CN107485610B - Application of the Miconazole in prevention angiorrhoxis - Google Patents

Abstract

The present invention relates to application of the Miconazole in preparation prevention angiorrbagia drug.The invention further relates to Miconazoles in perivascular cell, the application as MMP9 inhibitor.The invention further relates to Miconazoles in perivascular cell, the application as ERK inhibitor.The invention further relates to a kind of pharmaceutical composition for preventing angiorrbagia, main pharmacodynamics ingredient is Miconazole.The invention further relates to a kind of pharmaceutical compositions of MMP9 expression in inhibition perivascular cell, and main pharmacodynamics ingredient is Miconazole.The invention further relates to a kind of pharmaceutical compositions of ERK phosphorylation in inhibition perivascular cell, and main pharmacodynamics ingredient is Miconazole.The invention further relates to a kind of pharmaceutical compositions, contain U0126 and Miconazole.The invention further relates to application of the Miconazole in prevention aberrant angiogenesis rupture.The present invention provides new application of the Miconazole in prevention aberrant angiogenesis bleeding, can effectively prevent a variety of diseases caused by angiorrbagia.

Description

Application of the Miconazole in prevention angiorrhoxis

Technical field

The present invention relates to application of the Miconazole in prevention angiorrhoxis.

Background technique

Aberrant angiogenesis ruptures the generation that will lead to a variety of diseases.Cardiovascular disease is always that whole world disease causes for a long time The main reason for dead, wherein cerebral apoplexy ranks first three.2012 whole world, which are greater than, has 6,700,000 people to die of cerebral apoplexy, accounts for non-infectious 17% or so (WHO, 2015) of property disease general mortality rate.Hemorrhagic apoplexy only accounts for the 10-15% of all cerebral apoplexies, but it is sent out Raw rate and the death rate but want high more.

Although hemorrhagic apoplexy has quite high incidence and the death rate, compared with cerebral arterial thrombosis, people couple Its molecular pathology is but known little about it.Recent study points out that inflammatory reaction is that the main cell after hemorrhagic apoplexy occurs is thin Born of the same parents' event invades swashing for profit and microglia/macrophage and astroglia with neuronal death, leucocyte Living, main inflammatory regulatory factor includes matrix metalloproteinase (MMPs), nuclear factor 2 (Nrf2), heme oxidase (HO) and iron ion (Fe3+) (Wang, 2010).It is many to treat tasting for hemorrhagic apoplexy in preclinical and clinical test Examination be based on anti-inflammatory principle, such as mediate microglia/macrophage inhibition factor, MMP inhibitor, Nrf2 inducer, HO inhibitor or Deferoxamine (Egashira et al., 2015；Wang,2010).However all these treatments based on target spot Trial all fails to expand to clinical application.

So far, based on the above principles and in the drug or operation method of discovery, not yet there is effective enough, Ke Yijin Enter the treatment means of three phases clinic.This is perhaps not yet clear due to the therapy target of hemorrhagic apoplexy, also or this complexity Disease be by controlled by multiple genes, it is little for responding well to treatment for single target spot.In recent years, the medicament research and development based on phenotype is gradually Of interest by people, academia and industry put into very great dynamics and utilize the animal model development research and development including zebra fish (MacRae and Peterson,2015)。

It is more than 20 kinds of protein members that metallo-matrix proteases (MMPs) family, which includes five major class, in mammal, wherein MMP2 Belong to gelatinase (Cuzner and Opdenakker, 1999) with MMP9.Various kinds of cell in neural blood vessel unit can be secreted MMP albumen plays a crucial role during brain development --- and the height of MMPs expresses loose extracellular matrix with reality The neurite outgrowth of existing neuronal cell and cell migration (Yong et al., 2001).Experimental animal cerebral arterial thrombosis, In hemorrhagic apoplexy and head injury model, the level of MMP has up-regulation (Lo et al., 2003), while cerebral apoplexy patient MMP level in brain and blood plasma also obviously up-regulation (Clark et al., 1997；Montaner et al.,2001).Continue MMP activity be harmful for organism, such as direct injection MMP9 can cause cell death and inflammation into brain tissue Disease reaction (Anthony et al., 1998).MMP9 knock out mice can play protection to encephalic ischemic and head injury Effect (Asahi et al., 2001；Wang et al., 2000), and MMP2 knock out mice can not resist ischaemic Generation (Asahi et al., 2001).Illustrate during cerebral apoplexy and head injury, MMP9 plays more leading effect (Lo et al.,2003)。

Clinical data is shown, if Ischemic Stroke is very easy to out with higher blood plasma MMP9 concentration The transition (Montaner et al., 2001) of hemorrhagic cerebral apoplexy.In addition, commonly using fibre in embolic type ischaemic patient Plasminogen activator (PA), since MMP9 can be activated by activator of plasminogen, is caused in patient body with dissolving embolism The up-regulation of MMP9 level is easy to induce the hemorrhagic transition (Lo et al., 2003) of PA induction.

Miconazole is a kind of imidazoles antifungal drug of broad-spectrum high efficacy, plays the role of inhibiting fungi by a variety of accesses (Quatresooz et al.,2008).Miconazole is safe and efficient with its, multiple-effect and the unique multi-faceted spy for inhibiting fungi Point, and be widely used in dermatomycosis.The chemical structural formula of Miconazole is shown in formula I, the change of the nitrate of Miconazole Structural formula is learned as shown in Formula II.

Summary of the invention

In order to solve in the prior art, the status of various disease prevention effect differences caused by aberrant angiogenesis bleeding, the present invention Embodiment provides the following technical solutions.

As one aspect of the present invention, it is related to application of the Miconazole in preparation prevention angiorrbagia drug.It is specific next It says, the application includes application of the Miconazole in preparation prevention cerebral hemorrhage headstroke drug.Specifically, in cerebral hemorrhage brain Wind refers to that MMP9 is overexpressed caused cerebral hemorrhage headstroke.

As another aspect of the present invention, it is related to Miconazole in perivascular cell, as answering for MMP9 inhibitor With.Specifically, the application includes application of the Miconazole in perivascular cell, as MMP9 expression inhibiting agent.

As another aspect of the invention, it is related to Miconazole in perivascular cell, as answering for ERK inhibitor With.Specifically, the application includes application of the Miconazole in perivascular cell, as ERK phosphorylation inhibitor.

As an additional aspect of the present invention, it is related to a kind of pharmaceutical composition for preventing angiorrbagia, main pharmacodynamics ingredient For Miconazole.

As an additional aspect of the present invention, it is related to a kind of pharmaceutical composition for inhibiting mmp9 expression in perivascular cell Object, main pharmacodynamics ingredient are Miconazole.

As an additional aspect of the present invention, it is related to a kind of pharmaceutical composition for inhibiting ERK phosphorylation in perivascular cell Object, main pharmacodynamics ingredient are Miconazole.

As an additional aspect of the present invention, it is related to a kind of pharmaceutical composition, containing U0126 and Miconazole, specific ratio It can be 1:1.

As an additional aspect of the present invention, it is related to application of the Miconazole in prevention aberrant angiogenesis rupture.Specifically, The application includes application of the Miconazole in prevention cerebral hemorrhage headstroke.

The present invention provides new application of the Miconazole in prevention aberrant angiogenesis bleeding, can effectively prevent angiorrbagia and lead The a variety of diseases caused.

Detailed description of the invention

Fig. 1: bleeding inhibitory effect figure of the Miconazole to zebra fish cerebral hemorrhage mutant.

Fig. 2: bleeding inhibitory effect figure of the Miconazole to rat mesentery Hemorrhage Model.

Fig. 3: Miconazole influences result figure to the expression of MMP9 in zebra fish.

Fig. 4: MMP9 inhibitor is in zebra fish to the suppression result figure of MMP9 expression.

Fig. 5: Miconazole influences result figure to the expression of MMP9 in rats.

Fig. 6: Miconazole and PD inhibit the effect picture of ERK phosphorylation in zebra fish.

Fig. 7: Miconazole and U0126 inhibit the effect picture of ERK phosphorylation in zebra fish.

Fig. 8: Miconazole and U0126 are used in mixed way the effect picture for inhibiting ERK phosphorylation in zebra fish.

Specific embodiment:

Embodiment 1:

The quantity of red blood cell and position in living body embryo can be marked using o-dianisidine dyeing, thus can be compared The difference of zebra fish cerebral hemorrhage mutant and no bleeding embryo in hematocrit level more out.In 2 days embryos of after fertilization, no bleeding The red blood cell of embryo is distributed in heart, cuvierian duct, aorta dorsalis and other Major Vessels；In contrast, control group The red blood cell of mutant is mainly gathered in brain, and cuvierian duct red blood cell significantly reduces, and Miconazole processing group embryo brains Erythrocyte accumulation phenomenon is obviously improved, and cuvierian duct erythrocyte number increases.Thus illustrate, Miconazole is prominent to zebra fish cerebral hemorrhage The bleeding phenotype of variant has significant inhibiting effect, and does not influence its erythrocyte number.

Miconazole increases the bleeding inhibitory effect of zebra fish cerebral hemorrhage mutant with drug concentration and enhances, 5 μM of concentration Miconazole handle mutant, its Bleeding rate about 80% (Fig. 1) can be significantly reduced.

Effect is similar in zebra fish with it, and Miconazole also can inhibit the bleeding phenotype in mammal.For convenience of research, We establish rat mesentery Hemorrhage Model and detect the effect of Miconazole in the model.When experiment, to postanesthetic big Mouse intravenous injection physiological saline makees model group as control or 30000UI/kg urokinase；Two groups are respectively divided into three groups again, use respectively DMSO- physiological saline, 5mg/kg Miconazole-physiological saline and 10mg/kg Miconazole-physiological saline shift to an earlier date 30 minutes pre- places Reason observes the variation of blutpunkte quantity and total bleeding area on mesenterium.In physiological saline processing group, DMSO- physiological saline pair According to group without obvious bleeding phenotype in two hours of observation, 5mg/kg or 10mg/kg Miconazole-physiological saline group is not also observed To obvious bleeding.In the model group of urokinase processing, occur bleeding phenotype from about 60 minutes after modeling, and blutpunkte quantity and Bleeding area all extends at any time and increases.After 5mg/kg or 10mg/kg Miconazole-physiological saline pretreatment, bleeding points Amount and bleeding area are increased with Miconazole concentration and are significantly reduced.Biometric is carried out to it, measures visual field internal haemorrhage point Quantity and bleeding area, respectively divided by total length of vessel, every group of size of animal is greater than six, obtains statistical result as shown in the figure (Fig. 2).Illustrate to guard unanimously with effect in zebra fish, Miconazole can protect the mesenterium bleeding that urokinase induces in rat.

Specific experiment process is as follows:

Zebra fish experiment

Raw material: o-dianisidine (Sigma-Aldrich, D9143), sodium acetate (Beijing Chemical Plant), 30% peroxidating Hydrogen (Beijing Chemical Plant)

Experimental animal: wild-type zebrafish (TL is bought from zebra fish international resource center ZIRC), cerebral hemorrhage mutant (ENU induced mutation obtains)

Experimental procedure:

1. configuring o-dianisidine dyeing liquor

Component	Volume
		14%o-dianisidine	2ml
1M pH4.5 sodium acetate	500μl
		Deionized water	2ml
30% hydrogen peroxide	100μl

2. collecting wild type and cerebral hemorrhage mutant embryos, 5 μM of Miconazoles or one were added into embryo at after fertilization 6 hours Quantitative DMSO is compared, and embryo cultivates in 28 DEG C；Drug is washed away within after fertilization 24 hours, fresh embryos culture solution, embryo's training are changed to It supports in 28 DEG C.

3. after fertilization 2 days, taking wild type-DMSO, wild type-Miconazole, mutant-DMSO and mutant-miaow health Each 20 pieces of azoles embryo or so, egg membrane is carefully peelled off with tip tweezers, rinses well and is moved back into glass groove through embryo medium And embryo medium is neatly removed as far as possible.

4. the o-dianisidine dyeing liquor of the fresh configuration of 500-800 μ l is added dropwise to embryo, it is protected from light dyeing 20 minutes.

5. dyeing finishes, is softly washed with deionized water three times, directly observe, take a picture and count.

Rat experiment

Raw material: urokinase (Aladdin, 9039-53-6), physiological saline, dimethyl sulfoxide (DMSO, Amresco, 0231), Urethane (Sinopharm Chemical Reagent Co., Ltd., 30191228)

Experimental animal: 200 grams of male Wistar rats (from Beijing, dimension tonneau China is bought)

Experimental procedure:

1.200g male Wistar rat injects 20% urethane anesthetic of 2ml by leg muscle.

2. thoroughly so that Rat Right is lain on one's side in sighting disk in left neck vein and the double intubations of left femoral vein after anesthesia, soft point Mesenterium is separated out, selection suitable view places it in observation window and obtains suitable location.

3. the rat of placement, which is connected sighting disk, is transferred to 37 DEG C of insulating boxs, interruption is added dropwise physiological saline and keeps on mesenterium It is wet.Insulating box is connected with microscope, directly can observe and shoot.

4. 30 minutes in advance pass through physiological saline or Miconazole (5mg/kg or 10mg/kg) of the femoral venous catheter containing DMSO Pretreatment.

5. after the reference images (baseline) of typing Mesentery microcirculation, being intubated saline injection by jugular vein and making Control group or 30000UI/kg urokinase (uPA) establish model group.

Every the image of typing in 30 minutes after 6., amount to typing 2 hours.

7. image is analyzed and is counted in each time point after whole typings, blutpunkte quantity and bleeding on mesenterium Area.

Embodiment 2

Metallo-matrix proteases (MMPs) in extracellular matrix protein are one of the important components for maintaining vascular stability (Xing et al.,2012).Extracellular MMPs expression quantity increases the proteolysis process in enhancing extracellular matrix (ECM), has Conducive to the cell division and migration in growth course.MMPs is also vital inflammation-modulating factor during cerebral injury (del Zoppo,2010)。

There are many members in MMP family in zebra fish genome, wherein well annotated only there are six: mmp2, mmp9, Mmp13b, mmp14a, mmp14b and mmp23aa.Thus firstly, we use transcription of the method for quantitative PCR to this six genes Situation is detected.The mutant embryos of 2 days wild types of after fertilization, cerebral hemorrhage mutant and Miconazole processing, warp are taken respectively Quantitative PCR discovery is crossed, the only expression of mmp9 is significantly raised in mutant, and the expression quantity in the mutant of Miconazole processing It is down to and the comparable level of wild type group；The expression of other mmp changes without statistical discrepancy.Further experiment show with it is wild Type control group is compared, and the protein expression and rna transcription level of Mmp9 has obvious up-regulation in zebra fish cerebral hemorrhage mutant；Miaow health Azoles can lower the MMP9 expression increased extremely in mutant, and have no effect on the normal expression (Fig. 3) of wild type.With MMP9 inhibitor is in 6-24 hours processing zebra fish cerebral hemorrhage mutant of after fertilization, compared with the control group, can also by Bleeding rate from About 100% is down to 50% or so (Fig. 4).

Existing research shows that urokinase shearing precursor plasminogen (pro-plasminogen) is plasminogen (plasminogen), the latter can shear precursor MMP9 (pro-MMP9) as active MMP9；MMP9 content in extracellular matrix After raising, the enhancing of proteolysis process keeps matrix structure loose (Smith and Marshall, 2010).It is induced in urokinase Rat mesentery Hemorrhage Model in, in urokinase activated extracellular matrix MMP9 degrade extracellular matrix, reduce vascular stability from And generate bleeding.Compared with physiological saline group, MMP9 expression up-regulation in model group；In model group, 5mg/kg or 10mg/kg miaow health The MMP9 of azoles pretreatment sample expresses to obtain obvious downward recovery, and the pretreatment sample of physiological saline group is without significant changes (figure 5)。

In conclusion in the mesenterium Hemorrhage Model that urokinase induces in zebra fish cerebral hemorrhage mutant and rat, bleeding Phenotype is the overexpression due to MMP9 caused by leading to extracellular matrix degradation extremely, and Miconazole is by lowering MMP9 Expression and play the role of bleeding inhibition.

Specific experiment process is as follows:

Zebra fish experiment

Raw material: TriZol (Invitrogen), PrimeScript RT reagent kit (Takara, RR037A), SYBR Premix DimerEraser (Takara, RR091a)

Experimental animal: wild-type zebrafish (TL is bought from zebra fish international resource center ZIRC), cerebral hemorrhage mutant (ENU induced mutation obtains)

Experimental procedure:

1. collecting wild type and cerebral hemorrhage mutant embryos, 5 μM of Miconazoles or one were added into embryo at after fertilization 6 hours Quantitative DMSO is compared, and embryo cultivates in 28 DEG C；Drug is washed away within after fertilization 24 hours, fresh embryos culture solution, embryo's training are changed to It supports in 28 DEG C.

2. after fertilization 2 days, 50 pieces or so each group embryos is taken to be rinsed with embryo medium.No RNA is transferred to after rinsed clean In the centrifuge tube of enzyme, removal liquid as much as possible is rapidly added 1ml TriZol lysis at room temperature, can be aided with grinding if needed Stick or syringe.Sufficiently after cracking, 200 μ l are added without the chloroform of RNA enzyme and shake 15s, are stored at room temperature 5 minutes.4 DEG C, 10000g Centrifugation 15 minutes, supernatant is transferred in new centrifuge tube, is careful not to sucking albumin layer.500 μ l are added into supernatant without RNA The isopropanol of enzyme, it is soft to be uniformly mixed, precipitation at room temperature 10 minutes.4 DEG C, 10000g is centrifuged 5 minutes, abandons supernatant.Precipitating uses 1ml The rinsing of 70% ethyl alcohol, 4 DEG C, 7500g is centrifuged 5 minutes.Supernatant is abandoned, after sufficiently drying, is dissolved and is precipitated without RNA enzyme water with 10-20 μ l, Up to total serum IgE.It is stored in -80 DEG C of refrigerators.

3. designing and synthesizing quantification PCR primer, make PCR fragment length 100-200bp, annealing temperature is close.

4. total serum IgE obtains single stranded cDNA library with PrimeScript RT reagent kit kit reverse transcription.37℃ Reaction 20 minutes, 85 DEG C of heat treatments inactivate reverse transcriptase in 5 seconds, 4 DEG C of coolings.It can be reserved in -20 DEG C.

5. carrying out quantitative PCR experiment with SYBR Premix DimerEraser kit, and experimental result is counted Analysis.

Rat experiment

Raw material: anti-MMP9 (Sigma-Aldrich, HPA001238), urokinase (Aladdin, 9039-53-6), physiology Salt water, dimethyl sulfoxide (DMSO, Amresco, 0231), urethane (Sinopharm Chemical Reagent Co., Ltd., 30191228)

Experimental animal: 200 grams of male Wistar rats (from Beijing, dimension tonneau China is bought)

Experimental procedure:

1. 200g male Wistar rat injects 20% urethane anesthetic of 2ml by leg muscle.

2. thoroughly in left neck vein and the double intubations of left femoral vein after anesthesia, and being injected to rat containing DMSO's by femoral vein Physiological saline or Miconazole (5mg/kg or 10mg/kg) pretreatment.After 30 minutes, saline injection is intubated by jugular vein and is made Control group or 30000UI/kg urokinase (uPA) establish model group.

3. after 2 hours, rat puts to death and draws materials rapidly.The small intestine of appropriate length is intercepted, to be advisable close to caecum end.With The physiological saline of pre-cooling sufficiently cleans, and after cutting off intestinal tube along intestines middle line, softly removes mucous layer with swab stick, moves on blotting paper, It is sufficiently absorbed through moisture.Placenta percreta is scraped with elbow tweezers at blotting paper drying, moves to cryopreservation tube, it is cold with liquid nitrogen quick freeze - 80 DEG C of refrigerators are transferred to after freezing completely to save.All operations carry out on ice, are swift in motion.

4. the extraction of protein.

Appropriate intestinal tissue is taken sufficiently to be ground in liquid nitrogen with mortar.After weighing, 100 μ l albumen are added into 100mg tissue Lysate, appropriate ultrasonic vibration lytic cell.4 DEG C of 12000g are centrifuged 10 minutes, take supernatant, -80 DEG C of refrigerators are stored in after packing.

5. protein blot is tested.

The protein lysate dispensed in right amount is taken, is uniformly mixed, boiling water with 5X albumen sample-loading buffer (containing mercaptoethanol) It boils 10 minutes, it is cooling rapidly, it is spare.

Prepare 8% PAGE gel.Constant pressure electrophoresis after loading is concentrated in glue and keeps 90V, separation gel 120V.Electrophoresis To appropriate location, low temperature constant current 200mA is transferred to pvdf membrane.Pvdf membrane containing protein sample is face-up, with closing After liquid chamber temperature is closed 2 hours, with 4 DEG C of the confining liquid overnight incubations containing anti-MMP9.After washing away primary antibody, TBST is rinsed 3 times, Every time 10 minutes.It is incubated at room temperature 2 hours with the confining liquid containing secondary antibody.TBST is rinsed 3 times, every all over 10 minutes.Finally, ECL is used Luminescent solution develops the color and takes pictures.Gained protein band is quantitative with ImageJ.

Embodiment 3

Before studies have shown that in MMP9 and extracellular signal-regulated kinase 1 and 2 (ERK1/2) in transcription and post-transcriptional level Exist in adjusting and is quite closely connected (Garavello et al., 2010).MMP9 can be induced to express, and expression is much turned The record factor is regulated and controled, including Activating protein-1 and 2 (AP1/2), specificity protein-1 (SP1), NF-B and polyoma enhancer A binding protein 3/Ets(PEA3/Ets)(St-Pierre et al.,2004).Pass through Ras/Raf/MEK/ERK access, these transcription factor quilts Activate, be transferred into nucleus, so regulate and control MMP9 expression (Chang et al., 2011；Garavello et al., 2010；Wu et al.,2013).ERK1/2 is sufficiently complex to the regulation of MMP9, even shows in different types of cell The regulation of complete opposite trend out.However compare it is confirmed that MEK/ERK swashs in many neurological diseases such as cerebral arterial thrombosis The overexpression (Maddahi et al., 2009) of MMP9 living.Thus it is speculated that in zebra fish cerebral hemorrhage mutant, Erk1/2 Also the expression of Mmp9 is enhanced.

In order to verify this point, detection wild type and mutant zebra fish are under with or without Miconazole disposition first The activity of Erk1/2, the i.e. expression of p-ERK1/2.Immunoblotting analysis experiment shows and wild type control group (DMSO processing) phase Than p-ERK1/2 Zhan total Erk1/2 ratio is significantly raised in the embryo of cerebral hemorrhage mutant, illustrates Erk1/ in mutant 2 increased activities；After Miconazole handles mutant, raised p-ERK1/2 levels, which obtain lowering, to be restored, and nothing in wild type Significant difference illustrates that Miconazole reduces the Erk1/2 activity enhanced extremely in zebra fish cerebral hemorrhage mutant

Specific experiment process is as follows:

Raw material: anti-ERK (anti-P42/44, cell signaling technology, #4695), anti-pERK (anti-pP42/44, cell signaling technology, #4370), Goat anti-rabbit IgG-HRP (Abmart, M21001)

Experimental animal: wild-type zebrafish (TL is bought from zebra fish international resource center ZIRC), cerebral hemorrhage mutant (ENU induced mutation obtains)

Experimental procedure:

1. collecting wild type and cerebral hemorrhage mutant embryos, 5 μM of Miconazoles or one were added into embryo at after fertilization 6 hours Quantitative DMSO is compared, and embryo cultivates in 28 DEG C；Drug is washed away within after fertilization 24 hours, fresh embryos culture solution, embryo's training are changed to It supports in 28 DEG C.

2. the extraction of full embryo's albumen.

After fertilization 24 hours, 50-100 pieces of zebrafish embryo is taken, the PBS being pre-chilled after egg membrane with 4 DEG C is removed and rinses twice, turn Move into 1.5ml centrifuge tube.Embryo is gently blown and beaten with 21G syringe, ruptures complete yolk, 4 DEG C of 500g are centrifuged 6 minutes, to the greatest extent may be used It can mostly discard supernatant.100 μ l protein lysates are added into every pipe, sufficiently being blown and beaten with 25G syringe ruptures embryo, on ice Make within 30 minutes sufficiently to crack.4 DEG C of 12000g are centrifuged 3 minutes, take supernatant, -80 DEG C of refrigerators are stored in after packing.

3. protein blot is tested

The protein lysate dispensed in right amount is taken, is uniformly mixed, boiling water with 5X albumen sample-loading buffer (containing mercaptoethanol) It boils 10 minutes, it is cooling rapidly, it is spare.

Prepare 8% PAGE gel.Constant pressure electrophoresis after loading is concentrated in glue and keeps 90V, separation gel 120V.Electrophoresis To appropriate location, low temperature constant current 200mA is transferred to pvdf membrane.Pvdf membrane containing protein sample is face-up, with closing After liquid chamber temperature is closed 2 hours, with 4 DEG C of the confining liquid overnight incubations containing primary antibody (anti-ERK or anti-pERK).Wash away primary antibody Afterwards, TBST is rinsed 3 times, every all over 10 minutes.It is incubated with the confining liquid room temperature containing secondary antibody (goat anti-rabbit IgG HRP) It educates 2 hours.TBST is rinsed 3 times, every all over 10 minutes.It is developed the color and is taken pictures with ECL luminescent solution.Gained protein band is quantitative with ImageJ And it counts.

Since Miconazole can reduce the activity of ERK, thus further probe into whether ERK inhibitor has and Miconazole class As bleeding inhibiting effect.Choosing two kinds of ERK inhibitor --- U0126 and PD0325901 detects it to zebra fish cerebral hemorrhage The bleeding inhibitory effect of mutant.Experiment shows that going out for zebra fish cerebral hemorrhage mutant can be effectively reduced in two kinds of inhibitor Blood rate, and efficiency is inhibited to increase and enhance with inhibitor concentration.On the other hand, a certain low concentration is chosen, so that low dense herein ERK inhibitor and Miconazole cannot generate significant bleeding inhibitory effect when spending, and work as the ERK inhibitor and miaow of this concentration Mutant, available very significant inhibitory effect (Fig. 6,7) are reprocessed after the mixing of health azoles.This prompt Miconazole may pass through Erk access plays the function of inhibiting bleeding.

Analysis of protein is carried out with the embryo that U0126 and Miconazole are handled, is found in wild type regardless of whether having at small molecule Reason, the total Erk1/2 specific gravity of p-ERK1/2 Zhan are not much different, and illustrate that Erk1/2 activity is not much different.With wild type control group (DMSO processing) is compared, and the total Erk1/2 specific gravity of p-ERK1/2 Zhan of zebra fish cerebral hemorrhage mutant (DMSO processing) obviously rises Height illustrates Erk1/2 increased activity.After low concentration U0126, Miconazole or both mixed processing, p-ERK1/2 of up-regulation The total Erk1/2 specific gravity of Zhan is restored, and U0126 and the recovery effects of Miconazole mixed processing are best, illustrates U0126 and Miconazole It is active (Fig. 8) that the Erk1/2 enhanced extremely in fn40a mutant can be reduced.

Specific experiment process is as follows:

Raw material: anti-ERK (anti-P42/44, cell signaling technology, #4695), anti-pERK (anti-pP42/44, cell signaling technology, #4370), anti-Mmp9 (AnaSpec, 55345), anti- Tubulin (Easybio, BE0031), Goat anti-rabbit IgG-HRP (Abmart, M21001), Goat anti- MouseIgG-HRP (Abmart, M21002)

Experimental animal: wild-type zebrafish (TL is bought from zebra fish international resource center ZIRC), cerebral hemorrhage mutant (ENU induced mutation obtains)

Experimental procedure:

1. collecting wild type and cerebral hemorrhage mutant embryos, 5 μM of Miconazoles or one were added into embryo at after fertilization 6 hours Quantitative DMSO is compared, and embryo cultivates in 28 DEG C；Drug is washed away within after fertilization 24 hours, fresh embryos culture solution, embryo's training are changed to It supports in 28 DEG C.

2. the extraction of full embryo's albumen.

After fertilization 24 hours, 50-100 pieces of zebrafish embryo is taken, the PBS being pre-chilled after egg membrane with 4 DEG C is removed and rinses twice, turn Move into 1.5ml centrifuge tube.Embryo is gently blown and beaten with 21G syringe, ruptures complete yolk, 4 DEG C of 500g are centrifuged 6 minutes, to the greatest extent may be used It can mostly discard supernatant.100 μ l protein lysates are added into every pipe, sufficiently being blown and beaten with 25G syringe ruptures embryo, on ice Make within 30 minutes sufficiently to crack.4 DEG C of 12000g are centrifuged 3 minutes, take supernatant, -80 DEG C of refrigerators are stored in after packing.

3. protein blot is tested

The protein lysate dispensed in right amount is taken, is uniformly mixed, boiling water with 5X albumen sample-loading buffer (containing mercaptoethanol) It boils 10 minutes, it is cooling rapidly, it is spare.

Prepare 8% PAGE gel.Constant pressure electrophoresis after loading is concentrated in glue and keeps 90V, separation gel 120V.Electrophoresis To appropriate location, low temperature constant current 200mA is transferred to pvdf membrane.Pvdf membrane containing protein sample is face-up, with closing After liquid chamber temperature is closed 2 hours, with the envelope for containing primary antibody (anti-ERK, anti-pERK, anti-Mmp9 or anti-Tubulin) Close 4 DEG C of liquid overnight incubations.After washing away primary antibody, TBST is rinsed 3 times, every all over 10 minutes.With contain secondary antibody (goat anti-rabbit IgG HRP or goat anti-mouse IgG HRP) confining liquid be incubated at room temperature 2 hours.TBST is rinsed 3 times, every all over 10 points Clock.Finally, it is developed the color and is taken pictures with ECL luminescent solution.Gained protein band is quantified and is counted with ImageJ.

Claims (7)

1. application of the Miconazole in preparation prevention angiorrbagia drug.

2. being applied described in claim 1, which is characterized in that the application refers to that Miconazole is in preparation prevention cerebral hemorrhage brain Application in expelling wind drug object.

3. Miconazole is preparing the application in perivascular cell MMP9 inhibitor.

4. being applied described in claim 3, which is characterized in that the perivascular cell MMP9 inhibitor refers to perivascular cell MMP9 expression inhibiting agent.

5. Miconazole is preparing the application in perivascular cell ERK inhibitor.

6. being applied described in claim 5, which is characterized in that the perivascular cell ERK inhibitor refers to perivascular cell ERK phosphorylation inhibitor.

7. a kind of pharmaceutical composition, which is characterized in that contain U0126 and Miconazole.