h, followed by incubation in a tri-gas incubator for 48 h, for subsequent experiments.

[0067] 8. Immunofluorescence assay was used to detect the expression of CD31, VE-cadherin, and NG2 proteins in cells of each group. Cells were washed with PBS to remove culture medium and fixed with 4% paraformaldehyde for 30 minutes. Washed three times with PBS, 5 minutes each time. Treated with 0.2% Triton X-100 for 5 minutes. Washed three times with PBS, 5 minutes each time. Incubated with 3% hydrogen peroxide in a humidified chamber at room temperature for 10 minutes. Washed three times with PBS, 5 minutes each time. Blocked with 3% BSA for 30 minutes. Incubated with primary antibody at 37°C for 2 hours. Washed three times with PBS, 5 minutes each time. Incubated with secondary antibody anti-rabbit-HRP in a humidified chamber at room temperature for 40 minutes. Washed three times with PBS, 5 minutes each time; incubated with Cy3Tyramide working solution at room temperature for 10 minutes. Washed three times with PBS, 5 minutes each time. Incubated with DAPI in a humidified chamber at room temperature in the dark for 5 minutes. Washed twice with PBS, 5 minutes each time. Mounted with anti-fluorescence quencher. Examined under a microscope and photographed.

[0068] 9. Transwell assay to detect cell migration ability in different intervention groups Cell plating: Collect cells from each group, digest with trypsin, and adjust the cell density to 2.5 × 10⁻⁶ cells / mL using serum-free medium. 4 Cells / mL were cultured in Transwell chambers. 200 μL of serum-free culture medium was added to the upper chamber, and 600 μL of complete culture medium containing 20% ​​FBS was added to the lower chamber. Cell fixation and staining: After 12-24 h of cell culture, the upper chamber was removed, cells were wiped off, and the cells were fixed with 4% paraformaldehyde for 15 min, then washed three times with PBS. The upper and lower chambers were then incubated with crystal violet in the dark for 30 min, followed by three rinses with PBS. Cells were observed, photographed, and counted under an inverted microscope. ImageJ image processing software was used to count the number of migrated cells in each group.

[0069] 10. Statistical Analysis Statistical analysis was performed using GraphPad Prism 10.0. Levene's test was used to test for homogeneity of variance among groups. One-way ANOVA was used for overall comparisons of each group and each indicator. Post-hoc multiple comparisons were performed. Tukey's test was used for homogeneity of variance, and Games-Howell's test was used for heterogeneity of variance. P < 0.05 was considered statistically significant.

[0070] II. Human umbilical vein endothelial cells Part One 1 Experimental Methods 1.1 Huvecs resuscitation, medium replacement, passage, and cryopreservation Preparation: Disinfect the laminar flow hood with alcohol swabs beforehand, prepare the necessary consumables, and irradiate the laminar flow hood with UV light for 30 minutes; prepare and preheat the necessary reagents. Thawing: Turn on the water bath beforehand, prepare a PE glove, turn on the centrifuge, remove Huvecs from the liquid nitrogen tank and place them in the PE glove (to avoid contamination), thaw in a 37°C water bath, aspirate 1 mL of complete culture medium (containing 10% serum and 1% penicillin antibiotics), place it in a cryovial and mix well, transfer the cell suspension to a 5 mL centrifuge tube, balance, and centrifuge (1000 rpm, 5 min); aspirate 4 mL of complete culture medium into a T25 cell culture flask and label with the date, name, and cell name; remove the centrifuged cells, aspirate the supernatant with a pipette, add 1 mL of complete culture medium to resuspend the cells, add to the T25 culture flask, mix well, and shake in a figure-eight or cross pattern; observe cell morphology under an inverted microscope and transfer to a cell culture incubator. Medium Change: Remove the T25 cell culture flask from the cell culture incubator and observe the cell status under a microscope (if there are many floating cells or the culture medium color changes, the medium needs to be changed). After aspirating the old culture medium from the culture flask, wash twice with 3 mL of PBS, add 5 mL of complete culture medium, and place it in the cell culture incubator. Passaging: Remove the T25 culture flask from the incubator and observe the cell confluence under a microscope to ensure it reaches 80%-90%. After aspirating the old culture medium, wash twice with 3 mL of PBS, add 1 mL of trypsin to the culture flask, shake the flask, and place it in the incubator for 2-3 minutes. During this time, observe the cell morphology under a microscope. Once the cells become round, immediately add 2 mL of complete culture medium to stop digestion and mix well. Transfer to a centrifuge tube and centrifuge (1000 rpm, 5 minutes). Then, add 4 mL of complete culture medium to a new T25 culture flask and label it. After centrifugation, discard the supernatant, add 2 mL of complete culture medium to resuspend the cells, and transfer them to two separate T25 culture flasks. Mix well and shake in a figure-eight or cross-shaped motion. Observe the cell morphology under a microscope and transfer the cells to the cell culture incubator. Cryopreservation: Prepare cell cryopreservation solution in advance (55% basal medium + 40% serum + 5% DMSO) and pre-cool at 4°C. Remove the T25 culture flask from the incubator and observe under a microscope that the cell confluence reaches 80%-90%. Aspirate the old culture medium and wash twice with 3 mL PBS. Add 1 mL trypsin to the culture flask, shake the flask, and place it in the incubator for 2-3 minutes. During this time, observe the cell morphology under a microscope. Once the cells become round, immediately add 2 mL of complete culture medium to stop digestion and mix well. Transfer to a centrifuge tube and centrifuge (1000 rpm, 5 minutes). Label the cryopreservation tube (date, name, cell name, and passage number). Remove the centrifuge tube, discard the supernatant, add 1 mL of pre-cooled cryopreservation solution, mix well, and transfer to a cryopreservation tube. Then place it in a -80°C freezer. After 2-3 days, transfer it to a liquid nitrogen tank.

[0071] 1.2 Microscopic observation of Huvecs cell morphology P3 generation Huvecs cells were removed from liquid nitrogen, thawed in a 37°C water bath, and cultured in ECM medium containing 10% fetal bovine serum. The cells were then cultured in a 37°C, 5% CO2 incubator. When the cell adhesion density reached 80%-90%, the morphology of Huvecs cells was observed under an inverted microscope, photographed, and used for subsequent experiments.

[0072] 1.3 Establishing a Huvecs oxidative stress model and grouping them. With 5×10 4 Huvecs were seeded at a density of cells / mL in 96-well plates. Different concentrations (10-1000 μM) of TBHP were used to treat Huvecs for different durations (12-72 h). Cell viability was measured, and suitable TBHP concentrations and treatment times were screened to establish an oxidative damage model. The experiment was divided into a Control group (untreated) and a TBHP group (treated with TBHP for 24 h).

[0073] 1.4 CCK-8 assay for cell viability Huvecs cells were digested with trypsin and then adjusted to a cell density of 5 × 10⁶ cells using complete culture medium. 4 Cells / mL, 100 μL of cell suspension was seeded in each well of a 96-well plate, and a blank control group was set up; Drug preparation: TBHP was diluted with DMSO to an initial concentration of 1M, and then diluted with complete culture medium to the required concentrations (10, 20, 50, 100, 200, 500 and 1000 μM); 100 μL of the above-prepared drug-containing culture medium was added to each well, with 4 replicates for each concentration, and a control group without drug and a blank culture medium group were set up. The culture plates were placed in a 37℃ incubator with 5% CO2 for 12, 24, 48 and 72 h respectively; At the detection time point, the culture plates were removed, the old culture medium was discarded, and the culture medium and CCK-8 reagent were prepared at a ratio of 10:1, mixed well, and 100 μL of CCK-8 culture medium was added to each well. The plates were incubated in a 37℃ incubator in the dark for 2 h, and the absorbance (A) value at 450 nm was measured using an ELISA reader. Cell viability (%) = (Experimental group A - Blank group A) / (Control group A - Blank group A) × 100%.

[0074] 1.5 ROS detection of reactive oxygen species levels in each group Huvecs cells were digested with trypsin and then adjusted to a cell density of 1×10⁶ cells in complete culture medium. 5Cells / mL were seeded into each well of a 12-well plate with 1 mL of cell suspension and incubated at 37°C in a 5% CO2 incubator for 24 h. Cells were then treated with 161 μM TBHP and incubated for 24 h. Positive control wells (pretreated with 50 μM positive control reagent for 2 h) and negative control wells (cells only, without working solution) were set up. The cell culture medium was removed, and the cells were washed once with serum-free cell culture medium. The DCFH-DA reagent (10 mM) was diluted to a concentration of 10 μM with complete culture medium, and 1 mL was added to each well. The cells were incubated at 37°C in the dark for 30 min. The working solution was removed, and the cells were washed 2-3 times with serum-free cell culture medium to thoroughly remove any DCFH-DA that had not entered the cells. The cells were observed under a fluorescence microscope and photographed at 200× magnification using an excitation wavelength of 488 nm and an emission wavelength of 525 nm to detect the fluorescence intensity before and after stimulation.

[0075] 1.6 qPCR was used to detect the expression of endothelial cell markers, endothelial-mesenchymal transition markers, TGF-β1, and Snai1 mRNA in cells of each group. Primer design: All primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer concentration was diluted to 10 μM before being dispensed.

[0076] RNA extraction: ① Before use, add 20 μL of β-mercaptoethanol (<5 × 10⁻⁶) to each 1 mL of TRK Lysis Buffer. 6 Cells: 350 μL TRK Lysis Buffer / β-mercaptoethanol mixture, <1 × 10⁻⁶ 7Cells: 700 μL TRK Lysis Buffer / β-mercaptoethanol mixture); ② Adherent cell culture: Discard the culture medium, add TRK Lysis Buffer to the culture flask, lyse the cells by pipetting, transfer the lysate to a homogenization column, insert the homogenization column into a 2 mL enzyme-free centrifuge tube, centrifuge at 14000×g for 2 min at room temperature to remove insoluble impurities, and collect the filtrate; ③ Add an equal volume of 70% ethanol to the filtrate and vortex to mix; ④ Insert the RNA binding column into the collection tube, transfer the mixture obtained in step ③ (each transfer ≤700 μL of mixture), centrifuge at 10000×g for 1 min at room temperature, and discard the filtrate; ⑤ Repeat step ④ until all the mixture is bound to the RNA binding column; ⑥ Insert the RNA binding column into the collection tube, add 500 μL RNA Wash Buffer I to the binding column, centrifuge at 10000×g for 30 s, and discard the filtrate; ⑦ Insert the RNA binding column back into the collection tube, add 500 μL RNA Wash Buffer I. Add the RNA to the binding column, centrifuge at 10000×g for 1 min, and discard the filtrate; ⑧ Repeat step ⑦; ⑨ Put the RNA binding column back into the collection tube, centrifuge at 10000×g for 2 min; ⑩ Put the RNA binding column into a new 1.5 mL enzyme-free centrifuge tube, add 30-70 μL of Nuclease-free Water to the binding column, centrifuge at 10000×g for 2 min to elute the RNA, and store the product at -80℃.

[0077] RNA purity detection and quantification: 1 μL of extracted RNA was taken, and the concentration (ng / μL) of the sample was determined using an ultra-micro spectrophotometer. An A260 / A280 ratio between 1.8 and 2.2 indicates good RNA quality.

[0078] cDNA Synthesis: ① RNA DNA Desorption: Prepare the RNA DNA desorption reaction mixture in 200 μL RNase-free microcentrifuge tubes on ice, mix well by pipetting, total volume 10 μL, incubate at 42°C for 2 min, then store at 4°C. ② cDNA Synthesis: Prepare the cDNA synthesis reaction mixture in 200 μL RNase-free microcentrifuge tubes on ice, mix well by pipetting, total volume 20 μL. ③ Perform the reaction using a gradient PCR instrument according to the standard procedure, and store at 4°C. qPCR: ① Prepare the PCR reaction mixture for cDNA products in RNase-free microcentrifuge tubes, 3 replicates per template, on ice. ② Perform the PCR reaction using a two-step procedure. 1.7 Immunofluorescence staining was used to detect changes in the levels of CD31 and α-SMA proteins in cells of each group.

[0079] Huvecs cells were digested with trypsin and then adjusted to a cell density of 1×10⁶ cells in complete culture medium.5 Cells / mL were seeded into 12-well plates with 1 mL of cell suspension per well and incubated at 37°C with 5% CO2 for 24 h. Cells were then treated with TBHP at a concentration of 161 μM and incubated for 24 h. At the detection time point, the culture medium was discarded, and each well was washed 3 times with 1 mL of PBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 1 h, followed by 3 washes with PBS. The cells were permeabilized with 0.5% Triton X-100 (prepared in PBS) at room temperature for 20 min, followed by 3 washes with PBS. Blocking was performed with 1% BSA at room temperature for 30 min, and the blocking solution was discarded. CD31 (1:1000) and α-SMA (1:200) primary antibodies diluted in PBS were added, and the plates were incubated overnight at 4°C in a humidified chamber, followed by 3 washes with PBS. Alexa Fluor488 fluorescently labeled goat anti-mouse secondary antibody (1:500) diluted in PBS was added, and the plates were incubated at room temperature for 2 h, followed by 3 washes with PBS. Alexa Fluor488 fluorescently labeled goat anti-mouse secondary antibody diluted in PBS was added. Fluor594-labeled goat anti-rabbit secondary antibody (1:500) was incubated at room temperature for 2 h, followed by 3 washes with PBS; DAPI dye was added and incubated in the dark for 5 min, followed by 3 washes with PBS; anti-fluorescence quenching mounting solution was added, and then images were observed and acquired under a fluorescence microscope.

[0080] Part Two 2.1 Huvecs resuscitation, medium replacement, passage, and cryopreservation Same as the above solution.

[0081] 2.2 Model Establishment and Cell Grouping Cells were cultured in ECM medium containing 10% fetal bovine serum, with the medium changed every other day. When cell confluence reached 80%-90%, cells were passaged, resuspended in serum-containing medium, and thoroughly mixed before cell counting. Cells were plated or treated accordingly based on subsequent experimental requirements. An oxidative damage model was established by treating cells with 161 μM TBHP for 24 hours. The experimental groups were as follows: Control group: untreated; TBHP group: TBHP treated for 4 h; TBHP+NC siRNA group: cells were transfected with siRNA for 48 h, then treated with TBHP for 24 h; TBHP+Snai1 siRNA group: cells were transfected with siRNA for 48 h, then treated with TBHP for 24 h; TBHP+TGF-β1 group: cells were pretreated with TGF-β1 for 24 h, then treated with TBHP for 24 h; TBHP+TGF-β1+Snai1 siRNA group: cells were transfected with siRNA for 48 h, then pretreated with TGF-β1 for 24 h, then treated with TBHP for 24 h.

[0082] 2.3 Screening of TGF-β1 concentration and treatment time Huvecs cells were digested with trypsin and then adjusted to a cell density of 5 × 10⁶ cells using complete culture medium. 4 Cells / mL were seeded into each well of a 96-well plate with 100 μL of cell suspension, and a blank control group was set up. Drug preparation: TGF-β1 was briefly centrifuged and diluted with 100 μL of sterile water to an initial concentration of 100 μg / mL, and then diluted with complete culture medium to the required concentrations (1, 2.5, 5, 10, 15, 20 and 30 ng / mL). 100 μL of the above-prepared drug-containing culture medium was added to each well, with 4 replicates for each concentration. A control group without drug and a blank culture medium group were set up. The culture plates were placed in a 37℃ incubator with 5% CO2 for 24 h and 48 h, respectively. At the detection time point, the culture plates were removed, the old culture medium was discarded, and the culture medium and CCK-8 reagent were prepared at a ratio of 10:1, mixed well, and 100 μL of CCK-8 culture medium was added to each well. The plates were incubated in a 37℃ incubator in the dark for 2 h, and the absorbance (A) value at 450 nm was measured using an ELISA reader. Cell viability (%) = (Experimental group A - Blank group A) / (Control group A - Blank group A) × 100%.

[0083] 2.4 qPCR screening of Snai1 interference targets and verification of interference efficiency Same as Part 1.

[0084] 2.5 Western blotting screening of Snai1 jamming targets and verification of jamming efficiency Preparation of main reagents: ① 10×TBS (pH 7.6): Components: 200 mM Tris base, 1.37 M NaCl, 2 mM KCl. Preparation method: Weigh 15.0 g Tris base, 40.0 g NaCl, 1.0 g KCl. Place the above reagents in a beaker, add 400 mL of deionized water, adjust the pH to 7.2-7.4 by adding concentrated HCl dropwise, and bring the volume to 500 mL with deionized water. Store at room temperature. Before use, dilute 10 times with deionized water and add 0.1% Tween-20. ② 10× electrophoresis buffer: Components: 0.125 M Tris base, 1.25 M Glycine, 0.5% (w / v) SDS. Preparation method: 30.2 g Tris base, 188 g Glycine, 10 g SDS. Weigh the above reagents and place them in a beaker. Add 800 mL of deionized water, stir thoroughly to dissolve, and bring the volume to 1000 mL with deionized water. Store at room temperature. Dilute 10 times with deionized water before use. ③ 10× Transfer Buffer: Components: 25 mM Tris base, 200 mM Glycine, 3 mM SDS, 20% methanol. Preparation: 30 g Tris base, 144 g Glycine, 3.7 g SDS. Weigh the above reagents and place them in a beaker. Add 800 mL of deionized water, stir thoroughly to dissolve, and bring the volume to 1000 mL with deionized water. Store at room temperature. Dilute 10 times with deionized water before use and add 20% methanol. ④ 5% Skim Milk Blocking Solution: Preparation: Weigh 5 g skim milk powder and place it in a 100 mL beaker. Add TBST to dissolve, then bring the volume to 100 mL. Store at 4℃.

[0085] Protein extraction: Take centrifuged cells or 0.2g of tissue, transfer to a centrifuge tube, add 200µL of RIPA tissue lysis buffer (containing PMSF, final concentration of 1 mM), grind and lyse thoroughly on ice; centrifuge at 12000rpm for 15min at 4℃; transfer the supernatant to a new centrifuge tube.

[0086] Protein concentration determination: ① Preparation of working solution: Mix BCA Reagent and Cu Reagent at a ratio of 50:1. ② Preparation of standard solution: Add 20 µL of BSA standard (5 mg / mL) to the protein standard wells of a 96-well plate, add 30 µL of PBS, mix well, and then add 25 µL to the next well. Repeat this serial dilution process to achieve final concentrations of 2000, 1000, 500, 500, 250, 125, and 62.5 µg / mL. ③ Dilute the sample appropriately, adding 25 µL to the sample wells of a 96-well plate. Ensure the sample spot falls approximately halfway to the standard line. ④ Add 200 µL of BCA working solution to each well and incubate at 37℃ for 15-30 min. Measure the protein concentration at 570 nm using a microplate reader and calculate the protein concentration based on the standard curve. When using an incubator for incubation, care should be taken to prevent moisture evaporation from affecting the test results.

[0087] Protein denaturation: Mix 120 µL of protein sample with 30 µL of 5× loading buffer in a 200 µL centrifuge tube, boil at 98°C for 5 min on a PCR instrument, then immediately place on ice and incubate for 5 min. Repeat three times, and store at -80°C for later use.

[0088] Electrophoresis: ① Cleaning glass plates and beakers: Wash glass plates with laundry detergent and dry them for later use. ② Prepare separating gel of the corresponding concentration: Assemble the gel casting plate, pour in the separating gel, leaving a 2-3 cm gap from the top. Seal the liquid surface with anhydrous ethanol and let it stand for 30 minutes. ③ Prepare stacking gel: Pour off the ethanol from the gel casting plate, rinse repeatedly with distilled water, invert the gel casting plate to drain the water, and absorb the moisture with absorbent paper. Prepare a 5% stacking gel, pour it into the gel casting plate, and immediately insert the comb. ④ Sample loading: Take out the denatured protein sample at -80℃, centrifuge, and then load the sample. Centrifugation precipitates impurities in the sample. When loading, aspirate the upper layer of sample; otherwise, the protein bands will show abnormal phenomena such as dumbbell shape after exposure. ⑤ Electrophoresis: Assemble the electrophoresis apparatus, electrophoresis at 80 V for about 30 minutes. When the target protein reaches the separating gel, adjust the voltage to 120 V until the two marker bands above and below the target protein separate, then stop electrophoresis. If excessive heat is generated during electrophoresis, electrophoresis on ice is required.

[0089] Transfer: Place the gel after electrophoresis and dried filter paper in pre-chilled transfer buffer for a period of time. Cut a PVDF membrane to a certain size and soak it in anhydrous methanol for 15 seconds to activate the positively charged groups on the PVDF membrane. Transfer it to double-distilled water and soak it, then place it in pre-chilled transfer buffer to equilibrate. Place the membrane and filter paper in the middle of the semi-dry transfer apparatus in sequence. Use a glass rod to remove air bubbles. Cut off the upper left corner of the PVDF membrane from the side in contact with the gel to show the side with protein attachment. Assemble the transfer apparatus, add plenty of ice to the bottom of the apparatus, and transfer at a constant current of 0.2 A for 2 hours.

[0090] Protein denaturation: After transfer, place the PVDF membrane in 5% skim milk and incubate at room temperature for 1.5 hours. Alternatively, 5% BSA can be used, adjusting the ratio according to experimental results. Cut the PVDF membrane into strips according to the location of the target protein, and place each strip in the protein primary antibody dilution buffer, incubating overnight at 4°C with gentle shaking. Add GAPDH (1:1000) and Snai1 (1:1000). Wash the membrane with TBST at room temperature for 5×6 minutes with shaking. If the exposure background is high, the number of washes and the washing time can be increased. Add the appropriate secondary antibody according to the type of target protein and incubate at room temperature for 1 hour. Adjust the secondary antibody concentration (1:6000) according to specific experimental results. Wash the membrane with TBST at room temperature for 5×6 minutes with shaking. Place plastic wrap in a dark box, adding water to the bottom to fix the membrane, and place the PVDF membrane face up inside the box. Mix the two liquids in the chemiluminescence reagent kit at a 1:1 ratio and shake well. Add the solution dropwise onto the PVDF membrane and allow it to react for 5 minutes. Then, use tweezers to lift a corner of the PVDF membrane and allow the luminescent solution to flow down naturally. Expose and develop the solution for observation.

[0091] 2.6 qPCR detection of mRNA expression of endothelial cell markers and endothelial-mesenchymal transition markers in cells of each group Same as Part 1.

[0092] 2.7 Western blot analysis was performed to detect the expression of endothelial cell markers and endothelial-mesenchymal transition marker proteins in cells of each group. Protein denaturation: After transfer, place the PVDF membrane in 5% skim milk and incubate at room temperature for 1.5 hours. Alternatively, 5% BSA can be used, adjusting the ratio according to experimental results. Cut the PVDF membrane into strips according to the location of the target protein, and place each strip in the protein primary antibody dilution buffer. Incubate overnight at 4°C with gentle shaking. GAPDH (1:1000), P-AKT (1:1000), P-PI3K (1:1000). Wash the membrane with TBST at room temperature for 5×6 minutes with shaking. If the exposure background is high, the number of washes and the washing time can be increased. Add the corresponding secondary antibody according to the type of target protein and incubate at room temperature for 1 hour. The concentration of the secondary antibody can also be adjusted according to the specific experimental results. (1:6000). Wash the membrane with TBST at room temperature for 5×6 minutes with shaking. Place plastic wrap in a dark box, adding water to the bottom to fix the membrane. Place the PVDF membrane face up inside the box. Mix the two liquids in the chemiluminescence reagent kit at a 1:1 ratio and shake well. Add the solution dropwise onto the PVDF membrane and react for 5 minutes. Use tweezers to lift a corner of the PVDF membrane, allowing the luminescent solution to flow down naturally. Expose and develop the solution for observation. The rest is the same as in Part 2.5.

[0093] 2.7 TUNEL staining to detect apoptosis in each group Self-prepared reagents: 1×PBS, pH 7.2-7.6; neutral paraformaldehyde fixative (4% paraformaldehyde in PBS); permeation enhancer (0.5% Triton X-100 in PBS). Take cells in the logarithmic growth phase and seed them at an appropriate cell density in 12-well plates, then incubate at 37℃ for 24 h. Cell fixation and permeation enhancement procedures: ① Add 300 μL of fixative to each well and incubate at room temperature for 30 min, then discard the fixative; ② Add 300 μL of PBS washing buffer to each well and wash three times for 5 min each time, then discard the PBS; ③ Add 300 μL of permeation enhancer (0.5% Triton X-100 in PBS) to each well and incubate at room temperature for 20 min; ④ Wash cells three times with PBS for 5 min each time. Labeling: ① Add 300 μL of TdT Equilibration Buffer to each sample and equilibrate in a humidified chamber at 37°C for 30 min; ② Discard the TdT Equilibration Buffer, add 200 μL of labeling working solution to each well, and incubate in a humidified chamber at 37°C in the dark for 60 min; ③ Discard the labeling working solution and wash cells three times with PBS, 5 min each time; ④ Add DAPI working solution and incubate at room temperature in the dark for 5 min to counterstain cell nuclei; ⑤ Discard the DAPI working solution, wash cells four times with PBS, 5 min each time, and add 500 μL of PBS to each well. Image acquisition and analysis: Immediately after staining, observe and collect fluorescence signals under a fluorescence microscope and take pictures under 100x magnification.

[0094] The experimental results are as follows: Part 1: Experimental Results of TGF-β1 / Snai1 Pathway Regulation of EndoMT in RF / 6A Cells under Hypoxic Microenvironment 1. Effects of simulated hypoxic environment on the expression levels of CD31, VE-cadherin, NG2, FSP1 and α-SMA in RF / 6A.

[0095] 1.1 Expression levels of CD31, VE-cadherin, NG2, FSP1, and α-SMA in RF / 6A under hypoxic conditions: qPCR results: Compared with the control group, CD31 ( Figure 1 A) and VE-cadherin Figure 1 B) mRNA expression was highest at 6 h of hypoxia (both) p<0.0001), NG2 ( Figure 1 C), FSP1 ( Figure 1 D) and a-SMA ( Figure 1 E) mRNA expression was highest at 48 h of hypoxia (both) p<0.0001). Western blot results: Compared with the control group, CD31 ( Figure 1 H) and VE-cadherin Figure 1 I) Protein expression was highest at 12 h of hypoxia (both p<0.0001), NG2 ( Figure 1 J), FSP1 ( Figure 1 K) and a-SMA ( Figure 1 L) protein expression was highest at 48 h of hypoxia (both) (p<0.0001). The results indicated that with prolonged hypoxia, the expression of endothelial markers CD31 and VE-cadherin gradually decreased, while the expression of mesenchymal markers NG2, FSP1, and α-SMA gradually increased. RF / 6A cells under hypoxic conditions underwent EndoMT. Based on the above experimental results (mRNA and protein expression were significantly affected at 48 h), subsequent experiments used RF / 6A cells hypoxic for 48 h. The results are as follows... Figure 1 As shown.

[0096] 2. Effects of TGF-β1 on the dynamic changes of hypoxic RF / 6A cells and EndoMT-related proteins under a simulated 48h hypoxic environment.

[0097] 2.1 Cell morphological changes showed that after treatment with TGF-β1 (0, 1, 2.5, 5, and 10 ng / ml), RF / 6A cells exhibited a cobblestone phenotype and tended to bind tightly with increasing TGF-β1 concentration. The results are as follows... Figure 2 As shown in (AE).

[0098] 2.2 Protein Expression Detection: RF / 6A cells hypoxic for 48 h were treated with TGF-β1 (0, 1, 2.5, 5, and 10 ng / ml). Western blot was used to monitor the protein expression of CD31, VE-cadherin, NG2, FSP1, and α-SMA after TGF-β1 treatment. WB Results: Compared with the control groups at each concentration, CD31 (… Figure 2 H) Expression significantly decreased with increasing TGF-β1 concentration, and the difference was statistically significant. p<0.001), VE-cadherin ( Figure 2 I) Expression significantly decreased with increasing TGF-β1 concentration, and the difference was statistically significant. p<0.01), NG2 ( Figure 2 J) Expression significantly increased with increasing TGF-β1 concentration, and the difference was statistically significant. p<0.001), FSP1 ( Figure 2 K) expression significantly increased with increasing TGF-β1 concentration, and the difference was statistically significant. p<0.001), α-SMA ( Figure 2 L) expression significantly increased with increasing TGF-β1 concentration, and the difference was statistically significant. p<0.001). The results suggest that TGF-β1 induces EndoMT in hypoxic RF / 6A cells. (See results below.) Figure 2 As shown in (GL).

[0099] 2.3 CCK-8 assay results showed that the intervention effect of TGF-β1 on hypoxic RF / 6A cells was concentration-dependent. With increasing concentration, RF / 6A cells showed the highest expression at 10 ng / ml, indicating the strongest effect of TGF-β1 on hypoxic RF / 6A cells. p<0.0001). The results are as follows: Figure 2 As shown in (F).

[0100] 3. The effect of TGF-β1-Snai1 signaling pathway targeting EndoMT on CNV remodeling under simulated hypoxic conditions.

[0101] 3.1 Western blot analysis of protein expression: Snail protein expression was detected in hypoxic RF / 6A cells. Overexpression and knockdown vectors for Snail were constructed. Compared to the control group, the overexpression group showed the highest Snail expression. p<0.0001, the expression was lowest in the interference snail group ( p<0.01). The results showed that snail was expressed in hypoxic RF / 6A cells, and that hypoxia could induce snail expression. (See results below.) Figure 3 As shown in (IJ).

[0102] 3.2 Western blot analysis of protein expression: whether snail induces EndoMT. Compared with the control group, snail protein expression in CD31 was decreased ( p<0.001), decreased expression of snail protein in VE-cadherin, ( p<0.05), Snail protein expression was increased in NG2 (no statistically significant difference), and Snail protein expression was increased in FSP1 (p<0.05). p<0.01) and increased expression of snail protein in α-SMA ( p<0.05). The results show that snail promotes the occurrence of EndoMT. The results are as follows... Figure 3 As shown in (AF).

[0103] 3.3 Western Blot analysis of protein expression: Does TGF-β1 regulate snail protein expression? In RF / 6A cells treated with various concentrations of TGF-β1 (0, 1, 2.5, 5, and 10 ng / ml), snail expression significantly increased with increasing TGF-β1 concentration. (p<0.0001). The results showed that snail expression was upregulated with TGF-β1 dosage, indicating a positive promoting relationship between TGF-β1 and snail. Figure 3 As shown in (GH).

[0104] 3.4 Effects of different intervention groups on EndoMT mRNA expression: Compared with the control group and NC group, the expression of CD31 and VE-cadherin mRNA was highest in Snail after 48 h of hypoxia and knockdown (both... (p<0.0001) The expression of NG2, FSP1 and α-SMA mRNA was highest in the group hypoxic for 48 h + TGF-β1 + Snail overexpression group (all p<0.0001). (p<0.0001). The results suggest that the joint participation of overexpression of snail and TGF-β1 may jointly promote the occurrence of EndoMT.

[0105] 3.5 Effects of different intervention groups on EndoMT protein expression: Compared with the control group and NC group, the expression of CD31 and VE-cadherin proteins was highest in the Snail knockdown group after 48 hours of hypoxia. p<0.0001), the expression of NG2, FSP1 and α-SMA proteins was highest in the group hypoxic for 48 h + TGF-β1 + overexpression of snail (p<0.0001). (p<0.0001). CD31 and VE-cadherin protein expression were lower in the RF / 6A+TGF-β1+ knockdown snail group than in the NC group after 48 hours of hypoxia. These results suggest that intervention with overexpression of snail and TGF-β1 may jointly promote the occurrence of EndoMT.

[0106] 3.6 Effects of different intervention groups on EndoMT protein expression: Immunofluorescence results showed that CD31 and VE-cadherin protein expression was highest in the Snail knockdown group after 48 hours of hypoxia. p<0.05, NG2 protein expression was highest in the group hypoxic for 48 h + TGF-β1 overexpression snail (p<0.05). p<0.05). The results suggest that intervention with overexpression of snail and TGF-β1 may jointly promote the occurrence of EndoMT. Results are as follows... Figure 4 As shown in (LP).

[0107] 3.7 Effects of different intervention groups on cell function: Cell migration results showed that the group with hypoxia for 48 h + TGF-β1 + Snail overexpression had the strongest cell migration ability, and the differences were statistically significant. p<0.0001; while the RF / 6A+ knockdown Snail group under hypoxia for 48h showed the weakest migration ability, and the differences were statistically significant. p<0.05). The results suggest that TGF-β1 and snail can promote cell migration, and this dual mechanism of action jointly affects cell function. (Results are as follows...) Figure 5 As shown in (AH).

[0108] Part II: Experimental Results of TGF-β1 / Snai1 Pathway Regulation of EndoMT in Huvecs Cells under Oxidative Stress Microenvironment 1. Establish a TBHP-induced Huvec oxidative stress model Oxidative stress is another important characteristic of the local microenvironment of CNVs. To simulate the oxidative stress microenvironment of CNVs, an oxidative damage model was established by inducing Huvecs with tert-butyl hydroperoxide (TBHP). Cell viability after treatment with different concentrations of TBHP (0, 10, 20, 50, 100, 200, 500, 1000 μM) for 12 h, 24 h, 48 h, and 72 h was detected by CCK-8 assay to screen for optimal modeling conditions. Results are shown below. Figure 6-8 .

[0109] Observation of Huvec cell morphology showed that normal Huvec adhered well to the wall, were spindle-shaped, uniform in size, and had clear and regular cell boundaries, which is typical of endothelial cell morphology.

[0110] CCK-8 results showed that TBHP inhibited Huvecs viability in a concentration- and time-dependent manner: the half-maximal inhibitory concentration (IC50) was 223.5 μM in the 12h treatment group and 161.3 μM in the 24h treatment group. Significant cytotoxicity was observed even at low concentrations of TBHP in the 48h and 72h treatment groups, indicating that excessively low cell viability was detrimental to subsequent intervention experiments. Considering all factors, treatment of Huvecs with 161 μM TBHP for 24h was selected as the modeling condition for the oxidative stress model.

[0111] ROS fluorescent probe detection results showed that, compared with the normal control group, after treatment with 161 μM TBHP for 24 h, the ROS fluorescence intensity in Huvecs increased significantly (from 30.52±0.58 to 49.50±0.85, an increase of approximately 62%), and the difference was statistically significant. (p<0.0001), confirming the successful establishment of the oxidative stress model.

[0112] 2. Oxidative stress induces EndoMT in Huvecs and activates the TGF-β1 / Snai1 signaling pathway. To clarify the induction effect of oxidative stress on Huvecs EndoMT and its influence on the TGF-β1 / Snai1 pathway, q-PCR and immunofluorescence staining were used to detect the expression of EndoMT-related markers and TGF-β1 and Snai1 in an oxidative stress model. Results are shown below. Figure 9-10 .

[0113] q-PCR results showed that, compared with the normal control group, the mRNA expression of endothelial markers CD31 and VE-cadherin was significantly downregulated in the TBHP treatment group (CD31: p<0.001; VE-cadherin: p<0.05), the mRNA expression of mesenchymal markers α-SMA, FSP1, and NG2 was significantly upregulated (α-SMA: p<0.01; FSP1, NG2: p<0.001); meanwhile, the mRNA expression of TGF-β1 and Snai1 was also significantly upregulated (both p<0.05).

[0114] Immunofluorescence staining results showed that the normal control group of Huvecs exhibited a typical cobblestone pattern, with CD31 (green) continuously and highly expressed on the cell membrane, while α-SMA (red) was almost not expressed. In the TBHP-treated group, cell morphology changed to an elongated spindle shape, CD31 fluorescence signal weakened and became discontinuous, while α-SMA fluorescence signal significantly increased, with both showing co-localization. Quantitative analysis showed that the average fluorescence intensity of CD31 was significantly reduced in the TBHP group. p<0.01), the average fluorescence intensity of α-SMA increased significantly ( p<0.001).

[0115] The above results indicate that oxidative stress can induce typical EndoMT in Huvecs, accompanied by activation of the TGF-β1 / Snai1 signaling pathway, consistent with the results in hypoxic microenvironments, confirming that this pathway is the common core pathway for inducing EndoMT in CNV microenvironments.

[0116] 3. Screening the optimal concentration of TGF-β1 and the effective interfering target of Snai1 To conduct further mechanistic studies, we first screened the optimal concentration of TGF-β1 for Huvecs and the effective siRNA interference target for Snai1.

[0117] Screening for the optimal concentration of TGF-β1: The cell viability of Huvecs after treatment with 0, 1, 2.5, 5, 10, 15, 20, and 30 ng / mL TGF-β1 for 24 h and 48 h was detected by CCK-8 assay. The results showed that 5 ng / mL TGF-β1 had no cytotoxicity within 24 h and 48 h and could slightly promote cell proliferation (24 h: 106.10%, 48 h: 105.10%), which was the optimal concentration. Subsequent experiments used 5 ng / mL TGF-β1 for treatment for 24 h.

[0118] Screening for Effective Snai1 Interference Targets: Three siRNAs targeting Snai1 (Snai1-165, Snai1-650, and Snai1-829) were designed (primer sequences are shown in Table 2). After transfection with Huvecs, the interference efficiency was detected by q-PCR and Western blot. The results showed that Snai1-650 siRNA had the highest interference efficiency, with both mRNA and protein silencing efficiencies exceeding 50% (mRNA: 0.50±0.05, protein: 0.47±0.04), making it an effective interference target. This sequence was used in subsequent experiments. See Table 2 for the corresponding results. Figure 11-12 .

[0119] Table 2 siRNA fragment sequences

[0120] 4. The TGF-β1 / Snai1 pathway is a core regulatory pathway for oxidative stress-induced Huvecs EndoMT. To verify the regulatory role of the TGF-β1 / Snai1 pathway on EndoMT under oxidative stress microenvironment, Huvecs were divided into 6 groups: ① Control group, ② TBHP group, ③ TBHP+NC siRNA group, ④ TBHP+Snai1 siRNA group, ⑤ TBHP+TGF-β1 group, and ⑥ TBHP+TGF-β1+Snai1 siRNA group. The expression of EndoMT-related markers was detected by q-PCR and Western blot. Results are shown below. Figure 13-14 .

[0121] q-PCR results showed that, compared with the ①Control group, the ②TBHP group had significantly downregulated endothelial markers and significantly upregulated mesenchymal markers (both...). p<0.001). Compared with group ②TBHP, there was no statistically significant difference in mRNA expression of the above indicators in group ③TBHP + NC siRNA (all P>0.05), suggesting that the negative control siRNA had no significant effect on the expression of the above target genes; in group ④TBHP + Snai1 siRNA, the expression of CD31 and VE-cadherin mRNA significantly increased (all P>0.05). P<0.001), while the expression of α-SMA, FSP1, NG2, TGF-β1 and Snai1 mRNA was significantly downregulated (P<0.001). P<0.01), indicating that silencing Snai1 not only effectively knocks down its own expression, but also inhibits the transcriptional level of TGF-β1, and can partially alleviate the TBHP-induced EndoMT phenotype-related molecular changes; ⑤ The expression of CD31 and VE-cadherin mRNA in the TBHP + TGF-β1 group was further decreased, while the expression of α-SMA, FSP1, NG2, TGF-β1 and Snai1 mRNA was further increased (all P<0.01). (P<0.01) suggests that exogenous TGF-β1 intervention can further enhance the changes in molecular expression related to EndoMT under oxidative stress, in which TGF-β1 and Snai1 may play a key amplification role.

[0122] Further observation revealed that, compared with group ⑤ (TBHP + TGF-β1), the expression changes of various markers in group ⑥ (TBHP + TGF-β1 + Snai1siRNA) showed a slight reversal: CD31 and VE-cadherin mRNA expression increased (both...). P<0.001), while α-SMA, FSP1 and NG2 mRNA levels decreased (all P<0.001). P<0.01), TGF-β1 and Snai1 mRNA expression were significantly decreased (both P<0.01). (P<0.001). The results suggest that Snai1 may participate in the regulation of EndoMT-related molecule expression induced by oxidative stress under TGF-β1 intervention by regulating TGF-β1 expression.

[0123] The results of Western blot analysis were consistent with those of qPCR, showing that the overall differences in the expression of the above proteins in each group were statistically significant (all...). P<0.001).

[0124] Compared with the ①Control group, the ②TBHP group showed significantly downregulated expression of CD31 and VE-cadherin proteins, while significantly upregulated expression of α-SMA, FSP1, and NG2 proteins (all...). (P<0.001) indicates that HUVECs also showed phenotypic changes related to EndoMT at the protein level after TBHP treatment.

[0125] Compared with group ②TBHP, there were no statistically significant differences in protein expression of the above indicators in group ③TBHP + NC siRNA (all P>0.05), indicating that the negative control siRNA had no significant effect on the expression of the above target proteins; in group ④TBHP + Snai1siRNA, the expression of CD31 and VE-cadherin proteins was significantly increased, while the expression of α-SMA, FSP1 and NG2 proteins was significantly decreased (all P>0.05). P<0.001), indicating that silencing Snai1 can alleviate the above-mentioned protein expression changes induced by TBHP to some extent; ⑤ The expression of CD31 and VE-cadherin proteins in the TBHP + TGF-β1 group further decreased (both P<0.001), while the expression of α-SMA, FSP1, and NG2 proteins was further increased (all P<0.001). (P<0.05) suggests that exogenous TGF-β1 intervention can further enhance the changes in protein expression related to EndoMT under oxidative stress.

[0126] Further observation revealed that, compared with group ⑤TBHP + TGF-β1, group ⑥TBHP + TGF-β1 + Snai1 siRNA showed increased expression of CD31 and VE-cadherin proteins, while the expression of α-SMA, FSP1, and NG2 proteins decreased (all...). P<0.001). The above results are basically consistent with the trend of qPCR, suggesting that Snai1 may be involved in the regulation of EndoMT-related protein expression induced by oxidative stress under TGF-β1 intervention.

[0127] 5. TGF-β1 / Snai1 pathway-mediated EndoMT promotes apoptosis in Huvecs under oxidative stress. To clarify the effect of TGF-β1 / Snai1 pathway-mediated EndoMT on Huvec apoptosis, TUNEL staining was used to detect the apoptosis rate of the above six groups of cells. Results are shown below. Figure 15 .

[0128] The results showed that the overall difference in TUNEL positivity rates among the groups was statistically significant (F=438.30). p<0.001); ① The apoptosis rate in the Control group was extremely low (0.92±0.16). ② Compared with the TBHP group, the proportions of TUNEL-positive cells in the TBHP + NC siRNA group were 44.12% ± 2.94% and 43.89% ± 1.75%, respectively, with no statistically significant difference between the two groups (P>0.05), suggesting that transfection with the negative control siRNA had no significant effect on the level of apoptosis induced by TBHP. ③ Compared with the TBHP group, the proportion of TUNEL-positive cells in the TBHP + Snai1 siRNA group decreased significantly to 34.68% ± 1.63%, with a statistically significant difference (P<0.001). <0.001), indicating that silencing Snai1 reduced the level of apoptosis under oxidative stress. ④ Compared with the TBHP group, the proportion of TUNEL-positive cells in the TBHP + TGF-β1 group increased to 65.11% ± 1.36%, with a statistically significant difference (P < 0.001), suggesting that the level of apoptosis further increased after exogenous TGF-β1 intervention. ⑤ Further observation revealed that compared with the TBHP + TGF-β1 group, the proportion of TUNEL-positive cells in the TBHP + TGF-β1 + Snai1 siRNA group decreased to 54.40% ± 1.92%, with a statistically significant difference (P < 0.001). The value was <0.001, indicating that silencing Snai1 on the basis of TGF-β1 intervention reduced the level of apoptosis.

[0129] These results indicate that oxidative stress can induce Huvec apoptosis, and TGF-β1 / Snai1 pathway-mediated EndoMT can synergistically promote apoptosis under oxidative stress. Knocking down Snai1 can effectively inhibit apoptosis and partially rescue the apoptosis effect exacerbated by TGF-β1, further confirming the core role of this pathway in oxidative stress-induced EndoMT and abnormal cell function.

[0130] Summary of Experimental Results This invention utilizes in vitro cell models of two core CNV microenvironments—hypoxia and oxidative stress—using RF / 6A and Huvecs as research subjects, respectively, to systematically verify, from molecular, cellular phenotype, and functional levels, that the TGF-β1 / Snai1 signaling pathway is the core pathway mediating endothelial cell endoMT. This provides solid in vitro experimental evidence for the core hypothesis of the research project "ECs in CNV participate in vascular remodeling / fibrosis through TGF-β1 / Snai1-mediated EndoMT." The main conclusions are as follows: 1. The CNV local microenvironment can directly induce EndoMT in endothelial cells: Hypoxia (48h) and oxidative stress (161μM TBHP, 24h) can induce typical EndoMT in RF / 6A and Huvecs, which is characterized by loss of endothelial markers (CD31, VE-cadherin) and gain of mesenchymal markers (NG2, FSP1, α-SMA), and cell morphology transforming into a mesenchymal phenotype.

[0131] 2. TGF-β1 / Snai1 is a core signaling pathway mediating EndoMT: both hypoxia and oxidative stress can activate the TGF-β1 / Snai1 pathway. TGF-β1 positively regulates the expression of Snai1 in a dose-dependent manner. As a key downstream transcription factor, Snai1 directly mediates the occurrence of EndoMT. Knockdown of Snai1 can completely block the pro-EndoMT effect of TGF-β1, confirming the upstream and downstream regulatory relationship of this pathway.

[0132] 3. TGF-β1 / Snai1 pathway-mediated EndoMT has a clear cellular functional significance: EndoMT mediated by this pathway can significantly promote the migration ability of endothelial cells (RF / 6A) and synergistically promote the apoptosis of endothelial cells under oxidative stress (Huvecs), suggesting that EndoMT can participate in the vascular remodeling and fibrosis process of CNV by regulating endothelial cell migration and apoptosis.

[0133] 4. The findings of the two microenvironments corroborate each other: hypoxia and oxidative stress, as the core microenvironments of CNV, both induce EndoMT through the TGF-β1 / Snai1 pathway, confirming that this pathway is the common regulatory mechanism for EndoMT in CNV, laying the foundation for subsequent in vivo experiments and targeted intervention studies.

[0134] As illustrated in the above embodiments, this invention provides the application of substances that inhibit TGF-β1 / Snai1 pathway-mediated endothelial-mesenchymal transition (EMT) in the preparation of drugs for treating choroidal neovascularization, as well as methods for constructing hypoxia-induced RF / 6A and oxidative stress-induced Huvecs models and their applications in drug screening. Experimental results show that both hypoxia and oxidative stress, the core pathological microenvironments of choroidal neovascularization, can induce typical EMT in endothelial cells, and this process depends on the activation of the TGF-β1 / Snai1 pathway. By regulating this pathway, especially inhibiting Snai1 expression, EMT can be effectively reversed, and endothelial cell dysfunction can be improved. The established cell models can stably reproduce the key characteristics of EMT, and with the addition of biomarker detection, the intervention effects of candidate drugs can be accurately evaluated. The cell models and screening methods provided by this invention, as well as the drug applications based on TGF-β1 / Snai1 pathway targets, offer a novel strategy and experimental basis for the treatment of choroidal neovascularization fibrosis and vascular remodeling.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of an inhibitor in the preparation of a medicament for inhibiting choroidal neovascularization, fibrosis, or vascular remodeling; The inhibitor is a substance that inhibits the endothelial-mesenchymal transition mediated by the TGF-β1 / Snai1 pathway in endothelial cells.

2. The application according to claim 1, characterized in that, The inhibitor is a TGF-β1 inhibitor, a Snai1 inhibitor, or a TGF-β1 receptor inhibitor.

3. The application according to claim 1, characterized in that, The inhibition of endothelial mesenchymal transformation into inhibition of endothelial cell phenotype transformation.

4. The application according to claim 3, characterized in that, The inhibition of endothelial-mesenchymal transition manifests as upregulation of endothelial marker expression and downregulation of mesenchymal marker expression.

5. The application according to claim 4, characterized in that, The endothelial markers are selected from CD31 or VE-cadherin, and the mesenchymal markers are selected from α-SMA, FSP1, or NG2.

6. The application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization, fibrosis, or vascular remodeling, characterized in that, The hypoxia-induced RF / 6A model was obtained by a preparation method including the following steps: RF / 6A cells were cultured under mixed gas conditions for 24–72 hours to obtain a hypoxia-induced RF / 6A cell model. The mixed gas contains 0.5% to 5% oxygen by volume, 3% to 8% carbon dioxide by volume, and the balance is nitrogen. The sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

7. The application of an oxidative stress-induced human umbilical vein endothelial cell Huvecs model in the preparation of a model for screening candidate drugs that inhibit choroidal neovascularization, fibrosis, or vascular remodeling, characterized in that, The oxidative stress-induced Huvecs model was obtained by a preparation method including the following steps: Huvecs cells were treated in a medium containing tert-butyl hydrogen peroxide for 12–48 hours to obtain an oxidative stress-induced Huvecs cell model. The concentration of tert-butyl hydroperoxide in the culture medium is 100-200 μM.

8. The application of a hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model in the preparation of models for screening substances that inhibit endothelial-mesenchymal transition, characterized in that, The hypoxia-induced RF / 6A model was obtained by a preparation method including the following steps: RF / 6A cells were cultured under mixed gas conditions for 24–72 hours to obtain a hypoxia-induced RF / 6A cell model. The mixed gas contains 0.5% to 5% oxygen by volume, 3% to 8% carbon dioxide by volume, and the balance is nitrogen. The sum of the volume percentages of oxygen, carbon dioxide, and nitrogen is 100%.

9. The application of an oxidative stress-induced human umbilical vein endothelial cell Huvecs model in the preparation of a model for screening substances that inhibit endothelial-mesenchymal transition, characterized in that, The oxidative stress-induced Huvecs model was obtained by a preparation method including the following steps: Huvecs cells were treated in a medium containing tert-butyl hydrogen peroxide for 12–48 hours to obtain an oxidative stress-induced Huvecs cell model. The concentration of tert-butyl hydroperoxide in the culture medium is 100-200 μM.

10. A method for screening candidate drugs for inhibiting choroidal neovascularization, fibrosis, or vascular remodeling, characterized in that, Includes the following steps: The test drug was exposed to hypoxia-induced monkey choroidal retinal endothelial cell RF / 6A model or oxidative stress-induced human umbilical vein endothelial cell Huvecs model to detect the expression levels of endothelial markers and mesenchymal markers in the cells. If the test drug could upregulate endothelial markers and downregulate mesenchymal markers, the candidate drug was determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling. The endothelial marker is selected from CD31 or VE-cadherin, and the mesenchymal marker is selected from α-SMA, FSP1 or NG2. The expression level of Snai1 is also detected in the detection step. If the candidate drug can downregulate the expression of Snai1, the candidate drug is determined to be a candidate drug for inhibiting choroidal neovascularization fibrosis or vascular remodeling.

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