CN106377520B - Neuroprotective agent - Google Patents
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Abstract
The present invention relates to a kind of neuroprotective agents.The neuroprotective agent includes the compound for the formula that has the following structure:It is a kind of very effective neuroprotective agent that the compound of structure above, which can reduce cell Ca2+ influx to play the excitatory toxicity for protecting cortical neuron to cause from glutamic acid by the reversible inhibition to glutamate receptor,.
Description
Technical field
The present invention relates to drug field, in particular to a kind of neuroprotective agent.
Background technique
Cerebral apoplexy has become one of the three big diseases for seriously endangering human health.However currently, clinical treatment cerebral apoplexy
Drug other than the extremely limited logical bolt medicine that clinical time window uses, have no other effective nerve cell-protective agents,
Also without having the therapeutic agent for promoting neurological functional recovery.Although being induced cerebral apoplexy endogenous by the effort of decades
Nerve protects the understanding of molecule and mechanism still to limit to very much (Molecular mechanisms of cerebral
ischemia-inducedneuronal death.Hou ST,MacManus JP(2002),Int Rev Cytol 221:93-
148.；The science of stroke:mechanisms in searchof treatments.Moskowitz MA,Lo
EH,Iadecola C(2010).Neuron67:181-198)。
Phaseic acid (Phaseic Acid, PA) is point of abscisic acid (Abscisic Acid, ABA) terpenoid
Metabolite is solved, ABA generates phaseic acid ((+)-Abscisic acid under the catalytic action of cytochrome P-450 enzyme
8'-hydroxylase is a cytochrome P450monooxygenase.Krochko JE,Abrams GD,Loewen
MK,Abrams SR,Cutler AJ(1998).Plant Physiol118:849-860.).ABA higher plant growth and
Important physiological function is played in developmental regulation, including improves resistance (drought resisting, cold-resistant, salt resistance etc.), inhibit growth and is sprouted, and is promoted
Into falling off and suspend mode, accelerates aging and stomata is promoted to close etc..The contents level of plant endo ABA, by biosynthesis and generation
The variation control balanced between thanking.Calcium ion releases in the generation regulating cell that ABA passes through induction second messenger cADP-Ribose
It puts and (the Guard cellABA and CO that plays a role in plant2signaling network updates and Ca2+
sensor priming hypothesis.sraelsson M,Siegel RS,Young J,Hashimoto M,Iba K,
Schroeder JI(2006).CurrOpin Plant Biol 9:654-663.).(-)-PA and ABA effect is similar, and
Inhibit photosynthesis and promotes plant hormone (the ABA biosynthesis and degradation to fall off
contributing to ABAhomeostasis during barley seed development under control
and terminal drought-stressconditions.Seiler C,Harshavardhan VT,Rajesh K,
Reddy PS,Strickert M,Rolletschek H,Scholz U,Wobus U,Sreenivasulu N(2011).J
Exp Bot 62:2615-2632.)。
Summary of the invention
Based on this, it is necessary to a kind of neuroprotective agent is provided, so that phaseic acid (-)-PA is applied in neuroprotective agent
In.
A kind of neuroprotective agent, the compound including having the following structure formula:
It in one of the embodiments, further include pharmaceutically acceptable carrier.
The protein molecular is selected from bovine serum albumin molecule, ovalbumin molecule, keyhole blood in one of the embodiments,
One of azurin molecule, Fibrin molecules and human albumin's molecule.
The polypeptide is in poly-D-lysine, polyglutamic acid and two palmitin amic acids in one of the embodiments,
One kind.
It in one of the embodiments, further include pharmaceutically acceptable solvent.
The solvent is physiological saline or glucose solution in one of the embodiments,.
The neuroprotective agent is oral administration, drug administration by injection, respiratory tract administration, skin in one of the embodiments,
Administration, mucosa delivery or cavity/canal drug administration.
(-)-PA high level endogenous of structure above is present in ischemic brain, is different from other ABA derivatives, and
It is present in the area Ban Yin of ischemic perilesional to (-)-PA high level, by reversible inhibition glutamate receptor, reduces thin
Born of the same parents' Ca2+ influx can protect cortical neural cell from glutamate toxicity, so that (-)-PA of structure above becomes a kind of effective
Neuroprotective agent.
Detailed description of the invention
Fig. 1 a is the chromatogram of (-)-PA and (+)-PA of the PA synthetic standards product of embodiment；
Fig. 1 b is (-)-PA chromatogram in the brain test specimen of the mouse of embodiment；
Fig. 1 c is the chromatogram of (-)-PA in the blood test sample of the mouse of embodiment；
Fig. 2 is the brain test specimen of the mouse of embodiment and (-)-PA and its associated metabolites of blood test sample
Content histogram, wherein * in figure indicates P < 0.05, that is, has conspicuousness；* indicates P < 0.01, i.e. highly significant,
And it indicates to indicate above-mentioned meaning with the * in the following figure；
Fig. 3 is the histogram of the content of (-)-PA and its associated metabolites in the feed of embodiment；
Fig. 4 is 10 μM of the cortical neuron for the NMDA for being only added 100 μM, the NMDA of 100 μM of addition and addition of embodiment
(-)-PA cortical neuron, be added 100 μM NMDA and be added 100 μM (-)-PA cortical neuron, be added 1000 μ
The cortical neuron of (-)-PA of the NMDA and 10 μM of addition of M and only the mouse cortex neuron of addition 45mM potassium chloride
The ratio fluorescent figure of flow of calcium ions；
Fig. 5 is the current potential of the cortical neuron of NMDA and 100 μM (-)-PA for being added with 50 μM of embodiment at any time
Variation diagram；
Fig. 6 is the current potential of the cortical neuron of NMDA and 1000 μM (-)-PA for being added with 50 μM of embodiment at any time
Variation diagram；
The concentration and electric potential relation curve graph that Fig. 7 is (-)-PA in the cortical neuron of embodiment；
Fig. 8 is cortical neuron, the various concentration of the untreated cortical neuron of embodiment, 100 μM of NMDA processing
(-)-PA processing cortical neuron and DMSO processing cortical neuron survival rate histogram；
Fig. 9 be embodiment untreated cortical neuron, ALLN processing cortical neuron, 10 μM of (-)-PA and
100 μM NMDA processing cortical neuron, (-)-PA and 100 of 100 μM μM NMDA processing cortical neuron, with 1000
μM (-)-PA and 100 μM of NMDA processing cortical neuron survival rate histogram；
Figure 10 is experiment mice and control group mice 2 hours, 6 hours and 24 hours big after MCAO operation of embodiment
The histogram of (-)-PA content of cerebral ischemia side and opposite side sample；
Figure 11 is the photo of the mouse brain sample of embodiment；
Figure 12 is the histogram of the content of trizonal (-)-PA of Figure 11；
Figure 13 is perfusion (-)-PA of embodiment and mouse the depositing after 0.5 hour, 6 hours and 24 hours of physiological saline
Motility rate figure；
Figure 14 is perfusion (-)-PA of embodiment and the mouse of physiological saline carried out at 0.5 hour, 6 hours and 24 hours
Mice behavior scoring figure；
Figure 15 is perfusion (-)-PA of embodiment and the infarct size figure of the brain of the mouse of physiological saline；
Figure 16 be embodiment perfusion (-)-PA antibody and physiological saline mouse 0.5 hour, 6 hours and 24 hours into
Capable mice behavior appraisal result figure；
Figure 17 is perfusion (-)-PA antibody of embodiment and the mouse of physiological saline at 0.5 hour, 6 hours and 24 hours
The grasping reflex grip appraisal result figure of forefoot；
Figure 18 is the infarct size figure of the brain of perfusion (-)-PA antibody of embodiment and the mouse of physiological saline.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give preferred embodiment of the invention.But the invention can be realized in many different forms, however it is not limited to herein
Described embodiment.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more saturating
It is thorough comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term " and or " used herein includes one or more phases
Any and all combinations of the listed item of pass.
The neuroprotective agent of one embodiment, the compound including having the following structure formula:
Wherein, neuroprotective agent further includes pharmaceutically acceptable carrier.
Wherein, protein molecular is selected from bovine serum albumin molecule, ovalbumin molecule, keyhole limpet hemocyanin molecule, fiber egg
One of white molecule and human albumin's molecule.
Wherein, polypeptide is selected from one of poly-D-lysine, polyglutamic acid and two palmitin amic acids.
Wherein, neuroprotective agent further includes pharmaceutically acceptable solvent.
Wherein, solvent is physiological saline or glucose solution.
(-)-PA high level endogenous of structure above is present in ischemic brain, is different from other ABA derivatives, and
It is present in the area Ban Yin of ischemic perilesional to (-)-PA high level, by reversible inhibition glutamate receptor, reduces thin
Born of the same parents' Ca2+ influx can protect cortical neural cell from glutamate toxicity, so that (-)-PA of structure above becomes a kind of effective
Neuroprotective agent.
The following are specific embodiment parts:
Embodiment
One, detection (-)-PA endogenic presence of high level in mouse brain
(1) zoopery and feed:
Experimental animal: C57B/6 mouse 10, every 20~37g of weight, is male.
Mouse feed: the feed (LabDiet, Land O'Lakes Inc, Ontario, Canada) of market purchase.
(2) animal sample is collected and is pre-processed:
The collection of the cerebral tissue and blood of mouse: using isoflurane by mouse anesthesia, from the face of mouse after mouse anesthesia
Portion's venous collection blood, obtains the blood sample of mouse；Mouse is put to death, brain tissue is collected and obtains the brain samples of mouse, it will
Brain samples and blood sample are rapidly frozen in liquid nitrogen, and are saved at -80 DEG C.
The cerebral tissue sample and blood sample of mouse pre-process: the cerebral tissue sample and blood sample of mouse are distinguished
(Mini-BeadBeater-96, Biospec Products company, the city Bartels Wei Er, Russia are homogenized using multitube ball mill
Carat Homer state, the U.S.), it weighs every part of sample about 50mg and is respectively placed in multiple Falcon pipes.It will be (same comprising Isotopic Internal Standard
The plain internal standard compound in position includes: d3-DPA, d5-ABA-GE, d3-PA, d4-7'-OHABA, d3-neoPA, d4-ABA and d4-trans-
ABA, being synthetically prepared for these substances have been reported that in document Abrams et al., 2013, each 0.2ng/ μ of every kind of material concentration
L is dissolved in the mixed liquor of water and acetonitrile, and the volume ratio of water and acetonitrile adds in the mixed liquor of water and acetonitrile for the aliquot of 1:1)
Enter in every part of sample, and extracted using Extraction solvent (isopropanol: water: glacial acetic acid=80:19:1, v/v/v), extract exists
It is shaken 24 hours at being protected from light with 4 DEG C, is centrifuged, takes supernatant, volatilize solvent.Extract is re-dissolved in the methanol and ice of 100 μ L
In the mixed solvent (wherein, methanol: the volume ratio of glacial acetic acid is 99:1) of acetic acid, then sequentially adds 900 μ L volume basis and contain
Amount is 1% glacial acetic acid and 2mL n-hexane, and isolated water layer volatilizes solvent, obtains test specimen.Test specimen is in 100 μ L first
It is redissolved in the in the mixed solvent (methanol: the volume ratio of glacial acetic acid is 99:1) of pure and mild glacial acetic acid, 900 μ L, 1% ice vinegar is then added
The solution of sour water, then through upper Oasis HLB solid-phase extraction column (Waters, Mississauga city, Canada), eluting solvent is second
The mixed liquor (acetonitrile: water: glacial acetic acid=30:69:1, v/v/v) of nitrile, water and glacial acetic acid, then traditional vacuum uses concentrating instrument
(Labconco company, Kansas city, the U.S.) concentration collects and dry, respectively obtains the brain test specimen and blood of mouse
Liquid test specimen.
(3) UPLC/ESI-MS/MS (ultra performance liquid chromatography tandem mass spectrum method) is analyzed and is quantified
The UPLC/ESI-MS/MS of PA synthetic standards product is analyzed and is quantified: above-mentioned UPLC/MS equipment chiral separation PA is used,
The chiral column used for Regis (R, R) Whelko5/100Kromasil (4.6 × 150mm, 5 μm, REGIS Technologies
Company).Mobile phase A is the chromatographic grade water containing 0.1% glacial acetic acid, and Mobile phase B is the chromatographic grade first containing 0.1% glacial acetic acid
Alcohol.Sample volume is 15 μ L, flow velocity 0.40mL/min, gradient are as follows: 55% Mobile phase B of 0min~1min；1min~
55%~90% Mobile phase B of 18min；90%~100% Mobile phase B of 18min~19min；19min~20min 100% flows
Dynamic phase B；The Mobile phase B of 20min~21min100%~55%；55% Mobile phase B of 21min~25min balances pillar, under waiting
Secondary sample introduction.The chromatogram of (-)-PA and (+)-PA of isolated PA synthetic standards product, as shown in Figure 1a.
The brain test specimen of mouse and the UPLC/ESI-MS/MS of blood test sample are analyzed and are quantified: by above-mentioned (2)
The brain test specimen and blood test sample point of the mouse obtained after cerebral tissue sample and the blood sample pretreatment of mouse
It is not dissolved in the sample-adding recycling isotopic standard product of the methanol containing volumn concentration 40%, 0.5% acetic acid and 0.1ng/ μ L
The aqueous solution (v/v) of (d6-ABA and d2-ABA-GE), then by UPLC/ESI-MS/MS analysis and quantitatively.UPLC/ESI-MS/
When MS is analyzed and is quantitative, using Waters ACQUITY UPLC system, binary solvent transportation system, chromatographic column and sample managing
Device is coupled by Z-spray interface with Waters Micromass Quattro Premier XE quadrupole rods tandem mass spectrometry,
In, analyzing the chromatographic column used is ACQUITYHSS C18 (2.1 × 100mm, 1.8 μm), pre-column ACQUITY
HSS C18VanGuard Pre-column (2.1 × 5mm, 1.8 μm), mobile phase A are to contain volumn concentration 0.025%
The chromatographic grade water of glacial acetic acid, Mobile phase B are the chromatographic grade acetonitrile containing 0.025% glacial acetic acid.Sample volume is 10 μ L, and flow velocity is
0.40mL/min, gradient are as follows: 0min~0.2min2% Mobile phase B, the Mobile phase B of 0.2min~0.4min2%~15%；
15%~50% Mobile phase B of 0.4min~5.0min；The Mobile phase B of 5.0min~5.5min50%~100%；5.5min~
100% Mobile phase B of 6.2min；100%~2% Mobile phase B of 6.2min~6.5min；2% mobile phase of 6.5min~8.0min
B.Using acquiring data, negative ion mode, EFI under MassLynx v4.1 (Waters company) multiple-reaction monitoring pattern (MRM)
Mist ionization, condition are as follows: capillary voltage 1.75kV desolvation gas flow velocity 1100L/ hours, is bored air-flow 150L/ hours, from
Component and desolventizing temperature are respectively 120 DEG C and 350 DEG C.It is carried out using QuanLynx v4.1 software (Waters company) offline
Chromatographic data processing, calibration curve is created using all target compounds, and obtains (-)-in the brain test specimen of mouse
PA chromatogram, as shown in Figure 1 b；The chromatogram of (-)-PA in the blood test sample of mouse, as illustrated in figure 1 c；And pass through
The content of (-)-PA and its associated metabolites that the brain test specimen of mouse and the chromatogram of blood test sample obtain
Histogram, as shown in Figure 2.
The UPLC/ESI-MS/MS of mouse feed is analyzed and is quantified: using the brain test specimen and blood test sample of mouse
Method that is quantitative and analyzing identical UPLC/ESI-MS/MS feed (LabDiet, Land O' that above-mentioned market is commercially available
Lakes Inc, Ontario, Canada) detection, (-)-PA in feed is obtained from the chromatogram of the feed obtained by detection
And its histogram of the content of associated metabolites is as shown in Figure 3.
(4) interpretation of result
There are PA from the brain and blood that can be seen that mouse in Fig. 1 a~Fig. 1 c, existence form is (-)-PA, and
Non- (+)-PA.
From figure 2 it can be seen that with higher content of (-)-PA in the brain of mouse, is (-)-PA in blood
10 times, and the content of ABA illustrates that high level has (-)-PA in mouse brain lower than 30 times of content of (-)-PA in brain.
From figure 3, it can be seen that (-)-PA content is very low in feed, this illustrates that (-)-PA in mouse brain is not to come
Derived from food.Obviously explanation, (-)-PA of high-content is endogenous generation in mouse brain, rather than from food etc. it is exogenous because
Element.
Two, (-)-PA protects cortical neuron to test from glutamate toxicity
(1) cortical neuron culture: the CD-1 mouse being pregnant 15 or 16 days is used into mechanically decoupled cerebral hemisphere, removes brain
Film, then 25min is digested with the trypsin solution of 0.025% (w/v), it is then added in trypsin inhibitor and pancreatin is made
With adding 0.05% (w/v) Dnase (deoxyribonuclease), remove the DNA from dead cell.Pass through in the medium
A series of break process and mild centrifugation step isolate cortical neuron, then by neuron according to article (Collapsin
response mediatorprotein 3deacetylates histone H4to mediate nuclear
condensation and neuronal death.Hou ST,Jiang SX,Aylsworth A,Cooke M,Zhou L
(2013) .Sci Rep 3:1350.) used in cultural method, be added to B-27 replenishers and N2 replenishers
(Invitrogen) cultivated in culture medium 7~14 days (due to neuron in this culture medium full maturity, could be to paddy ammonia
The toxicity of hydrochlorate induction is reacted, and therefore, is needed first that neuron culture is mature).By cell with 1 × 106The density of cells/well
It is inoculated in the slide with poly-D-lysine 24 orifice plates handled in advance, then with 2 × 107Cell/10mL density is inoculated in
In 100mm Tissue Culture Dish.Cell is cultivated in 37 DEG C of cell incubators.
(2) the calcium ion ratio fluorescent measurement method of Fura-2 (the specificity fluorescent indicator of intracellular calcium) is used
(Abscisic acid does notevoke calcium influx in murine primary microglia and
immortalised murine microglial BV-2and N9cells.Jiang SX,Benson CL,Zaharia LI,
Abrams SR, Hou ST (2010) .BiochemBiophys Res Commun401:435-439.) measurement fluorescence ratio
It will be in 5 μM of Fura-2-AM of the addition of in vitro culture 7 days mouse cortex neurons in above-mentioned (1)
(Molecular Probes, Eugene, CA) and 0.02%pluronic's (Molecular Probes, Eugene, USA) is mixed
It closes in object, and in 37 DEG C of incubation 30min, then with no Mg2+PSS buffer (2mM pH be 7.2 HEPES, 140mMNaCl,
5mMKCl、2.3mM CaCl2With 10mM glucose) cleaning, then in no Mg2+PSS buffer in stablize 5min, use
Northern Eclipse Digital Ratio Image System (EMPIX, Mississauga, ON) system and
200 video camera of Axiovert detects the intensity of Fura-2, wherein launch wavelength 510nm, measurement Fura-2 exist
The fluorescence ratio of 340nm and 380nm wavelength.By DG-5system (Sutter Instrument Company, Novato,
CA) system carries out double stimuli screening.[Ca2+] i concentration by Fura-2 the two excitation wavelengths of R340/380 fluorescence intensity
Ratio after background correction indicates.R340/380 is the average of relatives of 20 cells in the same visual field on lid fragmentation
Value.[Ca is first recorded in detection process2+] i basal level 20s, no Mg is dissolved in 100 μM of NMDA2+PSS buffer, add
Enter in cortical neuron, then sequentially add (-)-PA (10 μM, 100 μM and 1000 μM) of various concentration, records [Ca2+] i water
Flat 60~100s；Then with no Mg2+PSS buffer solution for cleaning neuron 300s, be added the PSS buffer containing 45mM KCl into
Row depolarising processing, records [Ca2+] i level variation 60s, the vigor of neuron is indicated with this, all measurements at least repeat 3
Secondary, all data are analyzed using Microsoft Excel, and result is the average value of independent experiment three times, to be only added
The cortical neuron of (-)-PA of 10 μM of the cortical neuron of 100 μM of NMDA, the NMDA for being added 100 μM and addition is added 100
μM NMDA and be added 100 μM (-)-PA cortical neuron, 1000 μM of NMDA is added and 10 μM (-)-PA is added
Cortical neuron and only the ratio fluorescent figure of the flow of calcium ions of the mouse cortex neuron of addition 45mM potassium chloride, such as Fig. 4
It is shown.
(3) full cellular electrophysiologicalsensor record
Using Axopatch 700A patch-clamp amplifier (Axon Instruments, Inverurie,
Scotland) (22~25 DEG C) carry out Whole-cell recording to the cortical neuron that above-mentioned (1) is cultivated at room temperature.Data
By DigiData 1322A, 9.0 software collection of pClamp, sample frequency 10kHz, signal passes through 5kHz and filters.Electrode is used
Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA) drawing and polishing, note
It in liquid is 4~6M Ω that the resistance of electrode, which is recorded, in difference, during voltage-clamp recording, uses resistance capacitance circuit in the amplifier
Eliminate transient voltage.After the formation of full cell pattern, series resistance is usually < 15M Ω, and series resistance compensation is set as 70%
~90%, liquid junction potential is about 2mV, is automatically adjusted by electrode potential biasing adjustment, uses external solution (NaCl
150mM、KCl5mM、CaCl20.2mM, glucose 10mM and HEPES 10mM, NaOH adjust pH to liquid 7.4) and in electrode
(KCl 140mM、MgCl22.5mM, HEPES 10mM, EGTA 11mM and ATP5mM, KOH adjust pH to 7.3) recording NMDA/
AMPA activated current, unless stated otherwise, voltage-clamp recording film potential are clamped down in -70mV.Perfusion system is focused using the channel 8-
(ALA Scientific Instruments, Farmingdale, NY) by pressure by drug (50 μM of NMDA with it is difference dense
(-)-PA of degree) it is added on cortical neuron in external solution, wherein the concentration range of (-)-PA is at 100~1000 μM.Entire note
Record process cortical neuron is immersed in external solution always, completes drug solution replacement by electronic control.Patch clamp data uses
Clampfit 9.0 (Axon Instruments) processing, uses Origin 7.5 (OriginLab, Northampton, MA) point
Analysis, and obtain the potential change figure of the cortical neuron of NMDA and 100 μM (-)-PA added with 50 μM at any time, such as Fig. 5
It is shown, and the potential change figure of the cortical neuron of NMDA and 1000 μM (-)-PA added with 50 μM at any time is obtained,
As shown in Figure 6.And the current potential by record cortical neuron in (-)-PA of each concentration, it obtains in cortical neuron
The concentration and electric potential relation curve graph of (-)-PA, as shown in Figure 7, wherein dose-effect curve Logistic equation calculation: y
=(A1-A2)/(1+(x/x0)p]+A2, y is in response to, A1And A2It is minimum and maximum response, x respectively0Refer to and reaches maximum reaction
Corresponding concentration when half, x are drug concentrations, and p is hill coefficient.
(4) neuronal viability
The cortical neuron of external 37 DEG C of cultures 7 days of above-mentioned (1) is used to 100 μM of NMDA (N- methyl D-asparagus fern respectively
Propylhomoserin), concentration be respectively 10 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1000 μM, 2000 μM of (-)-PA and dimethyl sulfoxide
(DMSO) 15min is handled, and 37 DEG C are further cultured for 24 hours.After treatment process terminates, alma indigo plant is used
(Invitrogen) neuronal viability analysis is carried out to cortical neuron, is 1 by alma indigo plant and the volume ratio of culture medium:
10, it will be added in culture medium according to alma indigo plant, the culture medium for having alma indigo plant then will be added and be added in cortical neuron, so
The diluted alma indigo plant of 1:10 is added in cortical neuron afterwards, is incubated for 1 hour and is dyed in 37 DEG C.The cell of dyeing is used
Inverted microscope (Carl Zeiss, AX10vert 200M) is observed, and is taken pictures, is analyzed using Image J software.Root
The survival rate of cortical neuron is calculated according to stained area, to obtain untreated cortical neuron, 100 μM of NMDA is handled
The survival rate of the cortical neuron of the cortical neuron and DMSO processing of (-)-PA processing of cortical neuron, various concentration
Histogram, as shown in Figure 8.
The cortical neuron of external 37 DEG C of cultures 7 days of above-mentioned (1) is used into calpain inhibitor (ALLN), 10 μM respectively
(-)-PA and 100 μM of NMDA, (-)-PA and 100 of 100 μM μM of NMDA, (-)-PA and 100 of 1000 μM μM of NMDA
15min is handled, and 37 DEG C are further cultured for 24 hours.Neuronal viability point is carried out to cortical neuron using alma indigo plant decoration method
Analysis, the survival rate of cortical neuron is calculated according to stained area, to obtain the skin of untreated cortical neuron, ALLN processing
Cortical neuron, (-)-PA and 100 of 100 μM μM of the NMDA that layer neuron, (-)-PA and 100 of 10 μM μM of NMDA are handled
The cortical neuron of processing, with the column of the survival rate of 1000 μM of (-)-PA and 100 μM of the NMDA cortical neuron handled
Figure, as shown in Figure 9.
(5) interpretation of result:
Figure 4, it is seen that 100 μM of NMDA can induce intracellular Ca2+ ([Ca2+] flowing rapidly into i), work as use
When (-)-PA of 100 μM of the NMDA and concentration range from 10 μM to 1000 μM handles cortical neuron, make [Ca2+] i inflow holding
In reduced levels, and with the [Ca of dose dependent manner improvement cortical neuron2+] i inflow, potassium chloride is added to cortex neural
Member, which carries out depolarising processing, can cause a large amount of [Ca2+] i inflow.It is found from figure, (-)-PA can efficiently reduce NMDA and lure
Lead increased [Ca2+] i inflow.This explanation, (-)-PA is the potential reversible inhibitor of glutamate receptor.
A large amount of inward electric current out can be can induce on cortical neuron from the NMDA that can be seen that 50 μM in Fig. 5~7,
100 μM and 1000 μM of (-)-PA electric currents that can effectively inhibit NMDA to activate are added, after washing off NMDA, film potential can be restored again
To base level, illustrate that (-)-PA inhibits the electric current of NMDA activation to be reversible.And within the scope of 100~1000 μM, (-)-PA
The dose-dependent electric current for inhibiting NMDA to activate, IC50 are 175.4 ± 21.5 μM, and slope factor is 0.96 ± 0.3 (n=
10).It again demonstrates, (-)-PA may be the reversible inhibitor of a glutamate receptor.
As can be seen from Figure 8, the cortical neuron of 100 μM of NMDA processing culture will lead to cell mortality, and
The cortical neuron of (-)-PA processing of any concentration absolutely not influences cell, i.e. cortex mind of (-)-PA to culture itself
There is no toxicity through member, not will lead to the death of cell.
It can be seen in figure 9 that (-)-PA is used to pre-process cortical neuron, neuron can be improved to NMDA toxicity
Death, and (-)-PA concentration used is bigger, and the cell mortality of cortical neuron is lower, that is, is in dose dependent.
To sum up, above-mentioned experiment determines that (-)-PA has very important influence to the glutamate receptor of cortical neuron.And with
Upper experimental result clearly demonstrates that (-)-PA is a reversible glutamate receptor inhibitor, can draw in prevention ischemic brain
Key effect is played in the excitatory toxicity risen.
Three, cerebral ischemia induction (-)-PA is generated in cerebrum ischemia penumbra region, and (-)-PA has the brain of ischemic animal model
There is protective effect
(1) experimental animal: male C57B/6 mouse, every 20~37g.
(2) animal model and sample collection method:
Mouse MCAO (middle cerebral artery occlusion) modelling: by the mouse of MCAO group in isoflurane anesthetic effect in short-term
Under, operation (Collapsin response in left side is done to mouse brain respectively using invertibity MCAO line brush in lumen
mediatorprotein 3deacetylates histone H4to mediate nuclear condensation and
neuronal death.Hou ST,Jiang SX,Aylsworth A,Cooke M,Zhou L(2013).Sci Rep 3:
1350.).Cerebral ischemia 1 hour, after line bolt is taken out, restoration of blood flow was normal, is monitored by laser Doppler flowmetry, and hurt
Mouth suture.Mouse temperature is surveyed using rectal prob in the course of surgery, and maintains body temperature at 37 DEG C by heating cushion and heating lamp,
And the consistency in order to guarantee experimental implementation in the course of surgery, blood pressure, blood gas and pH value are all detected
(Increased Mdm2expression in rat brain after transient middle cerebral artery
occlusion.Tu Y,Hou ST,Huang Z,Robertson GS,MacManus JP(1998).J Cereb
BloodFlow Metab 18:658-669.)。
The collection of mouse brain sample: mouse euthanasia, separating mouse the two cerebral hemispheres brain.Selection is mainly comprising ischemic disease
The frontal lobe part (L1 and bregma 2.96-0.50) of the left hemisphere of stove tissue, obtains the brain samples of mouse, then by brain samples
It is rapidly frozen in liquid nitrogen and is saved at -80 DEG C.
(3) (after MACO operation, blood meeting nature is flow at ischemic blood reperfusion, as blood after MCAO ischemic
Perfusion) influence of the different time to mouse brain two sides (-)-PA content
Experimental animal: (1) mouse 12 described in, wherein control group 3, experimental group 9.
Experimental mice processing method: receiving (2) described identical surgical procedure, wherein 3 at 2 hours after surgery
Extremely, it puts to death within 6 hours after surgery for 3,3 are put to death for 24 hours after surgery.Mouse brain sample is collected using method described in (2).
Control group mice processing method: surgical procedure described in (2) is received, but without carrying out arterial embolism in brain.Art
It puts to death within 24 hours afterwards.Mouse brain sample is collected using method described in (2).
(-)-PA quantitative analysis is carried out to obtained cerebrum ischemia side and opposite side sample using UPLC/ESI-MS/MS, is obtained
Histogram is done to data, as shown in Figure 10.
(4) influence of the MCAO to ischemic core domain, penumbra region and contralateral regions in mouse brain
Experimental animal: (1) mouse 5 described in.
Experiment mice processing method: receiving (2) described identical surgical procedure, puts to death within postoperative 24 hours.Using (2) institute
The method stated collects mouse brain sample.
It is big to mouse respectively using Laser Microdissection Leica LMD6system (coming card microscopic system)
Brain sample carries out laser microprobe dating, and brain samples are divided into three regions, ischemic core (region 1), penumbra region (area
Domain 2) and opposite side (region 3) each region is cut as shown in figure 11, use 10 infrared rays and 2 ultraviolet pulses, wherein
All cells of same area are collected in the same LCM collecting pipe.Cell after cutting cracks immediately, and uses UPLC/
ESI-MS/MS carries out trizonal (-)-PA assay analysis, and the content data for obtaining trizonal (-)-PA does column
Shape figure, as shown in figure 12.
(5) (-)-PA injection experiments assessment mouse survival rate, neurological deficit assessment and infarction of brain area measurement
Experimental animal: (1) mouse 19 described in, wherein experimental group 11, control group 8.
Experimental group and control group mice processing method: receiving (2) described identical surgical procedure, after postoperative 24 hours,
According to content (the Directintraventricular delivery of drugs to the rodent of document
Centralnervous system.DeVos SL, Miller TM (2013) .J Vis Expe50326..), use injection speed
Degree be 1.0 μ L/hr micro pump and greater than 3 days discharge capacities catch basin (Alzet 1003D) (DURECT Corporation,
ALZET Osmotic Pumps, Cupertino, CA), (-)-PA (50mg/mL) injection, control group are carried out to experimental mice
Mouse carries out the injection of 100% physiological saline (placebo).First micro pump is immersed in 37 in sterile saline solution in advance
DEG C overnight, so as to after the implants rapidly will (-)-PA injection.0.5 hour, 6 hours and 24 hours progress mouse is deposited after injection
Motility rate statistics, the mapping of survival rate statistical result, as shown in figure 13.
The mouse progress neurological deficit of experimental group and control group was commented in 0.5 hour, 6 hours and 24 hours after injection
It assesses point, neurological deficit scoring is scored using six points of systems, evaluation criteria are as follows: 0, normally；1, mild steering behavior or do not have
Inconsistent curling is carried when tail, and 50% trial is crimped onto opposite side；2, slight consistent curling, 50% trial curling
To opposite side；3, it is powerful with it is direct it is consistent crimp, mouse holds that roll up posture be more than 1~2 second, and mouse nose almost encounters tail
Bar；4, severe curl causes roller, loses walking or righting reflex；5, stupor or on the point of dying.Every group of at least 8 mouse carry out
The average value of evaluation and scoring is for statisticalling analyze.Wherein, neurological deficit assessment appraisal result is shown in Figure 14.
Injection put to death mouse after 24 hours, carried out infarct size measurement to its brain.Measurement method is as follows: using 2,3,5-
Decoration method (the Chlortetracycline and demeclocycline inhibit of triphenyltetrazolium chloride (TTC)
calpains andprotect mouse neurons against glutamate toxicity and cerebral
ischemia.Jiang SX,Lertvorachon J,Hou ST,Konishi Y,Webster J,Mealing G,
Brunette E, Tauskela J, Preston E (2005) .J BiolChem280:33811-33818.) calculate injection (-)-
The infarct size of the brain of the mouse and saline injection mouse of PA, i.e., be cut into 4 2mm thickness for the brain samples of mouse respectively
Coronal section, dye 90min with the TTC of 5mL 2% at 37 DEG C.Then, with normal saline flushing, and it is subsequently exposed to second
To dissolve first a ceremonial jade-ladle, used in libation product in the mixture (volume ratio of ethyl alcohol and dimethyl sulfoxide is 1:1) of pure and mild dimethyl sulfoxide.It incubates in the dark
After educating 24 hours, the solvent extractable matter dissolved is in three Guan Zhongyong fresh ethanols and Me2SO's (wherein, Me indicates methyl)
Mixed solvent is 1:20 dilution according to volume ratio, and is placed on cuvette.With spectrophotometer test dilution at 485nm
The average value of absorbance value.TTC dyeing is calculated with contralateral brain loss late by following formula in cerebral ischemia side: loss late=
(absorbance of 1- ischemic side brain/opposite side brain absorbance) × 100%.The mouse of control group and experimental group is obtained according to above-mentioned formula
The mapping of infarct size measurement result, as shown in figure 15.
(6) assessment of (-)-PA antibody injection experiments assessment mouse Nerve functional defect, forelimb grip strength testing and infarction of brain
Area measurement
Experimental animal: (1) mouse 10 described in, wherein experimental group 5, control group 5.
Experimental group and control group mice processing method: receiving (2) described identical surgical procedure, after postoperative 24 hours,
According to content (the Directintraventricular delivery of drugs to the rodent of document
Centralnervous system.DeVos SL, Miller TM (2013) .J Vis Expe50326..), use injection speed
Degree be 1.0 μ L/hr micro pump and greater than 3 days discharge capacities catch basin (Alzet 1003D) (DURECT Corporation,
ALZET Osmotic Pumps, Cupertino, CA), (-)-PA antibody (50mg/mL) injection is carried out to the mouse of experimental group,
The mouse of control group carries out the injection of 100% physiological saline (placebo).Micro pump is first immersed in sterile saline in advance
It is stayed overnight for 37 DEG C in solution, after the implants rapidly to inject (-)-PA antibody.0.5 hour after injection, 6 hours and 24 small
When scoring, statistical result mapping, such as Figure 16 institute are assessed according to the neurological deficit in (5) to the mouse of control group and experimental group
Show.
0.5 hour, 6 hours and 24 hours progress mouse forelimb grip strength testing, test method are as follows after injection: forelimb is held
Power test grip tester-Columbus instrument (MyNeurolab, St.Louis, MO) measurement muscle power and related mind
The grasping reflex grip of forefoot through muscle integration.Crest value tension that peak value preamplifier is automatically stored simultaneously is shown in liquid crystal
On display screen.For each animal, at least 10 times measurements are carried out at specific time point, calculate average value and standard error.Institute
Result mapping is obtained, as shown in figure 17.
Injection put to death mouse after 24 hours, carried out infarct size to its brain according to the measurement method of infarct size in (5)
Measurement.The mapping of mouse infarct size measurement result is obtained, as shown in figure 18.
(7) interpretation of result:
It can be seen from fig. 10 that MCAO causes (-)-PA to lack in locality at MACO post-operative blood Reperfu- sion 2 hours
Content significantly increases in the brain of blood mouse.MCAO processing induction (-)-PA expression indicates (-)-PA in ischemic side brain and opposite side brain
It may play a protective role in opposite side brain.
In figure 12 it can be seen that (-)-PA level is significantly improved in penumbra region than ischemic core, (-)-of brain opposite side
PA expression is higher than ischemic focal zone.These are studies have shown that naturally occurring (-)-PA may play guarantor in ischemic brain
Shield effect.
As can be seen that (-)-PA processing mouse has more preferable compared with the mouse that physiological saline is handled from Figure 13~15
Survival rate, the infarct size of mouse that (-)-PA is perfused is significantly reduced, and in scores of nervous system, these mouse are flashed
Also there is better improvement.
As can be seen that the mouse of perfusion (-)-PA antibody is compared with the control group that physiological saline is handled from Figure 16~18,
TTC dyeing shows that experimental group ischaemic infarcted region is bigger, and the mouse performance neurologic impairment of perfusion (-)-PA antibody is commented
Divide and reduction fore paw tensile strength variation is more significant, this shows to reduce the behaviouristics knot that (-)-PA expression deteriorates cerebral ischemia in brain
Fruit.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (8)
1. having the following structure application of the compound of formula in the neuroprotective agent of preparation treatment cerebral arterial thrombosis:
2. application according to claim 1, which is characterized in that the neuroprotective agent further includes pharmaceutically acceptable load
Body.
3. application according to claim 2, which is characterized in that the carrier is polypeptide or protein molecular.
4. application according to claim 3, which is characterized in that the protein molecular is selected from bovine serum albumin molecule, egg white
One of protein molecular, keyhole limpet hemocyanin molecule, Fibrin molecules and human albumin's molecule.
5. application according to claim 3, which is characterized in that the polypeptide be selected from poly-D-lysine, polyglutamic acid and
One of two palmitin amic acids.
6. application described in any one according to claim 1~5, which is characterized in that the neuroprotective agent further includes pharmacy
Upper acceptable solvent.
7. application according to claim 6, which is characterized in that the solvent is physiological saline or glucose solution.
8. application according to claim 1, which is characterized in that the administration mode of the neuroprotective agent be oral administration,
Drug administration by injection, respiratory tract administration, percutaneous drug delivery, mucosa delivery or cavity/canal drug administration.
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