CN104825428B

CN104825428B - Purposes of the 4 methoxyl group benzylalcohols in blood-brain barrier protection medicine is prepared

Info

Publication number: CN104825428B
Application number: CN201510278259.7A
Authority: CN
Inventors: 段小花; 林青; 何芳雁
Original assignee: Yunnan University of Traditional Chinese Medicine TCM
Current assignee: Yunnan University of Traditional Chinese Medicine TCM
Priority date: 2015-05-27
Filing date: 2015-05-27
Publication date: 2017-08-04
Anticipated expiration: 2035-05-27
Also published as: CN104825428A

Abstract

The invention discloses purposes of the 4 methoxyl group benzylalcohols in blood-brain barrier protection medicine is prepared.The study find that 4 methoxyl group benzylalcohols can reduce the permeability of blood-brain barrier in During Ischemia; brain tissue NO, iNOS, nNOS expression are reduced, the high expression of AQP4 albumen is lowered and reduces TJ major structural proteins (Occludin, Claudin 5); there is good protective effect to blood-brain barrier; it can prepare and protect medicine as blood-brain barrier, potential applicability in clinical practice is good.

Description

Purposes of the 4- methoxyl groups benzylalcohol in blood-brain barrier protection medicine is prepared

Technical field

The present invention relates to purposes of the 4- methoxyl groups benzylalcohol in blood-brain barrier protection medicine is prepared.

Background technology

Blood-brain barrier (Blood-Brain Barrier, BBB) is found by German bacteriologist Pau1 Ehrhch, is It is present in the physiologic barrier together between blood and brain tissue, for maintaining the stabilization of intracerebral environment that there is important effect.Blood brain Barrier plays strict screening, under physiological status, blood-brain barrier phase between blood and brain tissue liquid in material exchange process To not penetrating, barrier function is mainly played, moreover it is possible to optionally intracerebral is harmful to or surplus substance is pumped out outside brain, the inner ring of brain is kept Border is stable.

Blood-brain barrier disruption is the important pathology that cerebral ischemia inspires encephaledema and cerebral infarction during cerebral ischemic injury One of change, be also the important step of pathophysiological change after cerebral ischemia, and influence the key factor of medication effect.In brain The damage and change and mutation functionally of blood-brain barrier structure occur after ischemic and cerebral ischemia re-pouring, it is main in structure Show as the influence to CMEC, astroglial foot processes and basilar memebrane, such as endothelial cell pyknosis, capillary Basement membrane of blood vessel is broken, and capillary tube chamber substantially deforms narrow, is destroyed the integrality of blood-brain barrier；Functionally make blood The increase of brain Barrier Permeability, ultimately results in vasogenic brain edema, even occurs hemorrhagic conversion, neurotrosis is aggravated, so as to go out The brain damage of existing Secondary cases.Therefore, blood-brain barrier is protected, there will be important meaning for the treatment and prognosis of ischemic cerebrovascular disease Justice.

4- methoxyl group benzylalcohols, colourless or slightly yellow liquid.23-25.5 DEG C of fusing point, 159 DEG C of boiling point, relative density 1.113 (15/15 DEG C), index of refraction 1.5442.Alcohol and ether are soluble in, water is practically insoluble in.Chemical formula is：C₈H₁₀O₂, structural formula isCurrent 4- methoxyl groups benzylalcohol is edible usually as flavorant, has no that 4- methoxyl groups benzylalcohol is used for what blood-brain barrier was protected Report.

The content of the invention

It is an object of the invention to provide purposes of the 4- methoxyl groups benzylalcohol in blood-brain barrier protection medicine is prepared.

The invention provides purposes of the 4- methoxyl groups benzylalcohol in blood-brain barrier protection medicine is prepared.

Wherein, the blood-brain barrier protection medicine is to reduce the medicine of blood-brain barrier permeability.

Wherein, the medicine be using 4- methoxyl groups benzylalcohol as active component, add pharmaceutically acceptable auxiliary material prepare and Into preparation.

Wherein, the preparation is oral formulations.

Wherein, the oral formulations are capsule, tablet.

Present invention also offers suppress blood-brain barrier protection medicine, it is characterised in that：It is using 4- methoxyl groups benzylalcohol as work Property composition, adds the preparation that pharmaceutically acceptable auxiliary material is prepared from.Wherein, the preparation is oral formulations.

Wherein, the oral formulations are capsule, tablet.

4- methoxyl groups benzylalcohol of the present invention can reduce the permeability of blood-brain barrier in During Ischemia, reduction brain tissue NO, INOS, nNOS expression, the high expression for lowering AQP4 albumen and reduction TJ major structural proteins (Occludin, Claudin- 5), there is good protective effect to blood-brain barrier, can prepare and protect medicine as blood-brain barrier, be the guarantor of clinical blood-brain barrier Shield provides a kind of new selection.

The present invention is described in further details below by embodiment, but is not the limit to the present invention System, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, not departing from, the present invention is above-mentioned Under the premise of basic fundamental thought, the modification of other diversified forms can also be made, replaces or changes.

Brief description of the drawings

Fig. 1 EB standard curves.

The influence that Fig. 2 4- methoxyl groups benzylalcohols score rat neuropathy.

Influence of Fig. 3 4- methoxyl groups benzylalcohols to rat cerebral tissue's EB contents.

Fig. 4 4- methoxyl groups benzylalcohols ooze out the influence of situation to rat cerebral tissue EB.

Fig. 5 BCA methods determine the standard curve of protein concentration.

The standard curve R2=0.9901 of Fig. 6 nNOS kits.

The standard curve R2=0.9969 of Fig. 7 iNOS kits.

The standard curve R2=0.9983 of Fig. 8 eNOS kits.

Fig. 9 BCA methods determine the standard curve R of protein concentration²=0.9955.

The standard curve R of Figure 10 nNOS kits²=0.9971.

The standard curve R of Figure 11 iNOS kits²=0.9960.

The standard curve R of Figure 12 eNOS kits²=0.9920.

The influence that Figure 13 4- methoxyl groups benzylalcohols are expressed rat ischemia side brain tissue NO and NOS.

The influence that Figure 14 4- methoxyl groups benzylalcohols are expressed rat ischemia side brain tissue AQP-4.

The influence that Figure 15 4- methoxyl groups benzylalcohols are expressed rat ischemia side brain tissue Occludin.

The influence that Figure 16 4- methoxyl groups benzylalcohols are expressed rat ischemia side brain tissue Claudin-5.

Embodiment

The blood-brain barrier protective effect of the 4- methoxyl group benzylalcohols of experimental example 1

1. experiment material

1.1 experimental animal

Adult healthy male SD rat, body weight 250-300g, SPF grade, by Sichuan Academy of Medical Sciences's Animal Experimental Study There is provided, production licence number：SCXK (river) 2013-12, product batch number：0016729.

1.2 experimental drug

4- methoxyl group benzylalcohols, purchased from Sigma Co., USA.

1.3 experiment reagent

1.4 laboratory apparatus

1.5 main agents are prepared

1.5.1 the configuration of 0.9% physiological saline:Sodium chloride 4.5g, plus distilled water is to 500mL, fully shakes up and puts 4 DEG C of ice Case is standby.

1.5.2 the preparation of physiological saline (O.9%) plus heparin (5 units/m1) solution：Sodium chloride 4.5g, plus distilled water is extremely 500mL, is added dropwise heparin 400uL, fully shakes up that to put 4 DEG C of refrigerators standby.

1.5.3 the configuration of 2%EB solution：Precision weighs EB2g, adds in physiological saline volumetric flask and is settled to 100mL, stirs Mix to being completely dissolved.

1.5.4 heparinized saline perfusion liquid：Sodium chloride (analysis is pure) 9g, injection liquaemin 25000u is with distilled water 1000mL is settled to constant volume bottle after 800mL complete miscibilities, is sub-packed in standby in 50OmL aseptic bottles.

1.5.5 the configuration (glutaraldehyde of 4% paraformaldehyde+0.5%) of Electronic Speculum cardiac perfusion fixer：Dissolve 2g paraformaldehydes In 25mL distilled waters, 80 DEG C of water-baths, shake is allowed to after dissolving plus 1-3 drops NaOH (1M), clarifies solution.Add 1mL after cooling 25% glutaraldehyde (Electronic Speculum is special), then add 24mL PBS (0.2M), adjust pH standby.

1.6 medicine ordinance

The configuration of 4- methoxyl groups benzylalcohol (20mg/kg)：Precision weighs 4- methoxyl groups benzylalcohol in right amount, and first add tween makes in right amount It is dissolved, and distilled water constant volume is used after being completely dissolved；The given low of rat is 1mL/kg, can be pre-configured to the solution of high concentration (2mg/mL), 4 DEG C of sealing preserves, using it is preceding according to experiment needs be diluted to suitable concentration.

2. experimental method

2.1 animal packets and medication

By body weight in 250-300g male (Sprague DawLey, SD) rat, SPF grades, random packet, i.e. sham-operation Group, model group, 4- methoxyl group benzylalcohol high dose groups (20mg/kg), low dose group (10mg/kg), every rat press 1mL/100g Volume gastric infusion, sham-operation group and model group gavage give isometric solvent (distilled water containing Tween-80), administration group difference Gavage gives institute's drug (1mL/100g), once a day, successive administration 5 days, on the operation same day, is administered in 0.5h before modeling.Often Group animal surgery ischemic 2h, Reperfu- sion 24h, carry out the detection of each index.

2.2 rat MCAO/R models

2.2.1 the making of paraffin line bolt

A diameter of 0.26mm import fishing nylon wire is taken, 5cm is about, respectively with black at its two ends 18mm, 10mm Color permanent pen blacking.It is 56 DEG C of one piece of solid paraffins to take fusing point, and heating fusing vertically exists one section of bolt line one end 5mm length Immerse and lift rapidly in the paraffin of fusing, cool down in atmosphere, the paraffin solidified immediately can firmly be attached on nylon wire one The surface at end, the other end of nylon wire makees identical processing, with being placed in 1 after 75% alcohol wipe：2500U heparin physiology salt It is standby in water.

2.2.2 the duplication of rat MCAO/R models

With reference to Longa, Kugal etc. and domestic report method, and improved, rat MCAO/R is replicated using line brush Model.Clone method is as follows：

By body weight in 250-300g male (Sprague DawLey, SD) rat, SPF grades, 10% chloraldurate is used (0.3mL/100g body weight) intraperitoneal injection of anesthesia, neck shaving simultaneously carries out routine disinfection collare and hits exactly at 0.5cm to the right otch about 1.5cm, separates rat right carotid (CCA) and vagus nerve to crotch, without separating external carotid artery with glass minute hand And internal carotid (ICA) (ECA).CCA proximal parts are ligatured, 1 venous clamp folder closes CCA distal ends, one is made a call to suture on CCA Slip-knot, eye scissors cuts a " V " shape breach at away from bifurcated 1cm, and bolt line is inserted through breach.Unclamp CCA on artery clamp, gently to Ligature on right drawing CCA proximal parts, to the left adjust inlet wire angle bolt line is successfully entered ICA, adjust to the right again into About 150 jiaos of line angle degree, and CCA is gently pulled, bolt line is entered brain, if entering bolt 12mm or so occurs as soon as the feelings for being difficult to enter Condition, is pointed out possibly into wing jaw artery (PPA), the segment distance of Outlet bolt one of now drawing back, and adjusts inlet wire to upper right side again Angle；If repeatedly can not still be correctly inserted into, illustrate the spasm for having resulted in ICA, blood is stayed again can eliminate its spasm, this Shi Kexian moves back Outlet bolt, treats restoration of blood flow 3-5min, then carry out the insertion of line bolt just have very high success rate.Line bolt insertion depth is 18-20mm (ECA is starting point with ICA turnofves), stops during micro- power of being hampered and (now can typically observe that face is twitched on the right side of rat), Make nylon wire head end by MCA section starts, reach thinner arteria cerebri anterior, now complete side middle cerebral artery occlusion (MCAO), the time of record now, it is easy to timing to implement Reperfu- sion.Then 1cm line bolt, MCAO are stayed outside skin suture, otch Slowly gently nylon wire is drawn its head end is returned in arteria carotis communis during 2h Reperfu- sions afterwards, you can to realize arteria cerebri media Reperfu- sion. Pull out after loop line bolt, recover blood supply in arteria cerebri media blood supply area, and arteria cerebri media can obtain arteria communicans anterior, posterior communicating artery and supply Blood, is conducive to causing Reperfu- sion.Room temperature is kept during post-ischemic reperfusion at 25 DEG C -30 DEG C.After rat is clear-headed, its god is observed Through afunction symptom, neurological scores are carried out in Reperfu- sion 24h, the detection that animal carries out each index is put to death after scoring.

2.3 neurological scores

Each group animal is scored by the following method when cerebral ischemia 2h Reperfu- sion 24h：

0 point：Impassivity functional impairment symptom；

1 point：Slight focal neurologic impairment, that is, carry fore paw on the left of the hanging not tensible of tail；

The focal land neurologic impairment of 2 points of moderates, walks turn-take to the left at once；

3 points：The focal flight of steps leading to a palace hall neurologic impairment of moderate, i.e. difficulty in walking, and toppling over to the left；

4 points：Spontaneous it can not walk, level of consciousness declines.

The measure of 2.4 brain tissue EB contents

EB osmosis is commonly used in the evaluations of BBB degrees of opening, and EB is a kind of azo group fluorescent dye, enter after blood almost all with Plasma albumin is combined, and can not enter brain tissue through BBB under normal circumstances, only when BBB is destroyed, and permeability is raised, This compound just can pass through BBB, and the elevated degree of BBB permeabilities is bigger, then the amount that EB is passed through is more, the blue journey of dye of brain tissue Degree is heavier；The organic solvents such as formamide are dissolved in into the EB in brain tissue, can be precisely measured out by AAS EB contents in extract solution, and then the permeability for the evaluation BBB that can be quantified.

The making of Evans blue (EvanSbLue, EB) standard curve：EB solution has maximum absorption band at 610nm.With Formamide solution returns to zero as blank control group, and the EB solution for preparing 1mg/mL is Cmax, and proportional diluted is configured to concentration For 1,1/2,1/4,1/8,1/16 (mg/mL) EB standard liquids, the OD values of above-mentioned EB standard liquids are detected on ELIASA, are entered EB standard curves are tried to achieve in row regression analysis.(the R of standard curve below figure 1²=0.9992)：

The measure of brain tissue EB contents：Each group randomly selects 6 rats, in after cerebral ischemia 15min, through vena femoralis injection 2%EB solution (is given) with 4mL/kg body weight, after cerebral ischemia 2h Reperfu- sions 24h, 10% chloraldurate of each group rat Thoracic cavity is opened after (0.3mL/100g) anesthesia, an osculum is cut in right auricle of heart portion, conduit is inserted to sustainer from left ventricle, to actively 400mL heparin-saline solution is slowly injected into arteries and veins, becomes limpid to atrium dextrum trickle.Broken end takes brain, removes olfactory bulb And pons, brain is divided into two hemisphere in left and right, brain hygrometric weight immediately is taken out, 105 DEG C are then put, constant temperature blast drying oven is dried After 24h, then rapid survey dry weight.Dry brain is put into formamide solution (1mL/ hemisphere), 50 DEG C of incubation 24h.Then 2000r/min centrifuges 30min, takes supernatant to be measured.Measure OD values under supernatant 200uL, 610nm are taken in ELISA Plate, are passed through EB standard curves calculate corresponding brain tissue EB contents (drying brain tissue with ug/g to represent).

The measure of 2.5 brain water contents

Each group randomly selects 6 rats, when cerebral ischemia 2h Reperfu- sion 24h, 10% chloraldurate of each group rat After (0.3mL/100g) anesthesia, quick broken end takes brain, and interface is cut off along along pons, removes olfactory bulb, cerebral hemisphere is separated, with dry Weight in wet base method measurement ischemic side water content, takes out brain hygrometric weight immediately, then puts 110 DEG C, constant temperature electric oven 24h to constant weight, then fast Speed surveys dry weight.Brain water content calculation formula：

Brain water content (encephaledema) (%)=(weight in wet base-dry weight)/weight in wet base × 100%

3. data processing

Experimental data carries out statistical analysis with GraphPad prism softwares, is as a result represented with mean ± standard deviation (), Using one-way analysis of variance (one-way ANOVE), first carry out needing to be compared between Dunnett analyses, such as other groups Tukey analyses are carried out, with P<0.05 is that difference has statistical significance.

4. experimental result

The influence that 4.1 4- methoxyl groups benzylalcohols score rat neuropathy

As a result as shown in table 1 and Fig. 2：

Influence that the 4- methoxyl groups benzylalcohol of table 1 scores rat neuropathy (N=6)

Note：With sham-operation group ratio：^▲▲▲P<0.001；With model group ratio：^**P<0.01

As a result show, compared with sham-operation group, model group rats have obvious neurological deficit symptom, difference has statistics Learn meaning (P<0.001)；Compared with model group, it is big that 4- methoxyl group benzylalcohol high and low dose groups can be obviously improved MCAO/R models Mouse neurological deficit symptom, the statistically significant (P of difference<0.01 and P<0.05).

Influence of the 4.2 4- methoxyl groups benzylalcohols to rat cerebral tissue's EB contents

As a result table 2 and Fig. 3~4 are such as referred to：

The 4- methoxyl groups benzylalcohol of table 2 to rat cerebral tissue's EB contents influence (N=6)

Note：With sham-operation group ratio：^▲▲▲P<0.001；With model group ratio：^***P<0.001,^**P<0.01

As a result show, each group rats with left brain tissue is not united without obvious EB bleedings, brain tissue EB content differences Meter learns meaning (P>0.05)；Compared with sham-operation, the right side brain tissue of model group rats ischemic has obvious EB bleedings, brain Organize EB contents significantly raised, the statistically significant (p of difference<0.001)；Compared with model group, high and low dose of 4- methoxyl group benzylalcohols Amount group can substantially reduce the EB contents of rat model ischemic side brain tissue, the statistically significant (p of difference<0.001 and p< 0.01)。

Experimental result illustrates that 4- methoxyl groups benzylalcohol can effectively reduce the permeability of blood-brain barrier, with significant blood brain Barrier protection is acted on, and blood-brain barrier protection class medicine can be made.

The blood-brain barrier protective effect of the 4- methoxyl group benzylalcohols of experimental example 2

Blood-brain barrier (BBB) is the architecture basics that body maintains intracerebral ambient stable, lacked in brain as the door of brain Occupy critical role in blood/reperfusion injury (CIRI).At present, the factor of influence BBB destructions is numerous, and its molecular mechanism is crisscross multiple Miscellaneous, CIRI causes BBB to damage, and the increased mechanism of permeability mainly has：Matrix metalloprotease protease (MMPs) expression rise；Water Channel protein 4 (AQP-4) is over-expressed；Close connection (TJ) between vascular endothelial cell is open, major structural protein (Occludin, Claudin-5) degrades；Radical damage, the cytotoxicity that NO excess accumulations are produced；Inflammatory reaction etc. it is a variety of because The common results of element interaction, are a multifactor complicated pathologic processes, it will usually be related to Multiple factors multiple pathogenic The interaction of the factor.

In view of NO, MMPs, AQP-4, Occludin, Claudin-5 unconventionality expression are to participate in cerebral ischemia/reperfusion injury The principal element of BBB destructions, therefore the angle discussion that regulates and controls from NO regulatory pathway and BBB correlative protein expressions of research of this experiment Protective effect of the 4- methoxyl groups benzylalcohol to rat MCAO/R Model Bs BB.

1. experiment material

1.1 experimental animal

1.2 experimental drug

4- methoxyl group benzylalcohols, purchased from the monomer component 4- methoxyl group benzylalcohols of Sigma Co., USA.

1.3 experiment reagent

1.4 laboratory apparatus

1.5 main agents are prepared

1.5.1 the reagent configured needed for Western-Blot experiments

1.5.1.1 10% Ammonium Persulfate 98.5 (AP)

Ammonium Persulfate 98.5 0.1g

Distilled water 1mL

Matching while using, first Ammonium Persulfate 98.5 dry powder can be weighed up and be placed on after component in 1.5mL centrifuge tubes (it is disposable claim it is several - 20 DEG C of pipe is saved backup), distilled water dissolving is added, what can be finished in 1 week is put into 4 DEG C of preservations, by remaining packing, It is put into -20 DEG C of refrigerator preservations.

1.5.1.2 4X sample-loading buffers (Loading buffer)

1.0moL/L Tris·HCL(pH6.8)

2mL SDS

0.8g bromophenol blues

0.04g glycerine

4mL deionized waters are settled to 10mL.

After mixing, it is sub-packed in 1.5mL centrifuge tubes, each 1mL, 4 DEG C of preservations.1X is diluted to when using.

1.5.1.3 1.0moL/L dithiothreitol (DTT)s (DTT) (10X)

3.09g DTT are dissolved with 20mL 0.01moL/L sodium acetate solutions (pH5.2), 1mL aliquots is distributed into and is stored in -20 DEG C, 1X is diluted to when using.

For example：The μ L of 20 5 μ L+DTT of μ L--4X sample-loading buffers of applied sample amount, 2 μ L+ samples albumen 13

1.5.1.4 5X electrophoresis liquid buffer solutions

Room temperature preservation after dissolving, the used time dilutes 5 times, generally takes 160mL to be configured to 800mL.

1.5.1.5 10X transferring film buffer solutions

Glycine (MW75.07) 151.1g

Tris(MW121.14) 30.3g

Distilled water is to 1000mL

Room temperature preservation after dissolving, the used time dilutes 10 times, and adds methanol to 20%.80mL mother liquors, plus 560mL is generally taken to steam Distilled water, most adds 160mL methanol, is configured to 800mL.(first plus methanol is also easy to produce precipitation)

1.5.1.6 10XTBS buffer solutions

Tris(MW121.14) 24.2g

NaCL 80g

Distilled water to 1000mL concentrated hydrochloric acids adjust pH to 7.6, room temperature preservation after dissolving.

1.5.1.7 1XTBST buffer solutions

10XTBS buffer solutions 100mL

Distilled water 900mL

Tween-20 1mL

10XTBS buffer solution 100mL are taken, distilled water 800mL is added, because Tween-20 is more sticky, should slowly draw dissolving Room temperature preservation afterwards.

1.5.1.8 confining liquid/antibody diluent (5% skim milk)

1XTBST buffer solutions 95-100mL

Skimmed milk power 5g

4 DEG C of preservations, can be used in 4 days after dissolving.

1.6 medicine ordinance

2. experimental method

The packet of 2.1 animals and medication

2.2 the duplication of rat MCAO/R models

2.2.1 the making of paraffin line bolt

2.2.2 the duplication of rat MCAO/R models

2.3 determine rat ischemia side brain tissue NO, NOS expression

Each group randomly selects 6 rats, in 10% chloraldurate of each group rat after cerebral ischemia 2h Reperfu- sions 24h Thoracic cavity is opened after (0.3mL/100g) anesthesia, an osculum is cut in right auricle of heart portion, conduit is inserted to sustainer from left ventricle, to actively 400mL heparin-saline solution is slowly injected into arteries and veins, becomes limpid to atrium dextrum trickle, whole operation is entered under ice bath OK.Broken end takes brain, removes olfactory bulb and pons, takes ischemic side brain tissue, blotted after surface moisture and weighed with filter paper, be cut into small pieces and put In the centrifuge tube for entering 10mL precoolings, in being homogenized under ice bath, per 100mg, tissue adds 1mL 1X PBS, and homogenate is made, then puts In -20 DEG C overnight.After 2 processing destruction cell membranes of multigelation, supernatant is taken within 5 minutes in 4 DEG C of 5000g centrifugations, by supernatant Packing is stored in -80 DEG C, and the sample after defrosting should be centrifuged again.A supernatant is respectively taken, is surveyed by the requirement of BCA protein determination kits Determine the content of albumen in each group brain tissue homogenate liquid, and containing for NO in kit requirements detection brain tissue homogenate liquid is determined by NO Amount；The expression of iNOS, eNOS, nNOS in brain tissue homogenate are detected by ELISA kit requirement.

The protein concentration of brain tissue homogenate's liquid is determined with BCA albuminimetries, and making is pointed out according to kit specification Standard curve, OD values are surveyed with ELIASA with 450nm, and its concentration is looked into by standard curve, and operation is said in strict accordance with ELISA kit Bright book is carried out.

The standard curve during influence that 2.3.1 measure 4- methoxyl groups benzylalcohol is expressed rat cerebral tissue NO, NOS, below figure 5 ~8, BCA method determine the standard curve R of protein concentration²=0.9974；The standard curve R of nNOS kits²=0.9901；iNOS The standard curve R of kit²=0.9969；The standard curve R of eNOS kits²=0.9983.

The standard curve during influence that 2.3.2 measure 4- methoxyl groups benzylalcohol is expressed rat cerebral tissue NO, NOS, below figure 9 ~12, BCA method determine the standard curve R of protein concentration²=0.9955；The standard curve R of nNOS kits²=0.9971；iNOS The standard curve R of kit²=0.9960；, the standard curve R of eNOS kits²=0.9920.

2.4 determine the expression of blood-brain barrier GAP-associated protein GAP

2.4.1 protein extraction

Rat continuously gives corresponding test medicine 5 days, after last dose after 30min, 10% chloraldurate intraperitoneal injection SD rats are anaesthetized, quick broken end takes brain, divides take ischemic side cerebral cortex on ice, the centrifuge tube of the prior precoolings of 10mL is placed in after weighing In, it is placed in and is ground into slurry with tissue grinder rod on ice.Added in right amount according to IP and cell pyrolysis liquid operation instructions Cell pyrolysis liquid (per 1mg organize add 8uL lysate), softly blown and beaten with micropipettor it is several under be allowed to mix, shake on ice 30min is swung, is moved into 1.5mL centrifuge tubes, 14000rpm/ turns 4 DEG C of centrifugation 5min, takes supernatant in be measured in another centrifuge tube.

2.4.2 BCA methods detect the concentration of total protein

1) appropriate 5mg/mL standard proteins are taken, final concentration of 2.5mg/mL is diluted to 0.9%NaCL or PBS.

2) according to sample size, 1 volume BCA reagents B (50 is added by 50 volume BCA reagent As:1) appropriate BCA work is prepared Liquid, is fully mixed.BCA working solutions room temperature is stablized in 24 hours.

3) by standard items by the method for proportional diluted be configured to 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.3125mg/mL, 0.156mg/mL, 0.078mg/mL, 0mg/mL standard solution, 96 orifice plates are added to by 20uL/ holes Standard sample wells in.

4) take sample 10uL to dilute 5 times with standard dilutions, the sample after 20 μ L dilutions is taken after mixing to 96 orifice plates In sample well.

5) each hole adds 200 μ L BCA working solutions, 37 DEG C of incubation 30min.

4) total protein concentration at 560nm is determined using machine on ELIASA, standard curve is formulated, then according to standard curve Calculate sample total protein concentration.

Note：Residual protein sample adds 100 DEG C of sample-loading buffer and DTT to boil 5-10min and be fully denatured, packing be stored in- 80 DEG C standby.

2.4.3 electrophoresis, transferring film, colour developing and imaging

1) Casting of gels

The groove cleaned up and the glass plate of un-grooved are alignd, clamping is put into electrophoresis tank.

Gel proportioning is as follows：

8% separation gel：

10% separation gel：

15% separation gel：

5% concentration glue：

Separation gel is recorded according to aforementioned proportion, after being well mixed, in the space that two glass plates are added slowly to liquid-transfering gun, plus To at upper strata about 3cm, upper strata is filled up with tri-distilled water, and room temperature places about 15min, after glue to be separated is irrigated, by upper strata Tri-distilled water fall it is dry, add afterwards record according to the above ratio upper strata concentration glue, be slowly added in gel groove, comb plugged rapidly Son.

2) electrophoresis

The offset plate filled is fixed in electrophoresis tank, appropriate 1 × electrophoretic buffer is added, gently extracts comb.To passage Interior addition testing protein sample and pre-dyed Marker, 80V electrophoresis about 30min, treat that Marker is clearly separated, and switching voltage is extremely 120V, electrophoresis about 1h, after bromophenol blue is run out of, terminate electrophoresis.

3) half-dried transferring film method

Appropriately sized pvdf membrane is cut according to the size of destination protein, 5min or so is soaked in methyl alcohol, by pvdf membrane And filter paper is put into 10min in transferring film buffer solution, transferring film sandwich, regulation are prepared according to the order of filter paper, glue, pvdf membrane, filter paper Appropriate electric current, the appropriate transferring film time is adjusted according to destination protein molecular size range.

4) close, colour developing and imaging

Transferring film terminates, and pvdf membrane is removed and is put into Ponceaux dyeing liquor, dyes 5min, and protein band is high-visible, uses Distilled water is cleaned 2-3 times, pvdf membrane is transferred in the confining liquid prepared before, closes 1h, plus primary antibody.Next day is put in shaking table use 1 × TBST is washed 3 times, each 5min, is then added the secondary antibody of HRP marks, is washed with 1 × TBST 3 times, each 5min, is prepared ECL chemistry reflective liquids, are added on pvdf membrane, are imaged with UVP gel imaging systems.

2.4.4 the influence that 4- methoxyl groups benzylalcohol is expressed rat ischemia side cortex AQP-4

AQP-4, beta-actin are determined with Western-Blot.The albumen applied sample amount of each sample is 61 μ g, uses AQP-4 (1:2000) 2h, secondary antibody (1 are incubated at room temperature:2000) 1h, colour developing are incubated.With beta-actin (1:1500) 2h, two are incubated at room temperature Anti- (1:2000) 1h, colour developing are incubated.

2.4.5 influence of the 4- methoxyl groups benzylalcohol to rat ischemia side cortex TJ protein expressions

Occludin, Claudin-5, beta-actin are determined with Western-Blot.The albumen applied sample amount of each sample For 61 μ g, with Occludin (1:300), stay overnight for 4 DEG C, secondary antibody (1:2000) 1h, colour developing are incubated；With Claudin-5 (1:200), 4 DEG C overnight, secondary antibody (1:2000) 1h, colour developing are incubated.With beta-actin (1:1500) 2h, secondary antibody (1 are incubated at room temperature:2000) incubate 1h is educated, is developed the color.

3. data processing

Experimental data carries out statistical analysis with GraphPad prism softwares, as a result with mean ± standard deviation Represent, using one-way analysis of variance (one-way ANOVE), first carry out needing to be compared between Dunnett analyses, such as other groups Compared with Tukey analyses are being carried out, with P<0.05 is that difference has statistical significance.Image procossing, profit are carried out using photshopCS5 Image intensity value processing is carried out with ImageJ.

4. experimental result

The influence that 4.1 4- methoxyl groups benzylalcohols are expressed ischemic side brain tissue NO and NOS

As a result show, compared with sham-operation group, model group rats ischemic side brain tissue NO, nNOS, iNOS expression have It is significantly raised, the statistically significant (P of difference<0.001、P<0.001 and P<0.01), eNOS expression is without significant change, difference Not statistically significant (P>0.05)；Compared with model group, 4- methoxyl group benzylalcohol high and low dose groups can substantially reduce rat model Ischemic side brain tissue NO (P<0.05 and P<0.001)、nNOS(P<0.01 and P<0.001)、iNOS(P<0.01) expression, difference Statistically significant, the expression to eNOS has not significant impact, no significant difference (P>0.05).It is shown in Table 3, Figure 13：

Influence that the 4- methoxyl groups benzylalcohol of table 3 is expressed rat ischemia side brain tissue NO and NOS (N=6)

Note：With sham-operation group ratio：^▲▲▲P<0.001,^▲▲P<0.01；With model group ratio：^***P<0.01,^**P<0.01,^*P< 0.05

The influence that 4.2 4- methoxyl groups benzylalcohols are expressed ischemic side brain tissue AQP-4

As a result show, compared with sham-operation group, model group rats ischemic side brain tissue AQP-4 expression is significantly raised, poor Different statistically significant (P<0.001)；Compared with model group, 4- methoxyl group benzylalcohol high and low dose groups can substantially lower model Rat ischemia side brain tissue AQP-4 high expression, the statistically significant (P of difference<0.01 and (P<0.001).Table 4 is referred to, is schemed 14。

Influence that the 4- methoxyl groups benzylalcohol of table 4 is expressed rat ischemia side brain tissue AQP-4 (N=6)

Influence of the 4.3 4- methoxyl groups benzylalcohols to rat ischemia side brain tissue TJ protein expressions

As a result show, compared with sham-operation group, brain tissue Occludin expression in model group rats ischemic side has obvious drop It is low, the statistically significant (P of difference<0.001)；Compared with model group, 4- methoxyl group benzylalcohol high doses group can significantly raised model Rat ischemia side brain tissue Occludin expression, the statistically significant (P of difference<0.05).Refer to table 5, Figure 15.

Compared with sham-operation group, model group rats ischemic side brain tissue Claudin-5 has obvious reduction, and difference has statistics Learn meaning (P<0.001)；Compared with model group, 4- methoxyl group benzylalcohol high doses group can significantly raised rat model ischemic side brain group Knit Claudin-5 expression, the statistically significant (P of difference<0.01).Refer to table 5, Figure 16.

Influence that the 4- methoxyl groups benzylalcohol of table 5 is expressed rat ischemia side brain tissue Occludin (N=6)

Note：With sham-operation group ratio：^▲▲▲P<0.001；With model group ratio：^**P<0.01,^*P<0.05

5. discuss

Influence of the 5.1 4- methoxyl groups benzylalcohols to NO and NOS paths

The effect that NO causes BBB to damage to Cerebral Ischemia/Reperfusion is widely recognized as by academia, using NOS inhibitor (L-NAME) it can obviously reduce the permeability of BBB after cerebral ischemia re-pouring.NO is messenger molecule important in organism and effect Molecule, is mainly synthesized and is discharged by vascular endothelial cell, vascular smooth muscle cells, nerve cell etc., with neurotransmitter and tune The function of matter, wide participation body physiological and pathologic event, endogenous NO participate in the regulation of BBB permeabilities；Damaged in cerebral ischemia During wound, N0 may play the double action of protection or damage, and the crucial rate-limiting enzyme that NOS is synthesized as mediation NO can be divided into Neuron pattern (nNOS), the NOS induced in type (iNOS) and endothelium in type (eNOS), endothelial cell are mainly eNOS, are produced by it The expansible cerebrovasculars of raw NO, increase cerebral blood flow (CBF) is so as to play a protective role, however, the NO vasorelaxation time is very short Temporarily, do not have neuroprotection then more than 2h；And NOS is mainly nNOS in nerve cell, the NO produced by it can then be produced Neurotoxicity；The iNOS produced is induced by environmental stimuli, astroglia and microglia, blood vessel is distributed mainly on Smooth muscle cell and vascular endothelial cell, extend with stimulus duration, and NO yields increase, its NO produced can aggravate ischemic Property brain damage.

This experiment detects the content of NO in the brain tissue of rat ischemia side using NO nitrate reductase methods, and uses enzyme linked immunological INOS, eNOS, nNOS activity, as a result show in the brain tissue of method detection model rat ischemia side, and 4- methoxyl groups benzylalcohol can be bright The NO contents of aobvious reduction rat model ischemic side brain tissue and the expression for suppressing iNOS and nNOS, illustrate 4- methoxyl group benzylalcohols It can protect blood-brain barrier by suppressing brain tissue NO content and NOS paths, reduce Blood Brain Barrier (BBB) permeability.

The influence that 5.2 4- methoxyl groups benzylalcohols are expressed AQP-4

Encephaledema is that ischemic cerebrovascular disease is common not only aggravates neurological deficit without negligible sequelae, its Intracranial hypertension, the hernia cerebri even threat to life of initiation, AQP4 serves vital in the formation and development of encephaledema. AQP-4 is the widest aquaporin of distribution in central nervous system, is distributed in BBB both sides, i.e. brain capillary endothelium is thin Born of the same parents and the astroglial foot processes around blood vessel, take part in BBB opening and the formation of encephaledema, disappear, and be encephaledema hairs Raw important molecule basis, is to maintain the key protein of the water balance of central nervous system, the increase of AQP-4 expressions can add The degree of weight encephaledema, its level declines or activity reduction can reduce or suppress the development of encephaledema.

As a result this experiment is shown, 4- first using Western Blot methods detection rat ischemia side brain tissue AQP-4 expression Epoxide benzylalcohol can substantially lower rat model ischemic side cortex AQP-4 high expression, illustrate that 4- methoxyl groups benzylalcohol can lead to The high expression for lowering AQP-4 is crossed, blood-brain barrier is protected, reduces Blood Brain Barrier (BBB) permeability.

Influence of the 5.3 4- methoxyl groups benzylalcohols to TJ protein expressions

Close connection (TJ) is to maintain the critical function unit of BBB integralities, is the key link of BBB permeabilities regulation. BBB functions change and TJ opening it is relevant with closure, and TJ whether open it is in close relations with correlative protein expression, at present grind Study carefully and show, one of increased main mechanism of BBB permeabilities that ischemia/reperfusion injury is caused is due to the structural proteins for constituting TJ Occludin, Claudin-5 synthesis and expression are reduced, and TJ integrality are destroyed, so that causing BBB permeability increases.

Occlusion albumen (Occludin) is TJ major structural protein, participates in the formation of TJ on brain microvessel endothelial cells in vitro, Intercellular permeability can be changed, or form aquaporin, so that uncharged solute flows, height expression can increase endothelium Intercellular resistance, its expression quantity in brain tissue blood vessel endothelium, apparently higher than the blood vessel endothelium of non-nervous tissue, is that BBB leads to Permeability is significantly lower than the major reason of other tissue barriers.Found in the research of rat MCAO/R models, when Occludin tables Up to when transcriptional level and translation skill are suppressed, TJ albumen disintegration, then TJ openings, BBB integralities are damaged.Therefore recognize BBB configuration state can be represented for Occludin expressions, it, which declines degree, can be used as the beautiful of BBB degree of injury.

Occludin -5 (Claudin-5) is the specific proteins in brain microvessel endothelial cells in vitro TJ, Claudin-5 genes Into the overexpression in fiber, the aggregation of cell is can result in, and forms similar TJ cord structures, only by single Claudin-5 gene expressions, it is possible to obtain TJ reconstruction, it is believed that it is the main transmembrane protein for constituting BBB, it is TJ shapes Into adequate condition.Claudin-5 exogenous expression can induce the formation of BBB features, its reduction expressed and BBB permeabilities Increase it is closely related.

This experiment detects rat ischemia side cortex Occludin and Claudin-5 table using Western Blot methods Reach, as a result show, 4- methoxyl groups benzylalcohol significantly raised rat model ischemic side cortex Occludin and Claudin-5 table Reach, illustrate that 4- methoxyl groups benzylalcohol can protect blood brain by reducing TJ GAP-associated protein GAPs (Occludin, Claudin-5) missing Barrier, reduces Blood Brain Barrier (BBB) permeability.

To sum up, 4- methoxyl groups benzylalcohol can reduce the permeability of blood-brain barrier in During Ischemia, reduction brain tissue NO, INOS, nNOS expression, the high expression for lowering AQP4 albumen and reduction TJ major structural proteins (Occludin, Claudin- 5), there is good protective effect to blood-brain barrier, Blood Brain Barrier (BBB) permeability can be reduced, can prepare and protect medicine as blood-brain barrier Thing, potential applicability in clinical practice is good.

Claims

Purposes of the 1.4- methoxyl groups benzylalcohol in blood-brain barrier protects medicine；The blood-brain barrier protection medicine is reduction blood brain screen Hinder the medicine of damage.
2. purposes according to claim 1, it is characterised in that：The blood-brain barrier protection medicine is that reduction blood-brain barrier leads to The medicine of permeability.
3. purposes according to claim 1 or 2, it is characterised in that：The medicine be using 4- methoxyl groups benzylalcohol as activity into Point, add the preparation that pharmaceutically acceptable auxiliary material is prepared from.
4. purposes according to claim 3, it is characterised in that：The preparation is oral formulations.
5. purposes according to claim 4, it is characterised in that：The oral formulations are capsule, tablet.