Application of lindelactone in preparation of medicine for inhibiting neointimal hyperplasia in blood vessel

Abstract

The invention relates to application of lindelactone in preparation of a medicine for inhibiting neointimal hyperplasia in blood vessels. Based on the natural source and biological activity of the lindelactone, the invention develops a pharmaceutical molecule with application prospects, and the lindelactone can be used for preventing and / or treating endometrial neogenesis, and is especially suitable for intervention of interventional postoperative restenosis. The linderane is used for preventing neointimal neogenesis by inhibiting abnormal proliferation and migration of vascular smooth muscle cells. Experimental data show that lindelactone does not show obvious systemic toxicity under the treatment dosage and has lower potential of side effects. The lindelinolide provides parameter data for preparation of the medicine for inhibiting endovascular neointimal hyperplasia, is expected to be applied to percutaneous coronary intervention postoperative complications, especially prevention and treatment of stent restenosis, and can become a potential medicine for intervening in restenosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of linderma lactone in the preparation of drugs that inhibit intravascular neointimal hyperplasia. Background Technology

[0002] Current treatments for cardiovascular diseases, particularly percutaneous coronary intervention (PCI) and stent implantation, have significantly improved patient outcomes. However, in-stent restenosis (ISR) remains a common and complex clinical challenge, severely limiting the long-term efficacy of interventional treatments. Intimal neoplasia is the main pathological basis for ISR, and its mechanisms involve vascular injury, inflammatory response, platelet activation, and abnormal proliferation and migration of vascular smooth muscle cells.

[0003] Existing treatments for intimal neoplasia have several limitations. For example, drug-eluting stents (DES) effectively reduce restenosis rates by releasing antiproliferative drugs (such as paclitaxel and rapamycin), but their polymer coatings may induce inflammation and thrombosis, and there is even a risk of allergic reactions to stent components, which could lead to delayed stent thrombosis and long-term adverse events. Furthermore, while endovascular radiotherapy can inhibit smooth muscle cell proliferation, it may cause persistent endothelial loss, fibrin deposition, and inflammatory cell infiltration of the vessel wall at the irradiation site, thus affecting the long-term function and integrity of the vessel. While these existing therapies address restenosis, they often introduce new complications or fail to completely eliminate the risk of recurrence, potentially leading to vascular complications such as recurrent angina, acute myocardial infarction, arteriovenous fistula or graft failure, transient ischemic attack, or stroke.

[0004] A deeper understanding of the pathophysiology of restenosis / intima neoplasia reveals it to be a complex, multifactorial, and multi-step process, not merely limited to the simple proliferation of smooth muscle cells. Endothelial dysfunction triggered by vascular injury, subsequent platelet activation, recruitment of inflammatory cells (such as monocytes, macrophages, and neutrophils), and the release of growth factors and cytokines from these cells collectively drive smooth muscle cell migration, proliferation, and collagen deposition. This complexity means that existing therapies targeting a single target or employing a single mechanism are insufficient to completely resolve the problem, leading to a high incidence and difficulty in managing restenosis. Existing treatments, such as drug-eluting stents and endovascular radiotherapy, have achieved some success in inhibiting smooth muscle cell proliferation, but their accompanying side effects, such as inflammation, thrombosis, or endothelial injury, reveal the inherent trade-offs of current treatment strategies. While addressing one problem, these therapies may introduce new complications or limit long-term efficacy, highlighting the urgent need to develop treatments with novel, safer, and more effective mechanisms. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention aims to provide the application of linderm in the preparation of drugs that inhibit intravascular neointimal hyperplasia. A second objective of the present invention is to provide a drug that inhibits intravascular neointimal hyperplasia.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of linderma lactone in the preparation of drugs that inhibit intravascular neointimal hyperplasia.

[0007] Preferably, the intravascular neointimal hyperplasia is caused by the proliferation and migration of vascular smooth muscle cells.

[0008] Preferably, the proliferation and migration of vascular smooth muscle cells refers to the increase in cell migration and proliferation capacity during the phenotypic transformation of vascular smooth muscle cells from a contractile state to a secretory state.

[0009] Preferably, the application is for the preparation of a drug for treating complications following percutaneous coronary intervention. More specifically, it is for vascular interventional therapies such as balloon angioplasty, stent implantation, and carotid endarterectomy.

[0010] Preferably, the complication is restenosis after intervention, especially in-stent restenosis.

[0011] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows: A drug that inhibits intravascular neointimal hyperplasia, said drug being made from lindera lactone and pharmaceutically or immunologically acceptable excipients, carriers or diluents.

[0012] Preferably, the drug comprises a compound pharmaceutical composition prepared from lindera lactone.

[0013] Preferably, the drug is administered via oral gavage, local incubation, intravenous injection, transdermal administration, intravascular local injection, or drug-eluting stent coating.

[0014] Preferably, the drug is formulated as granules, powders, capsules, tablets, mixtures, oral liquids, injections, liposomes, or aerosols.

[0015] The present invention has the following beneficial effects: 1. This invention, based on the natural source and bioactivity of linderonide, develops a promising drug molecule for the prevention and / or treatment of vascular intimal neoplasia, particularly suitable for interventional restenosis. Through a series of experiments, this invention has found that linderonide effectively inhibits the abnormal proliferation and migration of vascular smooth muscle cells, a key step in intimal neoplasia. Although its specific molecular mechanism is still under further investigation, preliminary results support its potential to target and regulate the vascular remodeling process. Unlike current strategies relying on mechanical dilation or non-specific anti-proliferative drugs, linderonide achieves its inhibitory effect by regulating cellular functional states, which is expected to reduce the impact on non-target tissues, improve the selectivity and safety of treatment, and achieve broad-spectrum and specific intervention effects. By significantly inhibiting key cellular behaviors in the intimal proliferation process, the intervention method provided by this invention is expected to reduce the risk of in-stent restenosis, thereby prolonging vascular patency time, improving the long-term effects of interventional treatment, and improving patients' quality of life.

[0016] 2. Compared with the best existing drug-eluting stents, the novel intimal neoplasia inhibitor based on Lindera lactone in this invention exhibits significant advantages in terms of safety, adaptability, and therapeutic efficacy. Drug-eluting stents effectively reduce restenosis rates by releasing antiproliferative drugs (such as paclitaxel or rapamycin), but their polymer coatings and drugs may induce inflammation, thrombosis, and hypersensitivity reactions, increasing the risk of delayed stent thrombosis. In contrast, this invention uses Lindera lactone, a sesquiterpene lactone extracted from the natural plant Lindera, which prevents intimal neoplasia by inhibiting the abnormal proliferation and migration of vascular smooth muscle cells. Experimental data show that Lindera lactone did not exhibit significant systemic toxicity at therapeutic doses and has a lower potential for side effects. Furthermore, Lindera lactone can be flexibly applied through various routes of administration (such as oral gavage and local incubation), offering greater adaptability compared to the single mode of stent implantation required by drug-eluting stents, and providing possibilities for developing novel delivery systems (such as stent coatings or perivascular sustained-release carriers).

[0017] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a diagram showing the experimental results of the effect of Lindera lactone on inhibiting PDGF-BB-induced vascular smooth muscle cell migration and proliferation; Figure 2This is a diagram showing the experimental results of the effect of Lindera lactone on the repair of endothelial cells and the promotion of reendothelialization in mice with carotid guidewire injury. Figure 3 This is a diagram showing the experimental results of the effect of linderm lactone on relieving intimal neoplasia in mice with carotid guidewire injury via gavage; Figure 4 This is an experimental result showing the effect of lindermide on relieving carotid intimal neoplasia in mice with carotid guidewire injury through local incubation administration. Detailed Implementation

[0019] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] The experimental methods used are as follows: I. Determination of cell migration and proliferation capacity 1. Transwell transfer experiments, including the following steps: 1) Hydration treatment of Transwell chambers (BD Falcon, 8.0 μm pore size) before use: Place the chamber in a well plate, add 100 μL of serum-free DMEM to the top layer, and hydrate in an incubator; 2) After treatment, trypsinize and centrifuge the cells, then resuspend them in 1 mL of serum-free DMEM to form a single-cell suspension; 3) Take 10 μL of the cell suspension for cell counting, and after proportional conversion, convert 1.0 × 10⁻⁶ cells to the appropriate concentration. 6 4) Add cells to the upper layer of Transwell; discard the hydration solution and adjust the volume of the added liquid according to the volume of the added cells: add 200 μL of cell suspension to the upper layer and 600 μL of serum-free DMEM to the lower layer; 5) Gently shake to avoid air bubbles interfering with the migration process and incubate in an incubator for 6 hours; 6) After incubation, discard the upper culture medium, wash with PBS and invert onto absorbent paper, then fix with paraformaldehyde; 7) Stain the nuclei of the migrating cells with DAPI and take pictures under dark conditions (10× inverted fluorescence microscope). Randomly collect 5 fields of view for each group and analyze the number of migrating cells using Image-Pro Plus.

[0021] 2. Cell proliferation assay (Ki67 staining), including the following steps: 1) Assess cell proliferation capacity using the proportion of Ki67-positive cells. Cells were pre-seeded in chambers and treated according to experimental requirements; 2) After cells reached a suitable density, the culture medium was discarded, and the cells were washed three times with PBS for 5 min each time; 3) Cells were fixed with paraformaldehyde at room temperature for 20 min; 4) The fixative was discarded, and the cells were washed three times again with PBS for 5 min each time; 5) Cells were permeabilized with 0.5% Triton X-100 membrane solution and incubated at room temperature for 30 min; 6) After permeabilization, the cells were washed three times with PBS; 7) Cells were blocked with blocking solution (e.g., 5% sheep serum) at room temperature for 30 min; 8) Ki67 antibody was diluted 1:100 with primary antibody dilution solution, mixed well, added to the chamber, and incubated overnight at 4°C in the dark; 9) The next day, the primary antibody solution was discarded, and the cells were washed three times with PBS for 5 min each time. Subsequent steps required operation in the dark; 10) Phalloidin staining of the cytoskeleton was performed, diluted 1:50, and incubated at room temperature for 30 minutes. min; 11) Wash 3 times with PBS; 12) Remove the chamber border, cover with DAPI and stain the nucleus, place in a humidified dark box, and store at 4°C for a short period of time.

[0022] II. Hematoxylin and eosin (HE) staining, including the following steps: 1. Tissue Section Preparation: Equilibrate tissue sections at room temperature for 30 minutes, then wash three times with PBS for 5 minutes each time. 2. Fixation: Fix with paraformaldehyde for 15 minutes, then wash three times with PBS for 5 minutes each time. 3. Hematoxylin Staining: Stain with hematoxylin for 2-3 minutes. Observe under a microscope until the cell nuclei turn blue-purple. 4. Rinsing and Blueing: Rinse with tap water for 1 minute to achieve blueing. Then react with differentiation solution for 15 minutes, then rinse with tap water. 5. Differentiation Solution Washing: Continue reacting with differentiation solution for 15 minutes to wash away excess stain, then rinse with tap water. 6. Eosin Staining: Stain with eosin for 10-15 seconds. Observe under a microscope until the cell cytoplasm turns red. 7. Gradient Dehydration: Immerse the sections sequentially in 75%, 85%, 95%, 100% (I), and 100% (II) ethanol, 5 minutes each time, for dehydration. 8. Xylene Clearing: Clear the slides by immersing them in xylene (I) and xylene (II) for 15 minutes each. 9. Mounting and Preservation: Mount the slides using a neutral resin. Place the prepared slides in a fume hood and allow them to dry completely. They can then be stored at room temperature for extended periods. Imaging and Analysis: Imaging was performed using an inverted microscope. Images can be analyzed using Image-Pro Plus software.

[0023] III. Immunofluorescence assay of tissue samples, including the following steps: 1. Tissue section preparation and cleaning: Equilibrate frozen tissue sections stored at -80℃ to room temperature for 30 minutes. Clean the sections with PBS for 5 minutes each time, for a total of 3 times, to thoroughly remove excess OCT embedding agent. 2. Fixation and cleaning: Fix the sections with paraformaldehyde at room temperature for 15 minutes. Then clean with PBS for 5 minutes each time, for a total of 3 times. 3. Permeabilization: Draw a hydrophobic zone around the tissue section using a histochemical pen. Add 0.5% Triton X-100 to the zone for permeabilization, and react at room temperature for 30 minutes. 4. Cleaning: Clean the sections with PBS for 5 minutes each time, for a total of 3 times. 5. Blocking: Add blocking sheep serum for blocking at room temperature and incubate for 30 minutes. 6. Primary antibody incubation: Dilute the primary antibody at a ratio of 1:200. Vortex to mix, centrifuge to remove the primary antibody dilution, discard the blocking solution, and add the diluted primary antibody directly. Incubate the sections at 4℃ overnight. 7. Mounting and Storage: After overnight incubation, wipe away excess liquid from the slides. Mount the slides using DAPI. The stained slides can be stored in a humidified darkroom at 4°C for short periods.

[0024] IV. Construction of a mouse model of left cervical guidewire sprain 1. Animal preparation: 8-10 week old C57BL / 6 mice (purchased from Spiefer Biomedical) were used to construct the endometrial neoplasia model. Mice were anesthetized by intraperitoneal injection of 1.2% tribromoethanol and then fixed to a simple operating table. The neck was fully exposed and hair was removed, and the skin was disinfected with povidone-iodine.

[0025] 2. Exposure of blood vessels: Make an incision along the midline of the mouse's neck, and use microforceps to bluntly dissect the connective tissue, adipose tissue, etc. along the left side of the neck until the left common carotid artery is fully exposed.

[0026] 3. Vascular dissection and ligation: Dissect the left common carotid artery and its upstream bifurcation, the internal carotid artery and the external carotid artery. Dissect the superior thyroid artery at the external carotid artery. Place a 6-0 suture distal to the external carotid artery and ligate it to block the blood flow back from above; place a 6-0 suture proximal to the external carotid artery (i.e., below the ligation point, above the bifurcation of the external carotid artery and the superior thyroid artery) for later use, but do not ligate it at this time.

[0027] 4. Incision preparation: Use arterial clips to clamp the blood flow in the internal carotid artery and common carotid artery. In the middle of the external carotid artery suture area, make a small incision as parallel as possible to the direction of the blood vessel with microsurgical scissors to allow the guidewire to enter.

[0028] 5. Vascular injury: A 0.38 mm diameter guidewire is used to treat vascular injury. The guidewire is inserted into the common carotid artery via the external carotid artery, and the hemostatic clamp on the common carotid artery is released to facilitate guidewire entry. The guidewire is moved back and forth 5 times before being withdrawn, and the blood flow is clamped again with an arterial clamp.

[0029] 6. Restoration of blood flow and wound closure: At this point, ligate the sutures placed proximal to the external carotid artery, release the arterial clamps of the common carotid and internal carotid arteries, observe the vascular condition, and wait for blood flow to be restored. The contralateral vessels are only exposed as a control group. Subsequently, the neck wound is sutured and disinfected with povidone-iodine.

[0030] 7. Tissue Collection and Subsequent Analysis: Tissue samples were collected at 3, 14, and 21 days post-modeling to obtain a mouse model of left cervical guidewire sprain. Blood vessels were preserved using different methods according to experimental requirements. Evans blue staining, HE staining, and immunofluorescence staining were performed on carotid artery tissue sections for verification.

[0031] V. Drug incubation protocol for the left common carotid artery of mice 1. Preparation of drug incubation matrix gel In this experiment, Pluronic® F-127 powder (Sigma-Aldrich; catalog number: P2443) and physiological saline were precisely prepared into a gel at a ratio of 30% (w / v). To ensure complete dissolution, the mixture was incubated overnight in a shaker at 4°C.

[0032] 2. Experimental Grouping and Procedures Control group: The left common carotid artery of mice was first injured by a guidewire, and after blood flow was restored, a matrix adhesive was applied to the periphery of the vessel. To provide an internal control, the contralateral right common carotid artery was only exposed without guidewire injury.

[0033] In the drug-treated group: After the left common carotid artery of mice was injured via a guidewire, a matrix gel was applied to the periphery of the vessel upon restoration of blood flow, but this gel contained 30 nM lindera lactone suspension. The contralateral right common carotid artery was similarly exposed without injury.

[0034] All mice were sampled 14 days after the modeling process was completed for subsequent analysis.

[0035] VI. Mouse drug gavage experiment protocol Eight- to 12-week-old C57BL / 6 mice were randomly divided into two groups and housed in an SPF-grade animal facility with free access to water and food. The control group received 0.5% CMC-Na solution by gavage every other day after a guidewire injury model was created, while the lindera lactone treatment group received 25 mg / kg of lindera lactone by gavage every other day using 0.5% CMC-Na as the solvent. Both groups received the treatment for two consecutive weeks.

[0036] VII. Evans Blue Staining Protocol for Mice As previously mentioned, an Evans blue assay was performed 3 days after guidewire injury in mice to assess the endothelial repair and re-endothelialization process of the damaged carotid artery. Thirty minutes before euthanasia, 50 μL of 2% dye solution was injected into the tail vein of the mice, the carotid artery was longitudinally opened and fixed with methanol, and then photographed.

[0037] Example 1: Lindera lactone inhibits PDGF-BB-induced vascular smooth muscle cell migration and proliferation. This embodiment aims to investigate whether lindera lactone can inhibit the migration and proliferation of vascular smooth muscle cells stimulated by platelet-derived growth factor-BB (PDGF-BB). This part uses Transwell assays to stain cell nuclei to observe migration ability and Ki67 immunofluorescence labeling to analyze proliferation status, further verifying the inhibitory effect of lindera lactone on vascular remodeling-related processes at the cellular level.

[0038] Experimental results are as follows Figure 1 As shown, where, Figure 1 In Figure A, human aortic smooth muscle cells were pretreated with DMSO or lindera lactone (10 μM) for 1 hour, then stimulated with PDGF-BB (20 ng / mL) for 6 hours, and their migration ability was assessed using Transwell assays. Cell nuclei were stained with DAPI, and microscopic imaging was used to count the number of migrating cells. Figure B shows the statistical analysis of the Transwell migration assay. The figure shows the number of migrating cells in each treatment group. Data are expressed as mean ± SEM. Differences between groups were analyzed using two-way ANOVA. * indicates p < 0.05, which is statistically significant (n = 6). In Figure C, aortic smooth muscle cells were pretreated with DMSO or lindera lactone (10 μM) for 1 hour, then stimulated with PDGF-BB (20 ng / mL) for 24 hours. Ki67 immunofluorescence staining was used to assess cell proliferation. Ki67-positive cells were marked in green, F-actin (Phalloidin staining) on ​​the cytoskeleton was in red, and DAPI staining of the cell nucleus was in blue. D represents the statistical analysis of Ki67 immunofluorescence staining. Data are expressed as Mean ± SEM. Statistical differences were determined by two-way ANOVA. * indicates p < 0.05, which is statistically significant. n = 6.

[0039] Figure 1As shown in Figure A, human aortic smooth muscle cells were pretreated with DMSO or lindera lactone (10 μM) for 1 hour, then stimulated with PDGF-BB (20 ng / mL) for 6 hours, and their migration ability was subsequently assessed using Transwell assays. DAPI staining showed that in the DMSO group, the smooth muscle cell nuclei in the Ctrl control group were relatively evenly distributed and the number of migrating cells was low; under PDGF-BB stimulation, cell migration ability was significantly enhanced, and the number of cell nuclei increased significantly. In the lindera lactone pretreatment group, the number of migrating cells in the PDGF-BB treatment group was significantly lower than that in the corresponding DMSO+PDGF-BB treatment group, but there was no significant difference in the Ctrl control group. These results suggest that lindera lactone treatment effectively inhibited the smooth muscle cell migration ability induced by PDGF-BB stimulation. Figure 1 (A, B)

[0040] Figure 1 Human aortic smooth muscle cells were pretreated with DMSO or lindera lactone (10 μM) for 1 hour, then stimulated with PDGF-BB (20 ng / mL) for 24 hours. Ki67 immunofluorescence staining was performed on the cells to assess cell proliferation. This figure shows the triple staining results for Ki67, F-actin, and the nucleus. In the DMSO group (Ctrl control group), the nuclei were clear, the cytoplasm was regular, and Ki67 positive signals were sparse. After PDGF-BB treatment, the Ki67 signal (green) significantly increased, indicating that the cells entered an active proliferative state. In contrast, in the lindera lactone intervention group, the number of Ki67 positive cells in the PDGF-BB treatment group was significantly reduced, indicating that it also inhibited PDGF-BB-induced proliferation, but there was no significant difference in expression levels in the baseline Ctrl control group. Figure 1 The proportion of Ki67-positive cells was further quantified by D, and the results showed that PDGF-BB-induced cell proliferation significantly increased, while treatment with lindera lactone effectively reduced the Ki67 positivity rate. Figure 1 (C, D)

[0041] This embodiment clearly demonstrates that linderonide has the ability to significantly inhibit PDGF-BB-induced migration and proliferation of vascular smooth muscle cells. In cell migration experiments, linderonide effectively reduced the number of cells migrating induced by PDGF-BB stimulation; and in Ki67 proliferation assays, linderonide also showed good anti-proliferative effects. Both experiments show that linderonide can reverse the effects of PDGF-BB on smooth muscle cell migration and proliferation, suggesting that it may achieve its vascular remodeling inhibitory function by inhibiting the migration and proliferation of smooth muscle cells.

[0042] Example 2: Lindera lactone is beneficial for endothelial repair and promotes the re-endothelialization process. This embodiment aims to verify whether lindera lactone has a positive effect on promoting the repair of endothelial damage induced by a mouse carotid guidewire injury model. Eight- to ten-week-old C57BL / 6 mice underwent a left common carotid artery guidewire injury model surgery, and the injured artery was locally incubated with lindera lactone. The Ctrl group (control group) received vector application; the Lind group received 30 nM lindera lactone application, and its effect on vascular endothelial repair was observed.

[0043] Three days after mouse modeling, vascular endothelial repair was assessed by Evans blue staining. Figure 2 In C57BL / 6 mice, the left common carotid artery was subjected to guidewire injury surgery, while the right common carotid artery was only exposed. The common carotid arteries were then locally incubated with lindera lactone for 3 days, as were the control group. Figure 2 Figure A shows a representative image stained with Evans blue, and Figure B shows the statistical analysis of the proportion of re-endothelialized areas. Data are expressed as Mean ± SEM, and statistical differences were analyzed by paired t-tests. * indicates p < 0.05, which is statistically significant. The sample size was n=5. The experimental results show that, compared with the control group, the treatment group with Lindera lactone promoted the endothelial repair process in mice, as evidenced by a reduction in the damaged area (blue area).

[0044] Example 3: Lindera lactone alleviates endometrial neoplasia This embodiment aims to verify the inhibitory effect of linderma lactone on intimal hyperplasia induced by a mouse carotid artery guidewire injury model. Eight- to ten-week-old C57BL / 6 mice underwent a left common carotid artery guidewire injury model surgery and were randomly divided into two groups (control group and linderma lactone treatment group). Mice were administered the drug via gavage, and its effects on vascular intimal hyperplasia, cell proliferation, and other related indicators were observed, in order to demonstrate the potential application value of linderma lactone in the treatment of diseases related to vascular intimal hyperplasia.

[0045] Fourteen days after mouse modeling, vascular intimal hyperplasia was assessed by HE staining. The experimental results are as follows: Figure 3As shown in the figures. A represents the left common carotid artery guidewire injury surgery in C57BL / 6 mice. Both the Lindera lactone gavage group and the control group received gavage for 14 days, with gavage administered every other day. This section presents representative histological sections stained with hematoxylin and eosin (HE). B presents the statistical analysis of the HE-stained histological sections. Data are expressed as Mean ± SEM and statistically significant were analyzed using paired t-tests. * indicates p < 0.05, which is statistically significant. The sample size was n = 5. C represents the left common carotid artery guidewire injury surgery in C57BL / 6 mice. Both the Lindera lactone gavage group and the control group received gavage for 14 days, with gavage administered every other day. This section presents the immunofluorescence staining of PCNA-positive cells. PCNA is labeled red, the smooth muscle cell marker α-SMA is labeled green, and the nuclear DAPI is labeled blue. D presents the statistical analysis of the PCNA immunofluorescence staining of the left common carotid artery. Data are presented as mean ± SEM. Statistical differences were analyzed using paired t-tests. * indicates p < 0.05, which is statistically significant. The sample size was n=5. E is a statistical graph of body weight after drug gavage treatment. Data are presented as mean ± SEM. Statistical differences were analyzed using paired t-tests. ns indicates no significant difference. The sample size was n=5. F is a statistical graph of left ventricular echocardiography after drug gavage treatment. Data are presented as mean ± SEM. Statistical differences were analyzed using paired t-tests. ns indicates no significant difference. The sample size was n=5. Compared with the control group, the degree of intimal hyperplasia in the blood vessels of mice treated with lindera lactone was significantly reduced, as evidenced by a decrease in intimal thickness. Figure 3 (A, B)

[0046] Immunofluorescence staining results showed that the number of proliferating cell nuclear antigen (PCNA) positive cells in the blood vessels of the linderma lactone treatment group was significantly lower than that in the control group. Further quantitative analysis indicated that the percentage of PCNA positive cells in the linderma lactone group was significantly lower than that in the control group, suggesting that linderma lactone can effectively inhibit the proliferation of vascular smooth muscle cells induced by the guidewire injury model. Figure 3 (C, D). Furthermore, throughout the experiment, there were no significant differences in cardiac function and body weight changes between the two groups, indicating that lindera lactone did not cause significant systemic toxicity or adverse effects on mouse growth at the administered dose. Figure 3 (E, F).

[0047] The results of this embodiment demonstrate that lindera lactone can significantly inhibit vascular intimal hyperplasia induced by a mouse guidewire injury model, and has good safety at therapeutic doses.

[0048] Example 4: Local incubation of Lindera lactone alleviates carotid intimal neoplasia Eight- to 12-week-old C57BL / 6 mice were randomly divided into three groups. All mice underwent a guidewire laceration surgery on the left common carotid artery, followed by local application of lindera lactone: the Ctrl group (control group) received the carrier application; the 30 nM group received 30 nM lindera lactone application; and the 50 nM group received 50 nM lindera lactone application. The experimental results are as follows: Figure 4 As shown, A represents guidewire injury surgery on the left common carotid artery of C57BL / 6 mice, while the right common carotid artery was only exposed. The carotid arteries were locally incubated with lindera lactone for 14 days in both the control and other groups. This section presents representative histological sections stained with hematoxylin and eosin (HE). B presents the statistical analysis of the HE-stained histological sections. Data are expressed as Mean ± SEM and statistical differences were analyzed using one-way ANOVA, where * indicates p < 0.05, indicating statistical significance. The sample size was n = 5. C represents guidewire injury surgery on the left common carotid artery of C57BL / 6 mice, while the right common carotid artery was only exposed. The carotid arteries were locally incubated with lindera lactone for 14 days in both the control and other groups. This section presents the immunofluorescence staining of PCNA-positive cells. PCNA is labeled red, the smooth muscle cell marker α-SMA is labeled green, and the nuclear DAPI is labeled blue. D presents the statistical analysis of PCNA immunofluorescence staining in the left common carotid artery. Data are presented as mean ± SEM. Statistical differences were determined by one-way ANOVA. * indicates p < 0.05, which is statistically significant. The sample size was n=5.

[0049] The experimental results showed that H&E staining was used to observe vascular morphological changes. In the control group of the left common carotid artery, significant intimal hyperplasia was observed, and the luminal structure was compressed to varying degrees. Dense cell arrangement and disordered structure were visible in the intimal region, significantly different from normal vascular morphology. In contrast, treatment with 30 nM and 50 nM linderma lactone alleviated the degree of intimal hyperplasia, thinned the intimal region, and showed a relatively regular cell arrangement, suggesting that linderma lactone can effectively reduce the surgically induced intimal neoplasia process. Figure 4 (A). The trend shows that lindera lactone has a significant inhibitory effect on endometrial hyperplasia, but the effect tends to stabilize at 30 nM, with no further significant decrease observed, suggesting that this dose may have reached the saturation point of its biological effect. Figure 4 (A, B)

[0050] Immunofluorescence staining was further used to assess cell proliferation in the blood vessels. The tricolor immunolabeling results showed that PCNA-positive cells (red) were relatively more abundant in the control group of the left common carotid artery, indicating that smooth muscle cells were in an active proliferative state. However, in the 30 nM and 50 nM groups, PCNA signaling was significantly reduced, and the cell number decreased. Figure 4 (C, D) This suggests that lindera lactone can effectively inhibit vascular intimal regeneration and reduce abnormal proliferation of smooth muscle cells.

[0051] Compared to the best existing drug-eluting stents, this invention, based on lindera lactone, demonstrates significant advantages in terms of safety, adaptability, and therapeutic efficacy as a novel inhibitor of intimal neoplasia. Drug-eluting stents effectively reduce restenosis rates by releasing antiproliferative drugs (such as paclitaxel or rapamycin), but their polymer coatings and drugs can induce inflammation, thrombosis, and hypersensitivity reactions, increasing the risk of delayed stent thrombosis. In contrast, this invention utilizes lindera lactone, a sesquiterpene lactone extracted from the natural plant Lindera strychnifolia, which prevents intimal neoplasia by inhibiting the abnormal proliferation and migration of vascular smooth muscle cells. Experimental data show that lindera lactone did not exhibit significant systemic toxicity at therapeutic doses and has a lower potential for side effects. Furthermore, lindera lactone can be flexibly applied through various routes of administration (such as oral gavage and local incubation), offering greater adaptability compared to the single mode of stent implantation required by drug-eluting stents, and providing possibilities for developing novel delivery systems (such as stent coatings or perivascular sustained-release carriers).

[0052] The advantages of this invention are further verified by experimental data. In vitro experiments showed that, under the condition of PDGF-BB (platelet-derived growth factor-BB, 20 ng / mL)-induced migration of human aortic smooth muscle cells, the number of migrating cells in the lindera lactone (10 μM) treatment group was significantly lower than that in the DMSO control group. Figure 1 In the middle AB), the proportion of Ki67 positive proliferating cells also decreased to the baseline level. Figure 1 (CD). In in vivo experiments, a mouse guidewire injury model showed that local incubation of lindera lactone in mice could promote endothelial repair and re-endothelialization after carotid artery injury. Figure 2 (A and B). The degree of endothelial hyperplasia and endothelial thickness in the Lindera lactone oral gavage group (25 mg / kg, administered every other day for 14 days) were significantly lower than those in the control group. Figure 3 In the middle AB), the proportion of PCNA-positive cells decreased ( Figure 3 (CD), and has no obvious toxicity ( Figure 3 (Central EF). Local administration experiments further confirmed that the intima / luminal area ratio in the 30 nM and 50 nM lindera lactone incubation groups was significantly lower than that in the control group ( Figure 4 In the middle B, the proportion of PCNA-positive cells decreased ( Figure 4 (D). In contrast, while drug-eluting stents can inhibit restenosis, literature reports that the risks of inflammation and thrombosis are difficult to completely avoid in clinical practice. Therefore, this invention not only effectively inhibits intimal neoplasia but also significantly improves upon the shortcomings of existing technologies through a safer and more flexible technical solution, providing a completely new option for vascular interventional therapy.

[0053] In addition to linderonide, this invention can also utilize other natural or synthetic compounds with similar biological activities. For example, sesquiterpene lactones extracted from Lindera or other plants (such as isolinderonide or related derivatives), or drugs known to have antiproliferative, anti-inflammatory, and antioxidant effects (such as rapamycin, paclitaxel, and statins), may inhibit intimal neoplasia by suppressing the migration and proliferation of vascular smooth muscle cells. The mechanisms of action of these compounds may be similar to or complementary to those of linderonide, providing diverse treatment options.

[0054] This invention employs two drug delivery methods: oral gavage and local incubation. Other routes of administration may also be included, such as: intravenous injection: delivering linderma lactone to the vascular lesion site via systemic circulation, potentially suitable for acute treatment scenarios; transdermal delivery: utilizing transdermal patches or gels to achieve slow drug release, suitable for long-term management; intravascular local injection: directly injecting the drug into the target vessel during interventional procedures to increase local concentration; and drug-eluting stent coating: integrating linderma lactone into the stent coating to achieve precise local drug release. These methods may enhance drug targeting or bioavailability.

[0055] This invention employed specific dosages (e.g., 25 mg / kg by gavage, or 30 nM or 50 nM local incubation) and dosing frequencies (every other day). This invention further enhances efficacy by optimizing dosage and frequency. For example, exploring low-dose long-term administration (e.g., 5-10 mg / kg daily) to maintain stable blood drug concentrations. Employing high-dose short-term administration (e.g., a single 50 mg / kg dose) to rapidly control acute endometrial neoplasia. Furthermore, linderon can be used in combination with existing anti-restenosis drugs or adjuvant drugs to enhance efficacy or reduce the dosage of a single drug. For example, synergistic effects may occur when combined with antiproliferative drugs (e.g., rapamycin, paclitaxel). Combination with anti-inflammatory drugs (e.g., aspirin, dexamethasone) may inhibit inflammation-driven endometrial neoplasia. Combination with antiplatelet drugs (e.g., clopidogrel) may reduce the risk of thrombosis. This multi-target intervention strategy may more comprehensively address the complex pathological process of endometrial neoplasia.

[0056] This invention can also employ improved drug delivery systems to enhance the local effects and bioavailability of linderon, for example: nanocarriers: encapsulating linderon in nanoparticles for targeted delivery to sites of vascular injury; sustained-release gels: improving the Pluronic® F-127 matrix gel formulation to prolong drug release time; stent coating technology: embedding linderon in biodegradable polymers for release on the stent surface. These technologies can reduce systemic side effects and improve efficacy.

[0057] Furthermore, the use of structural analogs or chemical derivatives of linderonide (such as compounds whose solubility or stability is improved through chemical modification) may retain the activity of inhibiting endometrial regeneration while improving the physicochemical properties of the drug. For example, adding a hydrophilic group may make it more suitable for aqueous formulations. In pharmaceutical compositions, the ratio of linderonide to other active ingredients can be adjusted according to therapeutic needs. For example, in combination therapy, the ratio of linderonide to rapamycin can be adjusted from 1:1 to 2:1 or 1:2 to optimize the balance between antiproliferative and anti-inflammatory effects. This invention can also combine linderonide with photodynamic therapy: after local administration, linderonide is activated by light of a specific wavelength, enhancing its inhibitory effect on smooth muscle cells while reducing damage to surrounding tissues. This approach may be suitable for clinical scenarios requiring high-precision intervention.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of Lindera lactone in the preparation of drugs that inhibit intravascular neointimal hyperplasia.

2. The application according to claim 1, characterized in that, The intravascular neointimal hyperplasia is caused by the proliferation and migration of vascular smooth muscle cells.

3. The application according to claim 1, characterized in that, The proliferation and migration of vascular smooth muscle cells refer to the increase in cell migration and proliferation capacity during the phenotypic transformation of vascular smooth muscle cells from a contractile state to a secretory state.

4. The application according to claim 1, characterized in that, The application is for the preparation of drugs to treat complications following percutaneous coronary intervention.

5. The application according to claim 1, characterized in that, The complication mentioned is restenosis after interventional procedures.

6. The application according to any one of claims 1-5, characterized in that, The aforementioned linderma lactone is used in combination with other therapeutic drugs.

7. A drug for inhibiting intravascular neointimal hyperplasia, characterized in that, The drug is made from lindera lactone and pharmaceutically or immunologically acceptable excipients, carriers or diluents.

8. The medicament according to claim 7, characterized in that, The drug comprises a compound drug composition prepared from lindera lactone.

9. The drug according to claim 7, characterized in that, The drug can be administered via oral gavage, local incubation, intravenous injection, transdermal administration, intravascular local injection, or drug-eluting stent coating.

10. The medicament according to claim 7, characterized in that, The drug is formulated as granules, powders, capsules, tablets, mixtures, oral liquids, injections, liposomes, or aerosols.

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