Application of gene inhibitor in preparation of medicine for preventing or treating restenosis after vascular injury

Abstract

The invention relates to the technical field of biomedicine, in particular to application of a gene inhibitor in preparation of a medicine for preventing or treating restenosis after vascular injury. Specific knockdown or knockout of TRIM65 or MYH9 can inhibit formation of neointima, excessive intima neogenesis is the core pathological feature of restenosis after vascular injury, and therefore the TRIM65 inhibitor or MYH9 inhibitor can be used for preparing the medicine for preventing or treating restenosis after vascular injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of gene inhibitors in the preparation of drugs for the prevention or treatment of restenosis after vascular injury. Background Technology

[0002] The latest report from the Global Burden of Disease (GBD) study shows that cardiovascular disease remains the leading cause of death worldwide, and its treatment faces significant challenges. Excessive intimal regeneration following vascular injury is a core pathological feature leading to restenosis and a key problem that urgently needs to be solved in the treatment of cardiovascular disease. After patients undergo angiogenesis or stent implantation, mechanical damage can trigger intimal regeneration, leading to restenosis and severely limiting recovery. Although modern drug-eluting stents have significantly reduced the restenosis rate, some patients still experience restenosis after the procedure.

[0003] Phenotypic conversion of smooth muscle cells is a key pathological mechanism in the development and progression of intimal neoplasia. Under physiological conditions, vascular smooth muscle cells (VSMCs) are in a quiescent, contractile state. When blood vessels are damaged, these cells undergo phenotypic conversion, transforming into a synthetic phenotype, with enhanced proliferation and migration capabilities. They abnormally migrate to the intima and secrete large amounts of extracellular matrix and synthetic markers, ultimately leading to abnormal intimal thickening and vascular stenosis.

[0004] Current clinical interventions have significant limitations. While drug-eluting stents can release anti-proliferative drugs locally, they easily lead to delayed endothelial repair and late-stage in-stent thrombosis. Systemic drugs have significant side effects and cannot fundamentally block the pathological process. Bioresorbable stents may induce inflammatory responses. Other methods, such as autophagy modulators, lack tissue specificity, and biotherapy suffers from low conversion efficiency. Existing technologies all suffer from limitations in their single mechanism and significant side effects, highlighting the urgent need to discover key molecular targets for specific regulation. Summary of the Invention

[0005] Based on this, the present invention provides the application of gene inhibitors in the preparation of drugs for the prevention or treatment of restenosis after vascular injury, thereby solving at least one problem in the prior art.

[0006] In a first aspect, the present invention provides the use of a gene inhibitor in the preparation of a medicament for the prevention or treatment of restenosis following vascular injury, wherein the gene inhibitor is a TRIM65 inhibitor or a MYH9 inhibitor.

[0007] Specific knockdown or knockout of TRIM65 or MYH9 can inhibit neointimal formation, and excessive intimal neoplasia is a core pathological feature of restenosis after vascular injury. Therefore, TRIM65 inhibitors or MYH9 inhibitors can be used to prepare drugs for the prevention or treatment of restenosis after vascular injury. TRIM65 inhibitors or MYH9 inhibitors can be biologically available substances that inhibit TRIM65 or MYH9 targets, such as siRNA, shRNA, small molecule compounds, and traditional Chinese medicine.

[0008] In some alternative embodiments, the drug for preventing or treating restenosis after vascular injury is a drug for preventing or treating restenosis after arterial injury.

[0009] In some optional embodiments, the TRIM65 inhibitor is a substance that downregulates TRIM65 expression in smooth muscle cells, and the MYH9 inhibitor is a substance that downregulates MYH9 expression in smooth muscle cells.

[0010] In some optional embodiments, the substance that downregulates TRIM65 expression in smooth muscle cells is TRIM65 siRNA or AAV9-SM22. cre -shTRIM65, the substance that downregulates MYH9 expression in smooth muscle cells is MYH9 siRNA or AAV9-SM22. cre -shMYH9. For example, the nucleotide sequence of the substance that downregulates the expression of TRIM65 in smooth muscle cells is AGCCAAGCCUGUGGACUUA, written as AGCCAGCCTGTGGACTTA according to WIPO standard ST.26 (SEQ ID No. 1); the nucleotide sequence of the substance that downregulates the expression of MYH9 in smooth muscle cells is GCAACATCGTCTTCAAGAA (SEQ ID No. 2).

[0011] In a second aspect, the present invention provides a drug for preventing or treating restenosis after vascular injury, comprising a TRIM65 inhibitor or a MYH9 inhibitor.

[0012] In some alternative embodiments, the TRIM65 inhibitor or MYH9 inhibitor may further include a pharmaceutically acceptable carrier.

[0013] In some alternative embodiments, the pharmaceutically acceptable carrier is water or physiological saline.

[0014] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: for the first time, the promoting effect of TRIM65 on intimal regeneration is revealed, and the molecular mechanism by which TRIM65 promotes intimal regeneration by regulating MYH9 is clarified. This not only provides a new perspective on the endogenous regulation of restenosis after vascular injury, but also provides a theoretical basis and experimental evidence for the development of drugs that target the TRIM65-MYH9 axis to inhibit intimal regeneration. Attached Figure Description

[0015] Figure 1 This is a graph showing the detection results of various markers of HASMCs in Embodiment 1 of the present invention.

[0016] Figure 2 This is a statistical analysis chart of the detection results of various markers of HASMCs in Example 1 of the present invention.

[0017] Figure 3 This is a statistical analysis chart of the detection results of HASMCs TRIM65 mRNA in Example 1 of the present invention.

[0018] Figure 4 In Embodiment 1 of the present invention, from Apoe - / - A schematic diagram of the process for isolating MASMCs from mice.

[0019] Figure 5 This is a statistical analysis chart of the detection results of various markers of MASMCs in Embodiment 1 of the present invention.

[0020] Figure 6 This is a schematic diagram of the process for establishing a mouse carotid artery ligation injury model in Embodiment 1 of the present invention.

[0021] Figure 7 This is a statistical analysis chart showing the detection results of various markers in the carotid artery of the model mouse in Example 1 of the present invention.

[0022] Figure 8 To construct Apoe in Embodiment 2 of the present invention - / - TRIM65 - / - A schematic diagram of the double knockout mouse procedure.

[0023] Figure 9 This is a diagram showing the H&E staining results of the carotid artery of a male mouse in Example 2 of this invention.

[0024] Figure 10 This is a quantitative statistical analysis diagram of the H&E staining results of the carotid artery of male mice in Example 2 of the present invention.

[0025] Figure 11 This is a diagram showing the H&E staining results of the carotid artery of a female mouse in Example 2 of this invention.

[0026] Figure 12 This is a quantitative statistical analysis diagram of the H&E staining results of the carotid artery of female mice in Example 2 of the present invention.

[0027] Figure 13 This is an image showing the immunofluorescence staining results of the mouse carotid artery in Example 2 of the present invention.

[0028] Figure 14 This is a statistical analysis chart of the immunofluorescence staining results of the mouse carotid artery in Example 2 of the present invention.

[0029] Figure 15 This is a Western blot result of the mouse carotid artery in Example 2 of the present invention.

[0030] Figure 16 This is a statistical analysis chart of the mouse carotid artery Western blot results in Example 2 of the present invention.

[0031] Figure 17 TRIM65 in Embodiment 3 of the present invention f / f and TRIM65 f / f SM22α cre A schematic diagram of the mouse construction process.

[0032] Figure 18 This is a diagram showing the H&E staining results of male mice in Example 3 of the present invention.

[0033] Figure 19 This is a statistical analysis chart of the H&E staining results of male mice in Example 3 of the present invention.

[0034] Figure 20 This is a diagram showing the H&E staining results of female mice in Example 3 of the present invention.

[0035] Figure 21 This is a statistical analysis chart of the H&E staining results of female mice in Example 3 of the present invention.

[0036] Figure 22 This is a diagram showing the results of immunofluorescence staining of the mouse carotid artery in Example 3 of the present invention.

[0037] Figure 23 This is a statistical analysis chart of the mouse carotid artery immunofluorescence staining results in Example 3 of the present invention.

[0038] Figure 24 This is a Western blot detection result of HASMCs in Example 4 of the present invention.

[0039] Figure 25 This is a statistical analysis chart of the Western blot detection results of HASMCs in Embodiment 4 of the present invention.

[0040] Figure 26 This is a diagram showing the scratch test results in Example 4 of the present invention.

[0041] Figure 27 This is a statistical analysis chart of the scratch test results in Example 4 of the present invention.

[0042] Figure 28 This is a statistical analysis chart of the CCK-8 experimental results in Example 4 of the present invention.

[0043] Figure 29 This is a diagram showing the experimental results of colony generation in Embodiment 4 of the present invention.

[0044] Figure 30 This is a statistical analysis chart of the colony generation experiment results in Embodiment 4 of the present invention.

[0045] Figure 31 This is a diagram showing the experimental results of Co-IP in Embodiment 5 of the present invention.

[0046] Figure 32 This is a diagram showing the immunofluorescence staining results in Example 5 of the present invention.

[0047] Figure 33 This is a diagram showing the Co-IP experimental results after overexpressing TRIM65 in Example 5 of the present invention.

[0048] Figure 34 The figure shows the results of the MYH9 knockdown experiment in Embodiment 5 of the present invention.

[0049] Figure 35 This is a statistical analysis chart of the MYH9 knockdown experiment results in Example 5 of the present invention.

[0050] In the figure, Ctrl represents the control, Sham represents the sham surgery, Left represents the left carotid artery, Right represents the right carotid artery, Male represents male, Female represents female, EV represents the empty vector plasmid, and GFP-TRIM65 represents the plasmid overexpressing TRIM65. Detailed Implementation

[0051] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0052] TRIM65, a member of the triple motif repeat protein family, possesses E3 ubiquitin ligase activity and can participate in pathological processes such as cellular metabolism, inflammatory responses, and tumorigenesis by mediating substrate protein ubiquitination. However, its role and mechanism in endometrial regeneration remain unclear. This invention, through the construction of a gene knockout mouse model and combined with cell experiments, reveals for the first time that TRIM65 can influence the biological behavior of vascular smooth muscle cells by regulating the ubiquitination modification of the substrate protein MYH9, thereby intervening in the process of endometrial regeneration. This discovery provides a new theoretical basis for the prevention and treatment of endometrial regeneration and the development of related targeted drugs.

[0053] Based on this discovery, the present invention provides the application of gene inhibitors in the preparation of drugs for the prevention or treatment of restenosis after vascular injury. The technical effects of the present invention are verified through the following examples.

[0054] In the following embodiments, the TRIM65 gene knockout mouse model was obtained by acquiring TRIM65 from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. f / f Mice and SM22α cre Tool mice, TRIM65 was obtained through hybridization f / f SM22α cre Mice (VSMC-specific TRIM65 knockout); simultaneously, systemic TRIM65 knockout mice were obtained from Cyagen (Guangzhou) Biotechnology Co., Ltd., and Apoe mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. - / - Mice, hybridized to construct Apoe - / - TRIM65 - / - Double knockout mice, with corresponding control mice from the same litter (Apoe) - / - ) and gene knockout mice (Apoe) - / - TRIM65 - / - (This is used for subsequent experiments.)

[0055] Example 1: Experimental Verification of the Role of TRIM65 in Endometrial Neoplasia Progression Human aortic smooth muscle cells (HASMCs) were treated with platelet-derived growth factor-BB (PDGF-BB, a potent inducer of vascular smooth muscle cell phenotypic transformation), and the expression of TRIM65 in HASMCs was examined. Results are as follows: Figures 1-3 As shown, PDGF-BB stimulation led to a significant dose-dependent increase in the protein and mRNA levels of TRIM65, while the protein levels of contraction markers (SM22α and CNN1) were also significantly downregulated in a dose-dependent manner.

[0056] according to Figure 4 The method shown is from Apoe - / -Primary mouse aortic smooth muscle cells (MASMCs) were isolated from mice and treated with PDGF-BB. Results are as follows: Figure 5 As shown, PDGF-BB treatment significantly increased TRIM65 mRNA expression while suppressing the mRNA levels of contraction markers (α-SMA, SM22α, and CNN1).

[0057] To investigate whether in vivo experiments are consistent with in vitro experiments, according to Figure 6 A mouse carotid artery ligation injury model was established as shown to induce neointimal formation in vivo. Apoe - / - mRNA levels were measured in the carotid artery of mice after surgery. Results are as follows: Figure 7 As shown, TRIM65 mRNA expression was significantly upregulated at 14 and 28 days post-ligation, while the mRNA levels of contraction markers (α-SMA, SM22α, and CNN1) were significantly reduced at both time points.

[0058] These data indicate that TRIM65 is upregulated during neointimal formation in vivo and in vitro, suggesting that TRIM65 may play a potential role in key processes of neointimal formation.

[0059] Example 2: Experimental Verification of the Effect of TRIM65 Knockout on Neointimal Membrane Formation like Figure 8 As shown, to investigate the role of TRIM65 in endometrial regeneration, Apoe was constructed. - / - TRIM65 - / - Double knockout mice, which served as the experimental group, were the same littermates as Apoe mice. - / - Mice served as the control group, and neointimal formation was induced by left carotid artery ligation. Twenty-eight days post-surgery, the effects of systemic TRIM65 knockout on neointimal formation were systematically evaluated through histological and molecular biological analyses.

[0060] Healthy 8-week-old male and female control and experimental mice were selected. Mice were anesthetized with isoflurane inhalation. The left carotid artery was isolated and ligated, while the right carotid artery served as a sham control. Mice were fed a normal diet for 28 days post-surgery, during which time their body weight, activity level, and wound healing were monitored. 28 days post-surgery, bilateral carotid arteries were collected, sectioned, and stained with Heteroscopic & Escherichia coli (H&E). ImageJ was used to quantitatively analyze the neointimal area, media area, neointimal / media area ratio, and perimeter of the extravascular elastic lamina. Immunofluorescence staining and quantitative fluorescence intensity analysis were performed to detect the levels of contractile markers (α-SMA), proliferative markers (OPN), and proliferation markers (Ki67) in the carotid artery tissue.

[0061] like Figures 9-12 As shown, H&E staining and quantitative analysis revealed that, in both male and female mice, the levels of Apoe...- / - Compared with the control group, Apoe - / - TRIM65 - / - The neointimal area of ​​the left carotid artery was significantly reduced, and the neointimal / media area ratio was also significantly decreased, while the media area and the perimeter of the extravascular elastic membrane showed no significant difference. This indicates that systemic TRIM65 knockout can alleviate intimal neoplasia, and this effect is not sex-dependent, showing consistent results in both male and female mice.

[0062] like Figure 13 and Figure 14 As shown, immunofluorescence staining results indicate that after carotid artery ligation in male mice, Apoe - / - TRIM65 - / - Double knockout mice compared to Apoe - / - The expression of the systolic phenotype marker SM22α in the carotid artery of mice was significantly increased, while the expression of the synthetic phenotype marker OPN and the proliferation marker Ki67 was significantly decreased.

[0063] like Figure 15 and Figure 16 As shown, proteins and RNA were collected from the left carotid artery of mice 28 days after ligation. Western blot analysis further confirmed that Apoe... - / - TRIM65 - / - The protein expression levels of key proteins for the shrinkage phenotype, α-SMA, SM22α, and CNN1, in VSMCs from double knockout mice were significantly higher than those in Apoe mice. - / - Control mice.

[0064] This demonstrates that systemic knockout of TRIM65 can effectively alleviate neointimal formation induced by carotid artery ligation, suggesting that TRIM65 may be a potential target for preventing and treating restenosis.

[0065] Example 3: Experimental Verification of the Role of TRIM65 in Vascular Remodeling like Figure 17 As shown, to investigate the role of vascular smooth muscle cells (VSMCs) TRIM65 in vascular remodeling, smooth muscle cell-specific TRIM65 knockout mice were constructed. f / f SM22α cre ) and littermate control mice (TRIM65) f / f The study induced intimal regeneration by ligation of the left carotid artery. Twenty-eight days post-surgery, the effects of smooth muscle cell-specific TRIM65 knockout on neointimal formation were systematically evaluated through histological and molecular biological analysis.

[0066] like Figures 18-21 As shown, H&E staining and quantitative analysis results indicated that, in both male and female mice, TRIM65... f / fCompared with the control group, TRIM65 f / f SM22α cre The neointimal area of ​​the left carotid artery was significantly reduced, and the neointimal / media area ratio was also significantly decreased. However, there was no significant difference in media area and perimeter of the extravascular elastic membrane. This indicates that smooth muscle cell-specific knockout of TRIM65 can alleviate intimal neoplasia, and this effect is not sex-dependent, showing consistent results in both male and female mice.

[0067] like Figure 22 and Figure 23 As shown, immunofluorescence staining results indicate that after carotid artery ligation in male mice, TRIM65... f / f SM22α cre Mouse vs. TRIM65 f / f The expression of the carotid artery contraction phenotype marker SM22α was significantly increased in mice, while the expression of the synthetic phenotype marker OPN and the proliferation marker Ki67 was significantly decreased.

[0068] This demonstrates that smooth muscle cell-specific knockout of TRIM65 can significantly alleviate carotid artery ligation-induced neointimal formation, indicating that TRIM65 mainly drives neointimal formation after vascular injury by regulating the phenotypic transformation of smooth muscle cells.

[0069] Example 4: Validation experiment on the effect of TRIM65 overexpression on smooth muscle cell phenotypic transformation at the cellular level

[0070] Overexpression of TRIM65 in human aortic smooth muscle cells (HASMCs) resulted in a significant phenotypic transformation. For example... Figure 24 and Figure 25 As shown in the results, Western blot analysis revealed that, compared with the empty vector control group, TRIM65 overexpression significantly downregulated the protein expression levels of the contractile phenotype markers α-SMA and CNN1, and this effect was further enhanced after PDGF-BB stimulation.

[0071] like Figure 26 and Figure 27 As shown in the scratch assay, TRIM65 overexpression significantly promoted the migration ability of HASMCs, with a significant increase in relative migration distance, and the migration ability was further enhanced after PDGF-BB treatment.

[0072] like Figures 28-30 As shown in the results of CCK-8 and colony generation experiments, overexpression of TRIM65 significantly increased the proliferation rate of HASMCs.

[0073] These results indicate that TRIM65 drives the transition of VSMCs from a contractile phenotype to a synthetic phenotype by inhibiting the expression of contractile markers and enhancing cell migration and proliferation in HASMCs.

[0074] Example 5: Molecular mechanism verification experiment of TRIM65 promoting endometrial regeneration by targeting MYH9 The interaction between TRIM65 and MYH9 was verified using Co-IP experiments, and the regulation of MYH9 ubiquitination levels by TRIM65 was analyzed to elucidate the molecular mechanism by which TRIM65 regulates MYH9. Simultaneously, by using the Lipo 3.0 cationic carrier from Abbkine Scientific Co., Ltd. to form a complex with siMYH9 (nucleotide sequence GCAACATCGTCTTCAAGAA), MYH9 protein expression was knocked down in human aortic smooth muscle cells (HASMCs). This further clarified that TRIM65 promotes smooth muscle cell phenotypic transformation by targeting MYH9, ultimately promoting intimal regeneration, thus elucidating the molecular mechanism by which TRIM65 promotes intimal hyperplasia.

[0075] like Figure 31 As shown, endogenous TRIM65 or MYH9 were enriched in HASMCs using co-immunoprecipitation (Co-IP), and Western blot analysis revealed that MYH9 and TRIM65 interact. Figure 32As shown, immunofluorescence staining revealed that TRIM65 and MYH9 were highly co-localized in the cytoplasm of HASMCs.

[0076] like Figure 33 As shown, in HASMCs, GFP-TRIM65 and HA-Ub plasmids were co-transfected, and endogenous MYH9 protein was enriched by co-immunoprecipitation (Co-IP). Western blot analysis showed that the ubiquitination level of MYH9 protein was significantly enhanced after overexpression of TRIM65.

[0077] like Figure 34 and Figure 35 As shown, knocking down MYH9 in HASMCs can reverse the VSMC phenotypic shift caused by TRIM65 overexpression.

[0078] In summary, this invention clarifies TRIM65 as a specific molecular target for preventing restenosis after arterial injury, establishes a precision drug screening system based on the TRIM65-MYH9 regulatory axis, and provides a novel technical solution for targeted therapy of restenosis.

[0079] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. The application of gene inhibitors in the preparation of drugs for the prevention or treatment of restenosis after vascular injury, characterized in that, The gene inhibitor is a TRIM65 inhibitor or a MYH9 inhibitor.

2. The application according to claim 1, characterized in that, The drugs mentioned are those for preventing or treating restenosis after vascular injury.

3. The application according to claim 1, characterized in that, The TRIM65 inhibitor is a substance that downregulates the expression of TRIM65 in smooth muscle cells, and the MYH9 inhibitor is a substance that downregulates the expression of MYH9 in smooth muscle cells.

4. The application according to claim 1, characterized in that, The substance that downregulates TRIM65 expression in smooth muscle cells is TRIM65 siRNA or AAV9-SM22. cre -shTRIM65, the substance that downregulates MYH9 expression in smooth muscle cells is MYH9siRNA or AAV9-SM22. cre -shMYH9.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the substance that downregulates TRIM65 expression in smooth muscle cells is shown in SEQ ID No.

1.

6. The application according to claim 4, characterized in that, The nucleotide sequence of the substance that downregulates MYH9 expression in smooth muscle cells is shown in SEQ ID No.

2.

7. A drug for preventing or treating restenosis after vascular injury, characterized in that, This includes TRIM65 inhibitors or MYH9 inhibitors.

8. The medicament for preventing or treating restenosis after vascular injury according to claim 7, characterized in that, The TRIM65 inhibitor or MYH9 inhibitor also includes a pharmaceutically acceptable carrier.

9. The medicament for preventing or treating restenosis after vascular injury according to claim 8, characterized in that, The pharmaceutically acceptable carrier is water or physiological saline.

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