Use of lycorine hydrochloride in the preparation of a medicament for inhibiting neointimal hyperplasia

Abstract

The application relates to the application of lycorine hydrochloride in the preparation of a medicine for inhibiting the proliferation of a neointimal in a blood vessel. Lycorine hydrochloride has the effect of inhibiting the proliferation of a neointimal in a blood vessel, and the effect is mainly realized by inhibiting the proliferation and migration of vascular smooth muscle cells. In the body, the results show that lycorine hydrochloride can significantly inhibit the intimal proliferation caused by left common carotid artery ligation, and in vitro experiments prove that lycorine hydrochloride can intervene in the proliferation, migration and phenotype transformation of VSMCs induced by PDGF-BB. Lycorine hydrochloride inhibits the proliferation, migration and phenotype transformation of VSMCs induced by PDGF-BB, and the proliferation of a neointimal in a blood vessel through a MAPKs signal path. The application provides data for preparing lycorine hydrochloride into a medicine for inhibiting the proliferation of a neointimal in a blood vessel, and is expected to be applied to the prevention and treatment of complications after percutaneous coronary intervention, such as restenosis or in-stent thrombosis, and can become a potential medicine for intervening in restenosis.

Description

Technical Field

[0001] This invention relates to the pharmaceutical use of lycorine hydrochloride, specifically to the use of lycorine hydrochloride in the preparation of drugs that inhibit intravascular neointimal hyperplasia. Background Technology

[0002] Percutaneous coronary intervention (PCI) plays a crucial role in the treatment of cardiovascular diseases, but related complications such as restenosis and in-stent thrombosis remain pressing clinical challenges. Restenosis is caused by neointimal hyperplasia (NIH) resulting from the proliferation and migration of vascular smooth muscle cells (VSMCs). Under physiological conditions, VSMCs are located in the vascular media, maintaining the integrity of the arterial structure and vascular tone, characterized by a resting state and a contractile phenotype. However, under various environmental stimuli, including PDGF-BB and inflammatory factors that damage blood vessels, VSMCs transform from a contractile to an anabolic state, disrupting the homeostasis that maintains vascular physiological function, rearranging the cytoskeleton, causing excessive proliferation of VSMCs, and their migration to the intima, leading to neointimal hyperplasia and restenosis.

[0003] Platelet-derived growth factor-BB (PDGF-BB), as a natural ligand of platelet-derived growth factor receptor-β (PDGFR-β), can activate downstream signaling pathways to promote the proliferation and migration of VSMCs and participate in angiogenesis, such as mitogen-activated protein kinases (MAPKs), eNOS-NO-cGMP, and phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathways. MAPKs regulate multiple cellular programs by transmitting extracellular signals to intracellular responses, mainly including extracellular signal-regulated kinases (ERK) 1 / 2, c-Jun terminal kinase (JNK), and p38 MAP kinase (p38MAPK) signaling pathways. Increasing evidence suggests that PDGF-BB triggers the MAPK signaling cascade, including the ERK1 / 2, p38 MAPK, and JNK signaling pathways, which participate in the proliferation and migration of VSMCs and promote VSMC phenotypic transformation.

[0004] Lycorine hydrochloride (LH) is an isoquinoline alkaloid extracted from the medicinal plant Lycoris radiata. As a derivative of lycorine, it has similar effects. Multiple studies have confirmed that lycorine possesses cardiovascular protective effects, including anti-inflammatory, anti-oxidative stress, improvement of myocardial injury, inhibition of myocardial fibrosis, and relief of cardiac dysfunction. However, whether LH affects injury-induced neointimal hyperplasia remains unclear. Therefore, this invention aims to investigate the role of LH in in vitro and in vivo intervention in neointimal hyperplasia and the regulation of VSMC proliferation, migration, and phenotypic transformation, providing valuable data for drug development in the prevention and treatment of restenosis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a pharmaceutical use of lycorine hydrochloride, specifically its application in the preparation of drugs that inhibit intravascular neointimal hyperplasia, and to explore the role of LH in regulating the proliferation, migration, and neointimal hyperplasia of VSMCs.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention relates to the application of lycorine hydrochloride in the preparation of drugs that inhibit intravascular neointimal hyperplasia.

[0008] Preferably, the intravascular neointimal hyperplasia is caused by the proliferation and migration of vascular smooth muscle cells.

[0009] Preferably, the vascular smooth muscle cell proliferation and migration refers to the proliferation and migration of vascular smooth muscle cells induced by platelet-derived growth factor-BB.

[0010] Preferably, the proliferation and migration of vascular smooth muscle cells refers to the phenotypic transformation of vascular smooth muscle cells from a contractile state to a synthetic state.

[0011] Preferably, the application is for the preparation of a drug for treating complications following percutaneous coronary intervention.

[0012] Preferably, the complication is restenosis or in-stent thrombosis.

[0013] Preferably, the pharmaceutical dosage of the lycorine hydrochloride is 0.125 μmol / L to 0.5 μmol / L.

[0014] Preferably, the drug comprises a compound pharmaceutical composition prepared from lycorine hydrochloride.

[0015] Preferably, the drug comprises a formulation prepared from lycorine hydrochloride.

[0016] Beneficial effects:

[0017] In vivo results showed that lycorine hydrochloride significantly inhibited vascular intimal hyperplasia induced by left common carotid artery ligation. In vitro experiments confirmed that lycorine hydrochloride intervened in PDGF-BB-induced VMSC proliferation, migration, and phenotypic transformation. Lycorine hydrochloride inhibits PDGF-BB-induced VSMC proliferation, migration, phenotypic transformation, and angiogenesis-induced intimal hyperplasia through the MAPK signaling pathway. Studies suggest that lycorine hydrochloride may be a potential drug for intervention in restenosis.

[0018] This invention provides pharmaceutical uses of lycorine hydrochloride, particularly evidence of its role in inhibiting intravascular neointimal hyperplasia. Lycorine hydrochloride inhibits neointimal hyperplasia primarily by suppressing the proliferation and migration of vascular smooth muscle cells. This invention provides reference data for the preparation of drugs using lycorine hydrochloride to inhibit intravascular neointimal hyperplasia, and holds promise for applications in the prevention and treatment of complications following percutaneous coronary intervention, such as restenosis or in-stent thrombosis. Attached Figure Description

[0019] 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. Wherein:

[0020] Figure 1 This is a detailed schematic diagram of the animal experiment provided in Embodiment 1 of the present invention. Wherein, Sham: sham-operated group; Ligation: left common carotid artery ligation group; AORA: aortic arch; ICA: internal carotid artery; ECA: external carotid artery; LCCA: left common carotid artery; LSA: left subclavian artery; RCC: right common carotid artery; RSA: right subclavian artery; BCT: brachiocephalic trunk; ip: intraperitoneal injection.

[0021] Figure 2 This is an H&E and Masson staining image of a paraffin section from Example 1 of the present invention.

[0022] The sample size was 6, and the scale bar was 100 μm.

[0023] Figure 3 This is a graph showing the ratio of the inner membrane area to the middle membrane area in Example 1 of the present invention.

[0024] Where *p<0.05, **p<0.01, ***p<0.001; the vertical axis in the figure represents the ratio of the inner membrane area to the middle membrane area.

[0025] Figure 4 This is an immunohistochemical staining image of ACTA2, OPN, and PCNA in Example 1 of the present invention.

[0026] The sample size was 6, and the scale bar was 100 μm.

[0027] Figure 5 This is an ImageJ analysis result of immunohistochemical staining in Example 1 of the present invention.

[0028] Where *p<0.05, **p<0.01, ***p<0.001; A represents the positive area ratio of ACTA2, B represents the relative positive area ratio of OPN, and C represents the relative positive area ratio of PCNA.

[0029] Figure 6 This is an immunofluorescence staining image used to detect ACTA2 expression in Example 1 of the present invention.

[0030] In this diagram, ACTA2 represents green, and DAPI represents the cell nucleus (blue); the sample size is 6, and the scale bar is 100 μm.

[0031] Figure 7 This is an ImageJ analysis result of immunofluorescence staining in Example 1 of the present invention.

[0032] Where *p<0.05, **p<0.01, ***p<0.001; the vertical axis represents the relative fluorescence intensity of ACTA2.

[0033] Figure 8 This is a graph showing the activity results of VSMCs detected by CCK-8 in Example 2 of the present invention.

[0034] In this diagram, Control represents the control group, and the vertical axis represents cell viability.

[0035] Figure 9 This is a diagram showing the CCK-8 assay results for PDGF-BB-induced MOVAS proliferation in Example 2 of this invention.

[0036] The vertical axis represents cell viability.

[0037] Figure 10 This is a graph showing the results of detecting the proliferation of PDGF-BB-induced MOVAS cells by different concentrations of LH using Edu in Example 2 of this invention. Edu represents green, Hoechst33342 represents the cell nucleus (blue), and the scale bar is 50 μm.

[0038] Figure 11 This is a bar chart showing the percentage of Edu-positive cells in Example 2 of the present invention. The vertical axis represents the percentage of Edu-positive cells.

[0039] Figure 12 This is a graph showing the results of a wound healing experiment in Example 2 of the present invention. Scale bar: 500 μm.

[0040] Figure 13This is a bar chart of cell migration rate in Embodiment 2 of the present invention. The vertical axis represents cell migration rate.

[0041] Figure 14 This is a Western blotting result from Example 3 of the present invention. MYH11, ACTA2, and SM22α are contractile proteins, OPN and PCNA are synthetic proteins, and GAPDH serves as a control.

[0042] Figure 15 This is a bar chart showing the relative protein levels of each treatment group in Example 3 of the present invention.

[0043] In this diagram, axis A represents the relative protein level of MYH11, axis B represents the relative protein level of ACTA2, axis C represents the relative protein level of SM22α, axis D represents the relative protein level of OPN, and axis E represents the relative protein level of PCNA.

[0044] Figure 16 This is a bar chart showing the relative mRNA levels of each treatment group in Example 3 of the present invention.

[0045] In this diagram, the vertical axis A represents the relative mRNA level of MYH11, the vertical axis B represents the relative mRNA level of ACTA2, the vertical axis C represents the relative mRNA level of SM22α, the vertical axis D represents the relative mRNA level of OPN, and the vertical axis E represents the relative mRNA level of PCNA.

[0046] Figure 17 This is a diagram showing the staining results of phalloidin in Example 3 of the present invention.

[0047] Figure 18 The image shows the Western blotting results and phosphorylation level bar chart in Example 4 of this invention.

[0048] In this figure, A is the result of Western blotting; B is the phosphorylation level bar chart.

[0049] Figure 19 This is a graph showing the results of CCK-8 assay for the activity of MAPKs inhibitors against MOVAS in Example 5 of this invention.

[0050] In this context, A represents the effect of PD98059 on MOVAS activity, B represents the effect of SP600125 on MOVAS activity, and C represents the effect of SB203580 on MOVAS activity.

[0051] Figure 20 This is a graph showing the results of CCK-8 assay in Example 5 of the present invention to detect the MAPKS inhibitor's effect on PDGF-BB-induced MOVAS proliferation.

[0052] Among them, A represents the effect of PD98059 on PDGF-BB-induced MOVAS proliferation, B represents the effect of SP600125 on PDGF-BB-induced MOVAS proliferation, and C represents the effect of SB203580 on PDGF-BB-induced MOVAS proliferation.

[0053] Figure 21 This is a graph showing the results of Edu's detection of the effect of MAPKS inhibitors on MOVAS proliferation in Example 5 of the present invention.

[0054] In this text, Edu represents green, Hoechst33342 represents the cell nucleus (blue), and the scale bar is 50 μm.

[0055] Figure 22 This is a bar chart showing the percentage of Edu-positive cells in Example 5 of the present invention.

[0056] Figure 23 This is a diagram showing the results of a wound healing experiment in Example 5 of the present invention.

[0057] Figure 24 This is a bar chart of cell migration rate in Example 5 of the present invention.

[0058] Figure 25 The images shown are Western blotting results and protein expression level histograms of MOVAS processed with PD98059 in Example 6 of this invention. In the image, A represents the Western blotting results, and B represents the protein expression level histogram.

[0059] Figure 26 The images shown are Western blotting results and protein expression level histograms of MOVAS processed with SP600125 in Example 6 of this invention. In the images, C represents the Western blotting results, and D represents the protein expression level histogram.

[0060] Figure 27 This is a Western blot analysis result and a bar chart showing the protein expression level of MOVAS processed with SB203580 in Example 6 of this invention. In this figure, E represents the Western blot analysis result, and F represents the protein expression level bar chart.

[0061] Figure 28 This is a diagram showing the staining results of phalloidin in Example 6 of the present invention.

[0062] Among them, fluorescent phalloidin stained green, and nuclear DAPI stained blue.

[0063] Figure 29This is a diagram illustrating the phenotype transformation and intima hyperplasia of VSMC induced by lycorine hydrochloride inhibiting the MAPKs signaling pathway, as well as by inhibiting MAPKs signaling pathway. Detailed Implementation

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0065] The present invention will now be described in detail with reference to embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0066] This invention addresses the existing problems by providing the application of lycorine hydrochloride in the preparation of drugs that inhibit intravascular neointimal hyperplasia.

[0067] In a preferred embodiment of the present invention, intravascular neointimal hyperplasia is caused by the proliferation and migration of vascular smooth muscle cells.

[0068] In a preferred embodiment of the present invention, the vascular smooth muscle cell proliferation and migration refers to the proliferation and migration of vascular smooth muscle cells induced by platelet-derived growth factor-BB.

[0069] In a preferred embodiment of the present invention, the proliferation and migration of vascular smooth muscle cells refers to the phenotypic transformation of vascular smooth muscle cells from a contractile state to a synthetic state.

[0070] In a preferred embodiment of the present invention, the application is for preparing a drug for treating complications following percutaneous coronary intervention.

[0071] In a preferred embodiment of the present invention, the complication is restenosis or in-stent thrombosis.

[0072] In a preferred embodiment of the present invention, the pharmaceutical dosage of lycorine hydrochloride is 0.125 μmol / L-0.5 μmol / L.

[0073] In a preferred embodiment of the present invention, the drug comprises a compound drug composition prepared from lycorine hydrochloride.

[0074] In a preferred embodiment of the present invention, the drug comprises a preparation made from lycorine hydrochloride.

[0075] The application of lycorine hydrochloride of the present invention in the preparation of drugs that inhibit intravascular neointimal hyperplasia is described in detail below through specific embodiments.

[0076] The lycorine hydrochloride used in this embodiment of the invention was purchased from MCE (HY-N0289). The chemical structure of lycorine hydrochloride is shown below. The molecular formula of lycorine hydrochloride is C1. 16 H 18 ClNO4, with a molecular weight of 323.77 g mol -1 .

[0077]

[0078] Example 1: Lycorine hydrochloride reduces neointimal hyperplasia induced by carotid artery ligation in mice.

[0079] This embodiment explores the effect of lycorine hydrochloride (LH) on neointimal formation in restenosis. A C57BL / 6J mouse model with left common carotid artery ligation was used to induce neointimal hyperplasia in mice. Figure 1 LH was injected intraperitoneally three days before surgery (at concentrations of 2.5 mg / kg and 5 mg / kg, respectively). Different concentrations of LH were injected intraperitoneally for three days after surgery. Samples were collected on 28 days after surgery, as detailed below.

[0080] The animal experiments involved in this embodiment were approved by the Animal Management Committee of the First Affiliated Hospital of Xinxiang Medical University (ethics approval number: 2020108 Henan Xinxiang). C57BL / 6 mice, male, 6–8 weeks old, weighing 20–22g, were purchased from Henan Skebest Biotechnology Co., Ltd. Housing conditions: room temperature 20–25℃, relative humidity 40%–70%. Free access to water and normal feed was provided under a 12-hour light-dark cycle.

[0081] For animal model construction, after one week of feeding, the mice were randomly divided into 4 groups (8 C57BL / 6J mice in each group): sham operation group, model group (Ligation), 2.5 mg / kg / day lycorine hydrochloride treatment group (ligation + LH 2.5 mg / kg / day group), and 5 mg / kg / day lycorine hydrochloride treatment group (ligation + LH 5 mg / kg / day group).

[0082] A mouse carotid artery ligation (CAL) model was established. Mice were anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg / kg) and placed in a supine position on the operating table. Hair on the neck was removed with depilatory cream, and the skin was disinfected with povidone-iodine. The skin was incised along the anterior midline of the neck, and the neck muscles were directly dissected. The left common carotid artery was separated and ligated proximal to the bifurcation using 6-0 silk suture, and the skin incision was sutured with 4-0 silk suture. In the sham-operated group, the left common carotid artery bifurcation was wrapped with 6-0 silk suture without ligation, while the model group underwent ligation. During drug administration, mice in both the sham-operated and model groups received an equal volume of physiological saline. Mice in the ligation + LH 2.5 mg / kg / day group and the ligation + LH 5 mg / kg / day group received intraperitoneal injections of different doses of lycorine hydrochloride, administered 3 days before and 3 days after surgery, for a total of 28 days. On the 28th day after surgery, carotid artery tissue was collected from 0.5 cm below the ligation point.

[0083] (1) H&E staining:

[0084] The collected carotid artery tissue was fixed with 4% paraformaldehyde and embedded in paraffin. The samples were sectioned transversely (4 μm). Sections were routinely dewaxed into water, stained with hematoxylin for 5 min, soaked in tap water for 15 min, stained with eosin for 2 min, washed, dehydrated, cleared, and mounted with neutral resin (G8590, Beijing Solarbio Science & Technology Co., Ltd.). Finally, the sections were observed and photographed under a microscope and analyzed using Image-J software.

[0085] Masson staining: Staining was performed according to the Masson trichrome staining kit (DC0033, Beijing Leigen Biotechnology). Carotid artery tissue sections were routinely dewaxed to water, then placed in Bouin's solution and incubated at 37°C for 2 hours for mordanting. The sections were then rinsed with running water until the yellow stain disappeared. Azurite blue staining solution was applied for 3 minutes; Mayer's hematoxylin staining solution for 3 minutes; acidic ethanol for differentiation for a few seconds; Ponceau S staining solution for 10 minutes; phosphomolybdic acid solution for 10 minutes; aniline blue staining solution for 15 minutes; weak acid solution for 2 minutes; rapid dehydration with ethanol and xylene; and mounting with neutral resin. Microscopic images were taken, and the intima to media ratio was calculated.

[0086] (2) Immunohistochemistry: Carotid artery tissue sections from different groups were dewaxed to water, immersed in 0.01M citrate buffer, heated to boiling in a microwave oven, cooled to room temperature, soaked in PBS containing 0.2% Triton X-100 for 15 min, treated with 3% H2O2 to quench endogenous peroxidase activity, blocked with 3% BSA for 1 hour, incubated with primary antibody ACTA2 at 4°C overnight, incubated with enzyme-labeled goat anti-rabbit IgG polymer secondary antibody at room temperature for 60 min, DAB (1:50) (GK500710, Genetech) was used for controlled staining, and hematoxylin counterstained the cell nuclei; the sections were dehydrated by anhydrous ethanol gradient, cleared with xylene, and mounted with neutral resin, photographed under a microscope, and analyzed using ImageJ. ACTA2 (GB111364), OPN (GB11500), and PCNA (GB11010) were purchased from Serviebio.

[0087] Immunofluorescence staining: Carotid artery tissue sections from different groups were dewaxed to water; washed in PBS for 5 minutes, then immersed in 0.01M citrate buffer, heated to boiling in a microwave oven, cooled to room temperature, and rinsed three times with PBS for 5 minutes each time; soaked in 0.2% Triton X-100 in PBS for 15 minutes, then soaked in 3% hydrogen peroxide, blocked with 5% BSA for 1 hour, then incubated with primary antibody (ACTA2) overnight at 4°C, and then incubated with secondary antibody (#4412S, CST) at room temperature for 60 minutes; and stained with DAPI for 10 minutes. Immunofluorescence images were captured using a confocal microscope and analyzed using ImageJ.

[0088] (3) Statistical analysis: Data were processed using GraphPad Prism 9.3.1 statistical software. Data were expressed as mean ± standard deviation. Independent samples t-test was used to compare two groups of continuous data. One-way ANOVA was used to compare multiple samples. P < 0.05 indicated that the difference was statistically significant.

[0089] (4) Results Analysis: H&E and Masson staining results showed that the intima area and the intima-to-media ratio were significantly increased in the model group, indicating collagen fiber deposition. Conversely, LH could inhibit neointima proliferation and collagen deposition. Figure 2-3 Immunohistochemical staining of ACTA2, OPN, and PCNA expression in carotid artery tissue showed that, compared to the sham-operated group, ACTA2 expression was decreased in the model group, while OPN and PCNA expression was upregulated. However, LH could reverse the expression trends of these marker proteins. Figure 4-5 The results of immunofluorescence detection were consistent with those of immunohistochemistry. Figure 6-7These results indicate that LH has an inhibitory effect on angiogenesis and intimal hyperplasia, and that LH reduces the formation of neointimal hyperplasia by inhibiting the phenotypic transformation of VSMCs.

[0090] Example 2: Lycorine hydrochloride inhibits PDGF-BB-induced proliferation and migration of VSMCs

[0091] In vitro experiments were conducted by constructing a PDGF-BB-induced phenotype transformation model of VSMCs and using LH intervention, as detailed below.

[0092] Reagents: PDGF-BB (#220BB-010, R&D Systems) was purchased from R&D; PD98059 (ERK inhibitor, 167869-21-8), SP600125 (JNK inhibitor, 129-56-6), and SB203580 (p38MAPKS inhibitor, 152121-47-6) were purchased from Selleck. Cell counting kit-8 (CCK-8) was purchased from Beijing Aoqing Biotechnology Co., Ltd. (AQ308).

[0093] Cell culture: MOVAS, mouse aortic smooth muscle cells (Beina Biotechnology, BNCC338213). MOVAS cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 humidity incubator. Cells were passaged when they reached 80-90% confluence. In all PDGF-BB-induced MOVAS phenotypic transformation experiments, all groups of cells were starved for 24 h using DMEM medium containing 0.5% FBS and 1% penicillin-streptomycin before the addition of PDGF-BB.

[0094] (1) CCK-8 detection:

[0095] MOVAS was seeded into 96-well plates (2 × 10⁶ per well). 3 Cells were pretreated with different concentrations (0.125 μM-2 μM) of lycorine hydrochloride for 2 hours, followed by stimulation with or without PDGF-BB (20 ng / ml) for 24 hours. The cell culture medium was changed before detection, and 10 μL of CCK-8 reagent was added to the plate. Cells were incubated for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula: (OD of treated group - OD of blank group) / (OD of control group - OD of blank group) × 100%.

[0096] First, the effect of LH on MOVAS activity was analyzed. CCK-8 assay results showed that at a concentration of 1 μM, the survival rate of VSMCs was significantly reduced. Figure 8The experimental groups were as follows: Control group: no lycorine hydrochloride treatment; Treatment groups: 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM lycorine hydrochloride treatment groups.

[0097] Therefore, this invention selected 0.625 μM, 0.125 μM, 0.25 μM, and 0.5 μM concentrations of LH to intervene in VSMCs. The CCK-8 assay was used to detect the effect of different concentrations of LH on PDGF-BB-induced MOVAS proliferation. LH dose-dependently inhibited PDGF-BB-induced VSMC proliferation, with 0.5 μM LH showing the most significant effect. Figure 9 The experimental groups were as follows: Control group: treated with PDGF-BB only; Blank group: treated with neither PDGF-BB nor lycorine hydrochloride; Treatment groups: treated with PDGF-BB and 0.625 μM, 0.125 μM, 0.25 μM, and 0.5 μM lycorine hydrochloride respectively.

[0098] (2) Edu detection:

[0099] Cell proliferation was determined using an Edu cell proliferation assay kit (KTA2030, Abbkine). Cells were seeded in 96-well plates. 2×Edu serum-free medium working solution (20 μM) was prepared in centrifuge tubes and added to the cells containing the same volume of medium to a final concentration of 10 μM. The cells were incubated for 2 h. 100 μl of 3.75% paraformaldehyde was added to each well for fixation at room temperature for 15 min. The cells were then incubated with 0.5% Triton X-100 at room temperature for 15 min. 100 μl of the prepared mixture (according to the manufacturer's instructions) was added, and the cells were incubated at room temperature for 30 min in the dark. Hoechst 33342 (5 μg / ml) was added for staining, and the cells were incubated at 37°C for 15 min. Images were taken using a confocal microscope (Nikon AX, Japan). The percentage of Edu-positive cells was calculated.

[0100] Further analysis was conducted on the inhibitory effect of LH on PDGF-BB-induced MOVAS proliferation. Edu experiments were performed on VSMCs. MOVAS were pretreated with different concentrations of LH (0.125 μM–0.5 μM) for 2 h, followed by stimulation with PDGF-BB (20 ng / ml) for 24 h. The results were consistent with those of CCK-8 proliferation experiments. Figure 10-11 The experimental groups were as follows: Control group: no PDGF-BB and lycorine hydrochloride treatment; 0.5 μM lycorine hydrochloride treatment alone; PDGF-BB treatment alone; and PDGF-BB and 0.125 μM, 0.25 μM, and 0.5 μM lycorine hydrochloride co-treatment groups, respectively.

[0101] (3) Wound healing experiment:

[0102] First, using a marker, draw evenly horizontal lines on the back of a 6-well plate, aligned with a ruler, ensuring each well has at least three lines. Seed MOVAS cells in the 6-well plates and culture to 90% confluence, then starve the cells with 0.5% fetal bovine serum (FBS) for 24 hours. Scrape the monolayer cells with a 200 μl pipette tip. Then treat the cells with PDGF-BB (20 ng / ml) and different concentrations of lycorine hydrochloride (0.125 μM–0.5 μM), respectively. Images were taken at 0 and 12 hours using an inverted microscope (Nikon TS2-S-SM, Japan). Cell migration rate = (0h scratch width - 12h scratch width) / 0h scratch width × 100%. Cell migration area was calculated using ImageJ.

[0103] Wound healing assays were conducted to analyze the migration rate of MOVAS after LH treatment, clarifying the effect of LH on smooth muscle cell migration. The results showed that LH significantly inhibited PDGF-BB-induced MOVAS migration. Figure 12-13 The experimental groups were as follows: Control group: no PDGF-BB and lycorine hydrochloride treatment; 0.5 μM lycorine hydrochloride treatment alone; PDGF-BB treatment alone; and PDGF-BB and 0.125 μM, 0.25 μM, and 0.5 μM lycorine hydrochloride co-treatment groups, respectively.

[0104] Example 3: Lycorine hydrochloride inhibits PDGF-BB-induced phenotype transformation of VSMCs

[0105] When blood vessels are injured, VSMCs transform from a contractile to a synthetic form (proliferation and migration). The expression of contractile proteins (such as ACTA2, SM22α, and MYH11) decreases, while the expression of synthetic proteins (OPN and PCNA) increases. In this example, Western blot and qRT-PCR were used to detect the effects of PDGF-BB (20 ng / ml) and different concentrations of LH (0.125 μM–0.5 μM) alone or in combination on MOVAS stimulated for 24 h. Details are as follows:

[0106] (1) RNA extraction and real-time quantitative PCR analysis

[0107] MOVAS from different treatment groups were lysed and extracted using TRIzol reagent (Invitrogen), and the concentration was determined using RNAIMPLEN (N80 Touch, Germany). RNA quantification was performed using reverse transcription reagents according to the instructions of the reverse transcription kit (Takara). qPCR was performed using SYBR Green (Takara), and detection was performed using a real-time quantitative PCR instrument (Quantstudio 6Flex). Gene primer sequences are shown in Table 1.

[0108] Table 1: Gene Primer Sequences

[0109]

[0110] Real-time PCR: The mRNA expression level of the target gene was detected using the TaKaRa qPCR kit TB Green Premix Ex Taq II, with ACTIN as an internal control. -ΔΔCt Calculate the relative expression of the target gene. ΔCt = Ct target gene - Ct internal reference, ΔΔCt = ΔCt treatment group - ΔCt control group.

[0111] (2) Western blot

[0112] MOVAs cells in different treatment groups were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. Cells were placed on ice and sonicated three times to ensure complete lysis, followed by centrifugation at 12000g for 15 min at 4°C. The supernatant was collected, and protein content was determined using a BCA protein assay kit. Equal volumes of protein were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF membrane via wet transfer and blocked in TBST blocking buffer containing 5% skim milk at room temperature for 1 h. Primary antibody was incubated overnight with gentle shaking on a horizontal shaker at 4°C. Secondary antibody was incubated at room temperature for 1 h, and finally, imaging was performed on a chemiluminescence imaging analyzer (Amersham ImageQuant 800, Cytiva). Relative quantification was performed using Image-J software.

[0113] ACTA2 (#19245) and GAPDH (#5174) were purchased from CST; MYH11 (ab53219), SM22α (ab14106), and OPN (ab8448) were purchased from Abcam; ERK1 / 2 (phospho-Y204, AP0490), ERK1 / 2 (phospho-T202, BS4759), and c-Jun (phospho-S73, BS4046) were purchased from Bioworld Technology; and phospho-p38 (Thr180 / Tyr182, 28796-1-AP), p38 (14064-1-AP), ERK1 / 2 (11257-1-AP), and JUN (24909-1-AP) were purchased from Wuhan Sanying.

[0114] (3) Phalloidin staining

[0115] MOVAS cells treated with different methods were fixed on ice with 4% paraformaldehyde for 15 min, then permeabilized in PBS with 0.5% Triton X-100 for 10 min at room temperature. Each well was stained with 200 μl of SF488-labeled phalloidin working solution (CA1640, Beijing Solarbio Science & Technology Co., Ltd.) diluted in PBS and incubated at room temperature for 20 min, followed by staining with DAPI (C1002, Beyotime) for 10 min. Cells were washed three times with PBS and photographed using a confocal fluorescence microscope to compare F-actin morphology.

[0116] (4) Results Analysis

[0117] The results showed that LH inhibited PDGF-BB-induced OPN and PCNA expression, but promoted PDGF-BB-induced ACTA2, SM22α, and MYH11 expression. Figure 14-16 The experimental groups were as follows: Control group: no PDGF-BB and lycorine hydrochloride treatment; 0.5 μM lycorine hydrochloride treatment alone; PDGF-BB treatment alone; and PDGF-BB and 0.125 μM, 0.25 μM, and 0.5 μM lycorine hydrochloride co-treatment groups, respectively.

[0118] Phalloidin staining showed that PDGF-BB promoted the rearrangement of the cytoskeletal protein F-actin, remodeling microfilaments from a sparse to a dense arrangement. LH, however, reversed PDGF-BB-induced MOVAS actin disorder. Figure 17 In vitro studies have shown that LH inhibits PDGF-BB-induced VSMC phenotypic transformation. The experimental groups were: Control group (no PDGF-BB and lycorine hydrochloride treatment); PDGF-BB alone treatment group; and PDGF-BB and 0.5 μM lycorine hydrochloride co-treatment group.

[0119] Example 4: Lycorine hydrochloride inhibits PDGF-BB-induced proliferation and migration of VSMCs via the MAPK signaling pathway.

[0120] Studies have shown that PDGF-BB activates the MAPK (ERK1 / 2, p38MAPK, JNK) signaling pathway, which is involved in the proliferation and migration of VSMCs. To clarify the role of LH in the above signaling pathway, the phosphorylation expression of each protein in the MAPK (ERK1 / 2, JNK, p38MAPK) signaling pathway was detected in PDGF-BB-stimulated MOVAS. After treating MOVAS with LH at a concentration of 0.5 μM for 2 h, PDGF-BB (20 ng / ml) was applied to MOVAS for 24 h. Western blotting was used to analyze the protein expression levels of p-ERK1 / 2 / ERK1 / 2, p-JNK / JNK, and p-P38 / P38, as well as the phosphorylation levels of ERK1 / 2, JNK, and p38MAPKS.

[0121] The results showed that LH treatment significantly reduced the phosphorylation of ERK1 / 2, JNK, and p38MAPK in MOVAS, but the total protein levels of ERK1 / 2, JNK, and p38MAPK remained unchanged. Figure 18 The experimental groups were as follows: Control group: no treatment with PDGF-BB and lycorine hydrochloride; PDGF-BB treatment alone; and PDGF-BB and 0.5 μM lycorine hydrochloride treatment together.

[0122] Example 5: Lycorine hydrochloride inhibits PDGF-BB-induced VSMC phenotypic transformation via the MAPK signaling pathway.

[0123] To investigate whether LH inhibits VSMC proliferation and migration through the ERK1 / 2, JNK, and p38MAPK signaling pathways, cells were treated with inhibitors of these pathways, PD98059, SP600125, and SB203580, respectively. The toxicity of PD98059, SP600125, and SB203580 to MOVAS was assessed using a CCK-8 assay. After pretreatment with 0.5 μL LH, PD98059, SP600125, and SB203580 for 2 h, MOVAS were incubated with PDGF-BB (20 ng / ml) for 24 h, and cell proliferation and migration were then evaluated.

[0124] The results showed that PD98059, SP600125, and SB203580 had no toxic effects on VSMCs when their concentrations were less than or equal to 10 μM, 5 μM, and 5 μM, respectively. Figure 19Therefore, 10 μM, 5 μM, and 5 μM were selected in subsequent experiments. Additionally, after 2 h of pretreatment with MAPKs inhibitors, MOVAS were stimulated with PDGF-BB for 24 h. The effect of the inhibitors on MOVAS proliferation was detected by CCK-8 assay. The results showed that, compared with the control group, inhibitor treatment could inhibit PDGF-BB-induced proliferation of VSMCs. Figure 20 ).

[0125] Compared with PDGF-BB treated MOVAS, in the presence of LH or PD98059, SP600125, SB203580, Edu assay and wound healing assay showed that MOVAS proliferation and migration were significantly reduced. Figure 21-24 The experimental groups were as follows: Control group: no treatment; PDGF-BB alone; PDGF-BB and 0.5 μM lycorine hydrochloride co-treatment group; PDGF-BB and 10 μM PD98059 co-treatment group; PDGF-BB and 5 μM SP600125 co-treatment group; PDGF-BB and 5 μM SB203580 co-treatment group.

[0126] Example 6: Mechanism of Lycorine Hydrochloride Inhibiting MAPK Signaling Pathway and Regulating PDGF-BB-Induced VSMC Phenotypic Transformation

[0127] To verify how LH inhibits phenotype transformation of VSMCs through the MAPK (ERK1 / 2, JNK, P38) signaling pathway. The protective effects of MAPKs and LH on VSMCs were confirmed using 10 μM PD98059, 5 μM SP600125, and 5 μM MSB203580, inhibitors of the MAPK (ERK1 / 2, JNK, P38) signaling pathway.

[0128] MOVAS were treated with PD98059, and the expression of MYH11, ACTA2, SM22α, PCNA, OPN, and p-ERK1 / 2 proteins was detected by Western blotting. The experimental groups were: Control group (no treatment); PDGF-BB alone; PDGF-BB and 0.5 μM lycorine hydrochloride co-treatment group; and PDGF-BB and 10 μM PD98059 co-treatment group.

[0129] MOVAS were treated with SP600125, and the expression of MYH11, ACTA2, SM22α, PCNA, OPN, and p-JNK proteins was detected by Western blotting. The experimental groups were: Control group (no treatment); 20 ng / ml PDGF-BB alone; 20 ng / ml PDGF-BB and 0.5 μM lycorine hydrochloride co-treatment group; and 20 ng / ml PDGF-BB and 5 μM SP600125 co-treatment group.

[0130] MOVAS were treated with SB203580, and the expression of MYH11, ACTA2, SM22α, PCNA, OPN, and p-P38 proteins was detected by Western blotting. The experimental groups were: Control group (no treatment); 20 ng / ml PDGF-BB alone; 20 ng / ml PDGF-BB and 0.5 μM lycorine hydrochloride co-treatment group; and 20 ng / ml PDGF-BB and 5 μM SB203580 co-treatment group.

[0131] Western blot results showed that MAPK inhibitors promoted the expression of ACTA2, SM22α, and MYH11 proteins, while the expression of OPN and PCNA proteins was inhibited. Figure 25-27 Furthermore, phalloidin staining revealed that MAPKs inhibitors reversed PDGF-BB-induced F-actin rearrangement in the MOVAS cytoskeleton. Figure 28 These results indicate that LH inhibits phenotypic transformation of VSMCs and is associated with the activation of ERK1 / 2, JNK, and p38MAPK signaling.

[0132] The present invention relates to the regulation of PDGF-BB-induced VSMC phenotypic transformation and intimal hyperplasia by lycorine hydrochloride inhibiting the MAPKs signaling pathway. See attached diagram for details. Figure 29 .

[0133] Discussion: Lycorine hydrochloride (LH), a derivative of lycorine, possesses significant anti-inflammatory, anti-oxidative stress, anti-cardiomyocyte fibrosis, and cardiac dysfunction-relieving effects. Although progress has been made in understanding the role of LH in cardiovascular diseases, its effects on the proliferation and migration of VSMCs and its underlying mechanisms remain unclear. This invention demonstrates that LH can inhibit neointimal hyperplasia induced by ligation of the common carotid artery in vivo and participate in regulating smooth muscle cell phenotypic transformation.

[0134] Phenotypic transformation of VSMCs is one of the early pathological factors in atherosclerosis and intimal hyperplasia. In healthy and mature VSMCs, α-SMA, SM22α, and MYH11 contractile proteins are highly expressed, regulating vascular tone and blood pressure. In vitro experiments have demonstrated for the first time that LH inhibits PDGF-BB-induced VSMC proliferation, migration, and phenotypic transformation. Mechanistic studies then suggest that LH may inhibit VSMC proliferation, migration, and phenotypic transformation through the MAPK signaling pathway. Intravascular restenosis is mainly due to excessive proliferation and migration of VSMCs to the intima, leading to neointimal hyperplasia, which alters the composition and structure of the vessel wall and accelerates restenosis after PCI and stent implantation. In this study, in vitro experiments involved LH intervention in PDGF-BB-induced MOVAS phenotypic transformation, and in vivo experiments used a mouse model of carotid artery ligation via intraperitoneal injection of LH. Therefore, LH inhibits VSMC phenotypic transformation and intimal hyperplasia.

[0135] MAPKs, members of the mitogen-activated serine / threonine kinase family including ERK1 / 2, JNK, and p38MAPK, are reported to regulate VSMC proliferation and migration, thereby participating in intimal hyperplasia. Won SM et al. found that catechins participate in VSMC proliferation and migration through MAPKs. Won KJ et al. demonstrated that desalted *Salicornia europaea* extract can regulate VSMC proliferation and migration in vitro via the MAPK pathway and regulate pathological intimal hyperplasia in vivo. Whether this pathway is involved in the anti-restenosis effect of LH remains unclear. In this invention, LH intervention in PDGF-BB-induced MOVAS showed that, compared with the control group, PDGF-BB induced the activation of ERK1 / 2, p38MAPK, and JNK. Compared with the PDGF-BB group, LH inhibited the phosphorylation activation of ERK1 / 2, p38MAPK, and JNK, but the total protein levels of ERK1 / 2, p38MAPK, and JNK did not change. This indicates that LH inhibits VSMC proliferation and migration by inhibiting the MAPK pathway. PD98059, SP600125, and SB203580 pathway inhibitors have been shown to suppress the MAPK signaling pathway. This invention found that treatment of VSMCs with PD98059, SP600125, and SB203580 promoted the expression of ACTA2, SM22α, and MYH11 proteins, respectively, while inhibiting the expression of OPN and PCNA proteins.

[0136] In summary, LH inhibits PDGF-BB-stimulated VSMC proliferation and migration by regulating the MAPK signaling pathway, thereby reducing endometrial hyperplasia. Figure 29This invention demonstrates for the first time that LH can inhibit the proliferation and migration of VSMCs in vitro and in vivo, effectively intervening in the progression of intimal hyperplasia. The key mechanism by which LH inhibits the proliferation and migration of PDGF-BB-stimulated VSMCs by regulating the ERK1 / 2, p38MAPK, and JNK pathways is its ability to prevent restenosis after arterial injury. Therefore, LH could be an effective drug for preventing restenosis after arterial injury.

[0137] Therefore, lycorine hydrochloride can be used to prepare drugs that inhibit intravascular intimal hyperplasia, as follows:

[0138] Application of lycorine hydrochloride in the preparation of drugs that inhibit intravascular neointimal hyperplasia.

[0139] The intravascular neointimal hyperplasia is caused by the proliferation and migration of vascular smooth muscle cells.

[0140] The proliferation and migration of vascular smooth muscle cells refers to the proliferation and migration of vascular smooth muscle cells induced by platelet-derived growth factor-BB.

[0141] The proliferation and migration of vascular smooth muscle cells refers to the phenotypic transformation of vascular smooth muscle cells from a contractile state to a synthetic state.

[0142] The application is for the preparation of drugs to treat complications following percutaneous coronary intervention.

[0143] The complications are restenosis or in-stent thrombosis.

[0144] In the aforementioned pharmaceutical applications, the pharmaceutical dosage of lycorine hydrochloride is 0.125 μmol / L to 0.5 μmol / L. The drug includes compound pharmaceutical compositions prepared from lycorine hydrochloride. The drug also includes formulations prepared from lycorine hydrochloride.

[0145] 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 lycorine hydrochloride in the preparation of drugs for treating restenosis caused by neointimal hyperplasia in blood vessels.

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

3. The application as described in claim 2, characterized in that, The proliferation and migration of vascular smooth muscle cells refers to the proliferation and migration of vascular smooth muscle cells induced by platelet-derived growth factor-BB.

4. The application as described in claim 2, characterized in that, The proliferation and migration of vascular smooth muscle cells refers to the phenotypic transformation of vascular smooth muscle cells from a contractile state to a synthetic state.

5. The application as described in claim 1, characterized in that, The restenosis mentioned is a complication of percutaneous coronary intervention.

6. The application as described in any one of claims 1-5, characterized in that, The pharmaceutical dosage of the lycorine hydrochloride is 0.125 μmol / L to 0.5 μmol / L.

7. The application as described in any one of claims 1-5, characterized in that, The drug includes a compound drug composition prepared from lycorine hydrochloride.

8. The application as described in any one of claims 1-5, characterized in that, The drug includes preparations made from lycorine hydrochloride.

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