CN108685896B - Application of oroxylin A in preparation of medicine for treating and/or preventing chronic peripheral vascular occlusive diseases - Google Patents

Abstract

The embodiment of the invention provides application of oroxylin A in preparing a medicine for treating and/or preventing chronic peripheral vascular occlusive diseases. The embodiment of the invention proves that oroxylin A has the following functions: 1) increasing the density of the blood capillaries of the ischemic tissue, thereby promoting the blood flow recovery of the ischemic tissue; 2) oroxylin A increases the density of the blood capillaries of ischemic tissues by promoting the migration of human vein endothelial cells; 3) oroxylin a can also reduce inflammation during revascularization. Because the oroxylin A has the functions, the oroxylin A can be used for preparing the medicine for treating and/or preventing chronic peripheral vascular occlusive diseases.

Description

Application of oroxylin A in preparation of medicine for treating and/or preventing chronic peripheral vascular occlusive diseases

Technical Field

The invention relates to the technical field of new application of oroxylin A, in particular to application of oroxylin A in preparation of a medicine for treating and/or preventing chronic peripheral vascular occlusive diseases.

Background

Chronic peripheral vascular occlusive diseases, such as limb arteriosclerotic occlusive disease (ASO), thromboangiitis obliterans (TAO), diabetic foot, etc., increase year by year, and when the disease condition becomes worse and cannot be treated by means of blood flow reconstruction, angioplasty, etc., the patient is often at risk of amputation. The rise of therapeutic angiogenesis (angiogenesis) in recent years has provided a new avenue for the treatment of such diseases. Vascular Endothelial Growth Factor (VEGFA) plays an important role as a specific vascular growth factor in therapeutic angiogenesis.

It has been shown that VEGF165 is added to a degradable composite material made of polylactide and polyglycolide (ratio 85:15) and placed in a lower limb ischemic mouse for sustained and slow release, so that the VEGF A sustained-release in the treatment group shows a more significant angiogenesis effect (Sun Q, Chen RR, Shen Y, et a1. Sustanated vascular growth and perfusion in ischemic tissue [ J ]. Pham Res,2005,22(7): 1110-). The investigators used VEGF A Gene therapy for limb ischemia in clinical trials in which the panel used hVEGF165 plasmid DNA coated on the surface of a balloon catheter, delivered it via the femoral artery to the distal femoral artery of a chronic lower limb arterial ischemic patient, expanded balloon transfected lesion vessel wall cells, and after 12 weeks, increased limb blood flow perfusion, and improved ischemic symptoms in the patient (Kusumanto YH, Van Weel V, Mulder NH, et A1.treatment with intravascular tissue wall cells, and patient ischemia symptoms improved with tissues with diabetes mellitus: a double-indendstabilized trial [ J ]. m Hu Gene Ther 2006,17 (6): 683 691.).

Oroxylin A (Oroxylin A, OA) also known as Oroxylin A and Oroxylin A belongs to flavonoid compounds, is a secondary metabolite of plants, mainly exists in oroxylum indicum and scutellaria baicalensis, is one of main drug effect components of scutellaria baicalensis of Labiatae, and has the activities of inhibiting tumor angiogenesis, resisting virus, allergy, oxidation stress, apoptosis, inflammation and the like. Molecular mass 284.26, moleculeFormula C₁₆H₁₂O₅The structural formula is shown as formula 1.

At present, the known pharmacological actions of oroxylin a are: (1) anti-tumor activity: inducing tumor cell apoptosis to directly act on Bcl-2 protein family and caspase family and present obvious dose-effect relationship; ② induction of apoptosis by mitochondrial pathway. Oroxylin A can remarkably promote cytochrome c to be released from mitochondria to cytoplasm without being influenced by a PTP (precision time protocol) specific inhibitor, so that Caspase-9 activation is promoted, PARP (para-amyloid peptide) is sheared, mitochondrial membrane potential is reduced, cell ROS (reactive oxygen species) is increased, and reduced GSH (glutathione) is reduced; ③ p53 apoptosis related gene; fourthly, the drug combination is used for inducing apoptosis; and inhibiting telomerase activity. Reversing drug resistance of tumor cells (i) affects P-glycoprotein (P-gp). Oroxylin A can reduce the effect of P-gp expression reversal 5-FU on human liver cancer drug-resistant cell strain BEL7402 by inhibiting NF-kB signal pathway, and the reversal multiple can be as high as 4.69; ② influence Integrin beta 1; inhibiting tumor cell invasion and metastasis, and inhibiting activity and expression of downstream proteins MMP-2 and MMP-9 by inhibiting ERK activation, thereby playing an anti-invasion role. Induction of tumor cell cycle arrest may be caused by the down-regulation of cyclin-dependent kinase activity kinase CDK7 expression, leading to the inhibition of cyclin-dependent kinase CDK2 expression, critical for the checkpoint in G2/M phase. (2) Neuroprotective effect: can reduce activated microglia, increase the expression of BDNF and promote the phosphorylation of CREB; can obviously improve the mouse dysmnesia caused by scopolamine, but the action can be inhibited by GABA receptor antagonist muscimol and diazepam, and in the electrophysiological experiment of the mouse, oroxylin A can block the inward chloride current mediated by GABA receptor; has protective effect on blood brain barrier permeability under the condition of cell hypoxia injury, and the action mechanism is related to the influence of the blood brain barrier tight junction protein ZO-1 expression. (3) Anti-inflammatory activity: raw264.7 macrophages stimulated by Lipopolysaccharide (LPS) are pretreated by oroxylin A, the expression of nitric oxide synthase iNOSmRNA and proteins is reduced, the generation of NO is reduced, and the dosage is dependent; in addition, oroxylin A can also inhibit the expression of COX-2 protein and mRNA and the production of inflammatory mediator PGE2, and the mechanism is to inhibit the combination and transcriptional activation of nuclear transcription factor NF-kB, thereby inhibiting the expression of iNOS and COX-2. (4) The anti-childbirth effect is as follows: oroxylin A is the main active ingredient in the anti-childbirth drug 'Angongning' clinically used at present, has obvious inhibition effect on spontaneous contraction of the uterus of rats and contraction induced by various agonists, and especially has the most obvious inhibition effect on the effect of oxytocin. (5) The effects of resisting itch, allergy and asthma are as follows: oroxylin A can effectively reduce the itch frequency of mice induced by histamine and simultaneously reduce the vascular permeability of the mice; the effect of the SRS-A on the guineA pig isolated ileum and the isolated lung strip can be obviously antagonized, the effect of the SRS-A on increasing the lung overflow of the guineA pig can be antagonized by intravenous injection, and A good antiallergic effect is prompted; the experimental result also shows that the traditional Chinese medicine has stronger effect of relieving asthma. (6) Anti-oxidative stress, oroxylin A relieves CS-induced oxidative stress and pulmonary inflammation by activating Nrf2 signaling pathway, down-regulates the expression of cytokines TNF-alpha, IL-1 beta and MCP-1, and down-regulates the levels of oxidation markers 3-nitrotyrosine and 8-isoprostane.

Disclosure of Invention

The formation of collateral circulation vessels around ischemic tissues or the formation of endogenous vascular bypasses can be used as therapeutic indicators for chronic peripheral vascular occlusive diseases, and the quantitative results are expressed as increased capillary density. The present inventors have made intensive studies on oroxylin a, and have unexpectedly found that oroxylin a can be used for treating and/or preventing chronic peripheral vascular occlusive diseases by up-regulating Vascular Endothelial Growth Factor (VEGFA) expression, increasing the capillary density of ischemic tissues, and improving and restoring the blood flow of ischemic tissues, and have completed the present invention based on the above finding. The specific technical scheme is as follows:

the invention provides application of oroxylin A in preparing a medicine for treating and/or preventing chronic peripheral vascular occlusive diseases.

The chronic peripheral vascular occlusive disease is vascular disease which occurs outside heart and cerebral vessels, is caused by vascular stenosis and occlusion due to long-term accumulation of various factors, further causes unsmooth blood flow and tissue ischemia. Including thromboangiitis obliterans, arteriosclerotic obliteration, arterial embolism, multiple Takayasu arteritis, aneurysm, diabetic foot, superficial thrombophlebitis, deep venous thrombosis, deep venous valvular insufficiency, varicosis, etc., and limb artery vasomotor dysfunction diseases such as Raynaud's disease and erythromelalgia. The chronic disease mainly refers to the disease with hidden disease, long course of disease and persistent disease, and is relative to the acute disease with rapid onset, rapid change of disease and serious symptom.

The oroxylin A disclosed by the invention can be used for treating and/or preventing chronic peripheral vascular occlusive diseases by increasing the capillary density of ischemic tissues so as to promote the blood flow recovery of the ischemic tissues.

The oroxylin A disclosed by the invention can be used for increasing the density of capillary vessels of an ischemic tissue by promoting the migration of human vein endothelial cells.

Oroxylin a according to the invention may also be used for the treatment and/or prevention of chronic peripheral vascular occlusive disease by reducing inflammation during revascularization.

In a second aspect, the invention provides a pharmaceutical composition for treating and/or preventing chronic peripheral vascular occlusive disease, comprising oroxylin a.

In a specific embodiment of the second aspect of the invention, the chronic peripheral vascular occlusive disease is selected from at least one of thromboangiitis obliterans, arteriosclerotic occlusive disease, arterial embolism, polyarteritis, aneurysm, diabetic foot, superficial thrombophlebitis, deep vein thrombosis, deep vein valvular insufficiency, varicose veins, raynaud's disease, erythematous limb pain.

In another specific embodiment of the second aspect of the present invention, wherein said oroxylin a is provided in the form of a monomer of oroxylin a or in the form of a plant extract comprising oroxylin a, mainly oroxylum indicum extract or scutellaria extract. Oroxylin a is commercially available; the oroxylin a extract or scutellaria extract as used herein can be prepared by known methods, and the present invention is not limited thereto.

In a particular embodiment of the second aspect of the invention, the pharmaceutically acceptable carrier or excipient is selected from the group consisting of solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, preservatives, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavorants, and antioxidants.

In another specific embodiment of the second aspect of the present invention, the pharmaceutical composition is formulated as any one of powder, tablet, capsule, pill, drop pill, injection, emulsion, suspension or tincture.

As used herein, the term "treatment" has its ordinary meaning and refers herein, in particular, to the treatment of an already afflicted chronic peripheral vascular occlusive disease, in order to effect a treatment, cure, mitigation, palliation, or the like, of said disease. Similarly, the term "prevention" as used herein has its ordinary meaning and refers herein in particular to the treatment of an animal subject, who may suffer from or is at risk of suffering from a chronic peripheral vascular occlusive disease as described herein, with a medicament according to the invention in order to produce a preventing, arresting, abrupting, etc. effect on said disease.

The pharmaceutical composition of the present invention can be prepared by conventional techniques in the pharmaceutical formulation field by obtaining the active ingredients of the raw materials of the pharmaceutical composition of the present invention by extraction, separation and purification means commonly used in pharmaceutical production, mixing with one or more pharmaceutically acceptable carriers, and then forming the desired dosage form.

As used herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.

The "pharmaceutically acceptable carrier" useful in the pharmaceutical compositions of the invention may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, antioxidants, and the like, which are conventional in the pharmaceutical field, may be included as the pharmaceutically acceptable carrier. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.

As used herein, the term "pharmaceutical composition" has its ordinary meaning. The pharmaceutical composition of the present invention can be prepared by obtaining the active ingredients of the raw materials of the pharmaceutical composition of the present invention by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers, and then forming a desired dosage form, using conventional techniques in the pharmaceutical field, particularly in the field of formulation. The pharmaceutical composition according to the present invention is a pharmaceutical formulation which may be suitable for oral, parenteral or topical, topical administration. The pharmaceutical composition can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field. Specifically, according to the pharmaceutical compositions of the present invention, the pharmaceutical dosage forms include, but are not limited to: tablet, capsule, granule, powder, injection, powder for injection, transdermal patch, ointment, gel, suppository, oral solution, oral suspension, emulsion for injection, oral emulsion, etc., sustained release tablet, and controlled release tablet. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.

Dosage forms for parenteral administration may include, for example, injections, drops for medical use, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches and the like, without being limited thereto.

The pharmaceutical compositions according to the present disclosure may be administered orally or parenterally, for example rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.

As used herein, the term "subject" or "animal subject" has its ordinary meaning and may refer herein to a subject or animal subject suffering from or at risk of suffering from a chronic peripheral vascular occlusive disease as described herein, and may also refer to a subject or animal subject used for some purpose, for example for scientific research purposes. Specifically, the subject is, for example, an animal subject, particularly a mammalian subject, such as a human, pig, dog, cat, cow, sheep, horse, rat, mouse, rabbit, guinea pig, monkey, and the like. More specifically, the subject of the invention is a human.

The pharmaceutically acceptable dose, i.e., the administration dose, of the active ingredient, oroxylin a, may vary according to the age, sex and weight of the subject to be treated, the particular disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration and the judgment of the diagnostician. Determining the dosage to be administered taking these factors into account is within the level of skill in the art. A typical dose may be 0.01-1000 mg/kg/day, specifically 1-100 mg/kg/day. However, the scope of the present disclosure is not in any way limited by the administration dosage.

The oroxylin A provided by the embodiment of the invention can increase the density of the capillary vessels of an ischemic tissue by up-regulating the expression of Vascular Endothelial Growth Factor (VEGFA), so as to improve and restore the blood flow of the ischemic tissue, and can also reduce the inflammatory reaction of the ischemic tissue during the vascular reconstruction, so that the oroxylin A has the effect of treating and/or preventing chronic peripheral vascular occlusive diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows that oroxylin A promotes the recovery of blood flow in the ischemic lower limbs of mice.

Figure 2 shows that oroxylin a increases capillary density in mouse ischemic tissue.

Figure 3 shows that oroxylin a promotes VEGFA expression in post-operative ischemic tissues in mice.

Figure 4 shows that oroxylin a promotes HUVEC cell migration.

FIG. 5 shows that oroxylin A promotes tube formation of HUVEC cells.

FIG. 6 shows that oroxylin A down-regulates the number of macrophages and neutrophils in the ischemic tissue.

FIG. 7 shows that oroxylin A down-regulates IL-1 β expression in ischemic gastrocnemius tissue and serum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: the oroxylin A promotes the blood flow recovery of the ischemic tissue of the lower limb of the mouse and increases the capillary density of the ischemic tissue

The experimental contents are as follows:

male C57BL/6J mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd., laboratory animals were raised in animal houses at 22-25 deg.C and 40-60% relative humidity in the institute of biomedical radiation engineering, national institute of medical sciences, Tianjin, free feeding), oroxylin A (purchased from Desite Biotech Co., Ltd., purity not less than 98%), simvastatin (Sigma-Aldrich, USA), Lectin Lectin I for injection (Vector Laboratories), goat anti-mouse Lectin I primary antibody (Vector Laboratories, USA), secondary antibody: (New England Biotechnology Co., Ltd.), and veterinary drug

594ANTI-GOAT IgG (H + L), Vector Laboratories, USA), DAPI (Solebao, 10. mu.g/ml ready-to-use), HEPES buffer (50mM, pH 8.5,0.1mM Ca²⁺)，

Lower limb ischemic surgery (HLI) embodiments: avertin (anesthetic) was intraperitoneally injected at a dose of 0.33ml/20g for anesthesia and the area was sterilized with 75% ethanol. Fix the supine position. A 5mm transverse incision was made in the flank of the abdomen and in the surface of the inguinal ligament, the abdominal femoral fat pad was separated from the peritoneal liner to expose the proximal femoral artery branching from the internal iliac artery, then the femoral artery and vein were separated from the membranous sheath, the upper and lower ends of the femoral artery were ligated with 6/0 # suture, the blood vessel was transected between the ligatures, and the skin was sutured. Limb perfusion was assessed by Laser Doppler Perfusion Imaging (LDPI). The blood flow rate after operation is reduced to less than 10 percent of that before the operation, the frequency spectrum of the blood flow is obviously changed, the frequency spectrum is used as a standard for successfully establishing a lower limb ischemia model, and then the mouse is placed on an electric blanket at 37 ℃ until the mouse is awakened.

Male C57BL/6J mice at 6-8 weeks were randomly divided into model group, oroxylin A group and simvastatin group, subjected to lower limb ischemic surgery, and the femoral artery was ligated. From the day of surgery (1 h after surgery), the model group, the oroxylin A group and the simvastatin group were administered by gavage daily, the model group was a physiological saline solution containing 0.2% sodium carboxymethylcellulose (administration amount: 0.01ml/g body weight), the oroxylin A group was administered at 10mg/kg body weight to the oroxylin A solution (use concentration: 1mg/ml), and the simvastatin group was administered at 10mg/kg body weight to the simvastatin solution (use concentration: 1 mg/ml). In the experiment, the oroxylin A solution and the simvastatin solution are both prepared by using normal saline containing 0.2 percent of sodium carboxymethyl cellulose. Simvastatin is a positive drug known in the art for promoting capillary regeneration in experiments.

Blood flow perfusion experiment of lower limbs of mice:

before HLI operation (before operation), after HLI operation (after operation), and 3, 7, 14, and 28 days after operation, the ratio of blood flow of the ischemic lower limb to blood flow of the non-ischemic lower limb, i.e., the blood flow recovery rate, was determined by using a laser doppler perfusion imager (USA), respectively.

The experimental result is shown in A picture in figure 1, the blood flow of lower limbs of mice in oroxylin A group is obviously improved compared with that of the mice in model group, and the improvement effect is similar to that of simvastatin group. The B picture in figure 1 is a quantification picture of the A picture, and it can be seen that the blood flow recovery rate of the ischemic tissues of the oroxylin A group and the simvastatin group is obviously improved compared with the model group at 28 days after the operation. Values are expressed as mean ± standard deviation (mean ± SD), and n is 5 to 6. P < 0.05. The experimental result shows that the blood flow velocity of the ischemic tissue of the mouse is remarkably recovered 28 days after the operation, and the oroxylin A can promote the blood flow recovery of the ischemic lower limb of the mouse.

Mouse ischemia tissue capillary density determination:

after 28 days of HLI operation, 5 mice in each of the model group and oroxylin A group were treated by caudal vein injection using Lectin I (using HEPES buffer at a concentration of 5mg/ml, administered at 50. mu.L/20 g body weight), decapitated after 10 minutes, paraffin sections were prepared from the gastrocnemius muscle on the operation side, stained with Lectin I primary antibody (1:200PBS dilution), secondary antibody (1:100PBS dilution), DAPI counterstained nuclei, sections were observed under a fluorescence microscope (DAPI:405nm, Lectin I:564nm), and the tubular structure positive to Lectin I was identified as capillaries, and the density of capillaries was expressed by the number of capillaries in a unit area under 4 random fields.

The results are shown in FIG. 2. Panel a in fig. 2 is the result of Lectin I staining under fluorescence microscopy, wherein the arrow indicates a Lectin I positive signal, Bar: 50 μm. Panel B is a quantification of capillary density and it can be seen that oroxylin a group Lectin I positive signals were significantly greater than the model group. Values are expressed as mean ± standard deviation (mean ± SD), and n is 5. P <0.01, compared to model group. The experimental result shows that oroxylin A can increase the density of the capillary vessels of the post-operation ischemic tissues of the mice.

Detecting VEGFA expression quantity:

the expression amounts of VEGFA mRNA and protein of ischemia side gastrocnemius tissue of a model group and a oroxylin A group of mice 7 days and 14 days after operation are respectively detected by RT-PCR, ELISA and Western blotting methods.

ELISA was performed using the VEGFA ELISA detection kit (cat. No.: E-EL-M1292c Elabasconce) according to the protocol.

And (3) RT-PCR detection: cutting the gastrocnemius tissue into small pieces of about 3mm × 3mm by using surgical scissors, removing non-target tissues such as fat tissue and connective tissue as much as possible, washing the small pieces twice by using PBS (phosphate buffer solution), centrifuging the small pieces to obtain precipitates, adding 1ml of TRIzol (Life technologies), transferring the precipitates into a sterilized glass homogenizer precooled in advance, manually homogenizing the slurry on ice until no obvious tissue pieces exist on the bottom wall of the homogenizer, transferring the homogenized slurry into a 1.5ml centrifuge tube, adding 1/5 volumes of chloroform into each tube, standing the mixture on ice for 15min, shaking the mixture 1 time every 5min, 30s every time, shaking the mixture 3 times, and centrifuging the mixture at 4 ℃ for 15min at 12,000 × g. The upper aqueous phase was taken, added with isopropyl alcohol (analytically pure; concategor), shaken well and then left overnight at-20 ℃. Centrifuging at 12000 Xg for 20min at 4 deg.C, discarding supernatant, adding 75% ethanol treated with DEPC water, shaking gently, centrifuging at 8000 Xg for 10min at 4 deg.C, discarding supernatant, drying, adding 20 μ LRNase to resuspend precipitate, washing in water at 55 deg.C for 15min, reading RNA concentration and 260/280 value with e-separator, and reverse-transcribing with Transcriptor First Strand Cdna Synthesis Kit (Roche) to prepare cDNA. With FastStart Universal SYBR Green Master (Roche) and

7500Real-time PCR System (Applied Biosystems) measures the expression of each target gene in different samples. Total 25. mu.L; the amplification conditions were: 95 ℃ for 15min, then 94 ℃ for 15s, 55 ℃ for 30s and 70 ℃ for 30s are used as a cycle, and the amplification is carried out for 40 cycles. The primer information is shown in Table 1.

TABLE 1

Western blotting: extracting a gastrocnemius tissue protein sample by adopting conventional operation, wherein the sample loading amount is 50 mu g; gapdh primary antibody (rabbit anti-mouse, CST #2118,) 1: 10005% BSA; VEGFA primary antibody (rabbit anti-mouse, Abcam ab46154)1: 10005% BSA; secondary antibody (HRP-labeled goat anti-rabbit IgG, zhongshan gold bridge): 10000 TBST.

The results are shown in FIG. 3. A-C in FIG. 3 are the expression levels of VEGFA mRNA and protein detected 7 days after operation by RT-PCR, ELISA and Western blotting, respectively; D-F is to detect the expression quantity of VEGFA mRNA and protein 14 days after operation by using RT-PCR, ELISA and Western blotting respectively. The experimental result shows that the expression of mRNA and protein of VEGFA of oroxylin A group ischemic tissues is remarkably increased after HLI is administrated for 7 days and 14 days after the HLI operation. Values are expressed as mean ± standard deviation (mean ± SD), and n is 3. P <0.05, P <0.01, compared to model group. The experimental result shows that oroxylin A has the function of promoting VEGFA expression of ischemic tissues.

Example 2: oroxylin A promotes HUVEC cell migration

During angiogenesis, endothelial cell proliferation and migration contribute to the spread of new vessels in the preexisting vessels, thus using HUVEC cells to explore the effect of oroxylin a on cell migration.

Human venous endothelial cells (HUVEC)

PCS-100-010^TM) Fetal bovine serum (FBS, lonza, CC-4147), EGM-2 medium (lonza, CC-3156), 0.25% pancreasEnzyme (Biological Industries, Israel), DAPI (Soilebo, 10. mu.g/ml ready-to-use).

HUVEC cell scratch test:

HUVEC cells were routinely cultured in EGM-2 complete medium containing 5% FBS, digested with 0.25% trypsin when 80% confluent, and then cultured at 1X 10⁴Individual cells/well were seeded in 96-well plates. Standing at 37 deg.C for 5% CO₂After overnight incubation in the incubator, the cells were divided into two groups, oroxylin a and control. At this time, the oroxylin A group medium was changed to contain 2.5X 10^-6The complete medium of M oroxylin A and 0.1% DMSO, the control medium was changed to complete medium containing 0.1% DMSO. HUVEC cells were subjected to cell-scratch experiments in IncuCyte (Thermo scientific 3111; USA). Putting the 96-hole plate into a scratching device, transversely scratching cells by using the 96-hole wound scratching device, taking down the 96-hole plate after the transverse scratching is finished, and cleaning the 96-hole plate by using Phosphate Buffer Solution (PBS) to remove the cells falling off during scratching; adding the corresponding culture media according to groups, and taking pictures under an inverted microscope at 40 multiplied by 0 hour (0 h); the cells are put back into the incubator to be continuously cultured for 8h, and the photographing is carried out again to record that the time is 8h (8 h); measuring the width of the scratch gap at 0h (before the cells start to migrate) and 8h, and calculating the mobility according to the width of the scratch gap and the width of the scratch gap, wherein the calculation formula is as follows: cell mobility ═ (W0h-W8h)/W0h × 100%; w refers to scratch gap width.

The results are shown in fig. 4, panel C, and the experimental results show that oroxylin a can significantly reduce the width of cell scratch after 8 h; panel D is a quantitative plot of panel C, showing a significant increase in the mobility of cells in oroxylin a group compared to the control group, with values expressed as mean ± SD, n ═ 6 and P < 0.001. Therefore, oroxylin A can enhance HUVEC cell migration and promote cell scratch healing

To exclude the cell migration results caused by cell proliferation, MTT and Brdu experiments were performed to detect cell proliferation. At 2.5X 10³Each cell was seeded in 96-well plates per well. Standing at 37 deg.C for 5% CO₂After 12h of culture in an incubator, the cells were grouped into oroxylin A groups (added to final concentrations of 2.5X 10, respectively)^-6M，5×10^-6M，1×10^-5Thousand of MPapatin a), a control group (complete medium containing 0.1% DMSO), a positive control group (VEGFA added to a final concentration of 100 ng/ml), dosing for 18h to detect MTT; adding the medicine for 24h to detect Brdu. In this experiment, oroxylin A and VEGFA were dissolved in complete medium containing 0.1% DMSO.

The MTT and Brdu results are shown in FIG. 4, panels A and B. Compared with the control group, the cell proliferation conditions of the oroxylin A group have no significant difference, which indicates that the oroxylin A does not influence the proliferation of the HUVEC cells. Values are expressed as mean ± standard deviation (mean ± SD), and n is 5 to 6. P < 0.05.

Stress fiber immunofluorescence staining experiment:

HUVEC cells at 5X 10 per well⁴The cells were seeded in 12-well plates, and the cells were divided into 2 groups, respectively, a control group (complete medium containing 0.1% DMSO), a oroxylin a group (complete medium containing 2.5 μ M oroxylin a and 0.1% DMSO), and after 24 hours of culture, HUVEC cells were subjected to a stress fiber immunofluorescence staining experiment. The specific experimental steps are as follows: washing with PBS for 2 times and 5 min/time; fixing with 3.7% paraformaldehyde for 10 min; washing with PBS for 4 times and 5 min/time; permeabilizing PBST buffer (PBS containing 0.1% TritonX-100) for 3-5 min; washing with PBS for 4 times and 5 min/time; blocking with 1% BSA in PBS buffer for 30 min; (the following steps need to be carried out in a dark place) rhodamine-labeled phalloidin actin dye is diluted by PBS buffer solution containing 1% BSA (volume ratio is 1: 40) and is stained for 20 min; washing with PBS for 3 times and 5 min/time; staining for 10min with DAPI; washing with PBS for 3 times and 5 min/time; fluorescence photography was performed. The results are shown in FIG. 4, Panel E.

The actin dye of rhodamine-labeled phalloidin actin can specifically label the stress fiber in the cell and takes the form of a shape (as indicated by an arrow in the figure) ending at a sharp edge under a microscope, which is a typical morphological characteristic of the migrating cell. As can be seen in panel E of fig. 4, 2.5 μ M oroxylin a induced actin cytoskeletal rearrangement in HUVEC cells and promoted the formation of stress fibers. Suggesting that oroxylin a might stimulate the migration of HUVEC cells by inducing stress fiber formation. (Bar: 100 μm)

Matrigel tube formation experiment:

matrigel (354234, corning) was spread on 96-well plates to avoid air bubbles, and the process was performed on ice. Put at 37 ℃ with 5% CO₂Incubate in incubator for 1 h. HUVEC cells were routinely cultured in EGM-2 complete medium containing 5% FBS, digested with 0.25% trypsin at 80% confluence, treated with complete medium containing 0.1% DMSO (control), 2.5. mu.M oroxylin A solution (oroxylin A group), 100ng/mL VEGFA solution (VEGFA group), 100ng/mL VEGFA and 2.5. mu.M oroxylin A solution (VEGFA + OA group), respectively, and treated with 2X 10⁴Each cell was seeded in 96-well plates plated with matrigel. Standing at 37 deg.C for 5% CO₂Culturing in an incubator, taking a picture after culturing cells for 18h, and calculating the number of the branch points of the blood vessels by photoshop. The solutions described in this experiment were all prepared using complete medium containing 0.1% DMSO.

The results are shown in FIG. 5, in which Panel A shows the results of matrigel tube formation experiments under the microscope (Bar: 100 μm), and Panel B shows the quantitative results. As shown in panel B, the number of vascular branch points was significantly increased compared to the control group (P <0.001) for both the oroxylin a and VEGFA groups, indicating that both oroxylin a and VEGFA have the ability to promote the vascularization of HUVEC cells; meanwhile, compared with the VEGFA group, the number of vascular branch points of the VEGFA + OA group is remarkably increased, which indicates that oroxylin A and VEGFA act synergistically to further improve the tube forming capability of HUVEC cells (P < 0.05).

Example 3: oroxylin A reduces inflammation during revascularization

The promotion of revascularization is generally accompanied by the activation of inflammatory response, which may increase the prevalence of atherosclerosis and limit the application of angiogenesis promoting drugs in ischemic diseases. In the transition phase from inflammation elimination to tissue repair, inflammatory cells continue to participate in subsequent repair reactions along with revascularization, and endogenous processes that partially compensate for tissue perfusion defects are tightly coupled with inflammatory reactions. Chronic inflammation is a key obstacle in revascularization, which impairs endogenous revascularization in peripheral arterial disease and limits the effectiveness of revascularization therapies, and the involvement of some inflammatory mediators, including IL-1 β, results in poor clinical efficacy in treating peripheral arterial disease and does not benefit patients with peripheral vascular disease. Surprisingly, the number of macrophages and neutrophils in ischemic tissues and the release condition of various inflammatory factors after the oroxylin A is perfused for 3 days are detected by using flow cytometry, and the result shows that the oroxylin A can regulate the polarization effect of the macrophages, reduce the infiltration of the neutrophils and relieve inflammatory reaction. The oroxylin A is suggested to have double regulation functions, on one hand, the angiogenesis is promoted, and on the other hand, the oroxylin A has anti-inflammatory activity.

To identify the leukocyte population in the muscle tissue, approximately 100mg of the gastrocnemius muscle on the HLI surgical side was excised, weighed, and an enzymatic digestion solution was made up by dissolving 500U/mL collagenase II (C6885, SIGMA) and 2.5U/mL dispase II (D4693, SIGMA) in 1800uL Hank's balanced salt solution (H1025, Solebao). Gastrocnemius muscles were placed in an enzymatic digest and the muscles digested at 37 ℃ for 1.5 hours, then the digest was filtered through a 40 micron cell filter and washed with 2mL PBS containing 1% BSA. Then stained with PE-F4/80, APC-Cy7-Ly6G and PerCP-CD45(565410, 560600, 561047, B & D). After 30 minutes, the cells were washed with 1mL of PBS-BSA and quantitatively analyzed using a BD Aria III flow cytometer and BD FACSDiva software, expressing the number of neutrophils as a Ly6G positive F4/80 negative cell to leukocyte ratio, expressing the number of macrophages as F4/80, CD45 double positive cell to leukocyte ratio, and BD Aria III flow cytometer and BD FACSDiva7.0 software. The expression of macrophages and neutrophils in the ischemic tissue 3 days after surgery is shown in figure 6.

Cell Quest software is used for establishing an acquisition template FSC/SSC two-parameter scatter diagram, and a P1 gate is set to select all cells except Cell debris in gastrocnemius tissues as target cells (A diagram). Establishing an analysis template APC-Cy7-Ly6G/PE-F4/80 fluorescence two-parameter cross gate scattergram, wherein the abscissa represents PE-F4/80 fluorescence intensity, the ordinate represents APC-Cy7-Ly6G fluorescence intensity, analyzing the proportion of neutrophil positive cells in gastrocnemius, the neutrophil is cells expressing Ly6G positive and F4/80 negative, as shown in a Q1 gate in a D picture, and a C picture is a quantitative result. The results showed that the number of neutrophils in the ischemia tissue of the model group was significantly increased (P <0.001) 3 days after the operation compared with the sham operation group, while the number of neutrophils in the oroxylin a group was significantly decreased (P <0.001) three days after the administration compared with the model group, suggesting that oroxylin a could decrease the number of neutrophils after the operation.

Then, an analysis template PerCP-CD45/PE-F4/80 fluorescent two-parameter cross-portal scatter diagram is established by the software, and the proportion of macrophages in gastrocnemius muscles is analyzed. The abscissa of the graph E represents the fluorescence intensity of PE-F4/80, and the ordinate represents the fluorescence intensity of PerCP-CD 45. Macrophages are double positive cells expressing both CD45, F4/80, as shown by the gate Q2-1 in panel E. Panel B is a quantitative result showing that the macrophage numbers in ischemic tissues of the model group were significantly increased (P <0.001) 3 days after the operation compared to those of the sham operation group, while the macrophage numbers in the oroxylin a group were significantly decreased (P <0.001) three days after the administration compared to the model group, suggesting that oroxylin a could decrease the number of macrophages after the operation.

Quantitative ELISA analysis of IL-1. beta. in gastrocnemius tissue reference is made to the instructions of the IL-1. beta. enzyme-linked immunosorbent assay kit (cat # E-EL-M003 0037c, Elapscience).

And (3) detecting IL-1 beta in serum, namely, standing a whole blood sample at room temperature for 2 hours, centrifuging at 1000 Xg for 20 minutes, and taking supernatant. IL-1. beta. detection was performed according to the instructions of the IL-1. beta. enzyme-linked immunosorbent assay kit (cat. E-EL-M0037c, Elapscience).

The results of IL-1. beta. expression in ischemic gastrocnemius tissue and serum 3 days after the operation are shown in FIG. 7. Compared with the model group, the expression of IL-1 beta (P <0.05 and P <0.01) in gastrocnemius muscle tissues and serum of the mice in the oroxylin A group is remarkably reduced (P <0.05 and P <0.01), which indicates that the expression of IL-1 beta in gastrocnemius muscle and serum can be remarkably reduced by the oroxylin A group. Values are expressed as mean + standard deviation (mean ± SD), and n is 3.

The treatment methods of the model group and the oroxylin A group in the embodiment are completely consistent with those of the corresponding group in the embodiment one, and the pseudo-operation group in the embodiment is not treated by operation and is infused with drugs according to the same formula, dosage and frequency as those of the model group.

The experimental results of the above examples show that: 1. oroxylin A can promote the blood flow recovery of the ischemic lower limbs of mice; 2. oroxylin A can increase the density of capillary vessels of ischemic tissues after the operation of the mice; 3. oroxylin A has the function of promoting the expression of VEGFA in ischemic tissues; 4. oroxylin A can enhance HUVEC cell migration and promote healing of cell scratches; 5. oroxylin a may stimulate the migration of HUVEC cells by inducing stress fiber formation; 6. oroxylin A can promote the tube forming capability of HUVEC cells; 7. oroxylin A down regulates the number of neutrophils, macrophages and the expression level of IL-1 beta after surgery. From the results, on one hand, oroxylin A can increase the density of the capillary vessels of the ischemic tissue by promoting the migration and the tube forming capability of vascular endothelial cells, and further promote the blood flow recovery of the ischemic tissue; on the other hand, oroxylin a is able to reduce the inflammatory response during revascularization; the oroxylin A achieves the effect of treating and/or preventing chronic peripheral vascular occlusive diseases through the two effects.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

<110> Tianjin Chinese medicine university

Application of oroxylin A in preparation of medicine for treating and/or preventing chronic peripheral vascular occlusive diseases

<130>PP184992

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ggtgctgagt atgtcgtgga 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ccttccacaa tgccaaagtt 20

<210>3

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tgcagattat gcggatcaaa cc 22

<210>4

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tgcattcaca tttgttgtgc tgtag 25

Claims (4)

1. Use of oroxylin a for the preparation of a medicament for promoting vascular endothelial growth factor expression, wherein the medicament is for the treatment and/or prevention of chronic peripheral vascular occlusive disease; the chronic peripheral vascular occlusive disease is at least one selected from the group consisting of arteriosclerotic obliteration, polyarteritis, aneurysm, diabetic foot, deep vein valve insufficiency, and varicose vein.

2. The use according to claim 1, wherein oroxylin A is used for treating and/or preventing chronic peripheral vascular occlusive disease by increasing the capillary density of ischemic tissue to promote the restoration of blood flow of ischemic tissue.

3. The use according to claim 2, wherein oroxylin A increases the capillary density of ischemic tissue by promoting migration of human venous endothelial cells.

4. Use according to claim 1, characterized in that the oroxylin A also treats and/or prevents chronic peripheral vascular occlusive disease by reducing inflammation during revascularization.

Images