CN106074468A - Cinnamic aldehyde purposes in preparation can induce the derivant of vegf expression and secretion - Google Patents

Abstract

The present invention relates to drug world, be specifically related to cinnamic aldehyde purposes in preparation can induce the derivant of vegf expression and secretion.The present invention passes through lot of experiments, is found that cinnamic aldehyde can be as effective ingredient to induce vegf expression and secretion first, it is possible to create good economic benefit and social benefit widely.

Description

Cinnamic aldehyde purposes in preparation can induce the derivant of vegf expression and secretion

The application is the divisional application of original application, the filing date of original application: 2014-5-22；Application No.: 2014102198731；Invention and created name is: cinnamic aldehyde application in preparing angiogenesis promoting medicine.

Technical field

The present invention relates to drug world, be specifically related to cinnamic aldehyde and can induce the derivant of vegf expression and secretion in preparation In purposes.

Background technology

Cinnamic aldehyde, English name: Cinnamic aldehyde, molecular formula: C₆H₅CHCHCHO, molecular weight: 132.16, CAS step on Record number: 104-55-2, chemical structural formula is:

Cinnamic aldehyde is a kind of aldehydes organic compound, sticks thick liquid for yellow, be naturally occurring in Oleum Cinnamomi, Oleum Cinnamomi, In the quintessence oils such as oil of Herba Pogostemonis, hyacinth oil and Oleum Rosae Rugosae.In nature, naturally occurring cinnamic aldehyde is transconfiguration, and this molecule is Connect a phenyl on one acrylic aldehyde, therefore can be considered as a kind of Propenal derivative.

The most frequently used cinnamic aldehyde synthetic method is in near-critical water, with benzaldehyde and acetaldehyde as raw material, without additional Under conditions of what catalyst, synthesize cinnamic aldehyde.Natural cinnamyl aldehyde can generate with sodium sulfite and add in Oleum Cinnamomi and Oleum Cinnamomi Become thing, then separate with caustic digestion, then refine and obtain.

The application in terms of medicine of the current published cinnamic aldehyde includes: 1. sterilization and anticorrosion, particularly has fungus significantly Curative effect.To escherichia coli, bacillus subtilis and golden yellow staphylococcus, Staphylococcus albus, dysentery bacterium, typhoid fever and secondary wound Cold first bacillus, streptococcus pneumoniae, aerobacteria, Bacillus proteus, anthrax bacillus, Salmonella enteritidis, vibrio cholera etc. has suppression to make With.And notable to gram positive bacteria bactericidal effect, can be used for treating the multiple disease caused because of antibacterial infection.Minimum suppression is dense Degree (MIC) is 0.02 0.07ul/ml, is 0.1 0.3ul/ml to deep pathomycete MIC.2. antiulcer, adds stomach strengthening, intestinal Road moves.Its mechanism of action is the suppression due to ulcer activities factor (gastric juice and pepsin) and defending factors (Mucosal blood Flow velocity rate) reinforcement, and suppression gastric mucosa current potential reduce and to caused by mucosa.In addition, cinnamic aldehyde can drop Low enzymatic activity.Cinnamic aldehyde system armaticity stomach invigorating carminative, has the stimulation of mitigation to the intestines and stomach, can promote that saliva and gastric juice divide Secrete, strengthen digestive function, release gastrointestinal smooth muscular spasm, alleviate intestinal cramps pain, have significant stomach invigorating, carminative effects. 3. steatolysis effect.Cinnamic aldehyde has suppression epinephrine and fatty acid is dissociated by ACTH, promotes the fat conjunction of glucose One-tenth effect, can be used in glycemic control medicine, strengthens insulin and replaces the performance of glucose, prevents and treats diabetes.4. antivirus action. Infected by influenza, the tumor inhibition effect that SV10 virus causes is powerful.5. antitumaous effect.The generation of tumor can be suppressed, and have anti- Mutation and radiation resistance.6. hypotensive effect.Adrenal cortex hypertension there is hypotensive effect.7. yang invigorating functions.U.S. sesame The a research adding brother's Therapy study center shows, cinnamic aldehyde has certain effect to male Yang tonifying.

But, at present about cinnamic aldehyde induction vegf expression and secretion in terms of effect have no report.

Summary of the invention

It is an object of the invention to open cinnamic aldehyde as promoting that angiogenesis effective ingredient is preparing angiogenesis promotion New application in agent, and discovery based on cinnamic aldehyde new application, it is provided that by promoting that angiogenesis produces the medicine of therapeutical effect Thing.

First aspect present invention discloses cinnamic aldehyde as promoting that angiogenesis effective ingredient is preparing angiogenesis promotion New application in agent.

Further, described angiogenesis accelerator play following active one or more:

A) endothelial cell proliferation is promoted；

B) endothelial cell damage healing is promoted；

C) endothelial cell migration is promoted；

D) endotheliocyte segment dislocation is promoted；

E) induction vegf expression and secretion；

F) PI3K/AKT signal path is activated；

G) c-Raf/MEK/Erk1/2 signal path is activated, and/or

H) promote that wound tissue's number of blood vessel increases.

Second aspect present invention discloses cinnamic aldehyde in preparation by promoting that angiogenesis produces in the medicine of therapeutical effect Purposes.

Further, described medicine is to promote wound healing and/or wound healing and/or the medicine of wound healing.

Preferably, described medicine is treatment skin acute wounds, skin chronic trauma or the medicine of skin ulcer.

It is furthermore preferred that described medicine is prevention and/or the medicine treating the skin injury that diabetes cause.

Further, described medicine is the medicine promoting collateral blood vessels to be formed.

Preferably, described medicine is prevention and/or the medicine for the treatment of coronary heart disease and myocardial ischemia.

Third aspect present invention also discloses by promoting that angiogenesis produces the pharmaceutical composition of therapeutical effect, described medicine Containing cinnamic aldehyde in the effective ingredient of thing.

Preferably, in aforementioned pharmaceutical compositions, cinnamic aldehyde is the sole active ingredient of this pharmaceutical composition.

Preferably, aforementioned pharmaceutical compositions also comprises pharmaceutically acceptable carrier.

" pharmaceutically acceptable " composition applies to people and/or animal and without excessive bad side reaction (such as toxicity, stimulation And allergy) i.e. there is the material of rational benefit/risk ratio." pharmaceutically acceptable carrier " is for by the meat of the present invention Cinnamic aldehyde sends acceptable solvent, suspending agent or the excipient pharmaceutically or on food of animal or people to.Carrier can be liquid Or solid.

Pharmaceutically acceptable carrier is various pharmaceutically conventional adjuvant and/or excipient, includes, but is not limited to sugar Class (such as lactose, dextrose plus saccharose), starch (such as corn starch and potato starch), cellulose and its derivates is (such as carboxymethyl Sodium cellulosate, ethyl cellulose and methylcellulose), tragacanth gum powder, Fructus Hordei Germinatus, gelatin, Talcum, kollag is (such as tristearin Acid and magnesium stearate), calcium sulfate, vegetable oil, such as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, Oleum sesami, olive oil, Semen Maydis oil and cupu oil, polynary Alcohol (such as propylene glycol, glycerol, Sorbitol, mannitol and Polyethylene Glycol), alginic acid, emulsifying agent is (such as Tween, polyoxyethylene Oleum Ricini), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, tablet agent, stabilizer, antioxidant, preservative, nothing Pyrogen water, isotonic saline solution and phosphate buffer etc.；This carrier can improve the stability of formula, active and biological as required Effectiveness etc..

When pharmaceutical composition of the present invention uses, described cinnamic aldehyde is as sole active ingredient, or described cinnamic aldehyde conduct One of effective ingredient, can be mixed and made into the medicine of different way of administration with one or more pharmaceutically acceptable carriers or excipient Agent type.

Preferably, the dosage form of described pharmaceutical composition be tablet, capsule, powder, granule, syrup, solution, Oral liquid, spirit, tincture, aerosol, powder spray, injection, injectable sterile powder, or suppository.Above-mentioned preparation type can To understand according to the related definition in pharmaceutics (sixth version, People's Health Publisher, Cui Fude), preparing of above-mentioned preparation is permissible Prepare according to the method for the related preparations in pharmaceutics (sixth version, People's Health Publisher, Cui Fude).

Preferably, the dosage form of described pharmaceutical composition is tablet or oral liquid.

Preferably, pharmaceutical composition of the present invention can be through oral, intravenous, intramuscular or subdermal routes of administration.

Preferably, can be degerming by membrane filtration when the pharmaceutical composition of the present invention produces, or carry out autoclaving.

Cinnamic aldehyde can change with mode of administration and the order of severity of disease as the effective dose of active component.To big portion For dividing large mammal, every day, the taking dose of effective ingredient cinnamic aldehyde was about 0.01～1000mg.Preferably, Cheng Renlin Bed dosage in the range of 0.01-200mg/ day, more preferably 0.05-100mg/ day.

The medicine of the present invention has the effect significantly promoting angiogenesis, can be used for the blood vessel injury that many reasons causes Treatment.

The invention have the benefit that the present invention passes through lot of experiments, be found that cinnamic aldehyde is as effectively becoming first Divide the new application in preparing angiogenesis accelerator and cinnamic aldehyde in preparation by promoting that angiogenesis produces therapeutical effect Medicine in new application.Cinnamic aldehyde abundant raw material, cheap, toxic and side effects is little, and dosage form is readily selected, and formulation ingredients is bright Really, preparation technology and quality standard thereof be easy to control, there is good market compliance and application prospect, it is possible to create good Economic benefit and widely social benefit.

Reference

The impact that HUVECs is bred by Fig. 1: cinnamic aldehyde, * * P < 0.01, * * * P < 0.001vs control.

The impact on HUVECs wound healing of Fig. 2: the cinnamic aldehyde, (A) (B) is scratch experiment result, and (C) is creep plate experiment knot Really, * * P < 0.01, * * * P < 0.001vs control.

The impact that HUVECs is migrated by Fig. 3: cinnamic aldehyde, a.control, b.0.1 μM, c.1 μM, d.5 μM, e.10 μM, * * * P<0.001vs control。

The impact on HUVECs segment dislocation of Fig. 4: the cinnamic aldehyde, a.control, b.0.1 μM, c.1 μM, d.5 μM, e.10 μ M, * * * P < 0.001vs control.

The impact on HUVECs secretion of VEGF of Fig. 5: the cinnamic aldehyde, * * * P < 0.001vs control.

Fig. 6: cinnamic aldehyde promotes AKT, eNOS protein phosphorylation, * * P < 0.05, * * P < 0.01, * * * P < 0.001vs control。

Fig. 7: cinnamic aldehyde promotes c-Raf, Erk1/2, p38 protein phosphorylation.

HUVECs propagation, VEGF secretion and AKT and the Erk1/2 protein phosphorylation that cinnamic aldehyde is induced by Fig. 8: inhibitor Impact, * * * P < 0.001vs control, ###P < 0.001vs CA.

Wound area after Fig. 9: wild-type mice and diabetic mice skin injury, P < 0.05vs control.

Figure 10: mice skin tissue pathology section (H&E dyeing), 4 × 10 amplifications.

The CD-31 immunofluorescence section of Figure 11: mice skin tissue, 10 × 20 amplifications, * * * P < 0.001vs control。

Figure 12: the VEGF expression in wild-type mice (A) and diabetic mice (B) skin histology, * * * P < 0.001vs control。

Detailed description of the invention

The present invention is expanded on further below in conjunction with specific embodiment, it should be appreciated that embodiment be merely to illustrate the present invention and not For limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip Part or according to the condition proposed by manufacturer.Unless otherwise indicated, the most all of percentage ratio and number are by weight.

Embodiment 1 cinnamic aldehyde promotes angiogenesis experiment in vitro

(1) experimental technique

Human umbilical vein endothelial's cell (HUVECs) is purchased from ALLCELLs company of the U.S., the purity equal 95% of every batch of cell Above, and using the expression of flow cytomery endotheliocyte Research of predicting markers (CD31, vWF, CD54), positive rate all reaches More than 99%, it is accredited as primary cell.

1. cinnamic aldehyde promotes HUVECs proliferation experiment

Take human umbilical vein endothelial's cell (HUVECs) of exponential phase of growth, with MCDB13 complete medium make 5 × 104/ml cell suspension, is inoculated into 96 orifice plates with 5 × 103/porocyte density, is placed in 37 DEG C, in 5%CO2 incubator；Treat After cell attachment is good, abandon former culture medium, change and (be purchased from China's pharmaceutical biological product inspection containing 1%FBS and variable concentrations cinnamic aldehyde Ding Suo company, purity is more than 98%) basal medium；Matched group is the not pastille culture medium containing equivalent DMSO, positive right It it is the not pastille culture medium adding VEGF (20ng/ml) according to group；After 24 hours, add CCK-8 solution (10 μ l/ hole), cultivating Case reacts 3 hours, chooses 450nm wavelength in microplate reader, detect each hole absorbance (OD value)；(OD gives rate of increase %= Medicine/OD comparison-1) × 100%.

2. cinnamic aldehyde promotes the experiment of HUVECs wound healing

In 24 orifice plates, add 0.5% gelatin (400 μ l/ hole), be placed in 37 DEG C, to hatch at least 2 in 5%CO2 incubator little Shi Hou, abandons gelatin, dries standby；Take human umbilical vein endothelial's cell (HUVECs) of exponential phase of growth, complete with MCDB131 Culture medium makes 1 × 105/ml cell suspension, is inoculated into coated 24 orifice plates of gelatin with 3 × 104/porocyte density, puts In 37 DEG C, in 5%CO2 incubator；After treating that cell attachment is good, with 200 μ l rifle heads in vertical standardized road, centre position, every hole Straight line, abandons former culture medium, and PBS washes floating cell (washing 2 times) off, changes containing 1%FBS and the basis of variable concentrations cinnamic aldehyde Culture medium；T0 and T24 time point is taken pictures under 100 power microscopes at same position respectively, records cut distance；Healing rate %= (1-T24 cut distance/T0 cut distance) × 100%.3. creep plate experimental evaluation cinnamic aldehyde promotes the energy of HUVECs wound healing Power

In 12 orifice plates, the round plastic coverslip of a piece of a diameter of 13mm is put in every hole, adds 0.5% gelatin (800 μ l/ Hole), after being placed in 37 DEG C, 5%CO2 incubator hatching at least 2 hours, abandon gelatin, dry standby；Take the primary of exponential phase of growth Human umbilical vein endothelial cells (HUVECs), makes 1 × 105/ml cell suspension with MCDB131 complete medium, with 3 × 104 Individual/porocyte density is inoculated into coated 24 orifice plates of gelatin, is placed in 37 DEG C, in 5%CO2 incubator；After 24 hours, use aseptic tweezer Son presss from both sides out the coverslip in every hole gently, abandons former culture medium, changes the basis cultivation containing 1%FBS and variable concentrations cinnamic aldehyde Base；After 24 hours, according to the method for cell dissociation every porocyte digested and move to respectively, in 1.5ml centrifuge tube, mix gently Even, with blood-counter system record every porocyte number；With cell number number evaluate cinnamic aldehyde promote HUVECs wound healing Ability.

4. cinnamic aldehyde promotes that HUVECs migrates experiment

In 24 orifice plates, every hole adds the (MCDB131 basal medium+50ng/ml VEGF+10% of room culture medium under 600 μ l FBS), Transwell cell is put in 24 orifice plates；Take human umbilical vein endothelial's cell (HUVECs) of exponential phase of growth, Make 5 × 105/ml cell suspension with MCDB131 basal medium, inoculate Transwell with 5 × 104/porocyte density In cell, add the cinnamic aldehyde of variable concentrations, be placed in 37 DEG C, in 5%CO2 incubator；After 24 hours, in one piece of 24 clean hole Plate adds PBS 600 μ l/ hole, cell is transferred in new 24 orifice plates, with cotton pellet wiping cell upper surface (note: power gently Degree wants light in order to avoid film air spots)；By 10 μMs of Hochest33258 10 minutes (room temperature, lucifuges) of dyeing；Further take out another block to do 24 clean orifice plates, add 4% paraformaldehyde 600 μ l/ hole, and cell fixes 15 minutes；Every hole random 5 under 40 times of fluorescence microscopies Take pictures in the individual visual field；IPP image analysis software is utilized to calculate the cell number of every pictures and carry out statistical analysis.

5. cinnamic aldehyde promotes the experiment of HUVECs segment dislocation

Pre-cooling 2 hours under the conditions of-20 DEG C by 96 orifice plates and 200 μ l aseptic rifle head, the defrosting of 4 DEG C of Matrigel glue.In nothing Under the conditions of bacterium, draw Matrigel glue (50 μ l/ holes, it is to avoid produce bubble) in 96 orifice plates, be placed in 37 DEG C, 5%CO2 incubator In 1 hour, standby after gelling is solid；Take human umbilical vein endothelial's cell (HUVECs) of exponential phase of growth, use MCDB131 base Basal culture medium makes 5 × 105/ml cell suspension, is inoculated in Matrigel glue coated 96 with 5 × 104/porocyte density In orifice plate, add the cinnamic aldehyde of variable concentrations, be placed in 37 DEG C, in 5%CO2 incubator；After 4 hours, every hole randomly selects 5 and regards Taking pictures under 40 power microscopes in open country, utilizes Image pro-plus (IPP) image analysis software to calculate the tube chamber number of every pictures Mesh also carries out statistical analysis.

6. enzyme linked immunosorbent assay (ELISA) detection cinnamic aldehyde induction VEGF release

Take human umbilical vein endothelial's cell (HUVECs) of exponential phase of growth, with MCDB131 complete medium make 1 × 105/ml cell suspension, is inoculated into 24 orifice plates with 3 × 104/porocyte density, is placed in 37 DEG C, in 5%CO2 incubator；Treat After cell attachment is good, abandoning former culture medium, change containing 1%FBS and the basal medium of variable concentrations cinnamic aldehyde, matched group is for containing The not pastille culture medium of amount DMSO, collected cell conditioned medium at 0,24,36,48,72 hours respectively, and 4 DEG C, 1500rpm is centrifuged 15 points Clock, takes supernatant to be measured in-20 DEG C of preservations.Detection method reference reagent box operating instruction, described test kit is Human VEGF ELISA kit (eBioscience company).

The 7.Western-Blot method detection cinnamic aldehyde induction each protein expression of HUVECs

Take the logarithm human umbilical vein endothelial's cell (HUVECs) of trophophase, make 1.5 with MCDB131 complete medium × 105/ml cell suspension, is inoculated into 6 orifice plates with 3 × 105/porocyte density, is placed in 37 DEG C, in 5%CO2 incubator； After cell attachment is good, abandon former culture medium, change the basal medium containing 1%FBS hungry two hours, add variable concentrations Medicine, extracts albumen after effect different time respectively；Abandon former culture medium, the PBS washed cell of horse back pre-cooling twice, finally will Residual liquid exhausts completely, adds lysate (90 μ l/ hole), places on ice 15 minutes and (shaked culture plate every 5 minutes once so that carefully Cellular lysate is complete)；Cracking homogenate being collected in 1.5ml EP pipe, 4 DEG C, 12000rpm is centrifuged 10 minutes, takes supernatant；Take a small amount of Albumen is used for determination of protein concentration, and remaining major part albumen adds sample-loading buffer, boils degeneration in 5 minutes, treats in-20 DEG C of preservations Survey.It is splined on 10%SDS-PAGE with every hole equal protein, after electrophoretic separation, albumen is gone to pvdf membrane, use corresponding monoclonal Antibody test, using β-actin as internal reference.Experimental result represents with β-actin expressing quantity ratio with target protein.

(2) experimental result

1. cinnamic aldehyde affects result to what HUVECs bred

Cinnamic aldehyde can induce HUVECs to breed significantly, and agent-feeding treatment, after 24 hours, uses CCK-8 method detection cell to increase Grow situation and calculate cell proliferation rate.Result such as Figure 1A shows, in concentration dependent, cinnamic aldehyde can promote that HUVECs breeds, 10 μMs of hourly growth rates reach 33%.Then further look in time dependence, whether cinnamic aldehyde promotes that HUVECs breeds, knot Fruit such as Figure 1B, number of viable cells can (6～24 hours) increase in time successively.Above experimental result as shown, cinnamic aldehyde in Between Xian Shi and the promotion endothelial cell proliferation of concentration dependent.

2. cinnamic aldehyde affects result to HUVECs wound healing

Cell cut result such as Fig. 2 A and Fig. 2 B shows, agent-feeding treatment damaged cell is after 24 hours, and cinnamic aldehyde is when 0.1 μM Can be obviously promoted cell injury healing, healing rate reaches 54.1%, and now matched group is then only 40.4%；When 10 μMs dense When spending, damaging cells heals the most completely.This ability of cell climbing sheet experiment proof cinnamic aldehyde further, as shown in Figure 2 C, Cinnamic aldehyde is remarkably improved cell quantity in 1 μM, for 1.15 times of matched group.Prompting, cinnamic aldehyde can damage by inducing endothelial cell Recover from injury and close and in concentration dependent.

3. cinnamic aldehyde affects result to what HUVECs migrated

Endothelial cell migration is the complicated process promoting angiogenesis factor and anti-angiogenesis factor interaction, It is demonstrated by the motor capacity of cell.Result is as it is shown on figure 3, compared with matched group, cinnamic aldehyde can induce HUVECs to move significantly Moving, when concentration is 0.1,1,5 and 10 μM, the cell number moved under film is respectively 22,28,35,33/visual field.Prompting meat Cinnamic aldehyde can migrate by inducing endothelial cell.

4. cinnamic aldehyde affects result to HUVECs segment dislocation

Extracorporeal blood vessel is newborn main by several steps such as endothelial cell proliferation, migration and segment dislocation, and segment dislocation is A most the most key step.As shown in Figure 4, compared with matched group, cinnamic aldehyde can induce HUVECs segment dislocation significantly, in When 10 μMs, luminal structure number increases by 35% (P < 0.001) than matched group.Prompting cinnamic aldehyde can promote that endotheliocyte tube chamber is tied It is configured to and presents concentration-dependent relation.

5. cinnamic aldehyde induction HUVECs secretion of VEGF

VEGF is important angiogenic factors, has played the regulating and controlling effect of key in angiogenic process.ELISA Testing result shows (such as Fig. 5), the inducing endothelial cell secretion of VEGF of cinnamic aldehyde presentative time and concentration dependent.6. cinnamic aldehyde PI3K/AKT signal path affected result

PI3K/AKT is an important signal path in angiogenic process, main maintenance endotheliocyte existence and increasing Grow.The result that affects of HUVECs intracellular PI3K/AKT signal path is shown by cinnamic aldehyde, cinnamic aldehyde induction AKT and downstream albumen thereof ENOS phosphorylation presentative time and concentration-dependent relation.As shown in Figure 6B, cinnamic aldehyde in 5 minutes time, induce AKT phosphorylation until 120 minutes.Simultaneously its can induce AKT downstream signaling molecule eNOS in 5 minutes time phosphorylation until 60 minutes (Fig. 6 D).Such as Fig. 6 A Shown in Fig. 6 C, along with the increase (1～10 μM) of cinnamic aldehyde concentration, the phosphorylation level of AKT, eNOS improves successively.Prompting meat Cinnamic aldehyde can activate PI3K/AKT signal path in significance ground.

7. cinnamic aldehyde affects result to MAPK signal path

Another major avenues of approach of angiogenesis is MAPK signal path, comprising: Erk1/2 signal path, p38 signal Path and JNK/SPAK signal path.As shown in figs. 7b and 7d, cinnamic aldehyde in 5 minutes significantly induction c-Raf phosphorylation until 120 minutes, and faint induction c-Raf downstream signaling molecule Erk1/2 is until 120 minutes.Meanwhile, along with the increasing of cinnamic aldehyde concentration Adding (1～10 μM), the phosphorylation level of c-Raf, Erk1/2 improves (Fig. 7 A and Fig. 7 C) successively.As shown in Figure 8 F, cinnamic aldehyde is in 5 Minute notable induction p38 phosphorylation is until 120 minutes.But its phosphorylation level does not change (figure because of the increase of concentration 7E).For JNK/SAPK signal path, cinnamic aldehyde does not has any impact to it.Prompting cinnamic aldehyde can activate c-in significance ground Raf/MEK/Erk1/2 signal path.

8.PI3K/AKT and Raf/MEK/Erk1/2 signal path participates in cinnamic aldehyde regulation extracorporeal blood vessel Newborn Process

Newborn with cinnamic aldehyde regulation extracorporeal blood vessel for proving PI3K/AKT and Raf/MEK/Erk1/2 signal path further Process is correlated with, and we use PI3K inhibitor LY294002 and mek inhibitor U0126, observes its blood outer to cinnamic aldehyde inductor The impact that pipe is newborn.As shown in Figure 8 A and 8 B, LY294002 and U0126 can substantially suppress HUVECs propagation and secretion VEGF.Meanwhile, Fig. 8 C and Fig. 8 D shows, LY294002 and U0126 can suppress HUVECs segment dislocation.Additionally, as illustrated in fig. 8e, LY294002 can block cinnamic aldehyde induction AKT phosphorylation and not affect cinnamic aldehyde induction Erk1/2 phosphorylation.Such as Fig. 8 F institute Showing, U0126 can block cinnamic aldehyde induction Erk1/2 phosphorylation and not affect cinnamic aldehyde induction AKT phosphorylation.By tying above Fruit draws, PI3K/AKT with Raf/MEK/Erk1/2 signal path is relevant to cinnamic aldehyde regulation extracorporeal blood vessel Newborn Process, but two The most mutual mediation between bar path.

Embodiment 2 cinnamic aldehyde promotes angiogenesis experiment in vivo

(1) experimental technique

1. laboratory animal

Wild type (C57BL/6) mice, male, body weight 18-22g；Fro diabetic (BSK.Cg-m+ /+Leprdb；db/db) Mice, male, body weight 55-60g, blood glucose value 27.3 ± 0.6mmol/L, public purchased from Shanghai Si Laike laboratory animal Limited Liability Department.

2. Animal Model

Mice, after pentobarbital sodium intraperitoneal injection of anesthesia, with house pet shaver against cant to back shaving, about exposes The skin (hair is shaved the shortest as far as possible, but can not injure skin) of the square size of 4cm, uniform application depilatory cream is at blank skin subsequently At skin, after 2 minutes, use cotton ball soaked in alcohol wiping, in order to the hair of Ex-all mouse back；The corneal trephine after sterilization is utilized to carry on the back mice A circular hole is made a call in portion, cuts the circular skin of 8mm diameter, i.e. forms skin injury model.

3. animal packet and administration

Respectively 64 C57 mices after modeling and 64 db/db mices are all randomly divided into 4 groups, often group 16, are respectively 100mg/kg cinnamic aldehyde administration group (high dose group), 50mg/kg cinnamic aldehyde administration group (middle dosage group), 25mg/kg cinnamic aldehyde are given Medicine group (low dose group), normal saline group (matched group)；Mice is divided cage feed (4/cage), every day intraperitoneal injection.

4. draw materials

Respectively at be administered after the 6th day and the 12nd day, often group four mices of random choose take off neck execution, cut wound tissue, It is averaged and is distributed into two parts: a part is tested for immunohistology, be placed in 4% paraformaldehyde fixing；Another portion Demultiplexing is in immunoblot experiment ,-80 DEG C of preservations.

5. Indexs measure

(1) wound diameter

Take pictures to every other day mouse back wound, utilize Image J image analysis software measure wound diameter size and calculate Wound healing rate.Healing rate (%)=(n is for taking pictures for (n-th day wound diameter size/0th day wound diameter size of 1-) × 100% The same day).

(2) immunoblotting surveys damage tissue vegf protein expression

Weigh-80 DEG C of frozen skin histologies, be cut into fine grained chippings as far as possible, load in 1.5mlEP pipe, be proportionally added into group Knit protein lysate (containing protease inhibitor), ground with electronic tissue homogenate instrument and (operate on ice, in case albumen becomes Property)；4 DEG C, 12000rpm is centrifuged 10 minutes, draws supernatant, takes a small amount of albumen for determination of protein concentration, remaining major part albumen Add sample-loading buffer, boil degeneration in 5 minutes, to be measured in-20 DEG C of preservations.It is splined on 10%SDS-with every hole equal protein PAGE, goes to pvdf membrane by albumen after electrophoretic separation, detects by corresponding monoclonal antibody, using β-actin as internal reference.Experiment Result represents with β-actin expressing quantity ratio with target protein.

(2) experimental result

1. cinnamic aldehyde accelerates mouse skin wound healing assay

Angiogenesis is the key link in wound healing process, and poor wound healing be diabetes serious the most also Sending out one of disease, its basic reason is owing to angiogenesis is inadequate.Whether this experiment can promote wild type to cinnamic aldehyde respectively Mice and the healing of diabetic mice skin injury are investigated.Result shows, wild-type mice skin wound healing speed (12 My god) apparently higher than diabetic mice (22 days).When, after lumbar injection cinnamic aldehyde, mouse skin speed of wound healing significantly improves. Wild-type mice skin wound reaches to heal completely after being administered 10 days, and diabetic mice then needs 16 days.For wild type Mice, is being administered the 4th day, and the wound area of dosing group is substantially little than matched group.As shown in Figure 9 A, low dose group wound healing rate (47%) compared with matched group (36%) (P < 0.01), middle dosage group (58%) (P < 0.001) compared with matched group (36%), high Dosage group (59%) is (P < 0.001) compared with matched group (36%).And for diabetic mice (Fig. 9 B), at the 4th day, low dosage Group there was no significant difference with matched group wound healing rate, middle dosage group (49%) (P < 0.001) compared with matched group (18%), high Dosage group (50%) is (P < 0.001) compared with matched group (18%).

2. histological observation mouse skin wound healing situation

For preferably proving that cinnamic aldehyde accelerates the activity of mouse skin wound healing, this experiment uses H&E dyeing to observe little The histologic effect of Mus skin wound healing.Such as Figure 10, within the 10th day, histopathologic slide shows, diabetic controls group mouse skin Defect area is the biggest, and the most a small amount of epidermis covers and seriality is poor, with the presence of substantial amounts of granulation tissue；And wild type pair The least according to group mouse skin defect area, a large amount of epidermises cover, it is seen that scar tissue, have a small amount of granulation tissue.Give Cortex Cinnamomi After aldehyde, diabetic mice wound epidermis covers but relatively thin, substantially still with the presence of granulation tissue；And wild-type mice wound is complete Repairing, granulation tissue disappears.Result shows, cinnamic aldehyde can repair mouse skin wound healing process well.

3. the cinnamic aldehyde impact on mouse skin wound tissue angiogenesis

For observing whether cinnamic aldehyde can promote mouse skin wound tissue angiogenesis, this experiment uses immunofluorescence CD31 antigen in method detection blood vessel wall, thus show the angiogenesis of wound tissue.As shown in figure 11, at the 10th day, wild Type mice wound tissue's number of blood vessel (56/visual field) is significantly more than diabetic mice (33/visual field) (P < 0.05).And give Wound tissue's number of blood vessel showed increased after cinnamic aldehyde, wild-type mice and diabetic mice wound tissue number of blood vessel are respectively It is the 166/visual field and the 156/visual field.Result above shows, cinnamic aldehyde can promote wild-type mice and diabetic mice skin Wound tissue's angiogenesis.

4. the impact that mouse skin wound tissue vegf protein is expressed by cinnamic aldehyde

Vitro Experimental Results shows that cinnamic aldehyde can promote HUVECs secretion of VEGF, then uses animal model to observe cinnamic aldehyde Whether can the expression of inducing mouse skin wound tissue vegf protein.As shown in figure 12, saline control group mouse skin Wound tissue's vegf expression amount is higher than intact skin histology, points out post-traumatic skin histology meeting secretion of VEGF itself.When giving After giving cinnamic aldehyde, mice wound tissue vegf expression amount is the most significantly raised.Result shows, cinnamic aldehyde promotes that mouse skin wound is more Close and induce to it up-regulated of vegf protein relevant.

Embodiment 3 experimental summary

In sum, the present invention have rated by setting up human umbilical vein endothelial's cell (HUVECs) culture system in vitro The impact of the cinnamic aldehyde endothelial cell proliferation activity of variable concentrations.

1. be respectively adopted CCK-8 proliferation experiment, cell cut and creep plate are tested, Transwell migrates experiment and external become The activity of the pipe experiment outer angiogenesis of detection cinnamic aldehyde inductor.Result shows, cinnamic aldehyde can lure when 0.01～100 μM of concentration Lead HUVECs propagation, wound healing, migration and segment dislocation, and present a certain amount effect relationship.

2. utilize enzyme linked immunosorbent assay (ELISA) to measure the VEGF of secretion in HUVECs culture supernatant (VEGF) content, it was found that cinnamic aldehyde can dramatically increase in the secretory volume of 24～48 hours induction VEGF.

3. detected by Western blot (Western-blot) testing result shows, cinnamic aldehyde can activate phosphatidylinositols 3 Kinases (phosphoinositide 3-kinase, PI3K)/AKT and mitogen activated protein kinase (mitogen- Activatedproteinkinases, MAPK) two signal paths, can in the time and concentration dependent raise master therein Want outside target protein: AKT, eNOS (endothelial nitric oxide synthase, eNOS), born of the same parents Signal regulated kinase (extracellular regulated protein kinases1/2, Erk1/2), c-Raf, p38 kinases (p38kinase,p38)；Additionally, PI3K and MEK specific inhibitor LY294002 and U0126 can block cinnamic aldehyde induction The phosphorylation of AKT and Erk1/2, and the secretion of inhibition of endothelial cell proliferation, segment dislocation and VEGF.

4. the present invention also uses wild-type mice and diabetic mice skin injury model, makees cinnamic aldehyde angiogenesis promoting Drug effect carried out internal checking.It was found that compared with matched group, every day distinguishes lumbar injection cinnamic aldehyde 10～100mg/ After kg, mouse skin wound is obviously reduced (P < 0.05).Additionally, cinnamic aldehyde can be by raising the expression of wound tissue's vegf protein Amount the formation of inducing wound surrounding tissue neovascularity, thus induced skin damage healing, and confirm that mice itself is not had by it Toxic and side effects.Showing the monitoring result of blood glucose in diabetic mice level, cinnamic aldehyde induced diabetes mouse skin damages simultaneously Healing is not rely on the change of blood glucose value.

As can be seen here, cinnamic aldehyde in 0.01～100 μM of concentration range can the notable outer angiogenesis of inductor, and in 10 ～during 100mg/kg dosage, accelerating mouse skin wound healing, its mechanism of action may lead to activating PI3K/AKT and MAPK signal It is correlated with in road.

Embodiment 4 cinnamic aldehyde new application in preparing medicine

The present invention passes through lot of experiments, finds that cinnamic aldehyde can be used for:

A) endothelial cell proliferation is promoted,

B) endothelial cell damage healing is promoted,

C) endothelial cell migration is promoted,

D) endotheliocyte segment dislocation is promoted,

E) induction vegf expression and secretion,

F) PI3K/AKT signal path is activated,

G) c-Raf/MEK/Erk1/2 signal path is activated, and/or

H) promote that wound tissue's number of blood vessel increases.

Therefore, cinnamic aldehyde new application in preparing angiogenesis promoting related drugs includes:

First, prepare wound healing/wound healing/wound healing medicine

Wound healing includes biology and the molecules process that cell proliferation, migration and extracellular matrix deposition etc. are complicated, its Need good dynamic circulation system, sufficient nutrition, and avoid the injury of mechanical external force, smoothly completing of this process as far as possible Typically require 3 to 14 days, be generally divided into three phases: inflammation phase, multiplicative stage, reconstructed tissue stage.The formation of neovascularity Occur mainly in the multiplicative stage, first fibroblast generation collagen stroma, the most newly-generated vascular invasion granulation tissue, Rear epidermis cell moves to injured surface and closes slight crack tissue.It is known that angiogenesis is the crucial rank of wound healing process Section, newborn blood vessel can be that enough oxygen and nutrition, simultaneously the peripheral cell metabolism of excretion tissue are transmitted in the growth of damaged tissues Produced by-product.The blood flow recovering affected area is conducive to the reparation of damage tissue, and skin injury agglutination Easily controllable with process, therefore it will be angiogenesis research ideal model.

Chronic trauma is one of the most serious clinical disease, and it has higher sickness rate and the most often lacks preferably Treatment means.It is a kind of disease blocking skin seriality and tissue integrity, reaching heals completely need to grow very much one section Time (at least 8 week), even can not heal or again recur.Wound healing is impaired will result directly in skin ulcer, is a kind of Serious diabetic complication, referred to as " diabetic foot ", it is not enough that its reason is probably angiogenesis.According to estimates, up to 200 Ten thousand Americans be suffering from lower limb wound heal disease.Report according to CDC, even if in terms of trauma care Improve, but intractable wound has still resulted in and had 72000 diabetes patients to face amputation every year.Today of 21 century, sugar The urine annual rate of increase of foot disease is between 1% to 4.1%, and amputation rate of increase is between 0.21% to 0.37%.Therefore, probe into Effective treatment means captures the top priority that the chronic trauma caused because angiogenesis is not enough is current researchers.

Second, prepare coronary heart disease and myocardial ischemia medicine

Coronary heart disease is many in such as hypertension, hyperlipidemia, smoking, diabetes, Bad Eating Habit and shortage motion etc. Planting under the effect of risk factor, coronary artery occurs and forms atherosis focus so that footpath, coronary artery chamber is narrow and small or blocks, And cause myocardial ischemia-anoxemia until the serious disease of cardiomyocyte cell death.Classic treatment bag to coronary heart disease and myocardial ischemia at present Include: (1) Drug therapy.Coronary artery dilator, increases coronary blood confession, such as nitrate esters medicine；Decreased heart rate, reduces blood pressure to subtract Few cardiac muscle oxygen bypass graft consumption, such as beta-blocker；Antiplatelet, anticoagulant therapy, stablize atherosis plate, such as low point Sub-heparin etc..(2) interventional therapy.(3) surgical intervention.And promote establishing and improve and arterial blood of ischemic myocardium region collateral circulation The new life of pipe, i.e. Therapeutic angiogenesis (therapeutic angiogenesis), be current international cardiovascular disease educational circles Study hotspot.The definition of Therapeutic angiogenesis is the generation promoting trophism collateral circulation blood vessel, is already blocking or narrow Periarterial composition in raw bypass circuit.It includes angiogenesis: refer to side shoot systemic vascular in cardiac muscle (< 200um) and hair Thin blood vessel (< 20um) increase；And tremulous pulse is newborn: refer in the epicardial coronary arteries blood supply region of stenosis or occlusion newly-generated Bigger arteries (> 200um).

Myocardial ischemia or the early stage of infarction, blood capillary and capillary density increase, and endotheliocyte or mononuclear cell are lived Changing, myocardial cell downright bad subsequently discharges into vascular somatomedin (Bfgf, VECF), cytokine with the inflammatory cell of infiltration (MCP-1, IL-6, IL-8), and the increase that intercellular adhesionmolecule1 (ICAM) is expressed, cause endotheliocyte and smooth muscle cell Mitosis increases, and ultimately forms normal muscular artery.Acid fibroblast growth factor (aFGF) mRNA after arteria coronaria obstruction Transcribing increase, aFGF has the mononuclear cell of activation to discharge, and is the strength mitosis of endothelium, connective tissue and smooth muscle cell Former, and can stimulating endothelial cell secretion urine plasminogen.In coronary artery or intravenous injection recombination basic fibroblast growth The factor (bFGF) also can accelerate the foundation of collateral circulation.VEGF (VEGF) is that specific blood vessels endotheliocyte has Silk mitogen, its mRNA is present in neighbouring side shoot reconstructing blood vessel or myocardial cell.Intracoronary injection VEGF can be gradual Coronary occlusion makes collateral circulation blood flow increase by 40% after 4 weeks.

3rd, preparation diabetic skin damage preventing/treating medicine

Invention also uses wild-type mice and diabetic mice skin injury model, to cinnamic aldehyde angiogenesis promoting effect Drug effect has carried out internal checking.It was found that compared with matched group, every day distinguishes lumbar injection cinnamic aldehyde 10～100mg/kg After, mouse skin wound is obviously reduced (P < 0.05).Additionally, cinnamic aldehyde can be by raising the expression of wound tissue's vegf protein And the formation of inducing wound surrounding tissue neovascularity, thus induced skin damage healing, and confirm that it does not has poison to mice itself Side effect.Showing the monitoring result of blood glucose in diabetic mice level, the damage of cinnamic aldehyde induced diabetes mouse skin is more simultaneously Merge the change not relying on blood glucose value.

Research contents based on the embodiment of the present invention 1 and 2, cinnamic aldehyde is a kind of activity having and promoting angiogenesis effect Material, skilled person will appreciate that, for can be reached the pathological process of rehabilitation or alleviation by angiogenesis means, as Above-mentioned wound healing, coronary heart disease and myocardial ischemia or diabetic skin damage, cinnamic aldehyde, as effective ingredient, promotees blood vessel in preparation new Raw related drugs aspect has great importance.

The above, only presently preferred embodiments of the present invention, the principle of above-described embodiment only illustrative present invention and Its effect, and not any formal and substantial to present invention restriction, it is noted that for the common skill of the art Art personnel, on the premise of without departing from the inventive method, also can make some improvement and supplement, and these improve and supplement also Should be regarded as protection scope of the present invention.All those skilled in the art, in the feelings without departing from the spirit and scope of the present invention Under condition, a little change of making when available disclosed above technology contents, the equivalent variations modified and develop, it is this The Equivalent embodiments of invention；Meanwhile, any equivalent variations that above-described embodiment is made by the substantial technological of all foundation present invention Change, modify and develop, all still fall within the range of technical scheme.

Claims (4)

1. cinnamic aldehyde purposes in preparation can induce the derivant of vegf expression and secretion.

Purposes the most according to claim 1, it is characterised in that described purposes is particularly as follows: cinnamic aldehyde can be induced in preparation Purposes in the derivant of vegf expression and secretion in endotheliocyte.

Purposes the most according to claim 1, it is characterised in that described purposes is particularly as follows: cinnamic aldehyde can be induced in preparation Purposes in the derivant of vegf expression and secretion in vascular endothelial cell.

Purposes the most according to claim 1, it is characterised in that cinnamic aldehyde is the sole active ingredient of described derivant or many One of individual effective ingredient.