Application of isoforskolin in preparation of medicine for inhibiting atherosclerosis

Abstract

The invention discloses an application of isoforskolin in inhibition of atherosclerosis, belongs to the technical field of medicines, and is used for preparing medicines for inhibiting atherosclerosis through the effect of adenylate cyclase agonist isoforskolin in inhibition of endothelial cells in atherosclerosis to interstitial substance. According to the medicine, the expression of endothelial markers CD31 and E-cadherin is increased through ISOF, the expression of N-cadherin and alpha-SMA of interstitial manufacturers is reduced, and in-vivo and in-vitro TGF-beta / Smad3 signal channels are inhibited; furthermore, expression of AC5 is up-regulated through ISOF, downstream PKA is activated, but exchange protein-1 (EPAC1) activated by cAMP is not activated. However, when AC5 is knocked out, the influence of ISOF on EndMT and cAMP / PKA-TGF-beta / Smad3 pathways is canceled, and a pathway and a medicine for treating atherosclerosis are provided.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of isofroskolin in preparing a drug for inhibiting atherosclerosis. Background Art

[0002] Atherosclerosis is a lipoprotein-driven chronic inflammatory disease involving multiple cell types, including endothelial cells (EC), smooth muscle cells (SMC), and immune cells, which are involved in pathological activation at different stages. Atherosclerosis has become a global public disease that threatens human health. At present, although the best treatment strategies, including risk factor control, lifestyle management, lipid-lowering therapy with statins and PCSK9 blocking antibodies, have been applied in clinical practice, atherosclerosis remains a global public health problem. Endothelial-to-mesenchymal transition (EndMT) has been identified as a key driver of atherosclerosis, and TGF-β is a key mediator of EndMT. Cyclic adenylate cyclase has the potential to regulate a variety of physiological responses because its catalytic product, cyclic adenosine monophosphate (cAMP), can regulate a variety of cellular functions. Some phosphodiesterase (PDE) inhibitors that degrade cAMP have been used clinically to treat atherosclerosis.

[0003] EndMT has emerged as a key driver of atherosclerosis, which may exacerbate plaque growth and instability. Vascular endothelial cells gradually lose their barrier function, but their proliferation and migration abilities are enhanced as those of mesenchymal cells. Subsequently, the accumulation of the mesenchymal cell pool secretes proinflammatory molecules, synthesizes extracellular matrix proteins (ECM) and metalloproteinases, thereby promoting plaque formation and vascular fibrosis. Currently, methods to inhibit EndMT or regulate pathways through drug inhibitors, miRNAs, nanoparticle-based approaches, or mesenchymal stem cell-based intervention strategies have been reported as a promising approach to alleviate atherosclerosis. However, several limiting factors, such as unnecessary side effects, poor selectivity, and heterogeneous responses in different organs, have hindered their clinical application. Therefore, the development of new effective targets or drugs that inhibit EndMT for the treatment of atherosclerosis is urgent.

[0004] Adenylate cyclases (ACs) are a family of enzymes responsible for catalyzing the conversion of adenosine triphosphate (ATP) to 3',5'-cyclic adenosine monophosphate (cAMP), an important second messenger in the living world. As the core effector molecule of βAR signaling, AC plays a crucial role in cardiovascular activation processes, including cardiac contractility, relaxation, left ventricular diastolic function, vascular reactivity, and tone. In mammals, cells express nine transmembrane AC isoforms (AC1-9) and one soluble AC isoform (AC10), which act as "bridges" to respond to stimuli and link extracellular signals to intracellular effectors. The expression of each AC isozyme varies in tissue distribution and regulatory properties, making them potential drug targets in different diseases. Intracellular cAMP concentrations are finely regulated by AC and phosphodiesterase (PDE) activities. PDE inhibitors such as vinpocetine, dipyridamole, and cilostazol have been shown to be effective drugs for the treatment of atherosclerosis because they promote vasodilation and inhibit lipid deposition and platelet aggregation by increasing cAMP concentration. Recently, studies have shown that specific PDE4B inhibitors inhibit EndMT by stimulating cAMP / PKA. However, there is still a lack of evidence that AC agonists can treat atherosclerosis by directly catalyzing cAMP generation.

[0005] Isoforskolin (ISOF) is an active compound extracted from Coleus forskohlii, which is native to Yunnan, China. It is an effector substance that enhances intracellular cAMP by activating AC. It has been reported that ISOF can downregulate inflammatory responses and proinflammatory cytokines by activating AC and cAMP, thereby alleviating lung diseases. However, whether ISOF can become a new type of anti-atherosclerotic drug candidate has not been studied. Chinese patent CN113244405A- discloses a pharmaceutical composition of forskolin-isoforskolin and pentacyclic triterpenoid compounds and its application, and the drug combination is used to prevent and treat metabolic diseases, fibrotic diseases and liver diseases.

[0006] Currently, there is insufficient evidence for the effectiveness and mechanism of AC agonists in treating atherosclerosis. We hypothesized that ISOF, as an AC agonist, could become a candidate drug for treating atherosclerosis through its ability to activate cAMP-related signaling pathways. We evaluated the efficacy of ISOF on the APOE- / - rat model of atherosclerosis induced by a high-fat diet to study the application of ISOF in the treatment of atherosclerosis. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention explores the therapeutic effect of ISOF on atherosclerosis, provides the use of isofroskolin in the preparation of a drug for inhibiting atherosclerosis, and provides a promising inhibitory drug for the treatment of atherosclerosis, as follows:

[0008] Apolipoprotein E - / - Rats were fed a high-fat diet for 18 months and then gavaged with ISOF for 12 months. Mouse aortic endothelial cells (MAECs) were injured with oxidized-LDL to establish a cell model. Oil red O staining and Masson staining were used to assess plaque area and collagen content. Aortic vasodilation function was analyzed by the DMT Myograph system. Immunofluorescence was used to determine the localization of CD31 and α-SMA. Proteomic analysis was used to determine the potential pharmacological mechanism of ISOF. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of AC(1-10) in aortic tissues. AC activity and cAMP concentration were detected using kits. CCK-8 assay, wound healing assay, and transwell assay were used to measure cell viability and proliferation. Mechanistically, Western blot analysis was used to detect the expression level of candidate proteins. Finally, pharmacological knockdown of AC5 was used to elucidate the potential mechanism of ISOF.

[0009] Results ISOF could effectively inhibit the expression of apolipoprotein E in high-fat diet - / - The development of atherosclerotic plaques in rats and improved aortic vasodilation. At the cellular level, ISOF promoted cell viability and proliferation of MAECs injured by ox-LDL. These phenotypic improvements were attributed to the inhibitory effect of ISOF on EndMT: ISOF increased the expression of endothelial markers CD31 and E-cadherin and decreased the expression of mesenchymal markers N-cadherin and α-SMA. Mechanistically, ISOF significantly inhibited the TGF-β / Smad3 signaling pathway in vivo and in vitro, which was consistent with the results of proteomic analysis. In addition, the expression level of AC5 in rat aorta was significantly higher than that of other AC isozymes. ISOF could upregulate the expression of AC5 and subsequently activate downstream PKA, but not cAMP-activated exchange protein-1 (EPAC1). However, when AC5 was knocked out, the effects of ISOF on EndMT and the cAMP / PKA-TGF-β / Smad3 pathway were suppressed.

[0010] In summary, ISOF can inhibit endothelial-mesenchymal transition by regulating the AC5-dependent cAMP / PKA-TGF-β / Smad3 axis, thereby effectively preventing and treating atherosclerosis, providing a promising method for the treatment of atherosclerosis.

[0011] Through the above experiments and conclusions, the present invention discloses the following applications of isofroskolin:

[0012] Application of isofroskolin in the preparation of drugs for inhibiting endothelial cell to mesenchymal transition.

[0013] Use of isofroskolin in preparing a drug for increasing the expression of endothelial marker CD31.

[0014] Use of isofroskolin in preparing a drug for increasing the expression of endothelial marker E-cadherin.

[0015] Use of isofroskolin in preparing drugs for inhibiting the expression of N-cadherin and / or α-SMA.

[0016] Use of isofroskolin in preparing a drug for promoting the expression of AC5.

[0017] Application of isofroskolin in the preparation of drugs for inhibiting atherosclerosis.

[0018] Furthermore, the dosage of isofroskolin is 2 mg / kg.

[0019] The working principle of the present invention is introduced as follows: Endothelial-to-mesenchymal transition (EndMT) is a key driving factor of atherosclerosis. Isoforskolin (ISOF) is a natural active compound of diterpenes, which can effectively activate adenylate cyclase (AC) and catalyze the production of cAMP. ISOF increases the expression of endothelial markers CD31 and E-cadherin, and reduces the expression of endothelial-mesenchymal markers N-cadherin and α-SMA; ISOF significantly inhibits the TGF-β / Smad3 signaling pathway in vivo and in vitro. ISOF also activates the cAMP / PKA signaling pathway and inhibits endothelial-mesenchymal transition by upregulating the expression of AC5, thereby effectively alleviating atherosclerosis.

[0020] Compared with the prior art, the beneficial effects of the present invention are: the intracellular cAMPs level is regulated by AC and PDE, and PDE inhibitors have been found to promote vasodilation, inhibit lipid deposition and platelet aggregation, and have been clinically used to treat atherosclerosis. However, there is a lack of evidence that AC agonists are used to treat atherosclerosis. This study first discovered that ISOF, an active ingredient of Coleus forskohlii produced in Yunnan, can excite AC5, a key AC subtype, excite the cAMP / PKA signaling pathway, inhibit the TGF-β / Smad3 signaling pathway, and then inhibit the endothelial-mesenchymal transition, thereby playing a role in alleviating atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The experimental results of Example 2 are shown in Figure 2 (Ach-induced relaxation is expressed as a percentage before contraction by U46199 (1 μmol / L) (n=7). Data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. AF and H are one-way ANOVA tests followed by Tukey's multiple comparison test; G is two-way ANOVA test followed by Tukey's multiple comparison test.);

[0022] in:

[0023] (AB) Representative images and quantification results of Oil Red O staining of whole aorta (n = 6);

[0024] (CD) Representative images and quantification of Oil Red O staining of aortic sinus (n = 6);

[0025] (EF) Representative images and Masson staining quantification of aortic sinus (n = 6);

[0026] (GH) Cumulative concentration-response curves and quantification of the maximum vasodilatory effect (Emax) of choline (Ach)-induced vascular relaxation in isolated aorta;

[0027] (IJ) Western immunoblotting results of p-eNOS and eNOS.

[0028] Figure 2 The figure is the experimental result of Example 3 (quantitative real-time polymerase chain reaction (qRT-PCR));

[0029] Among them: *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001; one-way ANOVA test followed by Tukey's multiple comparison test (AG);

[0030] (AE) Western immunoblotting results of CD31, E-cadherin, N-cadherin, and α-SMA in the aorta;

[0031] (F) Representative images of double immunofluorescence labeling of CD31 and α-SMA in aorta (n=6), and data are presented as mean±SEM.

[0032] Figure 3 It is the experimental result figure of Example 4;

[0033] in:

[0034] (A) CCK-8 was used to measure the cell viability of MAECs with different concentrations of ox-LDL (0-320 μg / ml, 24 h) (n=6);

[0035] (B) CCK8 was used to detect the cell viability of ox-LDL-damaged MAECs (40 μg / ml ox-LDL) with different concentrations of ISOF (n=6);

[0036] (CD) Representative images and quantitative results of in vitro wound healing assay (N=6). (EF) Representative images and quantitative results of Transwell assay (n=6);

[0037] (GH) Western blot analysis was used to detect the protein expressions of CD31, E-cadherin, N-cadherin and α-SMA in aorta (n=6).

[0038] Figure 4 This is a diagram of the experimental results of Example 5 of the present invention;

[0039] in:

[0040] (A) Heat map showing the correlation between samples;

[0041] (B) Volcano plot of differentially expressed proteins (DEPs) in different groups;

[0042] (C) Venn diagram showing that ISOF reversed 35 upregulated DEPs and 35 downregulated DEPs in the model group;

[0043] (D) Hierarchical clustering heat map of proteins;

[0044] (E) Bubble plot showing the top 15 enriched factors of KEGG pathways;

[0045] (FG) Expression of TGF-β, p-Smad3, and Smad3 in aortic tissue (n=6);

[0046] (HI) Expression of TGF-β, p-Smad3, and Smad3 in MAECs (n=6).

[0047] Figure 5 This is a diagram of the experimental results of Example 6 of the present invention;

[0048] Wherein: (A) mRNA levels of AC isozymes (1-10) in rat aorta were determined by quantitative real-time polymerase chain reaction (qRT-PCR; n = 4);

[0049] (BC) Western blot analysis showing AC5 protein levels (n=6);

[0050] (D) cAMP concentration in aortic tissue homogenate (n = 8);

[0051] (EG) Expression of EPAC1 and PRKACA in aortic tissues (n=6).

[0052] Figure 6 This is a diagram of the experimental results of Example 7 of the present invention;

[0053] (AB) Expression of AC5 in MAECs;

[0054] (C) Intracellular cAMP expression level (n=6);

[0055] (D) Concentration-response curve of ISOF stimulation of MAECs. (n=6);

[0056] (EG) Expression of EPAC1 and PRKACA in MAECs (n=6).

[0057] Figure 7 This is a diagram of the experimental results of Example 8 of the present invention;

[0058] Among them: (AJ) Expression of AC5, EPAC1, PRKACA, TGF-β, p-Smad3, Smad3, CD31, E-cadherin, N-cadherin, and α-SMA in MAECs (n=4);

[0059] (KL) Representative images and quantification results of in vitro wound healing assay (N = 6);

[0060] (MN) Representative images and quantification results of Transwell assay (n=6). DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0062] Example 1: Materials and methods:

[0063] 1. Experimental Animals:

[0064] 6-8 week old male SD rats and apolipoprotein E (APOE) knockout rats were obtained from Cyagen Biosciences. After one week of adaptation, SD rats were fed with regular feed and served as the control group. APOE knockout rats were fed with a high-fat diet containing 40% fat and 1.25% cholesterol for 18 weeks and then randomly divided into an atherosclerosis model group and an isofroskolin (ISOF) treatment group (ISOF dose was 2 mg / kg). The atherosclerosis model group continued to be fed with a high-fat diet for 12 weeks. All animals were housed in a controlled environment with a temperature of 20-22°C, a humidity of 50-60%, and a light / dark cycle of 12 hours (lights on from 7:00 to 19:00). Animals were free to eat and drink. All animal experiments were approved by the Animal Ethics Committee of Kunming Medical University.

[0065] 2. Experimental methods: Oil red O staining, Masson staining, aortic vasodilation assay, Astral-DIA proteomic analysis, cell culture and treatment, immunofluorescence staining, cell viability assay, wound healing assay, transwell migration and invasion assay, adenylate cyclase activation and cAMP detection, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis.

[0066] (1) Oil Red O staining

[0067] Oil Red O staining was used to detect atherosclerotic lesions in rat aorta and aortic sinus. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital after fasting for 8 hours. The rat aorta was quickly removed under anesthesia and washed with PBS buffer at 4°C. The samples were fixed with 4% paraformaldehyde overnight. The intact aorta was longitudinally incised to expose the intimal surface. The aortic root was embedded in OCT and sliced ​​with a microtome to prepare 4 μm frozen sections. Oil Red O staining was performed according to the standard protocol: the intact aorta and the prepared frozen sections were dehydrated with sucrose and dried at room temperature for about 15 minutes. The samples were placed in Oil Red O staining solution (G1015, Servicebio, China) for 8-10 minutes (protected from light and covered), and then stained with 60% isopropanol for 10 seconds. Images of the entire aorta were taken with a camera, and the sections were observed with a microscope. Finally, the area of ​​atherosclerosis was calculated using Image J.

[0068] (2) Masson staining

[0069] Aortic tissues from rats were fixed in 4% paraformaldehyde at 4°C and embedded in paraffin. According to experimental requirements, the tissues were cut into 4 μm slices using a paraffin slicer. After staining using the Masson staining kit (G1006, Servicebio, China), the tissues were observed and photographed using a microscope, and measured using Image J.

[0070] (3) Aortic vasodilation measurement

[0071] The isolated aorta of rats was cut into 1 mm long segment rings in a dissecting dish filled with Krebs Ringer bicarbonate solution (140 mM NaCl, 4.7 mM KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM MOPS, 1.4 mM Na2HPO4, 0.02 mM EDTA, 5.6 mM D-glucose; pH adjusted to 7.4, 37°C). The arterial rings were suspended by two tungsten wires in the chamber of a multi-electromyograph (Myo Technology A / S, Denmark). Each chamber was filled with 5 ml of Krebs solution, which was continuously ventilated with 95% oxygen and 5% carbon dioxide at 37°C. Before the start of the experiment, each ring was stretched to an optimal tension of 1 mN and allowed to equilibrate for 90 minutes. After 30 minutes of installation of the wire electromyograph, each ring was first contracted with 60 mM KCl and then rinsed three times with warm Krebs solution. Arterial rings with contraction amplitude less than 10% twice were used for the experiment. These selected arterial rings were contracted with 1 μmol / L U46619, and then acetylcholine (Ach, 0.001-100 μmol / L) was added cumulatively to test endothelium-dependent vasodilation. Isometric tension signals were recorded and collected by Powerlab data acquisition system (AD InstrumentsAsia, China).

[0072] (4) Astral-DIA proteomic analysis

[0073] Three rats were randomly selected from the three groups for Astral-DIA proteomics analysis. Total proteins in tissues were extracted using protein lysis buffer (8 M urea, 1% SDS) and quantified using the bicinchoninic acid (BCA) method. After protein degradation using triethylammonium bicarbonate buffer (TEAB), iodoacetamide (IAM) and trypsin, HLB was used for desalting and peptides were removed using a vacuum concentrator. Peptides were then quantified using a peptide quantification kit (#23275, Thermo Fisher Scientific, USA). Based on the quantification results of peptides, the peptides were analyzed using VanquishNeo equipped with an Orbitrap Astral mass spectrometer. Data-independent acquisition (DIA) data were acquired using an Orbitrap Astral mass spectrometer in DIA mode. Spectronaut software (version 18) was used to search DIA raw data. Common peptides and modified peptides were excluded, and peak areas were calculated and summed to obtain quantitative results. Only proteins with at least one unique peptide were used for protein identification. Finally, the proteomic data were analyzed using the Majorbio cloud platform for bioinformatics analysis. The P value and fold change (FC) of the two groups of proteins were calculated using the R package "t-test". Differentially expressed proteins (DEPs) were identified using fold change (>1.2 or <0.83) and P value <0.05 as thresholds. All identified proteins were functionally annotated using the KEGG pathway. The data were analyzed using the free online platform of majorbio choud platform (cloud.majorbio.com).

[0074] (5) Cell culture and treatment

[0075] Mouse aortic endothelial cells (MAECs) were purchased from BeNa Culture Collection and cultured in DMEM-H medium supplemented with 10% fetal bovine serum (C04001-500, Vivacell, China) and 1% penicillin-streptomycin solution (C3420-0100, Vivacell, China). The cells were kept in a humidified incubator with 95% O2 and 5% CO2 at 37°C. The culture medium was changed every two days. The effects of different concentrations of ox-LDL and ISOF on the survival rate of MAECs were detected by CCK8 assay to select the optimal concentrations of ox-LDL and ISOF. In the experiment, after stimulating MAECs with 40 μg / ml ox-LDL (bs-8574P, Bios Corporation, China), when the cell density grew to 70-80%, 20 μM ISOF solution was co-cultured with MAECs.

[0076] To knock down AC5, siRNA AC5 (forward: 5′-GGACCAGUUUCUGCUGAAATT-3′, reverse: 5′-UUUCAGCAGAAACUGGUCCTT-3′) and siRNA negative control (forward: 5′-UUCUUCGAACGUGUCACGUTT-3′, reverse: 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from GenePharma (Shanghai, China) and performed according to the manufacturer’s protocol. 5 The cells were seeded into 6-well plates at a density of 100 μg / well and cultured overnight. 75 pmol siRNA was diluted into 42.5 μl buffer and then 7.5 μl siRNA-mate plus (GenePharma, Shanghai, China) was added and mixed. Subsequently, the transfection mixture was added to MAECs treated with 40 μg / ml ox-LDL in 6-well plates. Total protein of MAECs was isolated 48 h after siRNA transfection.

[0077] (6) Immunofluorescence staining

[0078] Paraffin sections were deparaffinized and rehydrated before antigen retrieval. MAECs on glass slides were fixed with 4% paraformaldehyde for 15 min, and cell membranes were disrupted with 0.5% Triton for 20 min. Sections and MAECs were washed three times with PBS buffer and then sealed with 6% goat serum for 30 min at room temperature. Sections and MAECs were incubated with primary antibodies against α-SMA (1:250) and CD31 (1:250) overnight at 4°C, followed by incubation with fluorescein-labeled secondary antibodies for 2 h at room temperature. Cell nuclei were stained with DAPI and then photographed under a fluorescence microscope.

[0079] (7) Cell viability assay

[0080] Cell viability was detected using a cell counting kit-8 (proteintech, China). Cells were then treated with different concentrations of ox-LDL or ISOF, and the control group was treated with 0.1% DMSO. 10 μl of CCK8 reagent was added to each well and incubated at 37°C in the dark for 2 hours. After incubation at 37°C for 2 hours, the absorbance of the samples was measured at a wavelength of 450 nm using a spectrophotometer.

[0081] (7) Wound healing experiment

[0082] MAECs were seeded into 6-well plates and formed into a confluent endothelial monolayer after cell attachment. The endothelial monolayer was evenly scraped with a 200 μl pipette tip perpendicular to the bottom of the well plate, and then gently rinsed once with 1 ml of culture medium to remove all debris and displaced cells. An appropriate amount of DMEM-H culture medium containing 1% fetal bovine serum and 1% penicillin-streptomycin solution was added to each well, and the plate was then placed in an incubator at 37°C, 5% CO2, and 50% humidity for 24 hours.

[0083] (8) Transwell migration and invasion assay

[0084] A total of 5 × 10 4 Each treated MAECs was resuspended in 200 μl of serum-free medium and placed in the upper layer of a transwell chamber (Corning, USA; 24-well insert, pore size: 8 μm). A 10% FBS and 1% penicillin-streptomycin solution culture medium was added to the lower chamber as a chemotactic attractant. After 24 hours of culture, the infected cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, and the uninfected cells were removed with a cotton swab after staining with 0.5% crystal violet.

[0085] (9) Adenylate cyclase activation and cAMP detection

[0086] Aortic tissue with stimulation buffer (1×HBSS, 5mM HEPES, 0.5mM IBMX, 0.1% BSA, pH7.4) was pulverized with a tissue grinder, and the supernatant was taken by low-temperature centrifugation. MAECs were properly treated and used for experiments after reaching a density of 70%. The collected cells were discarded from the supernatant and resuspended in HBSS for cell counting, then centrifuged at 275g for 5 minutes and resuspended in stimulation buffer for cAMP detection. The cAMP content in cells and tissues was detected using the LANCE Ultra cAMP detection kit (TRF0263, PerkinElmer, USA). In the adenylate cyclase activation experiment, cells in stimulation buffer were treated with ISOF (0.003-100μM) in a 384-well plate and incubated at room temperature for 30 minutes. Then Eu-cAMP tracer and ULight-anti-cAMP solution were added and incubation continued for 60 minutes. Time-resolved fluorescence energy transfer (TR-FRET) was measured on a microwell fluorescence reader (Biotek Synergy 5, USA), and the measured TR-FRET signal was converted into the actual amount of cAMP produced in the cells by a cAMP standard curve.

[0087] (10) Quantitative real-time polymerase chain reaction (qRT-PCR)

[0088] Total RNA was extracted from rat aorta using TRIzol reagent (15596026CN, Invitrogen) and then expressed using PrimeScript TM Reverse transcription was performed using the RT kit (RR047A, Takara) at 37°C for 15 min and 85°C for 5 s. The reaction conditions were as follows: first stage, 95°C for 30 s; second stage, 95°C for 5 s and 60°C for 30 s for 40 cycles. The relative mRNA levels of AC1-10 were normalized to those of β-actin. Table 1 lists the specific primers used in this study.

[0089] Table 1 Primer sequences of rat AC1-AC10

[0090]

[0091] (11) Western blot analysis

[0092] Total protein was extracted from MAECs and tissues using RIPA buffer supplemented with cocktail / phosphatase inhibitors and quantified using the BCA method. Protein samples were separated by 8-10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore Corp). TMThe membrane was blocked with blocking buffer (Beyotime) for 2 hours. Anti-CD31 (bs-0468R, Bioss, 1:500), anti-E-Cadherin (20874-1-AP, Proteintech, 1:5000), anti-N-Cadherin (22018-1-AP, Proteintech, 1:2000), anti-α-SMA (#19245, Cell Signaling Technology, 1:1000), anti-TGF-β (ab215715, Abcam, 1:1000), anti-Smad3 (#9523, Cell Signaling Technology, 1:1000), anti-p-Smad3 (#9520, Cell Signaling Technology, 1:1000), anti-α-SMA (#19245, Cell Signaling Technology, 1:1000), anti-α-SMA (#19245, Cell Signaling Technology, 1:1000), anti-TGF-β (ab215715, Abcam, 1:1000), anti-Smad3 (#9523, Cell Signaling Technology, 1:1000), anti-α-SMA (#19245 ... Technology, 1:1000), anti-AC5 (ab66037, Abcam, 1:1000), anti-PRKACA (ab76238, Abcam, 1:20000), anti-EPAC1 (ab109415, Abcam, 1:1000), anti-eNOS (#32027, Cell Signaling Technology, 1:1000), and incubate at 4°C overnight. After washing three times with triphasic buffered saline containing 0.1% Tween (TBST), the cells were incubated with anti-mouse IgG-HRP (1:5000, CellSignaling Technology, 7076S) or anti-rabbit IgG-HRP (1:5000, Affinity, #S0001) at room temperature for 2 hours. Chemiluminescence detector (Merck Millipore Corp) and Amersham Image were used for detection. Images were collected using the 600 system. Quantification was performed using ImageJ software (version 1.47), and each lane was normalized to HSP90 or β-actin.

[0093] (12) Statistical analysis

[0094] All data are expressed as mean ± standard error (SEM) and analyzed using GraphPad Prism9.0 software (CA, USA). After normality and variance homogeneity tests, one-way ANOVA and Tukey post-test were used for comparison among multiple groups. When there were two experimental factors, two-way ANOVA was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

[0095] Example 2. ISOF can reduce the progression of atherosclerosis and improve vasodilation in apolipoprotein E gene knockout rats

[0096] To investigate the role of ISOF in atherosclerosis, male apolipoprotein E knockout rats were fed a high-fat diet for 18 weeks and then treated with ISOF for 12 weeks. The number of lipid-rich atherosclerotic plaques was assessed by Oil Red O staining. ISOF significantly reduced the number of atherosclerotic plaques throughout the aorta ( Figure 1 A, B) and aortic sinus ( Figure 1 C, D) Atherosclerotic plaques. The content of collagen is closely related to the stability of the plaque. Masson staining showed that ISOF increased the content of collagen ( Figure 1 E, F).

[0097] Atherosclerosis formation can affect vascular reactivity. In the aortic vasodilator assay, aortic vessels were isolated from rats and endothelium-dependent vasodilation was assessed using a DMT line electromyography system as follows:

[0098] The isolated aorta of rats was cut into 1 mm long segment rings in a dissecting dish filled with Krebs Ringer bicarbonate solution (140 mM NaCl, 4.7 mM KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM MOPS, 1.4 mM Na2HPO4, 0.02 mM EDTA, 5.6 mM D-glucose; pH adjusted to 7.4, 37°C). The arterial rings were suspended by two tungsten wires in the chamber of a multi-electromyograph (Myo Technology A / S, Denmark). Each chamber was filled with 5 ml of Krebs solution, which was continuously ventilated with 95% oxygen and 5% carbon dioxide at 37°C. Before the start of the experiment, each ring was stretched to an optimal tension of 1 mN and allowed to equilibrate for 90 minutes. After 30 minutes of installation of the wire electromyograph, each ring was first contracted with 60 mM KCl and then rinsed three times with warm Krebs solution. Arterial rings with two contractions less than 10% were used for the experiment. These selected arterial rings were contracted with 1 μmol / L U46619, and then acetylcholine (Ach, 0.001-100 μmol / L) was cumulatively added to test endothelium-dependent vasodilation.

[0099] The results showed that the isolated aorta of atherosclerotic rats could not show normal relaxation response induced by acetylcholine, and ISOF increased the vascular relaxation induced by acetylcholine ( Figure 1 G). Similarly, ISOF also enhanced the maximum vasodilation effect (Emax) ( Figure 1H). Since endothelial function mainly depends on the function and activity of endothelial nitric oxide synthase (eNOS), we also analyzed the effect of ISOF on eNOS expression. Consistent with the results of vasodilation, Western blot quantification showed that the protein expression of p-eNOS in the atherosclerosis group was lower than that in the control group, while ISOF significantly increased the expression of p-eNOS ( Figure 1 IJ). The results showed that ISOF can effectively inhibit the development of atherosclerotic plaques and improve vascular dilation function.

[0100] Example 3: ISOF can alleviate endothelial-mesenchymal transition in atherosclerotic apolipoprotein E knockout rats

[0101] Endothelial-mesenchymal transition (EndMT) is a biological process in which endothelial cells lose endothelial characteristics and acquire a mesenchymal phenotype, which is involved in the development of atherosclerosis. Western blot analysis of lysed aortas showed that compared with the control group, the expression of endothelial markers CD31 and E-cadherin in the atherosclerosis group was significantly decreased, while the expression of mesenchymal markers N-cadherin and α-SMA was increased, which could be restored to normal by ISOF ( Figure 2 AE). In addition, we co-stained CD31 with the mesenchymal marker α-SMA and found that the overlap percentage between CD31 and SMC markers in the ISOF group was much lower than that in the atherosclerosis group ( Figure 2 FG).

[0102] In summary, it was shown that ISOF could inhibit endothelial-mesenchymal transition in atherosclerotic apolipoprotein E knockout rats.

[0103] Example 4: ISOF protects MAECs from EndMT-associated ox-LDL damage

[0104] Cell viability was detected using a cell counting kit-8 (proteintech, China). Cells were treated with different concentrations of ox-LDL or ISOF to detect cell viability induced by different concentrations of ox-LDL and ISOF, and the control group was treated with 0.1% DMSO. 10 μl of CCK8 reagent was added to each well and incubated in the dark at 37°C for 2 hours. After incubation at 37°C for 2 hours, the absorbance of the sample was measured at a wavelength of 450 nm using a spectrophotometer.

[0105] like Figure 3 As shown in A, the CCK-8 test results showed that 40 μg / ml ox-LDL could significantly reduce the survival rate of MAECs ( Figure 3A). Then, after aortic endothelial cells (MAECs) were stimulated with 40 μg / ml ox-LDL, they were co-cultured with ISOF solutions at final concentrations of 0, 2.5, 5, 10, 20, 40, 80, and 160 μM. The results showed that the cell viability was most significantly improved when MAECs were co-cultured with 20 μM ISOF ( Figure 3 B).

[0106] The experiment selected 40μg / ml ox-LDL and 20μM ISOF for wound healing experiments. MAECs were seeded into 6-well plates, and cells were attached to form a confluent endothelial monolayer. The endothelial monolayer was evenly scraped with a 200μl pipette tip perpendicular to the bottom of the well plate, and then gently rinsed once with 1ml of culture medium to remove all debris and displaced cells. An appropriate amount of DMEM-H culture medium containing 1% fetal bovine serum and 1% penicillin-streptomycin solution was added to each well, and the plate was then placed in an incubator at 37°C, 5% CO2 and 50% humidity for 24 hours.

[0107] After treating MAECs under the above conditions, wound healing experiments showed that ISOF treatment promoted the migration and self-healing ability of MAECs ( Figure 3 CD) (The area reduction ratio of the middle black strip is calculated to reflect the migration and healing ability of MAECs)

[0108] 3. Transwell Migration and Invasion Assay

[0109] A total of 5 × 10 4 MAECs treated with 40 μg / ml ox-LDL and 20 μM ISOF were resuspended in 200 μl of serum-free medium and placed in the upper layer of a transwell chamber (Corning, USA; 24-well insert, pore size: 8 μm). 10% FBS and 1% penicillin-streptomycin solution culture medium was added to the lower chamber as a chemotactic attractant. After 24 hours of culture, the infected cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, and the uninfected cells were removed with a cotton swab after staining with 0.5% crystal violet.

[0110] The results are as follows Figure 3 As shown in Figure 2, ISOF reversed the effect of ox-LDL on the migration ability of MAECs, and in the transwell assay, the migration ability increased by 62% ( Figure 3 EF) (by calculating the area reduction ratio of the middle black band, compared with the ox-LDL group). Similarly, Western blot analysis showed ( Figure 3H), compared with the ox-LDL group, ISOF increased the expression of CD31 and E-cadherin, and decreased the expression of N-cadherin and α-SMA. Therefore, ISOF has a good protective effect on cell viability and migration.

[0111] Figure 3 Experiment G showed that compared with the ox-LDL group, ISOF increased the expression of CD31 and E-cadherin and decreased the expression of N-cadherin and α-SMA, indicating that ISOF can also inhibit EndMT in vitro.

[0112] Example 5: Mechanism of SOF in improving atherosclerosis

[0113] To further explore the mechanism of ISOF in improving atherosclerosis, Astral-DIA proteomics analysis of the aorta was performed. The correlation coefficient of protein expression was calculated and it was found that the samples were different between groups and similar within the groups ( Figure 4 A). Differentially expressed proteins (DEPs) were determined using fold change (>1.2 or <0.83) and p value <0.05 as thresholds. The volcano plot showed that a total of 636 DEPs were detected in the control and model groups, of which 337 were upregulated and 299 were downregulated. There were 275 DEPs in the model and ISOF groups, of which 99 were upregulated and 176 were downregulated ( Figure 4 B). Notably, 35 up-regulated DEPs and 35 down-regulated DEPs in the model group were reversed by ISOF ( Figure 4 C). Cluster heat map showing the different changes of a total of 70 DEPs in detail ( Figure 4 D). To reveal the biological pathways of DEPs, we performed KEGG pathway analysis. The results showed that DEPs regulated by ISOF were mainly involved in ECM-receptor interaction, TGF-β signaling pathway and lesion adhesion ( Figure 4 E). It is well known that TGF-β signaling is a major mediator of EndMT and accelerates the formation of atherosclerotic plaques. We paid special attention to the TGF-β signaling pathway because it is closely related to the progression of atherosclerotic plaques. To verify the proteomics findings, we performed Western blot analysis. The results showed that in vivo animal experiments ( Figure 4 FG) and in vitro cell experiments ( Figure 4 In HI), the expression of TGF-β and p-Smad3 increased significantly, while ISOF could reverse the expression of these two factors. These results suggest that ISOF may inhibit EndMT through the TGF-β signaling pathway.

[0114] Example 6: ISOF upregulates the expression of AC5 isozyme and activates the cAMP / PKA pathway

[0115] We analyzed the expression profiles of all AC isozymes (1-10) in aortic tissue to identify key AC isozymes that might be regulated by ISOF. Interestingly, real-time quantitative polymerase chain reaction revealed that AC5 mRNA expression was high compared with other AC isozymes and was selectively downregulated in atherosclerotic apolipoprotein E knockout rats, which was reversed by ISOF ( Figure 5 A). Western blot analysis also detected a parallel trend in AC5 protein expression. Figure 5 B, C). Therefore, we focused on AC5 in subsequent experiments because its changes were the most significant. In addition, ISOF increased the cAMP content in aortic tissue, indicating that ISOF can activate AC5 ( Figure 5 D). To investigate the downstream mechanism of ISOF's anti-atherosclerotic effect, we studied the downstream factors of cAMP, including PKA and EPAC. The results showed that ISOF could upregulate the protein expression of PKA catalytic subunit (PRKACA), but could not upregulate the protein expression of EPAC1 ( Figure 5 EG), which indicated that ISOF activated the cAMP / PKA signaling pathway by upregulating the expression of AC5.

[0116] Example 7: ISOF activates the enzyme activities of AC5 and cAMP / PKA pathways in vitro

[0117] To further validate these in vivo results, we also detected the expression of AC5 protein by Western blot analysis. Compared with the control group, the expression of AC5 was reduced in MAECs exposed to ox-LDL for 24 h. However, administration of ISOF upregulated the expression of AC5 ( Figure 6 AB). Accordingly, the results of the intracellular cAMP level detection in MAECs showed that ISOF could reverse the decrease in cAMP concentration induced by ox-LDL ( Figure 6 C). To further evaluate the ability of ISOF to activate AC, the cAMP levels in MAECs co-cultured with different concentrations of ISOF were detected. The results showed that ISOF stimulated the production of cAMP in a concentration-dependent manner, which means that ISOF has the ability to stimulate AC5 to produce cAMP. The EC50 value of the oxidized-LDL group (7.355±3.36) was lower than that of the control group (12.27±8.66), which is related to the damage of AC5 induced by oxidized-LDL ( Figure 6 D). Next, we studied the cAMP-mediated downstream pathway in vitro. Consistent with the in vivo results, ISOF increased the protein expression of PRKACA but not EPAC1 ( Figure 6EG). Taken together, these results suggest that ISOF-induced AC5 upregulation leads to activation of the downstream cAMP / PKA pathway.

[0118] Example 8: siRNA AC5 reversed the therapeutic effect of ISOF on ox-LDL-induced EndMT

[0119] To explore the important role of AC5 in ISOF's anti-EndMT, we knocked down the level of AC5 in MAECs using siRNA. The results showed that ISOF upregulated the PKA pathway and downregulated the TGF-β / Smad3 pathway, thereby inhibiting ox-LDL-induced EndMT. However, these effects were offset when AC5 was silenced in MAECs ( Figure 7 AJ). In addition, wound healing and Transwell assays also showed that siRNA AC5 could significantly inhibit the enhanced migration after ISOF treatment ( Figure 7 KN). Studies have shown that cAMP / PKA activation can inhibit the phosphorylation of Smad3. In summary, it is reasonable to speculate that ISOF inhibits EndMT through TGF-β / Smad3 signaling and plays a role in anti-atherosclerosis, and TGF-β / Smad3 signaling depends on AC5-induced PKA activation.

[0120] In summary, as described in Examples 2-8, ISOF can improve endothelial function and prevent atherosclerosis by inhibiting intimal hyperplasia. Mechanistically, ISOF can effectively upregulate the expression of AC5, which in turn activates cAMP / PKA and inhibits the TGF-β / Smad pathway (Figure 8). In summary, our research results support ISOF as a promising candidate drug for the treatment of atherosclerosis.

[0121] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

[0122] Sequence Listing Information:

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

Claims

1. Application of isofroskolin in the preparation of drugs for inhibiting atherosclerosis.

2. Application of isofroskolin in the preparation of drugs for inhibiting endothelial cell to mesenchymal transition.

3. Use of isofroskolin in the preparation of a drug for increasing the expression of endothelial marker CD31.

4. Use of isofroskolin in the preparation of a drug for increasing the expression of the endothelial marker E-cadherin.

5. Use of isofroskolin in the preparation of drugs for inhibiting the expression of N-cadherin and / or α-SMA.

6. Use of isofroskolin in the preparation of a drug for promoting the expression of AC5.

7. The use according to claims 1-6, characterized in that: The dosage of isofroskolin in the medicine is 2 mg / kg.

Images