Application of galloyl paeoniflorin in preparation of medicine for preventing and / or treating vascular occlusion diseases

Abstract

The invention relates to application of galloyl paeoniflorin in preparation of a medicine for preventing and / or treating vascular occlusion diseases. According to the invention, by detecting endothelial cell angiogenesis activity, lower limb angiogenesis of lower limb artery occlusion mice, vascular maturation and blood perfusion, the enhancement effect of galloyl paeoniflorin on endothelial cell angiogenesis activity is found; the effects of angiogenesis, blood vessel maturation and blood perfusion recovery of lower limbs of lower limb artery occlusion mice are achieved. A plurality of tests on the cell and animal level prove that the galloyl paeoniflorin can promote angiogenesis and improve blood flow recovery of the vascular occlusion disease, a new drug candidate is provided for improving the vascular occlusion disease through therapeutic angiogenesis, and the galloyl paeoniflorin has important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of galloylpaeoniflorin in preventing and / or preparing medicines for treating vascular occlusive diseases. Background Art

[0003] Blood vessels are essential to sustain life, supplying oxygen and nutrients to tissues and facilitating the critical transport of metabolic products. Despite advances in medicine, the prevalence of vascular occlusive diseases such as peripheral artery disease (PAD), chronic total coronary occlusions (CTOs), and stroke continues to rise. The impact of PAD goes beyond the threat of major adverse cardiovascular events; it also significantly reduces patients’ quality of life through pain, claudication, and limb dysfunction. In addition, CTOs are detected in approximately 20%-30% of patients undergoing invasive angiography.

[0004] Although great progress has been made in the prevention and treatment of vascular occlusive disease in recent years, its treatment measures still cannot meet the needs of all patients, and the mortality rate and adverse event rate of vascular occlusive disease remain high. Although interventions such as drug therapy, vascular intervention and bypass surgery can relieve symptoms, they are usually temporary rather than curative and may have significant risks, especially for patients with comorbidities. These limitations highlight the urgent need for innovative methods that can effectively address the basic pathophysiological problems of vascular occlusive disease.

[0005] Therapeutic angiogenesis is the use of the body's inherent ability to form new blood vessels in response to ischemia, and has become a promising strategy for the treatment of PAD, CTO, and stroke. Angiogenesis is the process of forming new blood vessels from existing blood vessels. It is a critical and complex process that plays an important role in the regeneration, growth, and development of tissues and organs. The main steps of angiogenesis include basement membrane degradation, cell proliferation, cell migration, cell lumen formation, cell differentiation, and maturation, all of which are completed by different endothelial cells. During angiogenesis, endothelial cells secrete metalloenzymes such as MMP2 and MMP9 under the action of signaling pathways such as VEGFA-VEGFR2 to dissolve the matrix membrane. Endothelial cells differentiate into "tip" cells to form filaments, which extend from the cell surface and migrate to the front end, leading the vascular buds to the angiogenic stimulus. At the same time, the endothelial "stalk" cells extend forward along the tip cells, and the endothelial "stem" cells arrange the static blood vessels, which are then surrounded by smooth muscle cells and pericytes, and finally form mature arterial vessels.

[0006] Chinese herbal medicine has a history of thousands of years of clinical practice, and its efficacy has been confirmed. In the history of Chinese medicine, many Chinese herbal medicines have been proven to have the effect of removing blood stasis and promoting blood circulation. With the development of modern pharmacology, many active compounds in these Chinese medicines have been found to have "angiogenic" effects in in vitro or in vivo models, thus explaining their activity of removing blood stasis and promoting blood circulation with modern pharmacology. Modern pharmacological studies on galloylpeonidin have shown that it has antioxidant, anti-tumor, anti-inflammatory, asthma-improving, osteoporosis-improving, anti-platelet aggregation, and reduced cerebral ischemia-reperfusion injury effects, but there are no reports on the effect of galloylpeonidin on improving vascular occlusive diseases.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide the use of galloylpaeoniflorin in the preparation of a drug for preventing and / or treating vascular occlusive diseases. Galloylpaeoniflorin prevents and / or treats vascular occlusive diseases by promoting angiogenesis.

[0009] The chemical structural formula of the galloylpeonidin (CAS No.: 122965-41-7) of the present invention is as follows:

[0010]

[0011] The source of the galloylpaeoniflorin of the present invention is not limited, and can be an extract of a natural product or a chemically synthesized product, for example, it can be derived from the roots of Paeoniae Radix Rubra, Paeoniae Radix Alba, and Paeonia suffruticosa Andr.

[0012] The first aspect of the present invention provides the use of galloylpaeoniflorin in the preparation of a drug for preventing and / or treating vascular occlusive diseases, wherein the galloylpaeoniflorin can be used to treat vascular occlusive diseases.

[0013] Specifically, the galloylpeonidin prevents and / or treats vascular occlusive diseases by promoting angiogenesis.

[0014] Specifically, the galloylpeonidin can upregulate the expression levels of angiogenesis-related proteins VEGFA and VEGFR2.

[0015] Specifically, the galloylpeonidin can upregulate the expression levels of angiogenesis-related genes VEGFA and VEGFR2.

[0016] Specifically, the galloylpeonidin can enhance the proliferation, migration, invasion and tube-forming ability of endothelial cells.

[0017] Specifically, the galloylpeonidin can promote blood flow recovery in the ischemic area, upregulate the density of new blood vessels and the number of arteries in the ischemic area, and promote vascular maturation.

[0018] Specifically, the vascular occlusive disease includes any one of peripheral arterial disease and chronic complete coronary artery occlusion.

[0019] Preferably, the administration concentration of the galloylpaeoniflorin is 2.5-10 μM.

[0020] A second aspect of the present invention provides a pharmaceutical composition for treating vascular occlusive diseases, wherein the main active ingredient of the pharmaceutical composition is galloylpaeoniflorin.

[0021] In the present invention, the pharmaceutical composition can be a drug for treating vascular occlusive diseases; the vascular occlusive diseases of the present invention include ischemic heart diseases, ischemic encephalopathy or ischemic peripheral diseases including PAD, CTO and stroke; examples of the vascular occlusive diseases of the present invention include but are not limited to coronary heart disease, thrombotic cerebral infarction or lower limb ischemic disease.

[0022] Specifically, the pharmaceutical composition promotes angiogenesis to prevent and / or treat vascular occlusive diseases through at least one of the following effects:

[0023] (Ⅰ) Upregulate the expression levels of angiogenesis-related proteins VEGFA and VEGFR2;

[0024] (Ⅱ) up-regulating the expression levels of angiogenesis-related genes VEGFA and VEGFR2;

[0025] (III) Enhance the proliferation, migration, invasion and tube formation ability of endothelial cells;

[0026] (IV) Promote blood flow recovery in the ischemic area, increase the density of new blood vessels and the number of arteries in the ischemic area, and promote vascular maturation.

[0027] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0028] The present invention has at least the following beneficial effects:

[0029] The present invention detects the angiogenesis activity of endothelial cells, angiogenesis, vascular maturation and blood perfusion of the lower limbs of mice with lower limb artery occlusion, and finds that galloylpeonidin has an enhancing effect on the angiogenesis activity of endothelial cells, as well as angiogenesis, vascular maturation and blood perfusion recovery of the lower limbs of mice with lower limb artery occlusion. The present invention proves that galloylpeonidin can promote angiogenesis and improve blood flow recovery in vascular occlusive diseases through multiple experiments at the cell and animal levels, providing a new drug candidate for improving vascular occlusive diseases through therapeutic angiogenesis, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is a statistical graph showing that different concentrations of galloylpeonidin promoted the proliferation of HUVECs as determined by CCK-8 experiment; GA represents galloylpeonidin, and C represents the blank control group.

[0032] Figure 2 These are the fluorescence images and statistical graphs of the promotion of HUVECs proliferation by different concentrations of galloylpeonidin measured by the EDU cell proliferation kit; the left image is the EDU fluorescence image, the red positive points are proliferating cells, the blue ones are cell nuclei, the right image is the statistical graph, GA represents galloylpeonidin, and C represents the blank control group.

[0033] Figure 3 The real-time and statistical graphs of the cells were used to detect the effect of different concentrations of galloylpeonidin on the migration of HUVECs at 24h and 48h in the scratch experiment; the left graph is the real-time graph of the scratch at 24h and 48h, and the right graph is the statistical result graph. GA represents galloylpeonidin, and C is the blank control group.

[0034] Figure 4 The real-time graphs and statistical graphs of the transwell invasion experiment to detect different concentrations of galloylpeonidin to promote the migration of HUVECs; the left graph shows the cells invading through the transwell membrane, and the right graph shows the statistical results. GA represents galloylpeonidin, and C represents the blank control group.

[0035] Figure 5The figures are statistical graphs showing that different concentrations of galloylpeonidin promote HUVECs tube formation; the left figure is a tube formation result graph, and the right figure is a statistical graph of the number of nodes, the number of tubes, the number of tube meshes, and the length of the tube. GA represents galloylpeonidin, and C represents the blank control group.

[0036] Figure 6 Different concentrations of galloylpaeoniflorin promoted the protein expression of VEGFR2 and VEGFA, key proteins in HUVECs angiogenesis; GA represents galloylpaeoniflorin, and C represents the blank control group.

[0037] Figure 7 Different concentrations of galloylpaeoniflorin promoted the protein expression of VEGFR2 and VEGFA, the key genes for angiogenesis in HUVECs; GA represents galloylpaeoniflorin, and C represents the blank control group.

[0038] Figure 8 The laser Doppler results and statistical results of galloylpeonidin improving blood flow recovery in occluded lower limbs of mice at different stages; the left figure is the laser Doppler blood flow diagram of the lower limbs of mice after intraperitoneal injection for different days, and the right figure is the statistical result diagram. Pre-operative refers to the laser Doppler blood flow diagram before ligation of the femoral artery of the lower limbs of mice, post-operative refers to the laser Doppler blood flow diagram 30 minutes after ligation of the femoral artery of the lower limbs of mice, 1 day, 3 days, 14 days, and 21 days are the laser Doppler blood flow diagrams 1 day, 3 days, 14 days, and 21 days after ligation of the femoral artery of the lower limbs of mice, respectively. GA represents galloylpeonidin, GA-L, GA-M, and GA-H refer to low, medium, and high (2.5, 5, and 10 mg / ml) concentrations of GA, respectively, and C represents the blank control group.

[0039] Fig. 9 This is a HE staining image of galloylpeonidin promoting angiogenesis in the gastrocnemius muscle of the ischemic area of ​​the lower limb of mice; the green arrows indicate the blood vessels in the tissue, GA represents galloylpeonidin, GA-L, GA-M, and GA-H refer to the three concentrations of GA, low, medium, and high (2.5, 5, and 10 mg / ml), respectively, and C represents the blank control group.

[0040] Fig.10 This is an immunofluorescence image of galloylpeonidin promoting angiogenesis and vascular maturation of the gastrocnemius muscle in the ischemic area of ​​the lower limb of mice. Angiogenesis was detected by staining of endothelial cell marker protein CD31, and vascular maturation was identified by co-staining of CD31 and smooth muscle cell maker protein α-SMA, where green fluorescence is α-SMA, red fluorescence is CD31, and blue fluorescence is DAPI cell nucleus. The left figure is a statistical graph of the fluorescence intensity of CD31 and α-SMA. GA represents galloylpeonidin, GA-L, GA-M, and GA-H refer to low, medium, and high (2.5, 5, and 10 mg / ml) concentrations of GA, respectively. C represents the blank control group.

[0041] Fig.11 Galloylpeonidin upregulates the protein expression level and mRNA gene expression level of angiogenesis-related proteins Vegfr2 and Vegfa in the gastrocnemius muscle of the ischemic area of ​​mice; wherein, GA represents galloylpeonidin, GA-L, GA-M, and GA-H refer to three concentrations of GA, low, medium, and high (2.5, 5, and 10 mg / ml), respectively, and C represents the blank control group. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In order to verify the angiogenic effect of galloylpeonidin, it is necessary to select appropriate cell models and animal models for testing. Endothelial cells are the main cells of blood vessels and the main participating cells of angiogenesis. Under the stimulation of angiogenic factors, endothelial cells need to undergo proliferation, migration, tube formation and other steps to complete the process of angiogenesis. For single endothelial cell culture, the three steps of endothelial cell proliferation, migration and tube formation are often verified separately when studying the mechanism of angiogenesis or evaluating the efficacy of drugs. The mouse lower limb artery occlusion model is a classic model for studying angiogenesis. By detecting the blood perfusion and vascular density of the lower limbs, the effect of drugs on improving vascular occlusive diseases through therapeutic angiogenesis can be verified. The experimental materials and instruments involved in the present invention are shown in Table 1.

[0046] Table 1 Experimental materials and instruments

[0047]

[0048]

[0049] Test Example 1

[0050] Endothelial cells are the main cells involved in angiogenesis, and endothelial cells need to complete the angiogenesis process through migration, proliferation, and tube formation. Therefore, when evaluating the angiogenesis-promoting activity of galloylpeonidin, this experiment verifies its ability to affect endothelial cell proliferation, migration, and tube formation.

[0051] 1. Extraction and culture of endothelial cells: Select the umbilical cord of a newborn baby born by cesarean section from a healthy pregnant woman without hepatitis B virus (HBV) or human immunodeficiency virus (HIV). Cut 20-30cm of umbilical cord tissue under sterile conditions and place it in a sterile container containing 100ml 0.9% saline (containing 200ug / mL P / S, 500ug / mL heparin sodium). Seal and store in a 4℃ refrigerator for later use. In the clean bench, place the umbilical cord in a 15cm culture dish containing 15ml PBS, fix the left end of the umbilical cord with the left hand, gently stroke the umbilical cord with the right hand, squeeze out the blood clot inside the umbilical cord, and clean the blood outside the umbilical cord. Trim both ends of the umbilical cord tissue flat, and cut off the tissue with pinch marks, damage, and hematoma. Use a flat-blade scalpel to cut off about 0.5-1cm of the umbilical cord at both ends to clearly expose the arteriovenous incision of the umbilical cord, identify the umbilical vein, carefully insert the gavage needle, insert the needle about 2cm, and seal it with a vascular clamp. Inject 25ml PBS into the umbilical cord, and temporarily pinch the other end of the umbilical cord with your fingers (vascular clamp) to make sure that the umbilical cord is not damaged, and then further flush the remaining blood in the umbilical cord. Use 25ml PBS to irrigate again until the outflow is completely clear. Fix the left end of the umbilical cord with your left hand, and gently stroke the umbilical cord with your right hand to remove the PBS remaining inside the umbilical cord. Inject 5-10ml of Dispase solution (37℃) into the umbilical cord. Inject slowly, and when the Dispase solution starts to come out from the other end of the umbilical cord, immediately clamp the umbilical cord with a clamp. Do not remove the syringe after the injection. Pour 37℃ PBS into a sterilized beaker. Then immerse the umbilical cord and incubate it in 37℃ PBS for 20min. Transfer the umbilical cord to a stainless steel plate and gently massage the umbilical cord with your thumb and index finger for 5 minutes to promote cell dissociation. Connect a syringe containing 20ml PBS to the gavage needle. Loosen the clamp on the other end and place the umbilical cord at that end into a 50ml centrifuge tube. Gently push the syringe containing PBS to inject the dissociated vascular endothelial cells from the umbilical cord into the centrifuge tube. Centrifuge at 300×g for 3 minutes at room temperature. Remove the supernatant and first use 1ml endothelial cell culture medium to resuspend the cells by flicking the tube wall. Add 15ml endothelial cell culture medium and blow gently 2-3 times with a 15ml pipette to mix and transfer the cells to a T75 culture flask and mark P0. After 6 hours (or overnight), when the cells have completely attached to the wall, wash them twice with PBS and then add endothelial cell culture medium (ECM culture medium for endothelial cells, which contains 5% fetal bovine serum, 1% penicillin-streptomycin double antibody and 1% endothelial cell growth factor). After 2-3 days, when the cells are confluent, subculture once and mark them as P1. When the cells grow to 90% confluent in the T75 culture flask, digest the cells and freeze 3 tubes per flask. Digest, count and freeze at 1x106 cells per tube (P1).

[0052] 2. Passaging of endothelial cells: When the cell growth confluence reaches about 90%, remove the waste liquid, add 3ml trypsin, incubate at 37℃ incubator for 30 seconds, observe the cells become round and shrink under the microscope, tap the side wall of the culture bottle to make the cells fall off the wall of the culture bottle, immediately add 3.5ml complete medium to terminate digestion, gently blow the bottle wall cells, collect the cells in a 15ml centrifuge tube, and centrifuge at room temperature 300×g for 5min. Discard the supernatant, add the corresponding culture medium in a ratio of 1:2 or 1:3, resuspend the cells, blow and evenly distribute the cells in the culture bottle or cell culture plate, and then place them in a 37℃ incubator for continued culture. P2-P8 cells were used during the experiment.

[0053] 3. Cell counting: After the cell suspension is evenly dispersed, take 10 μl and add it to the cell counting plate. Count the total number of cells in four large squares under a microscope. Calculate the cell concentration according to the following formula: Cell concentration (cells / ml) = total number of cells in 4 large squares / 4×104.

[0054] 4. Cell freezing: Select HUVEC in the logarithmic growth phase, and change the medium once a day before freezing. Digest and count the cells, resuspend the cells in cell freezing medium (92% fetal bovine serum + 8% DMSO) at a concentration of 1x106, divide the resuspended cells into cryopreservation tubes, tighten the bottle caps, mark them, place them in cell gradient freezing boxes, store them in a -80℃ refrigerator overnight, and transfer them to a liquid nitrogen tank for storage the next day.

[0055] 5. Cell recovery: Take 3mL of complete culture medium into a 15mL centrifuge tube, take out a tube of cells from liquid nitrogen, put it into a 37℃ water bath, melt the cells within 1 minute, sterilize with alcohol, use a pipette to aspirate the cells into a 15mL centrifuge tube filled with culture medium, centrifuge at 300×g for 5 minutes, add 10mL of new complete culture medium into the culture bottle, pour out the supernatant in the centrifuge tube, add 2mL of complete culture medium, blow evenly, then add 2mL of cell suspension into the culture bottle, shake in an "8" shape, and incubate in a 37℃, 5% CO2 incubator.

[0056] 6. Endothelial cell proliferation activity detection: In order to detect the effect of galloylpaeoniflorin on angiogenesis, the effect of galloylpaeoniflorin on HUVECs proliferation was detected by CCK-8 test and EDU test.

[0057] 6.1CCK-8 test: HUVECs were planted in a 96-well plate at a density of 2×105 / ml. After 24 hours of culture, the medium containing different concentrations (2.5, 5, 10μM) of galloylpeonidin was used without serum. The control group was given a serum-free medium. After 24 hours of culture, 10μl of CCK-8 reagent was added to each well. After incubation in a 37°C, 5% CO2 incubator for 3 hours, the absorbance value was detected at 480nm by a multifunctional microplate reader. The cell activity and number were determined based on the measured absorbance OD value. The larger the OD value, the stronger the cell activity and the greater the cell number. The experiment was repeated 6 times, and the results are as follows: Figure 1 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly promoted the proliferation of HUVECs.

[0058] 6.2 EDU cell proliferation test: The cells were seeded in a 24-well plate according to the above method, and after culturing for 24 hours, they were treated with different concentrations of galloylpeonidin according to the CCK-8 test method and then cultured for 24 hours. EDU staining of cells was performed according to the instructions of the Biyuntian EDU Cell Proliferation Viability Detection Kit. Images were collected using a Nikon fluorescence microscope, and imageJ was used to calculate the positive cells in each well, and finally mathematical statistics were performed. The results are shown in Figure 2 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly promoted the proliferation of HUVECs.

[0059] 7. Endothelial cell migration ability detection

[0060] To clarify the angiogenic activity of galloylated paeoniflorin, the effect of galloylated paeoniflorin on the migration of HUVECs was detected by scratch assay and transwell invasion assay.

[0061] 7.1 Scratch test

[0062] HUVECs were seeded in a 6-well plate according to the above method. After 24 hours, a scratch was made in the center of each well. Preheated PBS buffer was added, and the well was gently shaken and aspirated to remove floating HUVECs to prevent the scraped HUVECs from falling on the scratch and growing again. The above steps were repeated, and pictures were taken under a Zeiss ordinary microscope. The scratch width was recorded and measured as the baseline value. Different concentrations of galloylpeonidin (2.5, 5, and 10 μM) were given for treatment, and pictures were taken 12 hours and 24 hours after treatment. The area of ​​the scratch area was calculated using imageJ. Cell migration rate (%) = (scratch area before treatment - scratch area 12 hours or 24 hours after treatment) / scratch area before treatment * 100%. The experimental results are as follows. Figure 3 As shown in the figure, it can be seen that different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly promoted the migration of HUVECs.

[0063] 7.2 Transwell invasion assay

[0064] Dilute Matrigel with 4°C precooled incomplete medium to a final concentration of 1mg / ml (mix Matrigel with precooled incomplete medium at a ratio of 1:8), take 60μL of diluted Matrigel, vertically add it to the transwell chamber, evenly spread it on the bottom, and place the plate in an incubator for 3h to polymerize the Matrigel into a thin film. After 3h incubation, the Matrigel becomes solid, and the excess liquid in the upper chamber is aspirated. After adding 100μL of serum-free medium to each well, place it in an incubator for 30min to hydrate the basement membrane. Make cell culture medium suspension: one day before spreading the gel, treat with different concentrations of galloylpeonidin (2.5, 5, 10μM) for 24h. On the day of spreading the gel, take cells with a confluence of 70%-80%, add PBS to wash, then trypsinize, terminate digestion, centrifuge, collect cells, and discard the supernatant. Resuspend cells in incomplete medium and count cells; inoculate cells: remove the transwell chamber after hydration of basement membrane, aspirate the liquid in the upper chamber, add 500 μL of medium containing 20% ​​FBS to the lower chamber of the 24-well plate, place the transwell chamber with matrix gel in the 24-well plate (be careful to avoid bubbles between the chamber and the lower chamber), add 200 μL of cell suspension to the upper chamber, let it stand for 15 minutes to avoid aggregation of cells during the sedimentation process, and place the 24-well plate in the incubator for 24 hours. Remove the transwell chamber and aspirate the liquid in the upper and lower chambers. Wash 3 times with 1 ml PBS, then add 800 μL of 4% paraformaldehyde to the 24-well plate. Immerse the bottom of the chamber in paraformaldehyde and fix it at room temperature for 30 minutes. Discard the paraformaldehyde, wash the chamber three times (be careful to avoid touching the bottom of the chamber), then add 700 μL / well of crystal violet to the 24-well plate, immerse the bottom of the chamber in a crystal violet solution (0.1% crystal violet solution), and stain for 5-10 minutes; discard the crystal violet solution, wash the chamber three times with PBS, and gently wipe the chamber with a cotton swab to remove the cells on the membrane surface inside the upper chamber and the crystal violet that is not bound to the cells. Select 5 fields of view under a microscope to observe the cells and count them, and take the average. The results are as follows: Figure 4 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly promoted the migration and invasion ability of HUVECs.

[0065] 8. Detection of the ability of endothelial cells to form tubes in vitro

[0066] The tube formation experiment was used to detect the effect of galloylpaeoniflorin on the tubular structure formation of HUVECs.

[0067] Matrigel matrix gel was dissolved at 4°C overnight, and the tip box, EP tube, and Ebidi plate were pre-cooled at 4°C overnight. The gel was spread on an ice box the next day, and 10 μl of matrix gel was added to each well. The cells were incubated in a 37°C incubator for 30 min. HUVECs extracted and treated with different concentrations of galloylpeonidin (2.5, 5, and 10 μM) for 24 h were incubated at 1.5×10 4 The cells were seeded on ebidi plates pre-coated with matrix gel at 1000 μg / well and the formation of tubular structures was observed after culturing for 2-3 hours. ImageJ was used to analyze and count the number of pivot points and tubular length. The results are shown in the figure. Figure 5 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly promoted the in vitro tube-forming ability of HUVECs.

[0068] 9. Detection of angiogenesis-related gene and protein expression levels

[0069] Western blot and qRT-PCR were used to detect the effect of galloylpaeoniflorin on the expression levels of key proteins and genes in HUVECs angiogenesis.

[0070] 9.1 Western blot: HUVEC cells in the logarithmic growth phase (cell concentration of 2×105 / ml) were planted in 6-well plates and cultured for 24 hours, then divided into groups and cultured with different concentrations of galloylpeonidin (2.5, 5, 10 μM) for 24 hours. Collect cells: discard the old culture medium, add pre-cooled PBS to wash twice, scrape the cells and put them into a 1.5 ml EP tube. Centrifuge at 4℃, 2500rpm for 5min, discard the supernatant, add 100μl cell lysis buffer (Ripo cell lysis buffer: protease inhibitor: phosphatase inhibitor = 100:1:1), lyse on ice for 30min; centrifuge at 4℃, 12000rpm for 20min, collect the supernatant in a 1.5ml EP tube, divide 10μl for BCA protein quantification, add SDS-loading buffer to the remaining supernatant, mix well and heat in a 95℃ metal bath for denaturation for 10min, then store the sample at -20℃ after returning to room temperature. Then, gel preparation, electrophoresis, membrane transfer, incubation with primary antibodies for angiogenesis-related proteins VEGFA and VEGFR2, incubation with secondary antibodies, and film bursting were performed. ImageJ was used to perform mathematical statistics on the results of film bursting. The results are as follows Figure 6 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly upregulated the expression of HUVECs angiogenesis-related proteins VEGFA and VEGFR2.

[0071] 9.2qRT-PCR: HUVEC cells in the logarithmic growth phase (cell concentration of 2×105 / ml) were planted in 6-well plates and cultured for 24h, then divided into groups and cultured with different concentrations of galloylpeonidin ((2.5, 5, 10μM)) for 24h. Collect cells: discard the old culture medium, wash twice with pre-cooled PBS, add 1ml RNA extraction reagent (Trizol), scrape the cells into a 1.5ml RNAase-free EP tube, add 200μl chloroform, vortex for 30s, and let stand on ice for 10min; centrifuge at 4℃, 12000rpm for 15min, and collect the upper liquid in a 1.5ml RNAase-free EP tube. Add 500μl isopropanol, mix by inversion, incubate at minus 20℃ for 15min, and invert every 5min. Centrifuge at 4℃, 13000rpm for 20min. Discard the supernatant (the precipitate is RNA), add RNAase-free 75% ethanol, centrifuge at 4°C, 7500rpm for 5 minutes, repeat twice, discard the supernatant, dry the ethanol, add 50μl DEPC water to dissolve the RNA, measure the RNA concentration, and reverse transcribe the RNA according to the full-form gold reverse transcription kit. According to the instructions of the full-form gold qPCR kit, the expression of angiogenesis-related genes such as VEGFA, VEGFR2, and FGF in the sample was detected. The results are as follows Figure 7 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 μM) significantly upregulated the expression of HUVECs angiogenesis-related genes VEGFA and VEGFR2.

[0072] Test Example 2

[0073] Mouse lower limb artery occlusion model

[0074] The subjects of this experiment were male C57BL / 6J mice aged 8-12 weeks, with a body weight of 19-26 g. The experimental mice were provided by the Shanghai Southern Model Organism Research Center [Production License No. SCXK (Shanghai) 2017-0010]; the mice were adaptively raised for one week before the experiment. The mice were raised at room temperature and in a light-controlled environment with sufficient food and drinking water. All experiments were approved by the Ethics Committee.

[0075] Male mice can avoid the effects of cyclical changes of estrogen on the systemic vascular system (especially the lateral suture. A unilateral limb ischemia model of lower limbs in mice was established. The experimental group was matched with the control group in age. Erythromycin ointment was evenly applied to the incision to prevent infection, and then the mice were placed in a 37°C incubator until they woke up. 30 minutes after surgery, laser Doppler blood perfusion was measured, and there was almost no blood flow reflow under the femoral artery of the left limb, proving that the model was successful. After the operation, three concentrations of galloylpeonidin (2.5 mg / ml, 5 mg / ml, and 10 mg / ml) were intraperitoneally injected for 21 days, and the vital signs of the mice were closely observed during the period. After the operation, the mice had pain in the surgical area, and the surgical injury caused a significant decrease in the activity of the mice. They could not eat or drink normally, and needed to be fed with water in the cage. Sterile cotton balls soaked in distilled water were placed in the cage to prevent the mice from drinking water directly and causing suffocation. Mice may have a lower body temperature after surgery, and the indoor temperature needs to be raised to 24-25°C to reduce death caused by improper postoperative care.

[0076] 1. Laser Speckle Doppler Blood Perfusion Measurement

[0077] Before surgery and 1, 3, 7, 14, and 21 days after surgery, the animals were anesthetized with 3% isoflurane using a small animal gas anesthesia machine and placed on a 35°C constant temperature pad to reduce body temperature differences between individuals. The blood perfusion of the bilateral hind limbs was then measured using an RFLSIⅢ laser speckle blood flow imaging system (RFLSIPro, Shenzhen Ruiwode RWD), and the average perfusion value was recorded (10 animals in each group). The results are shown in the figure. Figure 8 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 mg / ml) injected intraperitoneally into mice significantly promoted the recovery of blood flow in the ischemic area on the 7th, 14th and 21st days.

[0078] 2. Histological examination of pathological tissue

[0079] After the modeling was completed and the experiment was over, the experimental animals were killed by overdose of anesthetic injection, and the gastrocnemius muscles were taken (after ligation of the femoral artery and its branches, these two muscles will be ischemic). The muscles were used for paraffin sections, and the rest were used for qRT-PCR detection and Western blot detection. After the tissue was removed, it was immersed in 4% paraformaldehyde solution, embedded in paraffin within 24 hours, and then sliced ​​with a thickness of 8 μm, stained with hematoxylin-eosin (HE), and the rest was prepared for immunofluorescence staining.

[0080] 2.1 HE staining:

[0081] Dewax the paraffin sections to water: make a note on the slide, insert it into the slide rack and place it in an oven at 60-65℃, bake for 1-1.5h, dehydrate the slides in the order of xylene 1, xylene 2, and xylene 3 for 15min, then dehydrate in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5min, and wash with distilled water for 5min; stain with hematoxylin for 0.5-2min, wash with running water for 3min, dip in hydrochloric acid ethanol, rinse with running water for 3min, reverse blue with 1% ammonia aqueous solution for a few seconds, rinse with running water for 3min, stain with eosin for a few seconds, and rinse with running water for 3min. Dehydrate with 85% ethanol, 95% ethanol, and anhydrous ethanol for 2min respectively, dehydrate with anhydrous ethanol for 4min, soak in xylene (1) and xylene (2) for 10min respectively, seal with neutral gum, dry and store at room temperature, and take pictures with a microscope. The results are as follows Fig. 9 As shown in the figure, different concentrations of galloylpeonidin (2.5, 5, 10 mg / ml) were intraperitoneally injected into mice for 21 days, which significantly increased the vascular density in the ischemic area.

[0082] 2.2 Immunofluorescence staining:

[0083] Dewaxing of paraffin sections: Make a note on the slide, insert it into the slide rack and place it in an oven at 60-65℃, bake for 1-1.5h, dehydrate the slides in the order of xylene 1, xylene 2, and xylene 3 for 15min, then dehydrate in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5min, and wash with distilled water for 5min; Antigen repair: Place the tissue sections in a repair box filled with citric acid antigen repair buffer in a microwave oven for antigen repair, medium heat for 3min, medium-high heat for 3min, and repeat 3 times. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be dried. After natural cooling, place the slides in PBS and shake on a decolorizing shaker to wash 3 times, 5min each time. After the sections are slightly dried, use a tissue pen to circle around the tissue, add sheep serum in the circle to evenly cover the tissue, and seal at 37℃ for 30min. Gently shake off the blocking solution, add primary antibodies (CD31, α-SMA) prepared with 5% BSA at a 1:300 ratio on the slices, and incubate the slices flat in a humidified box at 4°C overnight. Wash the slides in PBS three times, 5 minutes each time. Add fluorescent secondary antibodies prepared at a 1:500 ratio, incubate the slides at room temperature for 1 hour, wash them three times with PBS, 5 minutes each time; add anti-fluorescence quenching sealing agent containing DAPI, and cover with a coverslip. Use a Nikon laser confocal microscope to collect images of the resulting slides, and then use ImageJ for mathematical statistics. The results are as follows: Fig.10 As shown in the figure, galloylpeonidin significantly increased the density of new blood vessels and the number of arteries in the gastrocnemius muscle in the ischemic area, and promoted vascular maturation.

[0084] 3. Detection of angiogenesis-related gene and protein expression levels

[0085] Western blot and qRT-PCR were used to detect the effect of galloylpeonidin on the expression levels of key proteins and genes in angiogenesis of the gastrocnemius muscle in the ischemic area. The gastrocnemius muscle tissue in the ischemic area was taken, lysed and protein was extracted, and the expression of Vegfa, Vegfr2 and other proteins was detected by Western blot to verify the intervention of galloylpeonidin on the activation and expression of angiogenesis-related signaling pathway proteins in the ischemic area of ​​the lower limbs of mice. At the same time, the gastrocnemius muscle tissue in the ischemic area was taken, the RNA in the tissue was extracted, and reverse transcription was performed. The expression of angiogenesis-related genes Vegfa and Vegfr2 was detected by qRT-PCR. The results are as follows Fig.11 As shown in the figure, galloylpeonidin significantly upregulated the protein and mRNA gene expression levels of Vegfa and Vegfr2 in the gastrocnemius muscle of the lower limb ischemic area of ​​mice (p < 0.05).

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of galloylpeonidin in the preparation of a drug for preventing and / or treating vascular occlusive diseases, characterized in that: The galloylpeonidin can be used for preventing and / or treating vascular occlusive diseases.

2. The use according to claim 1, characterized in that: The galloylpeonidin prevents and / or treats vascular occlusive diseases by promoting angiogenesis.

3. The use according to claim 2, characterized in that: The galloylpeonidin can upregulate the expression levels of angiogenesis-related proteins VEGFA and VEGFR2.

4. The use according to claim 2, characterized in that: The galloylpeonidin can upregulate the expression levels of angiogenesis-related genes VEGFA and VEGFR2.

5. The use according to claim 2, characterized in that: The galloylpeonidin can enhance the proliferation, migration, invasion and tube-forming ability of endothelial cells.

6. The use according to claim 2, characterized in that: The galloylpeonidin can promote blood flow recovery in the ischemic area, increase the density of new blood vessels and the number of arteries in the ischemic area, and promote vascular maturation.

7. The use according to claim 1, characterized in that: The vascular occlusive disease includes any one of peripheral arterial disease, chronic coronary artery complete occlusion, and stroke.

8. A pharmaceutical composition for treating vascular occlusive diseases, characterized in that: The main active ingredient of the pharmaceutical composition is galloylpeonidin.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition promotes angiogenesis to prevent and / or treat vascular occlusive diseases through at least one of the following effects: (Ⅰ) Upregulate the expression levels of angiogenesis-related proteins VEGFA and VEGFR2; (Ⅱ) up-regulating the expression levels of angiogenesis-related genes VEGFA and VEGFR2; (III) Enhance the proliferation, migration, invasion and tube formation ability of endothelial cells; (IV) Promote blood flow recovery in the ischemic area, increase the density of new blood vessels and the number of arteries in the ischemic area, and promote vascular maturation.

10. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

Images