CN105232499B - 3,4- 4-dihydroxy benzaldehydes prepare treatment or/and prevent the purposes of cerebral ischemia re-pouring injured drug - Google Patents
3,4- 4-dihydroxy benzaldehydes prepare treatment or/and prevent the purposes of cerebral ischemia re-pouring injured drug Download PDFInfo
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- CN105232499B CN105232499B CN201510695078.4A CN201510695078A CN105232499B CN 105232499 B CN105232499 B CN 105232499B CN 201510695078 A CN201510695078 A CN 201510695078A CN 105232499 B CN105232499 B CN 105232499B
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Abstract
Treatment is used to prepare the invention discloses 3,4 4-dihydroxy benzaldehydes or/and prevents the purposes of cerebral ischemia re-pouring injured drug.Effect experiment shows that the compound has the function of significantly to protect blood-brain barrier, protection nervous function and NO toxic reactions is inhibited to occur, so as to achieve the effect that treatment or/and prevention are cerebral ischemia re-pouring injured.Compared to clinically common similar drugs and treatment means, 3,4 4-dihydroxy benzaldehydes derive from a wealth of sources, are cheap, is significant in efficacy at present, there is good potential applicability in clinical practice.
Description
Technical field
Treatment is used to prepare the present invention relates to 3,4- 4-dihydroxy benzaldehydes or/and prevents cerebral ischemia re-pouring injured drug
Purposes, belong to field of medicaments.
Background technology
After cerebral ischemia certain time restores blood supply, function not only fails to restore, and more serious brain but occurs
Dysfunction, referred to as cerebral ischemia re-pouring (cerebral ischemia-reperfusion, CIR) are damaged.At present, inhibit again
Perfusion injury has become the important link for the treatment of ischemic cerebrovascular disease.
Cerebral ischemia re-pouring injured is a kind of pathophysiological process of complexity, is mainly excessively formed with free radical, excitability
The number of mechanisms such as amino acid toxicity effect, intracellular calcium overload, inflammatory reaction are related.In recent years for cerebral ischemia re-pouring injured
Therapy study obtained certain effect, clinically often using mild hypothermia therapy, anti-inflammatory treatment, inhibit the means such as Apoptosis
Combination therapy, mitigate jointly cerebral injury (Chen Yumin etc., cerebral ischemia re-pouring injured mechanism and current treatment status [J], medical research with
Education, 2012,29：47-54).However, since the cause of disease is more, pathogenesis is complicated, prevention is cerebral ischemia re-pouring injured still to be lacked
Effective drug and method, it is extremely urgent to develop effective drug.
3,4- 4-dihydroxy benzaldehydes (also known as protocatechualdehyde) are a kind of important medicine intermediates, a variety of anti-available for synthesizing
Rhzomorph and anti-inflammation drugs are widely present in natural products (such as Rhizoma Gastrodiae, Radix Salviae Miltiorrhizae), and can pass through simple processing step
It is prepared.In recent years to protocatechualdehyde pharmacological activity and its mechanism of action etc. research shows that, it have anti-artery congee
The extensive pharmacological activity such as sample hardening, protection cardiac muscle, antithrombus formation, neuroprotection, anti-purulence blood, antiviral, anti-fibrosis (
Emerald green English etc., the pharmacological research progress [J] of protocatechualdehyde, Chinese experimental pharmacology of traditional Chinese medical formulae magazine, 2014,19 (23)：338~342).
However, there has been no have to prevent cerebral ischemia re-pouring injured pharmacological action about 3,4- 4-dihydroxy benzaldehydes so far
Open report.
Invention content
The purpose of the present invention is to provide 3,4- 4-dihydroxy benzaldehydes to be used to prepare treatment or/and prevention cerebral ischemia re-pouring
The purposes of the drug of damage.
Treatment is used to prepare the present invention provides 3,4- 4-dihydroxy benzaldehydes or/and prevents cerebral ischemia re-pouring injured medicine
The purposes of object.
Further, the drug is treatment or/and prevention nervous function damage, Blood Brain Barrier (BBB) permeability or nitric oxide
(NO) in toxic reaction one or more of diseases drug.
Further, the drug is lowered one in brain tissue Caspase-3, Caspase-9, AQP-4 or MMP-3
The drug of kind or several protein expressions；The drug be it is a kind of in raising brain tissue Occludin, Claudin-5 or GFAP or
The drug of several protein expressions；The drug is the drug for reducing NO contents in brain tissue；The drug is to inhibit nerve
The drug of one or two kinds of enzymatic activitys in type nitricoxide synthase (nNOS), nitric oxide synthase type (iNOS).
Further, the drug be by a effective amount of 3,4- 4-dihydroxy benzaldehydes be active constituent, add in pharmaceutically
The preparation that acceptable auxiliary material or complementary ingredient are prepared.
Preferably, the preparation is oral preparation.
Preferably, the preparation is capsule, granule or tablet.
The present invention also provides a kind for the treatment of or/and prevent cerebral ischemia re-pouring injured pharmaceutical composition, it be with 3,
4- 4-dihydroxy benzaldehydes are active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material is prepared.
Further, the preparation is oral preparation.
Further, the oral preparation is capsule, granule or tablet.
Treatment is used to prepare the present invention provides 3,4- 4-dihydroxy benzaldehydes or/and prevents cerebral ischemia re-pouring injured medicine
The purposes of object.Effect experiment shows that the compound has significant protection blood-brain barrier, protection nervous function and inhibits NO toxicity
The effect occurred is reacted, so as to achieve the effect that treatment or/and prevention are cerebral ischemia re-pouring injured.It is clinically normal compared at present
Similar drugs and treatment means, 3,4- 4-dihydroxy benzaldehydes derive from a wealth of sources, are cheap, is significant in efficacy, have good
Potential applicability in clinical practice.
Obviously, the above according to the present invention according to the ordinary technical knowledge and customary means of this field, is not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 EB standard curves.
Fig. 2 BCA methods measure the standard curve of protein concentration
The standard curve of Fig. 3 nNOS kits
The standard curve of Fig. 4 iNOS kits
Influence of Fig. 5 3,4- 4-dihydroxy benzaldehydes to rat cerebral tissue's EB contents
Influence of Fig. 6 3,4- 4-dihydroxy benzaldehydes to rat cerebral tissue EB exudation situations
The influence that Fig. 7 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue AQP-4
The influence that Fig. 8 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue Occludin
The influence that Fig. 9 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue Claudin-5
The influence that Figure 10 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue MMP-3
The influence that Figure 11 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue GFAP
The influence that Figure 12 3,4- 4-dihydroxy benzaldehydes score to rat neuropathy
The influence that Figure 13 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue Caspase-3
The influence that Figure 14 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue Caspase-9
Influence of Figure 15 3,4- 4-dihydroxy benzaldehydes to rat cerebral tissue's NO contents and NOS activity
Specific embodiment
Raw material, reagent and the equipment used in the specific embodiment of the invention is known product, by buying commercially available production
Product obtain.
Beneficial effects of the present invention are proved below by way of experimental example.
1. experiment material
1.1 experimental animal
Adult healthy male SD rat, weight 250-300g, SPF grade, by Sichuan Academy of Medical Sciences's Animal Experimental Study
It is provided, production licence number：SCXK (river) 2008-24, quality certification number：0017670.
1.2 experimental drug
3,4- 4-dihydroxy benzaldehydes, by Chinese Academy of Sciences's Kunming Institute of Zoology from EtOAc layers of extract of Rhizoma Gastrodiae separation and purification
Obtained by transparence solid crystal, this experiment monomer component for being available from Sigma Co., USA used after structure determination.
In addition it is also possible to using the other methods of document report from the plant (such as Radix Salviae Miltiorrhizae) for containing 3,4- 4-dihydroxy benzaldehydes
Middle extraction 3,4- 4-dihydroxy benzaldehydes；Or directly buy commercial product (No. CAS：139-85-5).
1.3 main agents are prepared
1.3.1 the preparation of 0.9% physiological saline:Sodium chloride 4.5g adds distilled water fully to shake up to 500mL and put 4 DEG C of ice
Case is spare.
1.3.2 the preparation of physiological saline (O.9%) plus heparin (5 units/m1) solution：Measure 0.9% physiological saline
2500U heparin 800uL are added dropwise in 1000mL, fully shake up that put 4 DEG C of refrigerators spare.
1.3.3 the preparation of 2%EB solution：Precision weighs EB2g, adds in physiological saline volumetric flask and is settled to 100mL, stirs
It mixes to being completely dissolved.
1.3.4 heparinized saline perfusion liquid：Sodium chloride (analysis is pure) 9g, injection heparin sodium 25000u is with distilled water
1000mL is settled to constant volume bottle after 800mL complete miscibilities, is sub-packed in spare in 500mL aseptic bottles.
1.3.5 the preparation (+0.5% glutaraldehyde of 4% paraformaldehyde) 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 the PBS (0.2M) of 24mL, adjust pH spare.
1.3.6Western-Blot the reagent of required preparation in testing
1.3.6.1 10% Ammonium Persulfate 98.5 (AP)
Ammonium Persulfate 98.5 0.1g
Distilled water 1mL
Matching while using is placed in 1.5mL centrifuge tubes after can Ammonium Persulfate 98.5 dry powder first being weighed up component and (disposably claims several more
- 20 DEG C of pipe saves backup), distillation water dissolution is added in, what can be finished in 1 week is put into 4 DEG C of preservations, by remaining packing,
- 20 DEG C of refrigerators are put into preserve.
1.3.6.2 4X sample-loading buffers (Loading buffer)
Deionized water is settled to 10mL.It after mixing, is sub-packed in 1.5mL centrifuge tubes, each 1mL, 4 DEG C of preservations.During use
It is diluted to 1X.
1.3.6.3 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, when use, is diluted to 1X.
Such as：20 5 μ L+DTT of μ L--4X sample-loading buffers of applied sample amount, 2 μ L+ samples albumen, 13 μ L
1.3.6.4 5X electrophoresis liquid buffer solutions
Room temperature preservation after dissolving, used time dilute 5 times, usually 160mL are taken to be configured to 800mL.
1.3.6.5 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 in methanol to 20%.80mL mother liquors are usually taken, 560mL is added to steam
Distilled water most adds 160mL methanol, is configured to 800mL.(first plus methanol is also easy to produce precipitation)
1.3.6.6 10XTBS buffer solutions
Tris(MW121.14) 24.2g
NaCL 80g
Distilled water is to 1000mL concentrated hydrochloric acid tune pH to 7.6, room temperature preservation after dissolving.
1.3.6.7 1XTBST buffer solutions
10XTBS buffer solutions 100mL
Distilled water 900mL
Tween-20 1mL
10XTBS buffer solution 100mL are taken, distilled water 800mL is added in, because Tween-20 is more sticky, should slowly draw dissolving
Room temperature preservation afterwards.
1.3.6.8 confining liquid/antibody diluent (5% skim milk)
1XTBST buffer solutions 95-100mL
Skimmed milk power 5g
4 DEG C of preservations, can use in 4 days after dissolving.
1.4 medicine ordinance
1.4.1 the configuration of 3,4- 4-dihydroxy benzaldehydes (20mg/kg)：It weighs in right amount, with distillation water dissolution and constant volume.
1.4.2 the configuration of 3,4- 4-dihydroxy benzaldehydes (10mg/kg)：The solution for taking 20mg/kg is appropriate, dilute with distilled water
Release 2 times.
2. experimental method
2.1 animal packets and medication
By weight in male (Sprague DawLey, SD) rat of 250-300g, SPF grades, random to be grouped, i.e. sham-operation
Group, model group, 3,4- 4-dihydroxy benzaldehydes high dose group (or for 8# high groups, 8#g groups, 20mg/kg), 3,4- dihydroxy benzenes first
Aldehyde low dose group (or be low group of 8#, 8#d groups, 10mg/kg), every rat is by 1mL/100g volume gastric infusions, sham-operation
Group and model group gavage give isometric distilled water, and gavage gives given tested material (1mL/100g) to administration group respectively, and daily one
Secondary, successive administration 5 days, on the operation same day, 0.5h is administered before modeling.Every group of animal surgery ischemic 2h, Reperfu- sion for 24 hours, carry out each
The detection of index.
The duplication of 2.2 rat MCAO/R models
2.2.1 the making of paraffin line bolt
The import fishing nylon wire of a diameter of 0.26mm is taken, is about 5cm, respectively with black at its both ends 18mm, 10mm
Color permanent pen blacking.Fusing point is taken as 56 DEG C of one piece of solid paraffins, heating fusing vertically exists one section of bolt line one end 5mm long
It immerses and lifts rapidly in the paraffin of fusing, cool down in air, the paraffin solidified immediately can firmly be attached on nylon wire one
The surface at end, the other end of nylon wire make identical processing, and 1 is placed on 75% alcohol wipe：The heparin physiology salt of 2500U
It is spare in water.
2.2.2 the duplication of rat MCAO/R models
It with reference to the method for Longa, Kugal etc. and domestic report, and is improved, rat MCAO/R is replicated using line brush
Model.Clone method is as follows：
By weight 250-300g male (Sprague DawLey, SD) rat, SPF grades, with 10% chloraldurate
(0.3mL/100g weight) intraperitoneal injection of anesthesia, neck shaving simultaneously carry out routine disinfection posterior neck and hit exactly at 0.5cm to the right notch about
1.5cm, with glass minute hand separation rat right carotid (CCA) and vagus nerve to crotch, without detaching external carotid artery
(ECA) and internal carotid (ICA).CCA proximal parts are ligatured, CCA distal ends are closed with 1 artery clamp folder, beaten on CCA with suture
One slip-knot, eye scissors cut a " V " shape notch at away from bifurcated 1cm, and bolt line is inserted into through notch.Artery clamp on CCA is unclamped, gently
The ligature on CCA proximal parts is pulled to the right, and adjusting inlet wire angle to the left makes bolt line be successfully entered ICA, adjusts to the right again
About 150 jiaos of inlet wire angle, and CCA is gently pulled, bolt line is made to enter brain, is difficult to what is entered if being occurred as soon as into bolt 12mm or so
Situation is prompted possibly into wing jaw artery (PPA), Outlet bolt a distance of drawing back at this time, again to upper right side adjust into
Line angle degree；If being repeated as many times cannot still be correctly inserted into, illustrating to have resulted in the spasm of ICA, blood resurgent can eliminate its spasm,
Outlet bolt can be first moved back at this time, treats restoration of blood flow 3-5min, then carries out the insertion of line bolt just to have very high success rate.Line bolt insertion depth
For 18-20mm (ECA with ICA crotches be starting point), when micro- power of being hampered, stops (generally observing Rat Right side face pumping at this time
Jerk), nylon wire head end is made to pass through MCA section starts, reaches thinner arteria cerebri anterior, completes the resistance of side arteria cerebri media at this time
It fills in (MCAO), the time at this time is recorded, convenient for periodically implementing Reperfu- sion.Then skin suture, notch stay the line bolt of 1cm outside,
Slowly gently draw nylon wire that its head end is made to return in arteria carotis communis during 2h Reperfu- sions after MCAO, you can to realize that arteria cerebri media fills again
Note.After pulling out loop line bolt, blood supply is restored in arteria cerebri media blood supply area, and arteria cerebri media can obtain arteria communicans anterior, posterior communicating artery
Blood supply is conducive to cause Reperfu- sion.Room temperature is kept during post-ischemic reperfusion at 25 DEG C -30 DEG C.After rat regains consciousness, it is observed
Neurological deficit symptom, Reperfu- sion for 24 hours when carry out neurological scores, the inspection that animal carries out each index is put to death after scoring
It surveys.
2.3 neurological scores
Each group animal in cerebral ischemia 2h Reperfu- sions for 24 hours when, score with reference to Longa and 5 point-scores improved：
0 point：Impassivity functional impairment symptom；
1 point：Slight focal neurologic impairment carries tail vacantly not tensible left side fore paw；
2 points：The focal neurologic impairment of moderate, walks turn-take to the left at once；
3 points：The focal neurologic impairment of moderate, i.e. difficulty in walking, and toppling over to the left；
4 points：It spontaneous cannot walk, level of consciousness declines.
The measure of 2.4 brain tissue EB contents
The making of Evans blue (EvanSbLue, EB) standard curve：EB solution has maximum absorption band at 610nm.Match
The EB solution of 1mg/mL processed is maximum concentration, and proportional diluted is configured to the EB of a concentration of 1,1/2,1/4,1/8,1/16 (mg/mL)
Standard solution is returned to zero using formamide solution as blank control group, the OD values of above-mentioned EB standard solution is detected in microplate reader, into
Row regression analysis acquires EB standard curves.
The measure of brain tissue EB contents：Each group randomly selects 6 rats, after cerebral ischemia 15min, through vena femoralis injection
2%EB solution (is given) with 4mL/kg weight, cerebral ischemia 2h Reperfu- sions for 24 hours after, 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 into aorta from left ventricle, to actively
The heparin-saline solution of 400mL is slowly injected into arteries and veins, until atrium dextrum trickle becomes limpid.Broken end takes brain, removes olfactory bulb
And pons, brain is divided into two hemisphere in left and right, takes out brain hygrometric weight immediately, then puts 105 DEG C, constant temperature blast drying oven drying
After for 24 hours, then rapid survey dry weight.Dry brain is put into formamide solution (1mL/ hemisphere), 50 DEG C are incubated for 24 hours.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 (being represented with ug/g drying brain tissues).
Influence of the 2.5 3,4- 4-dihydroxy benzaldehydes to rat ischemia side brain tissue NO contents, NOS activity
Each group randomly selects 6 rats, in cerebral ischemia 2h Reperfu- sions for 24 hours after each group rat 10% chloraldurate
Thoracic cavity is opened after (0.3mL/100g) anesthesia, an osculum is cut in right auricle of heart portion, conduit is inserted into aorta from left ventricle, to actively
The heparin-saline solution of 400mL is slowly injected into arteries and veins, until atrium dextrum trickle become it is limpid, whole operation under ice bath into
Row.Broken end takes brain, removes olfactory bulb and pons, takes ischemic side brain tissue, weigh after blotting surface moisture with filter paper, be cut into small pieces and put
It in the centrifuge tube for entering 10mL precoolings, is homogenized under ice bath, per 100mg, tissue adds in 1mL 1X PBS, and homogenate is made, then puts
In -20 DEG C overnight.After 2 processing of multigelation destroy cell membrane, centrifuged 5 minutes in 4 DEG C of 5000g and take supernatant, by supernatant
Packing is stored in -80 DEG C, and the sample after defrosting should centrifuge 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 contain by NO in NO assay kits requirement detection brain tissue homogenate liquid
Amount；The activity of iNOS, nNOS in brain tissue homogenate are detected by ELISA kit requirement.
2.6 measure the expression of GAP-associated protein GAP
2.7.1 protein extraction
Rat continuously gives corresponding test medicine 5 days, after the last administration after 30min, 10% chloraldurate intraperitoneal injection
SD rats are anaesthetized, quick broken end takes brain, divides take ischemic side cerebral cortex on ice, weigh and be placed on the centrifuge tube that 10mL is pre-chilled in advance
In, it is placed in and is ground into slurry with tissue grinder stick on ice.It is added according to IP and cell pyrolysis liquid operation instructions suitable
Cell pyrolysis liquid (per 1mg tissue add in 8uL lysate), softly blown and beaten with micropipettor it is several under be allowed to mixing, 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.
2.7.2 the concentration of BCA methods detection 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, add 1 volume BCA reagents B (50 by 50 volume BCA reagent As:1) appropriate BCA work is prepared
Liquid, abundant mixing.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,
The standard solution of 0.3125mg/mL, 0.156mg/mL, 0.078mg/mL, 0mg/mL are added to 96 orifice plates by 20uL/ holes
Standard sample wells in.
4) sample 10uL is taken 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 in 200 μ L BCA working solutions, 37 DEG C of incubation 30min.
4) using the total protein concentration in microplate reader at machine measure 560nm, 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 denaturalized, packing be stored in-
80 DEG C spare.
2.7.3 electrophoresis, transferring film, colour developing and imaging
1) Casting of gels
The groove cleaned up and the glass plate of un-grooved are aligned, 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 mixing, is added slowly in the gap of two glass plates, added with liquid-transfering gun
To at upper strata about 3cm, upper strata is filled up with tri-distilled water, is placed at room temperature for about 15min, after glue to be separated irrigates, by upper strata
Tri-distilled water falls to do, and adds in the upper strata concentration glue recorded according to the above ratio later, is slowly added in gel slot, plugs comb rapidly
Son.
2) electrophoresis
The offset plate filled is fixed in electrophoresis tank, appropriate 1 × electrophoretic buffer is added in, gently extracts comb.To channel
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 impregnated in methyl alcohol, by pvdf membrane
And filter paper is put into 10min in transferring film buffer solution, prepares transferring film sandwich according to the sequence of filter paper, glue, pvdf membrane, filter paper, adjusts
Appropriate electric current adjusts the appropriate transferring film time according to destination protein molecular size range.
4) it closes, 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 cleans 2-3 times, and pvdf membrane is transferred in pre-arranged confining liquid, closes 1h, adds primary antibody.Next day is put in shaking table use
1 × TBST is washed 3 times, each 5min, is then added in the secondary antibody of HRP labels, is washed 3 times, each 5min with 1 × TBST, is prepared
ECL chemistry reflective liquids, are added on pvdf membrane, are imaged with UVP gel imaging systems.
2.7.4 the influence that 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue AQP-4
AQP-4, beta-actin are measured 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) incubation at room temperature 2h, two
Anti- (1:2000) 1h, colour developing are incubated.
2.7.5 3,4- 4-dihydroxy benzaldehydes to rat ischemia side brain tissue anti-apoptotic GAP-associated protein GAP (Caspase-3,
Caspase-9) the influence of expression
Caspase-3, Caspase-9, beta-actin are measured with Western-Blot.The albumen applied sample amount of each sample
For 122 μ g, with Caspase-3 (1:200)、Caspase-9(1:100) it, stays overnight for 4 DEG C, secondary antibody (1:2000) 1h, colour developing are incubated；
The internal reference of Caspase-3, Caspase-9 beta-actin (1:1500) 2h, secondary antibody (1 are incubated at room temperature:2000) 1h is incubated, is shown
Color.
2.7.6 the influence that 3,4- 4-dihydroxy benzaldehydes express brain tissue star spongiocyte characteristic protein (GFAP)
GFAP, beta-actin are measured with Western-Blot.The albumen applied sample amount of each sample is 61 μ g, uses GFAP
(1:800) 2h, secondary antibody (1 are incubated at room temperature:2000) 1h, colour developing are incubated.With GAPDH (1:1500) 2h, secondary antibody (1 are incubated at room temperature:
2000) 1h, colour developing are incubated.
3. data processing
Experimental data carries out statistical analysis with GraphPad prism softwares, as a result with mean ± standard deviation
It represents, using one-way analysis of variance (one-way ANOVE), Dunnett analyses is first carried out, as compared between other groups
Compared with Tukey analyses are being carried out, with P<0.05 has statistical significance for difference.Image procossing, profit are carried out using photshopCS5
Gray value of image processing is carried out with ImageJ.
4. experimental result
Protective effect of 4.1 3, the 4- 4-dihydroxy benzaldehyde to blood-brain barrier
4.1.1 influence of 3, the 4- 4-dihydroxy benzaldehydes to rat cerebral tissue's EB contents
The experimental results showed that comparing difference is not statistically significant (P between each group rat normal side brain tissue EB contents>
0.05)；Compared with sham-operation, model group rats ischemic side brain tissue EB contents are increased significantly, the statistically significant (p of difference<
0.001)；Compared with model group, 3,4- 4-dihydroxy benzaldehyde high dose groups (20mg/kg) can be substantially reduced rat model ischemic side
EB contents (the p of brain tissue<0.01).Refer to table 1, Fig. 5, Fig. 6：
Table 13, influence of the 4- 4-dihydroxy benzaldehydes to rat cerebral tissue's EB contents
Note：With sham-operation group ratio：▲▲▲P<0.001；With model group ratio：★★P<0.01
4.1.2 influence of 3, the 4- 4-dihydroxy benzaldehydes to ischemic side Blood-Brain Barrier correlative protein expression
4.1.2.1 the influence that 3,4- 4-dihydroxy benzaldehydes express ischemic side brain tissue AQP-4
The results show that compared with sham-operation group, the expression of model group rats ischemic side brain tissue AQP-4 is significantly raised, poor
Different statistically significant (P<0.05)；Compared with model group, 3,4- 4-dihydroxy benzaldehyde high dose (20mg/kg) groups can significantly under
The high expression of mode transfer type rat ischemia side brain tissue AQP-4, the statistically significant (P of difference<0.05).Refer to table 2, Fig. 7.
Table 23, the influence that 4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue AQP-4
Note：With sham-operation group ratio：▲▲▲P<0.001；With model group ratio：★P<0.05
4.1.2.2 the influence that 3,4- 4-dihydroxy benzaldehydes express ischemic side brain tissue TJ
The results show that compared with sham-operation group, brain tissue Occludin expression in model group rats ischemic side has apparent drop
It is low, the statistically significant (P of difference<0.001)；Compared with model group, 3,4- 4-dihydroxy benzaldehydes height (20mg/kg), low
The expression of the significantly raised rat model ischemic side brain tissue Occludin of (10mg/kg) dosage group energy, difference have statistics meaning
Justice (P<0.05).Refer to table 3, Fig. 8.
Compared with sham-operation group, brain tissue Claudin-5 expression in model group rats ischemic side has apparent reduction, and difference has
Statistical significance (P<0.01)；Compared with model group, 3,4- 4-dihydroxy benzaldehydes height (20mg/kg) dosage group can significantly raised mould
The expression of type rat ischemia side brain tissue Claudin-5, the statistically significant (P of difference<0.05).Refer to table 3, Fig. 9.
Table 33, influence of the 4- 4-dihydroxy benzaldehydes to rat ischemia side brain tissue TJ protein expressions
Note：With sham-operation group ratio：▲▲▲P<0.001,▲▲P<0.01；With model group ratio：★P<0.05
4.1.2.3 the influence that 3,4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue MMP-3
The results show that compared with sham-operation group, the expression of model group rats ischemic side brain tissue MMP-3 is increased significantly,
Statistically significant (the P of difference<0.05)；Compared with model group, 3,4- 4-dihydroxy benzaldehydes height (20mg/kg), low (10mg/kg)
Dosage group can significantly lower the expression of rat model ischemic side cortex MMP-3, the statistically significant (P of difference<0.05).In detail
It is shown in Table 4, Figure 10.
Table 43, the influence that 4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue MMP-3
Note：With sham-operation group ratio：▲P<0.05；With model group ratio：★P<0.05
4.1.2.4 the influence that 3,4- 4-dihydroxy benzaldehydes express ischemic side brain tissue GFAP
The results show that compared with sham-operation group, the expression of model group rats ischemic side brain tissue GFAP significantly lacks, difference
Statistically significant (P<0.01)；Compared with model group, 3,4- 4-dihydroxy benzaldehyde low dose groups (10mg/kg) can be lowered significantly
The missing of rat model ischemic side brain tissue GFAP, the statistically significant (P of difference<0.05).Refer to table 5, Figure 11.
Table 53, the influence that 4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue GFAP
Note：With sham-operation group ratio：▲▲P<0.01；With model group ratio：★P<0.05
The neuroprotection of 4.2 3,4- 4-dihydroxy benzaldehyde
4.2.1 the influence that 3,4- 4-dihydroxy benzaldehydes score to rat neuropathy
The results show that compared with sham-operation group, model group rats have apparent neurological deficit symptom, and neurology is commented
Divide the statistically significant (P of difference<0.001)；3,4- 4-dihydroxy benzaldehydes high dose group (20mg/kg) can be bright compared with model group
It is aobvious to improve MCAO/R rat model neurological deficit symptoms, the statistically significant (P of difference<0.01).Refer to table 6, Figure 12.
Table 63, influence of the 4- 4-dihydroxy benzaldehydes to rat neuropathy
Note：With sham-operation group ratio：▲▲▲P<0.001；With model group ratio：★★P<0.01
4.2.2 influence of 3, the 4- 4-dihydroxy benzaldehydes to rat ischemia side brain tissue Caspase-3/-9
The results show that compared with sham-operation group, the table of model group rats ischemic side brain tissue Caspase-3, Caspase-9
Up to apparent increase, the statistically significant (P of difference<0.05 and P<0.01)；Compared with model group, 3,4- 4-dihydroxy benzaldehydes are low
(10mg/kg) dosage group can be substantially reduced the expression of rat model ischemic side brain tissue Caspase-3/-9, and difference has statistics
Meaning (P<0.01).Refer to table 7, Figure 13, Figure 14.
Table 73, the influence that 4- 4-dihydroxy benzaldehydes express rat ischemia side brain tissue Caspase
Note：With sham-operation group ratio：▲▲P<0.01,▲P<0.05；With model group ratio：★★P<0.01
The effect that the anti-NO toxicity of 4.3 3,4- 4-dihydroxy benzaldehyde occurs
4.3.1 influence of 3, the 4- 4-dihydroxy benzaldehydes to ischemic side brain tissue NO contents and NOS activity
The results show that compared with sham-operation group, model group rats ischemic side brain tissue NO contents, the activity of nNOS, iNOS
It is increased significantly, the statistically significant (P of difference<0.001 and P<0.05)；Compared with model group, 3,4- 4-dihydroxy benzaldehydes
High (20mg/kg), low (10mg/kg) dosage group can be substantially reduced rat model ischemic side brain tissue NO (P<0.05 and P<
0.01) content, nNOS (P<0.01)、iNOS(P<0.05) activity, difference are statistically significant.Refer to table 8, Figure 15.
Table 83, influence of the 4- 4-dihydroxy benzaldehydes to NO contents and NOS activity
Note：With sham-operation group ratio：▲▲▲P<0.001,▲P<0.05；With model group ratio：★★P<0.01,★P<0.05
5th, conclusion
Protective effect of 5.1 3, the 4- 4-dihydroxy benzaldehyde to blood-brain barrier
EB osmosis is commonly used in the evaluation of blood-brain barrier (Blood-Brain Barrier, BBB) degree of opening, and EB is a kind of
Azo group fluorescent dye, almost all is combined with plasma albumin after entering blood, cannot enter brain group through BBB under normal circumstances
It knits, only when BBB is destroyed, and permeability increases, this compound just can pass through BBB, and the raised degree of BBB permeabilities is got over
Greatly, then the amount that EB is penetrated is more, and the dye indigo plant degree of brain tissue is heavier；It is organic molten that formamide etc. is dissolved in into the EB in brain tissue
Agent can precisely measure out the EB contents in extracting solution by spectrophotometry, and then the evaluation BBB's that can be quantified is penetrating
Property.Current study show that BBB is caused to damage, the increased mechanism of permeability mainly has：Close connection between vascular endothelial cell is opened
It puts, major structural protein (Occludin, Claudin-5) degradation；Aquaporin 4 (AQP-4) over-expresses；Matrix metal egg
Albumin enzyme (MMPs) expression raising；Other factors：Star spongiocyte characteristic protein (GFAP) degradation etc..
The results show 3 of the present invention, 4- 4-dihydroxy benzaldehydes can reduce MCAO/R rat model ischemics side brain tissue EB
Content reduces the permeability of blood-brain barrier；The high expression of rat model ischemic side brain tissue AQP-4 can be significantly lowered, reduces TJ
The missing of GAP-associated protein GAP (Occludin, Claudin-5) lowers the high expression of MMP-3, reduces the missing of GFAP, show 3,4-
4-dihydroxy benzaldehyde is by the protective effect to blood-brain barrier, so as to have the function that anti-ischemia-reperfusion injury.
The neuroprotection of 5.2 3,4- 4-dihydroxy benzaldehyde
In cerebral ischemia/reperfusion injury generating process, the purpose of neuroprotection is to intervene central ischemic zone peripheral nerve
The pathological biochemistry cascade reaction of the cell still cerebral ischemic penumbra (ischemic penumbra) in " half function " state, prevents
Or delay cell death, it is avoided to develop into irreversible infarct.Mainly there is nerve cell apoptosis after cerebral ischemia/reperfusion injury
Two access mediations, one is intrinsic pathway, i.e. mitochondrial apoptosis access；Another is extrinsic pathway, i.e. death receptor
Access.Either which approach, they are performed by the downstream effector (Caspase families) of active cell apoptosis
Apoptosis, Caspase families are a kind of apoptosis regulating genes, are the initiator and executor of Apoptosis, in apoptotic process
In play a crucial role.Under normal circumstances, the Caspase in living cells exists with inactive zymogen forms, is lured when by apoptosis
It is activated after leading signal stimulus, causes Caspase cascade reactions, lead to Apoptosis.Caspase-9 is that mitochondria/CytC is situated between
Key enzyme on guided cell apoptosis pathway, Caspase-3 are most important protease in apoptotic process, are that Caspase cascades are anti-
The final executor answered, its activation are the marks that apoptosis enters the irreversible stage.
The results show 3 of the present invention, 4- 4-dihydroxy benzaldehydes can improve the missing of MCAO/R rat model nervous functions
Symptom lowers the high expression of Caspase-3 and Caspase-9, shows that 3,4- 4-dihydroxy benzaldehydes by anti-apoptotic, there is god
Through protective effect, so as to have the function that anti-ischemia-reperfusion injury.
The effect that the anti-NO toxicity of 5.3 3,4- 4-dihydroxy benzaldehyde occurs
NO is messenger molecule and effector molecule important in organism, mainly thin by vascular endothelial cell, vascular smooth muscle
Born of the same parents, nerve cell etc. are synthesized and are discharged, and have the function of neurotransmitter and quenched, wide participation body physiological and pathological activity；
During cerebral ischemic injury, NO may play the double action of protection or damage, crucial limits of the NOS as mediation NO synthesis
Fast enzyme, can be divided into neuron pattern (nNOS), induce type (iNOS) and endothelium in type (eNOS), and the NOS in endothelial cell is mainly
ENOS by the expansible cerebrovasculars of NO that it is generated, increases cerebral blood flow (CBF) so as to play a protective role, however, the expansion blood vessel of NO
Effect time is very of short duration, is not had neuroprotection then more than 2h；And NOS is mainly nNOS in nerve cell, is produced by it
Raw NO can then generate neurotoxicity；By environmental stimuli induction generate iNOS, be distributed mainly on astroglia and
Microglia, vascular smooth muscle cells and vascular endothelial cell, extend with stimulus duration, and NO yields increase, production
Raw NO can aggravate ischemic brain damage.
The results show 3 of the present invention, 4- 4-dihydroxy benzaldehydes can be substantially reduced MCAO/R rat ischemias side brain tissue
NO contents and the activity for inhibiting iNOS and nNOS, show that 3,4- 4-dihydroxy benzaldehydes can be by inhibiting containing for brain tissue NO
Amount and regulation and control NOS accesses, the toxicity for resisting NO occurs, so as to have the function that anti-ischemia-reperfusion injury.
Claims (5)
1. 3,4- 4-dihydroxy benzaldehydes are used to prepare treatment or/and prevent the purposes of cerebral ischemia re-pouring injured drug；It is described
Drug be treatment or/and prevent Blood Brain Barrier (BBB) permeability drug.
2. purposes as described in claim 1, it is characterized in that：The drug is to lower brain tissue Caspase-3, Caspase-
9th, in AQP-4 or MMP-3 one or more of protein expressions drug；The drug be raising brain tissue Occludin,
The drug of one or more of protein expressions in Claudin-5 or GFAP；The drug is the medicine for reducing NO contents in brain tissue
Object；The drug is to inhibit neuronal nitric oxide synthase（nNOS）, nitric oxide synthase type（iNOS）It is middle a kind of or
The drug of two kinds of enzymatic activitys.
3. purposes as claimed in claim 1 or 2, it is characterized in that：The drug is by a effective amount of 3,4- dihydroxy benzenes first
Aldehyde is active constituent, adds in the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared.
4. purposes as claimed in claim 3, it is characterized in that：The preparation is oral preparation.
5. purposes as claimed in claim 4, it is characterized in that：The preparation is capsule, granule or tablet.
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