CN108272787A - Purposes of 7,8 dihydroxyflavones in treating or preventing the relevant cognitive ability of aging and declining - Google Patents

Purposes of 7,8 dihydroxyflavones in treating or preventing the relevant cognitive ability of aging and declining Download PDF

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CN108272787A
CN108272787A CN201711169037.7A CN201711169037A CN108272787A CN 108272787 A CN108272787 A CN 108272787A CN 201711169037 A CN201711169037 A CN 201711169037A CN 108272787 A CN108272787 A CN 108272787A
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陈锦辉
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Suzhou glanke Pharmaceutical Technology Co.,Ltd.
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Abstract

This application provides purposes of 7,8 5,7-Dihydroxyflavones in the cognitive ability for treating or preventing the relevant cognitive ability decline of aging in subject or for improving aging subject.

Description

7,8 dihydroxyflavones are in treating or preventing the relevant cognitive ability of aging and declining Purposes
Background technology
Aging is the process that a whole body function slowly fails over time, and the memory that aging is brought subtracts The elderly's daily life quality (Hedden＆Gabrieli 2004) can be influenced with cognitive ability decline by moving back.According to statistics bureau of the U.S. Data, the elderly's population to the year two thousand fifty, over-65s will reach 83,700,000, close to 20% (Jennifer of total population M.Ortman 2014).We are badly in need of a treatment for capableing of effective immunotherapy targeted autoantibody and the relevant cognitive dissonance problem of aging Scheme.However, at this stage pair with the understanding of the molecular cell level principle of the relevant cognitive dissonance problem of aging is also opposite has Limit, thus hinder the development of effective clinical treatment.
The nerve regneration ability and synaptic plasticity of hippocampus are all related with cognitive function, both of which can greatly by The influence (Rao et al.2006) of aging process.Nerve regneration includes several committed steps, including neural stem cell or ancestral The breeding of cell (NSC), the survival of prematurity nerve cell, dendron growth, maturation and function integrate (Ming＆Song 2005). We confirm that aging process can targetedly reduce the split speed (Romine of active neural stem cell at previous research et al.2015).In addition to this, the survival rate of newborn neuron cell persistently rests on reduced levels, further hinders new The generation of raw neuronal cell, the whole of newborn neuron cell is unable to get so as to cause the existing neural circuit of aging animal Close supplement (Bondolfi et al.2004；Rao et al.2005).
In newborn neuron after hippocampus generation, they also need to integrate by further morphological development and cynapse It can just work orderly.Can the maturation in cellular morphology is Granule Neurons correctly be received dendritic spines by aixs cylinder Signal is transmitted to the basic premise in the regions CA3.In the hippocampus dentate fascia of adult, newborn neuron is usually in 3-4 weeks Initial development of the market goes out more complete aixs cylinder, dendron and dendritic spines form, and every structure is persistently carried out in the next several months It changes (Zhao et al.2006).Our research finds that aging process can not only interfere neuron regeneration, but will be by subtracting Few dendritic arborization quantity and the mode of total length greatly hinder dendron growth.It is provided largely for the formation of cynapse in view of dendron The reduction of surface area, dendron quantity and dendron total length will finally hinder the formation of newborn cynapse.So far, without effective hand Section can prevent or save aging destruction caused by neuron regeneration and dendron growth.
Neuron regeneration and by adult generate newborn neuron morphological maturity be all by brain-derived neurotrophy because Sub (BDNF) regulation and control.BDNF combined by the special receptor TrkB with it come activated receptor, and plays regulating and controlling effect with this (Tolwani et al.2002；Binder&Scharfman2004).The effect of BDNF include promote neure growth, survival, It migrates and cynapse adjusts (Alderson et al.1990；Horch&Katz 2002；Gorski et al.2003； Cohen-Cory et al.2010).BDNF is sent out in regulation and control hippocampus dendron growth and later stage synaptic function and structure plasticity Wave effect (the Tolwani et al.2002 being even more important；Vigers et al.2012；Wang et al.2015).Institute With the function of BDNF forms (Vigers et al.2012) related with memory consolidation with memory.But BDNF-TrkB systems are non- It is often easy to be influenced by aging, this may can explain why memory related with hippocampus can be influenced by the age And it is damaged (Calabrese et al.2013；Budni et al.2015) and provide one for the memory function for improving the elderly Therapeutic purpose.Although BDNF is the key factor of neurotrophic factor, the limited transitivity of BDNF, shorter half-life period and can not The validity that it is used as drug is reduced by the characteristic of blood-brain barrier.
Therefore, there is the demand of the drug for the cognitive ability decline for treating or preventing aging object in this field.
Invention content
Inventor has found that aging can damage newborn neuron dendron growth under study for action.These damages are all to cause always The reason of age people's cognitive ability declines.There is presently no effective method can aging brain in promote nerve regneration or The development of newborn neuron dendron.
Inventor it is further surprisingly found that, can show to 7,8 dihydroxyflavone (DHF) of aged mouse systemic injection The length for increasing aged mouse newborn neuron dendron is write, to save damage of the aging to newborn neuron dendron growth. Therefore, DHF can slow down the brain cognitive ability that aging is brought by promoting the development of hippocampus newborn neuron dendron Decline.
Present invention comprises treatment or prevention methods and purposes that cognitive ability caused by aging declines.Implement at one In scheme, the present invention relates to 7,8- 5,7-Dihydroxyflavones (DHF) or derivatives thereof or its pharmaceutically acceptable salt, ester, solvate, Tautomer, optical isomer, inclusion compound, hydrate, polymorph or pro-drug prepare for treat or prevent by Use in examination person in the composition of the relevant cognitive ability decline of aging or the cognitive ability for improving aging subject On the way.
In one embodiment, the present invention relates to 7,8- 5,7-Dihydroxyflavones (DHF) or derivatives thereof or its can pharmaceutically connect Salt, ester, solvate, tautomer, optical isomer, inclusion compound, hydrate, polymorph or the pro-drug received exist Prepare the purposes in the composition of the dendron length for extending newborn neuron in aging subject.
The present invention includes giving DHF or derivatives thereof to aging subject.DHF or derivatives thereof also can be in clinical setting Lower administration.In one embodiment, DHF or derivatives thereof is administered by way of abdominal cavity or intravenous injection.
In one embodiment, the present invention include it is preventative give DHF or derivatives thereof, thus prevent aging from causing Cognitive ability decline.
In one embodiment, composition of the invention is drug or pharmaceutical composition.
In one embodiment, DHF of the invention or derivatives thereof or its pharmaceutically acceptable salt, ester, solvation Object, tautomer, optical isomer, inclusion compound, hydrate, polymorph or pro-drug also join at least one therapeutic agent Close application.
In one embodiment, subject is mammal, preferably people.
Description of the drawings
Fig. 1 display agings reduce the quantity of prematurity newborn neuron in aged mouse hippocampus and damage its dendron shape State is developed.Figure 1A-B：Immunostaining is carried out using antibody to mark microtubule associated protein (doublecortin [Dcx] is red) Show the prematurity neuron in the hippocampal dentate (HDG) of 3 monthly age mouse (A) and 12 monthly age mouse (B).Use 4 ', 6- bis- Amidino groups -2-phenylindone (DAPI, blue) dyes HDG structures.Fig. 1 C-D：Magnification at high multiple image is shown in Figure 1A and B The structure (being indicated by white box) of Dcx positive prematurity neurons.Fig. 1 E：Quantify 3 monthly age mouse (n=3) and 12 monthly ages are small The newborn prematurity neuronal quantity of the Dcx positives in mouse (n=4) HDG.Fig. 1 F-G：It rebuilds 3 monthly age mouse (F) and 12 monthly ages is small The dendron of the positive newborn prematurity neurons of Dcx in mouse (G) HDG.Fig. 1 H-I：Quantify 3 monthly age mouse (n=3) and 12 monthly ages The average dendritic arborization quantity (H) of the newborn prematurity neuron of the Dcx positives and total dendron length are shown in mouse (n=4) HDG (I)(*p<0.05,**p<0.01)
Fig. 2 shows that DHF processing does not significantly improve the quantity of newborn prematurity neuron in aged mouse hippocampus.Figure 2A：Experimental method schematic diagram.Fig. 2 B-D：Receiving is shown respectively using immunostaining processing microtubule associated protein (Dcx, red) Prematurity neuron in 12 monthly age hippocampus of mice body dentate fascias (HDG) of different injection treatments：Phosphate buffered saline Injection group (PBS；N=4, Fig. 2 B), dimethyl sulfoxide (DMSO) injection group (DMSO；N=4, Fig. 2 C) and 7,8 dihydroxyflavone injection groups (DHF；N=4, Fig. 2 D).HDG structures are dyed using 4 ', 6- diamidinos -2-phenylindone (DAPI, blue).Fig. 2 E- G：Magnification at high multiple picture (being indicated by the white box) display of Fig. 2 B, C, D receive PBS injections (Fig. 2 E), DMSO injections respectively The prematurity neuron of the Dcx positives is individually shown in 12 monthly age mouse HDG of (Fig. 2 F) and DHF injection (Fig. 2 G).Fig. 2 H：Quantization Receive to show the prematurity neuronal quantities of the Dcx positives in 12 monthly age mouse HDG of PSB, DMSO and DHF processing respectively.
Fig. 3 shows that DHF processing improves the dendron morphological development of newborn prematurity neuron in Aged Mice hippocampus.Figure 3A-C：Immunostaining is carried out using antibody to mark microtubule associated protein (doublecortin [Dcx] is red) to show respectively Receive the single prematurity neuronal structure in 12 monthly age hippocampus of mice body dentate fascias (HDG) of different injection treatments：Phosphate Buffer saline injection group (PBS；N=4, Fig. 3 A), dimethyl sulfoxide (DMSO) injection group (DMSO；N=4, Fig. 3 B) and 7,8 dihydroxy Flavones injection group (DHF；N=4, Fig. 3 C).Using 4 ', 6- diamidinos -2-phenylindone (DAPI, blue) to granular cell layer into Row dyeing is to show structure.Fig. 3 D：It rebuilds in the HDG for the 12 monthly age mouse for receiving PSB, DMSO and DHF processing respectively and shows Dcx The dendron of positive prematurity neuron.Fig. 3 E-G：Quantization receives 12 monthly age mouse of PSB, DMSO and DHF processing respectively Shown in HDG the average dendritic arborization quantity (Fig. 3 E) of the prematurity neuron of the Dcx positives, average dendron length (Fig. 3 F) and Total dendron length (Fig. 3 G).Fig. 3 H：The Dcx sun in receiving the hippocampus of 12 monthly age mouse of PSB, DMSO and DHF processing respectively The dendron total length of sex immature neuron accounts for each group of other ratio (* p<0.05,**p<0.01,***p<0.001).
It is described in detail
Definition
Term " joint " refers to one or more therapeutic agents to be added in DHF or derivatives thereof and is administered, including same When administration or in any order in succession administration.
Term " 7,8- 5,7-Dihydroxyflavones " or " DHF " refer to 7,8- dihydroxy -2- phenyl -4H-1- chromenes -4- Ketone is following formula: compound：
DHF is a kind of tyrosine kinase receptor B (TrkB) agonist, it combines the extracellular domain (Kd=of this receptor 320nM), promote receptor dimer and autophosphorylation.
For the purpose for handling or treating, term " mammal " refers to any animal for being defined as mammal, Include the animal of the mankind, domestic animal and zoo, sport category or pets, such as dog, horse, cat, milk cow.Preferably, lactation Animal is the mankind.
Term " processing " or " treatment " are a kind of means for obtaining beneficial or desired clinical effectiveness.Herein In, beneficial or desired clinical effectiveness includes the mitigation, the mitigation of disease degree, the state of an illness of (but being not limited only to) clinical symptoms Stabilization (not deteriorating), the development for postponing or slowing down the course of disease, improvement or alleviate morbid state and mitigate (part or all of), It is no matter detectable or undetectable.Herein, " treatment " does not include " preventing ", " avoiding " or " prevention ".
Term " compound " refers to DHF or derivatives thereof or pharmaceutically acceptable salt, esters, solvate, change Isomers, optical isomer, inclusion compound, hydrate, polycrystalline or pro-drug, while also including its protective derivative. Compound can include therefore one or more chiral centres and/or double bond exist with following form：Stereoisomer is for example Double bond isomer (that is, geometric isomer), enantiomter or diastereoisomer.According to the present invention, mention herein Chemical constitution, including the compound of the present invention, including the enantiomer of all respective compounds, diastereomer and geometrical isomerism Body, that is to say, that either the pure form of spatial chemistry is (such as：Geometric isomer is pure, enantiomeric pure or diastereo-isomerism The pure form of body) and isomer mixture is (such as：Geometric isomer, enantiomter and non-enantiomer mixture).One In the case of a little, a kind of enantiomter, diastereoisomer or geometrical isomerism know from experience than other isomers have it is preferably active Or improved toxicity or dynamic characteristic.In this case, these enantiomters, diastereomeric of the compound of the present invention are different Structure body and geometric isomer then become preferred.
Term " polycrystalline " refers to originally asking disclosed compound or the solid crystalline form of its compound.Same chemical combination The different polycrystal of object can express out different physics, chemistry and/or spectral quality.Different physical properties includes (but not only It is limited to) stability is (such as：To the stability of heat or light), compressibility and density (prepare and product manufacturing in it is critically important), with And dissolution rate (this can influence bioavilability).The difference of stability can be due to chemically reactive variation (such as：Otherness oxygen Change so that rapider than the dosage form colour fading formed by another polycrystal by a kind of dosage form that polycrystal is formed) or mechanical property (such as：As the advantageous polycrystal of dynamics is transformed into the more stable polycrystal of thermodynamics, tablet is broken in storage) or both (such as：The tablet of single polycrystal more easily ruptures at high humidity) cause.The different physical properties of polycrystal can influence its processing. For example, a kind of polycrystal may be than another polycrystal it is more likely to form solvate, or it is more difficult to be filtered, washed miscellaneous Matter, because their grain shapes or size distribution are different.
Term " hydrate " refers to compounds as disclosed herein or its salt, further comprises stoichiometry or non-chemical The water of metered amount combined by non-covalent intermolecular forces.
Term " inclusion compound (clathrate) " refers to the compound or its salt disclosed herein in form crystal lattice, the crystalline substance Lattice include space (for example, channel), have the guest molecule (for example, solvent or water) of capture wherein.
Term " pro-drug " refers to the drug molecule of the compound of the present invention, is no biological activity until by generation Journey of apologizing for having done sth. wrong activates.Pro-drug includes the derivative of compound, can under biotic factor (in vitro or in vivo) hydrolyze, aoxidize or It otherwise reacts to provide the compound of the present invention.Pro-drug can have after carrying out the reaction under biotic factor Activity or they in the form of its unreacted in the presence of can be active.The example of pro-drug for use in the present invention includes The analog or derivative of (but not limited to) compounds as disclosed herein, it includes can biological hydrolysis part, such as can Biological water Solve amide, can biological hydrolysis ester, can biological hydrolysis carbamate, can biological hydrolysis carbonic ester, can biological hydrolysis Uride and can biological hydrolysis phosphate analog.Other examples of pro-drug include containing for compounds as disclosed herein There is -- NO, -- NO2, -- ONO or -- ONO2Partial derivative.Pro-drug can usually be prepared using well-known method.
Officinal salt
In the case where compound has enough acid-base values to form stable, nontoxic ackd salt or basic salt, chemical combination Object can be suitable as officinal salt application.Officinal salt includes being originated from pharmaceutical inorganic or those of organic base and acid salt.It closes Suitable salt includes derivative by alkali metal such as potassium and sodium, the well-known acid of alkaline-earth metal such as calcium and magnesium and other drugs field Object.In particular, the example of officinal salt is the organic acid addition salt formed with acid, acceptable anion on physiology, than Such as：Toluene fulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, resists mesylate Bad hematic acid salt, α-ketoglutaric acid and α-glycerophosphate.Suitable inorganic salts, including sulfate, nitrate, carbon can also be formed Sour hydrogen salt and carbonate.
Officinal salt can be obtained by standard, well known preparation method, such as by by the compound (example of enough alkalinity Such as amine) it is reacted with suitable acid, acceptable anion on physiology.Can also prepare carboxylic acid alkali metal (for example, sodium, Potassium or lithium) or alkaline-earth metal (such as calcium) salt.
Composition
Composition based on DHF or officinal salt can with for any indication described herein therapeutically effective amount with Pharmaceutically acceptable additive is prepared with any carrier or excipient joint.Therapeutically effective amount can according to illness to be treated, its Severity, the therapeutic scheme taken, the pharmacokinetics of medicament and individual patients situation and change.
On the one hand, preferably mixture is made with pharmaceutical acceptable carrier in DHF.Under normal circumstances, preferably with Orally-administrable Form applies pharmaceutical composition, but preparation can also by parenteral, intravenous, intramuscular, percutaneous, oral cavity, subcutaneous, suppository or Other approach administrations.Intravenous and intramuscular formulations are preferably administered in Sterile Saline.It is unstable in the composition that will not make the present invention In the case of determining or influencing its therapeutic activity, those of ordinary skill in the art can adjust preparation to carry under the introduction of this specification For the several formulations for specific administration method.More specifically, for example, being adjusted to desired compound so that it is in water In or other excipient in it is more stable, can by general adjustment (salt formula, esterification etc.) simple realization.
Under normal circumstances, therapeutically effective amount of the compounds of this invention in pharmaceutical dosage form is typically about 0.1mg/kg to about 100mg/kg or higher depend on used compound, the degree and administration route that cognitive ability declines.For this hair Improving eyesight, the prevention of composition according to the present invention or to prevent effective quantity from falling into above-mentioned for identical described in therapeutically effective amount It is in range and usually identical as above-mentioned effective quantity.
The application of DHF or derivatives thereof can be continuous (intravenous drip) to oral medication several times daily (for example, QID, BID, etc.), and may include taking orally, part, parenteral is intramuscular, and intravenously, subcutaneously, percutaneous (it may include infiltration enhancing Agent), oral cavity and suppository administration and other administration route.Enteric oral tablet can be used for enhancing and be administered by oral route Compound bioavilability and stability.Most effective dosage form is by the pharmacokinetics depending on selected particular agent Learn and patient in traumatic brain injury severity.Peroral dosage form is particularly preferred, because being easy to be administered and conducive to trouble Person's compliance.
To treat pharmaceutical composition according to the present invention, preferably by the chemical combination of one or more present invention of therapeutically effective amount Object is mixed with pharmaceutical carrier according to conventional pharmaceutical mixed technology.Carrier can take many forms, this takes The certainly dosage form needed for administration, for example, oral and parenteral administration.In the pharmaceutical composition for preparing peroral dosage form, appoint What common drug media can use.Therefore, suitable to carry for liquid oral medicine such as suspension, elixir and solution Body and additive include that water, glycol, oil, alcohol, flavoring agent, preservative, colorant and analog can be used.For Peroral solid dosage form Preparation such as powder, tablet, capsule and solid dosage forms such as suppository, suitable carrier and additive include starch, sugar carrier such as Portugal Grape sugar, mannitol, lactose and relevant carriers, diluent, granulating agent, lubricant, adhesive, disintegrant and analog can be made With.If necessary, tablet or capsule can carry out enteric coating for being sustained by standard technique.It can using these dosage forms Significantly affect the bioavilability of compound in patients.
For parenteral administration, carrier generally includes sterile water or sodium-chloride water solution, although other ingredients, including Those dispersing aiies may also be included in that interior.When that will use sterile water or keep the germ-free condition of sterile water, the combination Object and carrier must also sterilize.Also it can be prepared to injection suspension, in this case, liquid-carrier appropriate, suspension Agent etc. can also be used.
In one embodiment, compound and composition are for treating, preventing or delaying the relevant cognition energy of aging Power declines.Preferably, be treatment, prevent or the relevant cognitive ability of aging is delayed to decline, composition with peroral dosage form below The amount of row is administered：About 250 micrograms are to about 1 gram or more, at least once a day or one day up to four times.Compound preferably take orally to Medicine, but can also be in the form of parenteral administration, local administration or suppository etc..
In one embodiment, DHF or derivatives thereof can be configured to a part for beverage, including sports drink (example Such as,), energy drink (e.g., red ox Red), nutritious drink (for example,) or any types Supplement or addition vitamin, electrolyte and analog beverage.
Embodiment
1. aging of embodiment significantly reduces the quantity of newborn prematurity neuron in hippocampus and compromises dendron Form.
Cognitive ability decline is the mark of aging process, but specific principle not yet defines (Bishopet al.2010).In a variety of possible principles, is reduced by neuron regeneration rate and the hippocampus somatic nerves of decrease of synapses representative is plastic Property reduce be principal element (Burke＆Barnes 2006).Neuron regeneration, including nerve stem cell proliferation, prematurity god It survives, shift through member, is ripe, dendron growth and function integrate (Ming＆Song 2005), by old-age group in a variety of levels The influence of change.Before us is that aging damage nerve cord is thin the main reason for researches show that aging influences neuron regeneration Born of the same parents are proliferated, proliferation (Romine et al.2015) of the especially active neural progenitor cell in hippocampus of mice body.This time study In, we further have evaluated the newborn prematurity neuron of 3 monthly age mouse (n=3) and 12 monthly age mouse (n=4) quantity and Their dendron growth situation.Whenever the 6th brain sections are all handled with immunostaining, using for microtubule associated protein The antibody of (doublecortin, Dcx) is marked, this is a kind of marker (Gleeson for newborn prematurity neuron et al.1999).In 3 monthly age mouse, the newborn prematurity neuron of the display Dcx positives is mainly evenly distributed on hippocampus tooth Shape returns at (HDG) granular cell layer (GCL) interior one third (Figure 1A).3 monthly age mouse are compared, it is observed that 12 monthly age mouse Newborn prematurity neuronal quantity significantly decrease, show the Dcx positives cell quantity it is less, be sporadically distributed in December At age hippocampus of mice body dentate fascia (HDG) granular cell layer (GCL) interior one third (Figure 1B).Under high magnified glass, we The cell (Fig. 1 C, Fig. 1 D) of each Dcx positives can be clearly observed on granular cell layer (GCL).It is every in 3 monthly age mouse A hippocampus dentate fascia (HDG) is containing about 11,098 ± 2,841 newborn prematurity neurons, this number is in 12 monthly age mouse In fall below 341 ± 54, show in aging animal, (p=is greatly reduced in the number of newborn prematurity neuron 0.022, Fig. 1 E).
It will be clear that the dendron of prematurity neuron (Fig. 1 C, is schemed to molecular layer growth under high magnified glass 1D).The mouse at 3 monthly ages is compared, the Dcx positive cells of 12 monthly age mouse show poor dendron form.It is rebuild by dendron, Our quantitative analysis adult mice (46 neuronal cells in 3 mouse) and Aged Mice (91 god in 4 mouse Through first cell) the dendron form (Fig. 1 F, 1G) of newborn prematurity neuron in HDG.In the mouse at 3 monthly ages, each newborn god It is 297.0 ± 65.4 microns (Fig. 1 H, 1I) usually to have 4.1 ± 0.4 dendritic arborizations, total length through member.In the mouse at 12 monthly ages In, the quantity of newborn neuron dendron significantly has dropped 58%, and average each neuron has 1.7 ± 0.2 dendritic arborization (p =0.004, Fig. 1 H), dendron total length significantly drops to averagely each 70.6 ± 18.2 microns of neuron (p=0.022, figure 1I), fall 76%.To sum up, it is observed that the newborn prematurity neuronal quantity in Aged Mice HDG substantially Degree declines, and newborn neuron dendron form is damaged.
Effects of the embodiment 2.DHF for aged rats neonatal neuron survival rate
Until up to now, there are no effective drugs can slow down or prevent under the cognitive ability brought by aging Drop, so working out, a kind of can to promote the method for nerve regneration and/or dendron morphological development be necessary.
First, we are dedicated to verifying whether DHF can improve neonatal neuron survival rate in Aged Mice HDG.December Age mouse is divided into three groups, receives DHF injections (5mg/kg is dissolved in dimethyl sulfoxide (DMSO) [DMSO], n=4), 70% diformazan respectively Base sulfoxide injects (n=4) or phosphate buffered saline injection (PBS, n=4), continues two weeks once a day.All mouse It is put to death behind 24 hours after last time is injected, and takes out brain (Fig. 2A) to assess number and the tree of prematurity neuron Prominent morphological development situation.One is chosen by permanent order use microtubule associated protein in every 6 big brain section Newborn neuron in (doublecortin, Dcx) antibody test hippocampus is to carry out immunostaining.Receiving PBD and DMSO In the mouse brain slice of injection, it is observed that minimal amount of Dcx positive cells (Figure 1B, 2B, 2C).Receiving DHF injections Afterwards, the quantity of Dcx positive cells does not have acute variation (Fig. 2 D).Under high magnified glass, we can count each Dcx sun Property cell (Fig. 2 E-G).In DHF injection groups, the par of the average Dcx positive cells per HDG is 330 ± 111；DMSO is noted The quantity for penetrating group is 293 ± 191；The quantity of PBS control group is 341 ± 54 (Fig. 2 H), without notable statistics between three groups of data Difference.Therefore, a length of two weeks DHF injection treatments cannot improve the newborn prematurity neuron survival rate of Aged Mice when.Phase Than under, before us researches show that when a length of two weeks same dose of DHF injection treatments can improve young animals at The quantity (Zhao et al.2015) of the newborn prematurity neuron generated after year.This shows the relevant cognition for the treatment of aging The difficulty and complexity of decline ability.
Embodiment 3.DHF improves Aged Mice dendron form
Although DHF does not improve the survival rate of Aged Mice newborn neuron, microexamination shows the dendron shape of mouse State produces variation (Fig. 2 E-G, Fig. 3 A-C).It is positive thin that each hippocampus Dcx is obtained using Zeiss (Zeiss) microscopic system The image of 40 times of amplifications of born of the same parents.Using the tracer of Neurolucida softwares and rebuilds cell body and dendron (Fig. 3 D) and use NeuroExplorer analyzes (Fig. 3 E-H).We have found that the average number of the dendritic arborization of each neuron slightly improves, from 1.7 ± 0.2 (91 neurons of 4 mouse) of PBS groups and 1.6 ± 0.04 (94 neurons of 4 mouse) of DMSO groups 2.1 ± 0.4 (109 neurons of 4 mouse) of DHF groups are increased to, but this difference does not reach notable statistics Difference (Fig. 3 E).Nevertheless, the average length of each dendritic arborization is by 32.1 ± 3.4 microns of PBS groups and DMSO groups 38.3 ± 5.5 microns of 47.6 ± 3.4 microns (Fig. 3 F) for improving significantly to DHF groups.Therefore, total dendron length of each neuron The 110.3 ± 23.7 micro- of DHF groups is improved significantly to by 70.6 ± 18.2 microns of PBS groups and 67.0 ± 9.8 microns of DMSO groups Rice (Fig. 3 G).By the way that further newborn prematurity neuron is grouped according to the different of dendron total length, it has been found that in PBS groups In DMSO control groups, newborn prematurity neuron major developmental goes out the very short dendron (PBS in 50 microns of cell body Group mouse 67.8 ± 8.8%, DMSO groups mouse 61.4 ± 2.4%, Fig. 3 H).In DHF groups, have short within 50 microns of total length The ratio of the newborn neuron of dendron considerably reduces to 45.9 ± 5.7% (Fig. 3 H).As a result, the mouse in DHF groups has more More newborn neurons develop relatively longer dendron (200 microns away from cell body or more), in all new life nerves measured 18.7 ± 2.6% are accounted in member, this ratio is only 8.4 ± 3.3% in PBS groups in comparison, in DMSO groups for 5.1 ± 2.0% (Fig. 3 H).To sum up, although DHF does not promote newborn prematurity neuronal development to go out greater number of dendritic arborization, But DHF promotes dendron growth by way of extending dendron, this effect shows as DHF and extends the flat of Aged Mice dendron Equal length and total length, while the newborn prematurity neuron for possessing longer total dendron length is also improved on a general level Ratio.
It discusses
It is brought to our society in view of general who has surrendered under current elderly population rapid growth, and the relevant cognitive ability of aging Increasing burden (Jennifer M.Ortman 2014).Although situation is so severe, aging brings cognition energy The molecular mechanism that power declines is still uncertain, hinders the development that can delay or prevent the therapy that this cognitive ability declines.By Neural plasticity representated by hippocampus nerve regneration ability and synaptic plasticity is a ring important in ability of learning and memory. There is research to confirm that the neural plasticity of muroid and the mankind can all be influenced (Burke＆Barnes 2006 by age growth；Rao etal.2006).In the present invention, some are observed we used the mouse at 12 monthly ages and with them to grind with before The similar feature of older animals studied carefully, most notable one are the newborn prematurity neurons compared to 3 monthly age young mices Number reduces and dendron form damage (Fig. 1).These observation results represents the nerve regneration scarce capacity of aged experimental group with Prominent plasticity is insufficient.Being dedicated to enhancing the method for nerve regneration ability and/or synaptic plasticity perhaps can become and potentially control Treat target.
So far, without effective therapeutic scheme can enhance geriatric animals nerve regneration ability or cynapse it is plastic Property.
In this experiment, we have injected two weeks DHF to mouse, it is surprising that DHF is not significantly increased always The neonatal neuron survival rate (Fig. 2) of year mouse.Nevertheless, we are same it was unexpectedly found that between experimental group and control group There are notable dendron morphological differences.Our data are shown, by continuing two weeks injection DHF daily, although newborn neuron Dendritic arborization quantity does not significantly change, and the average dendron length of newborn neuron compares PBS groups and increased substantially 48%, phase 24% (Fig. 3 F) is increased compared with DMSO groups.Similarly, total dendron length of newborn neuron compares PBS groups and increases substantially 53%, it compares DMSO groups and has increased substantially 64% (Fig. 3 G).In addition, it is observed that possessing the newborn neuron of longer dendron Ratio increases, and dendron total length is more than that the number of 200 microns of neuron increases 2.2 times (Fig. 3 H).
BDNF has been found that the shape and quantity of dendritic spines can be controlled, to influence the development of synapse circuit, especially Synapse circuit (Ji et al.2005 in hippocampus；Cohen-Cory et al.2010).Research before us is direct The formation (Gao et al.2009) of postpartum hippocampal dentate granular cell cynapse can be damaged by demonstrating the missing of BDNF.In this hair In bright, it has been found that can acutely be reduced with the total dendron length of each newborn neuron of process of aging.
From the mouse at 2 monthly ages to the mouse at 12 monthly ages, the dendron total length of each neuron has decreased to original 23.7%.In this experiment, DHF processing significantly improves dendritic arborization quantity and tree in each newborn neuron of aged mouse Prominent total length.Nevertheless, compared to the Young adult mouse at 3 monthly ages, received the dendron length of the aged mouse of DHF injections Or it is short very much, the 37% of the Young adult mouse at only 3 monthly ages.
Experimental method
Animal is handled
Male C57BL/6 mouse (Jackson Laboratories) group rearing at any time may be used under 12/12 hour light and shade recycles To obtain food and water.Mouse Age used in experiment is 3 monthly ages and 12 monthly ages.It is big that all programs meet Indiana The regulation learned animal welfare and ratified using the committee.
DHF processing
The mouse at 12 monthly ages receives one kind in three kinds of processing respectively, and (5mg/kg is dissolved in dimethyl sulfoxide (DMSO) for DHF injections [DMSO], n=4) or 70% dimethyl sulfoxide (DMSO) injects (n=4) or phosphate buffered saline injects (PBS, 50mg/kg, n =4, intraperitoneal injection [i.p.]), continue two weeks once a day.All mouse are behind 24 hours after last time is injected Extremely in the form of the dendron of the quantity and assessment newborn neuron that calculate newborn neuron (Fig. 2A).
Tissue treatment
With 2.5% ethobrom deep anaesthesia experimental animal, and heart perfusion is passed through with 0.9% cool brine.Next it uses Cold 4% paraformaldehyde (Paraformaldehyde, [PFA]) fixer is fixed.After fixing overnight, brain is taken out, is placed in 30% sucrose solution 48 hours is cut into 30 microns of brain piece with slicer (Leica, CM1900), stores -20 DEG C of refrigerators.These Slice is then by carry out immunohistochemical analysis.
Cell count
Immunohistochemistry staining method is taken to detect Dcx positive cells to all slices simultaneously.Each hippocampus slice Whenever the 6th slice (30 microns of thickness, at a distance of 180 microns) is all handled in series.With reference to (Gao＆Chen 2013) institute State carry out cell count.In brief, the newborn nerve cell in each Dcx positives of hippocampal dentate granular cell layer is counted.Carefully Born of the same parents' sum is further corrected (Coggeshall＆Lekan 1996).
Micro-imaging technology
Slice is analyzed (Zeiss Axiovert 200M) by inverted microscope system and is combined simultaneously with ApoTome Using computer-controlled digital camera (Zeiss AxioCam MRc5) as interface.Image is captured and is combined by ApoTome AxioVision v4.8 softwares (ZeissAxioVision, v4.8) finally use 7.0 (Adobe of Photoshop Systems picture mosaic and label) are carried out to picture.
Dendron tracer and analysis
The pictures of 40 times of amplifications of each Dcx positive cells by Neurolucida softwares (MBFBioscience, Williston, VT) it is used for tracer neuron cell body and dendron.Finally use NeuroExplorer softwares (Next Technologies, Madison, AL) analyze neuron traces.
Statistical analysis
The data being collected into are expressed in the form of average ± standard deviation.Using student t examine (Fig. 1 E, 1H, 1I) come into The data of 3 monthly age of row and 12 monthly ages mouse neonatal prematurity neuron number, average dendron number and total dendron length compare.Make Receive PBS, DMSO and DHF treated 12 monthly age mouse (figure with one-way analysis of variance method (one-way ANOVA) to analyze 2H, 3E, 3F, 3G) the number of newborn prematurity neuronal cell, average dendron number, average dendron length and total dendron it is long It spends and Fisher least significant differences method (LSD) is used to be used as subsequent comparing check.
Receive PBS, DMSO and DHF treated 12 monthly age mouse using two-way analysis of variance (two-way ANOVA) The ratio of the total dendron length of (Fig. 3 H) each different types of newborn prematurity neuron simultaneously uses one-way analysis of variance method (one-way ANOVA) and LSD are as subsequent comparing check.Statistical analysis is carried out using SPSS softwares (IBM Corporation) and by p Value is set as<0.05.

Claims (6)

1.7,8- 5,7-Dihydroxyflavones (DHF) or derivatives thereof or its pharmaceutically acceptable salt, ester, solvate, tautomer, Optical isomer, inclusion compound, hydrate, polymorph or pro-drug are being prepared for treating or preventing old-age group in subject Change the purposes in the composition of relevant cognitive ability decline or the cognitive ability for improving aging subject.
2.7,8- 5,7-Dihydroxyflavones (DHF) or derivatives thereof or its pharmaceutically acceptable salt, ester, solvate, tautomer, Optical isomer, inclusion compound, hydrate, polymorph or pro-drug are being prepared for extending new life in aging subject Purposes in the composition of the dendron length of neuron.
3. the purposes in claims 1 or 2, wherein the composition is drug or pharmaceutical composition.
4. the purposes of claims 1 or 2, wherein described DHF or derivatives thereof or its pharmaceutically acceptable salt, ester, solvation Object, tautomer, optical isomer, inclusion compound, hydrate, polymorph or pro-drug also join at least one therapeutic agent Close application.
5. the purposes of claims 1 or 2, wherein the subject is mammal.
6. the purposes of claim 5, wherein mammal position people.
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