CN108721311A - Astragaloside is preparing the application in promoting the regenerated drug of neural stem cell - Google Patents

The present invention relates to a kind of astragalosides to prepare the application in promoting the regenerated drug of neural stem cell.Research has shown that, astragaloside can effectively facilitate self-renewing and the proliferation of neural stem cell, it can also promote neural stem cell differentiating in rat brain after ischemical reperfusion injury to generate new neuronal cell, astroglia or cynapse spongiocyte etc., the effective power of regeneration for restoring neural stem cell.

Astragaloside is preparing the application in promoting the regenerated drug of neural stem cell

Technical field

The present invention relates to drug fields, and the promotion regenerated medicine of neural stem cell is being prepared more particularly to a kind of astragaloside Application in object.

Background technology

Apoplexy is general designation of the traditional Chinese medicine to acute cerebrovascular diseases, and because of hurried symptoms include multiterminal of falling ill, change of illness state is rapid, It is similar to the benefaction number of wind change feature, therefore named apoplexy, palsy.Apoplexy is a kind of brain blood circulation disorder disease, is often accompanied by sudden So faint, be senseless, the distortion of commissure that occurs together, dysphonia and there are the cardinal symptoms such as hemiplegia.Due to Apoplexy Morbidity It is high, the death rate is high, disability rate is high, the feature more than high recurrence rate and complication, so medical field its same coronary heart disease, cancer simultaneously It is classified as one of the three big diseases for threatening human health.Apoplexy Chang Liuyou sequelae, age of onset also tend to rejuvenation, therefore, are Threaten the great illness of human life and quality of life.Clinically headstroke can be divided into ishemic stroke and hemorrhagic stroke Two kinds of hypotypes, and wherein ishemic stroke accounts for about 80% or so of all apoplexy incidences.Ishemic stroke is also referred to as ischemic brain Infarct is that brain blood supply obstacle causes brain lesions, pathogenic process to generally comprise cerebral thrombosis, lacunar infarction and brain bolt Plug etc..Ischemic Cerebral Infarction generation after 4.5 hours in, using recombinant tissue-type plasminogen activator (r-tPA) into Thrombolysis in row vein is the mainstream therapy of current acute ischemic stroke, but r-tPA often has thrombolysis that symptomatic brain goes out The serious complication such as blood, and need, in stringent administration time window innerlich anwenden, to limit its extensive use, therefore be badly in need of grinding Study carefully new therapy and drug.

The research of neural stem cell is that the treatment of apoplexy brings new opportunity, in recent years the hair of stem cell transplantation technology Exhibition provides a new thinking for treatment apoplexy, but its high medical expense and involved problems of morals principles all hinder The technology has been hindered to be widelyd popularize in clinical.It is nearest researches show that：Endogenic neural stem cell is distributed widely in people and moves The brain of object, they are usually in opposite silence state.Hypoxic-ischemic environment can activate the neural stem cell of big intracerebral, promote The regeneration of neural stem cell, therefore Endogenous neural stem cells become ishemic stroke hindbrain structure and function reparation and reconstruction Critical treatment target cell.Regrettably, these endogenic neural stem cell being activated are in low-level proliferation and differentiation State, most of neural stem cell finally stop Proliferation, Differentiation because of apoptosis, therefore very there is an urgent need to obtain can It expands the proliferation of Endogenous neural stem cells and promotes neural stem cell differentiating drug.

Invention content

The study found that astragaloside can be effectively promoted the neural stem cell regeneration after apoplexy in animal body.Carry out After specification, science, rigorous experiment and pharmacological experiment, it is regenerated in preparation promotion neural stem cell to provide a kind of astragaloside Application in drug.

Astragaloside, astragaloside medicine can prepared with salt, the solvate of astragaloside or astragaloside derivative Promote the application in the regenerated drug of neural stem cell.

In the application of an embodiment, which is characterized in that the astragaloside be astragaloside I, astragaloside II, Astragaloside III, astragaloside IV, astragaloside V or astragaloside VI.

In the application of an embodiment, the astragaloside is astragaloside VI, and the astragaloside VI has such as Lower structure formula (6)：

In the application of an embodiment, the astragaloside can promote nerve stem cell proliferation or differentiation.

In the application of an embodiment, it is thin that the astragaloside can promote stem cell of cranial nerve to be divided into neuron At least one of born of the same parents, astroglia and cynapse spongiocyte.

In the application of an embodiment, the astragaloside can promote the endogenous neural of ischemic cerebral after death Stem cell regenerating.

In the application of an embodiment, the astragaloside can promote the regions brain SVZ, regions brain LV and big Neural stem cell regeneration in the regions brain DG at least one region.

Astragaloside, astragaloside medicine can use salt, the solvate of astragaloside and astragaloside derivative in extremely A kind of few composition with EGF receptor is preparing the application in promoting the regenerated drug of neural stem cell.

A kind of regenerated drug of promotion neural stem cell, including the medicine of astragaloside, astragaloside can use salt, astragaloside Solvate or astragaloside derivative.

The regenerated pharmaceutical composition of a kind of promotion neural stem cell, which is characterized in that including EGF receptor and above-mentioned promotion The regenerated drug of neural stem cell.

Research has shown that astragaloside can effectively facilitate self-renewing and the proliferation of neural stem cell, moreover it is possible to promote to lack Endogenous neural stem cells differentiation after blood reperfusion injury in rat brain generate new neuronal cell, astroglia or Cynapse spongiocyte etc., the effective power of regeneration for restoring neural stem cell.

Description of the drawings

Fig. 1 is spectrograms of the astragaloside VI after ESI/MS is analyzed；

Fig. 2 is that Cs of the astragaloside VI after ESI/MS is analyzed composes spectrogram；

Fig. 3 is that Hs of the astragaloside VI after ESI/MS is analyzed composes spectrogram；

Fig. 4 is collection of illustrative plates of the astragaloside VI after HPLC-LESD is analyzed；

Fig. 5 is the result that BrdU fluorescent stainings after the astragaloside VI of various concentration are added in neural stem cell C17.2；

Fig. 6 is positive in BrdU after BrdU fluorescent stainings after the astragaloside VI of neural stem cell C17.2 addition various concentrations The statistical result of the cell percentages of property；

Fig. 7 is the regions the SVZ frozen section using rat brain after BrdU and Sox2 marker fluorescent stainings；

Fig. 8 is the statistical result of the regions brain SVZ BrdU and Sox2 positive cell；

Fig. 9 is the regions the LV frozen section using rat brain after BrdU and Sox2 marker fluorescent stainings；

Figure 10 is the statistical result of the regions brain LV BrdU and Sox2 positive cell；

Figure 11 is the regions the DG frozen section using rat brain after BrdU and NeuN marker fluorescent stainings；

Figure 12 is the statistical result of the regions brain DG BrdU and NeuN positive cell；

Figure 13 is to use the regions the rat brain SVZ frozen section after BrdU and GFAP marker fluorescent stainings；

Figure 14 is the statistical result of the regions brain SVZ BrdU and GFAP positive cell；

Figure 15 is the regions the LV frozen section using rat brain after BrdU and GFAP marker fluorescent stainings；

Figure 16 is the statistical result of the regions brain SVZ BrdU and GFAP positive cell；

Figure 17 is the diameter of neural stem cell under different culture media treatment conditions；

Figure 18 is the result of the average bulb diameter statistics of neural stem cell under different culture media treatment conditions；

Figure 19 is the brain tissue immunoblot results of rat under the conditions of different disposal；

Figure 20 is the schematic diagram for the interaction that molecular simulation software Autodock simulates astragaloside VI and EGF receptor；

Figure 21 is the diameter of neural stem cell under different culture media treatment conditions；

Figure 22 is the statistical result of the average bulb diameter of neural stem cell under different culture media treatment conditions；

Figure 23 is result of the neural stem cell after BrdU is dyed under different culture media treatment conditions；

Figure 24 is the statistics knot of neural stem cell BrdU positive cells after BrdU is dyed under different culture media treatment conditions Fruit；

Figure 25 is that the rat platform of different disposal condition in water maze laboratory passes through preclinical statistical result；

Figure 26 is the statistical result of the rat target quadrant residence time of different disposal condition in water maze laboratory；

Figure 27 is the statistical result of the rat escape latency of different disposal condition in water maze laboratory.

Specific implementation mode

In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment and Attached drawing is described in detail the specific implementation mode of the present invention.Elaborate in the following description many details in order to Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation Limitation.

The astragaloside of one embodiment, the medicine of astragaloside can use the solvate or astragaloside of salt, astragaloside Derivative is preparing the application in promoting the regenerated drug of neural stem cell.

Astragaloside can be extracted from Chinese herbal medicine astragalus, can also be to be prepared by chemically synthesized method.There is research table Bright astragaloside has good therapeutic effect to illnesss such as ephritis, hypertension.However about astragaloside to neural stem cell Mechanism of action research it is less, having not yet to see it can promote the research in terms of neural stem cell regeneration to report.And this hair Bright inventor has been surprisingly found that astragaloside can effectively facilitate self-renewing and the proliferation of neural stem cell after experimental study, It can also promote neural stem cell differentiating in rat brain after ischemical reperfusion injury to generate new neuronal cell, astroglia Or cynapse spongiocyte etc., the effective power of regeneration for restoring neural stem cell, the drug to treat apoplexy provide one newly Treatment thoughts.

Specifically, the medicine of astragaloside can further be increased with salt, the solvate of astragaloside or astragaloside derivative The performances such as stability, the solubility of strong drug.The medicine of astragaloside for example can be able to be hydrochloric acid astragaloside or trifoliate orange acid with salt Astragaloside etc..

In the application of an embodiment, astragaloside is astragaloside I, astragaloside II, astragaloside III, Huang Stilbene saponin I V, astragaloside V or astragaloside VI.

Specifically, astragaloside is shown in the structural formula such as following formula (1) of astragaloside I：

Shown in the structural formula of astragaloside II such as following formula (2)：

Shown in the structural formula of astragaloside III such as following formula (3)：

Shown in the structural formula of astragaloside IV such as following formula (4)：

Shown in the structural formula of astragaloside V such as following formula (5)：

Shown in the structural formula of astragaloside VI such as following formula (6)：

In the application of an embodiment, astragaloside is astragaloside VI.Result of study shows that astragaloside VI promotees It is stronger into neural stem cell power of regeneration, self-renewing and the proliferation of neural stem cell can be effectively facilitated, moreover it is possible to promote ischemic The new neuronal cell of neural stem cell differentiating generation, astroglia or cynapse colloid are thin in rat brain after reperfusion injury Born of the same parents etc..

Astragaloside I, astragaloside II, astragaloside III, astragaloside IV and astragaloside VI all have similar Agent structure.In other embodiments, in addition to astragaloside VI.Astragaloside I, astragaloside II, astragaloside III, Huang Stilbene saponin I V and astragaloside V also shows the regenerated performance of stronger promotion neural stem cell.Herein mainly with astragaloside It is illustrated for the result of study of astragaloside VI.

Further, it is the monomer extracted from Chinese herbal medicine astragalus to have astragaloside VI, which is referred to as HQ22, chemical molecular formula C₄₇H₇₈O₁₉, molecular weight 946, white cotton-shaped powder.It is, of course, also possible to using artificial synthesized tool There is the astragaloside of structure above.

In the application of an embodiment, neural stem cell regeneration includes proliferation or differentiation of neural stem cell etc..It should Astragaloside can promote nerve stem cell proliferation or differentiation.Specifically, the dosage of astragaloside is 1nmol~200nmol, Such as 5nmol~100nmol.Result of study shows in certain concentration range, increases with the drug concentration of astragaloside, Nerve stem cell proliferation ratio is higher.

Specifically, astragaloside can promote stem cell of cranial nerve to be divided into neuronal cell, astroglia and dash forward Touch at least one of spongiocyte.More specifically, astragaloside can promote stem cell of cranial nerve to be divided into neuron simultaneously Cell, astroglia and cynapse spongiocyte.Result of study shows that after astragaloside is added neural stem cell can be restored Power of regeneration, further promote it is neural stem cell differentiating be neuronal cell, astroglia and cynapse spongiocyte.

Further, astragaloside can promote the regeneration of the Endogenous neural stem cells of ischemic cerebral after death.Research The result shows that in Ischemia and Reperfusion in vivo in Rats model, astragaloside can effectively facilitate the endogenous god of ischemic cerebral after death Proliferation through stem cell or differentiation.Result of study shows that endogenic neural stem cell is usually in opposite silence state, is added Astragaloside can effectively facilitate proliferation or the differentiation of the Endogenous neural stem cells of ischemic cerebral after death, to treat ischemic Apoplexy provides a new therapeutic scheme.

More specifically, astragaloside can effectively facilitate proliferation or the differentiation of neural stem cell C17.2.

Specifically, astragaloside can promote the regions brain SVZ (sub-ventricular zone, telocoele region pipe The areas Mo Xia), the regions brain LV (lateral ventricle telocoeles region) and the regions brain DG (dentate gyrus, sea Horse dentate fascia) in neural stem cell regeneration at least one region.More specifically, astragaloside can promote big brain area simultaneously Neural stem cell regeneration in domain, the regions brain LV and the regions brain DG.

To sum up, research has shown that, astragaloside can effectively facilitate self-renewing and the proliferation of neural stem cell, moreover it is possible to promote It is neural stem cell differentiating in rat brain after into ischemical reperfusion injury to generate new neuronal cell, astroglia or cynapse Spongiocyte etc., the effective power of regeneration for restoring neural stem cell, new therapy mechanism is provided for apoplexy.

In addition, the application also provides the regenerated drug of promotion neural stem cell of an embodiment, which includes yellow Stilbene saponin(e, astragaloside medicine can use salt, the solvate of astragaloside or astragaloside derivative.

In one embodiment, the astragaloside in drug be astragaloside I, astragaloside II, astragaloside III, Astragaloside IV, astragaloside V or astragaloside VI.

Further, the astragaloside in drug is astragaloside VI, shown in structural formula such as formula (I).

Specifically, in the regenerated drug of promotion neural stem cell, the effective dose of astragaloside can be 1nmol~ 200nmol, such as 5nmol~100nmol.

The experimental results showed that astragaloside can effectively facilitate neural stem cell regeneration, and it is damaged after repairing rat stroke Learning ability and spatial memory capacity, can be used as promote the regenerated drug of neural stem cell.

In addition, the application also provides the regenerated pharmaceutical composition of promotion neural stem cell of an embodiment, the composition The solvation of salt, astragaloside can be used including EGF receptor (EGF-R ELISA) and the medicine of astragaloside or astragaloside Object or astragaloside derivative.

In one embodiment, the astragaloside in pharmaceutical composition is astragaloside VI.Further, the Radix Astragali Shown in the structural formula of saponin(e VI such as formula (I).

Specifically, EGF receptor is the polypeptide chain containing 1186 amino acid residues.

In one embodiment, the molar ratio of astragaloside and EGF receptor is 1：0.5~2, such as 1:1.

The astragaloside of one embodiment, the medicine of astragaloside can use the solvate and astragaloside of salt, astragaloside At least one of derivative and the composition of EGF receptor are preparing the application in promoting the regenerated drug of neural stem cell.

Research has shown that astragaloside has the function of skins Porcine HGF, and it is aobvious by molecular simulation data To show, the binding force of astragaloside and EGF receptor is strong, and when use EGF receptor antagonist, astragaloside promotes neural stem cell again Raw effect is suppressed.It is in close relations with EGF after illustrating that astragaloside enters in vivo, Wnt, AKT, MAPK/ERK are may participate in, The a plurality of signal pathway such as VEGF and JAK/STAT.Aforementioned pharmaceutical compositions, including the medicine of astragaloside, astragaloside are available Salt, the solvate of astragaloside and astragaloside derivative and EGF receptor, astragaloside can effectively facilitate neural stem cell Regeneration generates synergistic effect with EGF receptor, improves the combination of astragaloside and EGF receptor, further promotes neural stem cell again It is raw.

It is specific embodiment below.

In embodiment if not otherwise indicated using drug and instrument, it is this field conventional selection.It is not specified in embodiment The experimental method of actual conditions, condition usually according to normal condition, such as described in document, books or kit factory The method that family is recommended is realized.

In following embodiment, to illustrate, Sham indicates that the rats in sham-operated group of non-modeling also non-dosing, MCAO indicate The operation group rat (control group) after middle cerebral artery occlusion model treatment is carried out, HQ22 indicates to carry out middle cerebral artery occlusion mould The operation dosing group rat (experimental group) of injection astragaloside VI after type processing.

Embodiment 1

Extraction obtains astragaloside VI monomers from Chinese herbal medicine astragalus, obtains white cotton-shaped powder.Further to extraction Ingredient carry out spectrogram verification.Wherein, ESI/MS (electrospray ionization mass spectrometry, EFI Mist ionization-mass spectrometry) analysis collection of illustrative plates as shown in Figure 1, C spectrums as shown in Fig. 2, H spectrums are as shown in Figure 3.HPLC-LESD (efficient liquid phases Chromatography) analysis collection of illustrative plates as shown in figure 4, the result of Fig. 4 is as shown in table 1 below.

Table 1：HPLC-LESD analysis results

The above result shows that verified, the ingredient of extraction is really astragaloside VI.The chemical molecular formula of astragaloside VI For C₄₇H₇₈O₁₉, molecular weight 946, shown in structural formula such as formula (6).

Embodiment 2

Neural stem cell C17.2 original cuitures

Pregnant 14 days~15 days rats of female are taken, cranial cavity is opened under aseptic condition after anesthesia is put to death and takes out brain, be placed in ice-cold PBS liquid culture dish in, with PBS liquid rinse for several times.Blood vessel and meninx are carefully removed under the microscope, are cut under two telocoeles Area's lateral wall, tissue shear shred, and centrifuge 5min.Supernatant is abandoned, the addition etc. after 37 DEG C of water-baths digest 10min of enzyme mixed liquor is precipitated Trypsin inhibitor is measured, bubble formation is softly blown and beaten and avoided with 1mL liquid-transfering guns, 110g centrifuges 7min later, abandons supernatant, sinks It forms sediment and mixing after DMEM/F12 is blown and beaten is added, slowly slowly grind and sieve with 100 mesh sieve after sedimentation centrifugation, cell suspension after collection sieving, Be added after 110g centrifugations nerve stem cell proliferation culture solution (DMEM/F12, the bFGF containing final concentration of 20ng/mL and EGF, 1% B27,1% green chain is dual anti-, 2mmol/L glutamine), it is blown and beaten repeatedly with internal diameter sequence reduction pipette tips and single cell suspension is made, passed through Trypan blue staining counts cell, with 2 × 10⁵/ mL cell densities are inoculated into 25mm³In culture bottle.In 37 DEG C, saturated humidity, 5%CO₂It is cultivated in incubator, a culture solution is changed in inoculation afterwards for 24 hours, adds fresh medium every 2d or liquid is changed in centrifugation.With Set growth, division and the proliferative conditions of the daily routine observation neural stem cell (NSCs) of microscope.Cell culture to 7~10 days, When the neural bulb diameter of formation is about 100 μm or so (<150 μm) when, nerve ball is collected, is passed on after being digested to single cell suspension. 2~5 generation cells of selection are for testing.God is determined by the immunofluorescence staining of neural stem cell marker Nestin before experiment Purity through stem cell.

Embodiment 3

Astragaloside VI promotes the neural stem cell C17.2 proliferation of in vitro culture

Nerve ball analysis (Neurosphere assay) is first used to measure the self-renewal capacity of neural stem cell：Specifically It is as follows, the nerve ball in 2~5 generations is dispelled to single cell suspension, is inoculated in 96 orifice plates with the density of 500 cells in every hole, Using the proliferated culture medium culture that growth factor is added, it is separately added into 5nmol/L, 10nmol/L, 20nmol/L, 100nmol/L Astragaloside VI (HQ22), control group (ctrl) is not added with.And determine nerve using BrdU (cell proliferation markers) incorporation methods The proliferative capacity of stem cell, processing count the nerve ball number formed under different disposal under the microscope after 7 days, and the ball that is averaged is straight Diameter.The results are shown in Figure 5 by BrdU fluorescent stainings for different adding consistencies.The cell percentages statistical result of the BrdU positives is as schemed Shown in 6.The above result shows that the quantity and ratio of the cell of the BrdU positives are stepped up with astragaloside VI drug concentrations, Astragaloside VI has the ability of good promotion neural stem cell C17.2 proliferation.Cell proliferation marker Ki67 is used simultaneously It is also consistent to contaminate the result further proved altogether with Nestin.Illustrate that astragaloside VI promotes neural stem cell in vitro The ability of C17.2 proliferation is stronger.

Embodiment 4

Astragaloside VI promotes nerves within the body stem cells hyperplasia

Rat cerebral ischemia realizes that specific as follows, rat is sucked with isopentane using middle cerebral artery occlusion model (MCAO) Anesthesia makees stringer notch in the slightly biased right side of neck, exposes and free right carotid, internal carotid, external carotid artery and fan walk Nerve etc. will be pushed through the pretreated 3-0 lines of silica gel through external carotid artery to internal carotid direction, until encountering light resistance not Until capable of readvancing.Make stringer notch in the slightly biased right side of neck after rats in sham-operated group anesthesia, exposes and free right side neck always moves Arteries and veins, internal carotid, external carotid artery, vagus nerve etc., conventional skin suture.Bolt money is carefully extracted after ischemic 1.5h, assessment is every The only nervous function grade of operation mouse, continues to raise.Nervous function grade evaluation method is as follows：After surgery the 0th day and Neuroscore is carried out within 7th day, by 6 grades 5 points of Bederson.5 grades (0 points) normally, 4 grades of (1 point) offside upper limbs cannot be complete Stretching, extension, resistance declines when 3 grades (2 points) are pushed away to offside, and 2 grades (3 points) turn-take when carrying tail to offside, and 1 grade (4 points) turn-take automatically, and 0 Grade (5 points) is without spontaneous activity, the disturbance of consciousness.Start continuous 7 days to treatment group's tail vein within second day after dosing group rat operation Inject the astragaloside VI (HQ22) of 2mg/kg, the physiological saline of control group and sham-operation group injection same dose.Rat is in quilt It puts to death first 2 days, BrdU is injected intraperitoneally.After reaching feeding time, it is perfused and is fixed with 4% paraformaldehyde after the ice-cold PBS perfusions of rat, Frozen section.

The regions SVZ of separation rat brain are fabricated to frozen section, use BrdU and Sox2 marker (stem cell labelings Object) fluorescent staining is carried out, the results are shown in Figure 7, and (a) figure indicates the sham-operation rat of non-modeling also non-dosing in wherein Fig. 7 BrdU fluorescent stainings are as a result, (b) figure indicates the sham-operation rat Sox2 fluorescent stainings of non-modeling also non-dosing as a result, (c) chart Show non-modeling also non-dosing sham-operation rat BrdU/Sox2 fluorescent stainings fusion after as a result, (d) figure indicate MCAO modelings after The rat BrdU fluorescent stainings of injecting normal saline are as a result, (e) figure indicates the rat Sox2 of injecting normal saline after MCAO modelings Fluorescent staining as a result, (f) figure indicate MCAO modelings after injecting normal saline rat BrdU/Sox2 fluorescent stainings fusion after knot Fruit (g) injects the rat BrdU fluorescent stainings of astragaloside VI as a result, (h) figure indicates MCAO modelings after figure expression MCAO modelings The rat Sox2 fluorescent stainings of injection stilbene saponin(e VI are as a result, (i) figure indicates the rat of injection stilbene saponin(e VI after MCAO modelings afterwards Result after BrdU/Sox2 fluorescent stainings fusion.Statistical result such as Fig. 8 institutes of BrdU and Sox2 positive cells after fluorescent fusion Show.

The regions LV of separation rat brain are fabricated to frozen section, and fluorescence dye is carried out using BrdU and Sox2 markers Color, the results are shown in Figure 9, and (a) figure indicates the sham-operation rat BrdU fluorescent staining knots of non-modeling also non-dosing in wherein Fig. 9 Fruit, (b) figure indicate non-modeling also non-dosing sham-operation rat Sox2 fluorescent stainings as a result, (c) figure indicate non-modeling also not plus After the sham-operation rat BrdU/Sox2 fluorescent stainings fusion of medicine as a result, (d) figure indicates injecting normal saline after MCAO modelings Rat BrdU fluorescent stainings as a result, (e) figure indicate MCAO modelings after injecting normal saline rat Sox2 fluorescent stainings as a result, (f) figure indicate MCAO modelings after injecting normal saline rat BrdU/Sox2 fluorescent stainings fusion after as a result, (g) figure indicate The rat BrdU fluorescent stainings of astragaloside VI are injected after MCAO modelings as a result, (h) figure injects stilbene saponin(e after indicating MCAO modelings The rat Sox2 fluorescent stainings of VI are as a result, (i) figure indicates the rat BrdU/Sox2 fluorescence dye of injection stilbene saponin(e VI after MCAO modelings Result after color fusion.The statistical result of BrdU and Sox2 positive cells after fluorescent fusion is as shown in Figure 10.

The above result shows that astragaloside VI dramatically increases the ischemia-reperfusion regions SVZ of rat brain and LV after 7 days The quantity of the neural stem cell in region, effectively facilitates nerve stem cell proliferation.

Embodiment 5

Astragaloside VI promotes nerves within the body stem cell to be divided into neuronal cell

Middle cerebral artery occlusion model (MCAO) is established to rat brain using the method for embodiment 4.Second after experimental group operation It starts continuous 7 days to treatment group tail vein injection astragaloside VI (HQ22), and control group injects the physiological saline of same dose. BrdU is injected intraperitoneally 2 days before being condemned to death in rat.

The regions DG of separation rat brain are fabricated to frozen section, use BrdU and NeuN marker (neuronal cells Marker) fluorescent staining is carried out, as a result as shown in figure 11, (a) figure indicates the sham-operation of non-modeling also non-dosing in wherein Figure 11 Rat BrdU fluorescent stainings are as a result, (b) figure indicates the sham-operation rat NeuN fluorescent stainings of non-modeling also non-dosing as a result, (c) Figure indicate non-modeling also after the sham-operation rat BrdU/NeuN fluorescent stainings fusion of non-dosing as a result, (d) figure indicates that MCAO makes The rat BrdU fluorescent stainings of injecting normal saline are as a result, (e) figure indicates the rat of injecting normal saline after MCAO modelings after mould NeuN fluorescent stainings are as a result, (f) after figure expression MCAO modelings after the rat BrdU/NeuN fluorescent stainings fusion of injecting normal saline As a result, (g) figure indicates the rat BrdU fluorescent stainings of injection astragaloside VI after MCAO modelings as a result, (h) figure indicates MCAO The rat NeuN fluorescent stainings of stilbene saponin(e VI are injected after modeling as a result, (i) figure injects the big of stilbene saponin(e VI after indicating MCAO modelings Result after mouse BrdU/NeuN fluorescent stainings fusion.The statistical result of BrdU and NeuN positive cells after fluorescent fusion is as schemed Shown in 12.

The above result shows that astragaloside VI dramatically increases the nerve in ischemia-reperfusion regions DG of rat brain after 7 days The quantity of first cell, it is neuronal cell to effectively facilitate neural stem cell differentiating.

Embodiment 6

Astragaloside VI promotes nerves within the body stem cell to be divided into astroglia and cynapse spongiocyte

Middle cerebral artery occlusion model (MCAO) is established to rat brain using the method for embodiment 4.Second after experimental group operation It starts continuous 7 days to treatment group tail vein injection astragaloside VI (HQ22), and control group injects the physiological saline of same dose. BrdU is injected intraperitoneally 2 days before being condemned to death in rat.

The regions SVZ of separation rat brain are fabricated to frozen section, use BrdU and GFAP markers (astroglia mark Remember object) fluorescent staining is carried out, as a result as shown in figure 13, (a) figure indicates that the sham-operation of non-modeling also non-dosing is big in wherein Figure 13 Mouse BrdU fluorescent stainings are as a result, (b) figure indicates the sham-operation rat GFAP fluorescent stainings of non-modeling also non-dosing as a result, (c) figure Indicate non-modeling also after the sham-operation rat BrdU/GFAP fluorescent stainings fusion of non-dosing as a result, (d) figure indicates MCAO modelings The rat BrdU fluorescent stainings of injecting normal saline are as a result, (e) figure indicates the rat of injecting normal saline after MCAO modelings afterwards GFAP fluorescent stainings are as a result, (f) after figure expression MCAO modelings after the rat BrdU/GFAP fluorescent stainings fusion of injecting normal saline As a result, (g) figure indicates the rat BrdU fluorescent stainings of injection astragaloside VI after MCAO modelings as a result, (h) figure indicates MCAO The rat GFAP fluorescent stainings of stilbene saponin(e VI are injected after modeling as a result, (i) figure injects the big of stilbene saponin(e VI after indicating MCAO modelings Result after mouse BrdU/GFAP fluorescent stainings fusion.The statistical result of BrdU and GFAP positive cells after fluorescent fusion is as schemed Shown in 14.

The regions LV of separation rat brain are fabricated to frozen section, and fluorescence dye is carried out using BrdU and GFAP markers Color, as a result as shown in figure 15, (a) figure indicates the sham-operation rat BrdU fluorescent staining knots of non-modeling also non-dosing in wherein Figure 15 Fruit, (b) figure indicate non-modeling also non-dosing sham-operation rat GFAP fluorescent stainings as a result, (c) figure indicate non-modeling also not plus After the sham-operation rat BrdU/GFAP fluorescent stainings fusion of medicine as a result, (d) figure indicates injecting normal saline after MCAO modelings Rat BrdU fluorescent stainings as a result, (e) figure indicate MCAO modelings after injecting normal saline rat GFAP fluorescent stainings as a result, (f) figure indicate MCAO modelings after injecting normal saline rat BrdU/GFAP fluorescent stainings fusion after as a result, (g) figure indicate The rat BrdU fluorescent stainings of astragaloside VI are injected after MCAO modelings as a result, (h) figure injects stilbene saponin(e after indicating MCAO modelings The rat GFAP fluorescent stainings of VI are as a result, (i) figure indicates the rat BrdU/GFAP fluorescence dye of injection stilbene saponin(e VI after MCAO modelings Result after color fusion.The statistical result of BrdU and GFAP positive cells after fluorescent fusion is as shown in figure 16.

The above result shows that astragaloside VI dramatically increases the ischemia-reperfusion regions SVZ of rat brain and LV after 7 days The quantity of the astroglia in region, it is astroglia to effectively facilitate neural stem cell differentiating.

Meanwhile we also inject BrdU and phosphorylation NF-H (aixs cylinder marker) and synaptic versicle egg in rat abdominal cavity White synaptophysin (cynapse marker), is used in combination BrdU, NF-H and synaptophysin marker to carry out fluorescent staining As a result similar for the measurement of axon function, astragaloside VI dramatically increases the areas SVZ of ischemia-reperfusion rat brain after 7 days The quantity of the cynapse spongiocyte in domain and the regions LV, it is cynapse spongiocyte to effectively facilitate neural stem cell differentiating.Illustrate Huang Stilbene saponin(e VI has good promotion power of regeneration to neural stem cell.

Embodiment 7

Astragaloside VI has the function of class Porcine HGF (EGF) to the neural stem cell C17.2 of in vitro culture

Nerve ball in 2~5 generations is dispelled to single cell suspension, respectively using the proliferated culture medium that growth factor is added (EGF+) it is cultivated with the proliferated culture medium (EGF-) of removal growth factor.The proliferated culture medium (EGF+) of growth factor is wherein added Ingredient it is as follows：The green chains of the EGF, 1%B27,1% of DMEM/F12, bFGF and 1uL/mL containing final concentration of 20ng/mL are dual anti-, 2mmol/L glutamine.The proliferated culture medium (EGF-) of growth factor is removed without outside EGF, remaining is identical.Removal growth because Son proliferated culture medium (EGF-) culture culture neural stem cell be separately added into 5nmol/L, 10nmol/L, 20nmol/L, The astragaloside VI (HQ22) of 100nmol/L, control group (ctrl) are not added with.Processing observes different disposal under the microscope after 7 days Under the conditions of neural stem cell average bulb diameter.As a result as shown in figure 17, the result of statistics is as shown in figure 18.

The neural stem cell diameter of proliferated culture medium (EGF-) culture of removal growth factor is significantly less than to be cultivated in EGF+ Neural stem cell under the premise of depriving EGF factor cultures, increase with astragaloside VI concentration, the nerve cord of original cuiture is thin The diameter of born of the same parents' ball gradually increases, and high concentration astragaloside VI is added, and cell dia is straight close to the cell restored under EGF cultivation conditions Diameter.The lower cell bulb diameter of maximum concentration astragaloside VI processing and cell bulb diameter under recovery EGF recovery states are poor without statistics It is different.Illustrate that astragaloside VI has the function of class Porcine HGF (EGF) to the neural stem cell C17.2 of in vitro culture.

Embodiment 8

Astragaloside VI improves EGF receptor concentration in ischemia-reperfusion rat brain simultaneously with EGF receptor binding force height

Albumen is carried out after the brain tissue of sham-operation group, control group and dosing experimental group rat in embodiment 4 is crushed respectively Immunoblot experiment.Marker is Nestin (neural stem cell marker), pEGFr (EGF receptor marker), tEGFr respectively (EGF receptor marker) and GAPDH (internal reference), immunoblot results are as shown in figure 19.The result shows that astragaloside VI can Improve neural stem cell (Nestin) and the content of EGF receptor in ischemia-reperfusion rat brain.As shown in figure 20, using molecule The interaction of simulation softward Autodock simulations astragaloside VI and EGF receptor, as a result show astragaloside VI and EGF receptor Affinity be -7.6kcal/mol, the binding force of astragaloside VI and EGF receptor is stronger.

Embodiment 9

EGF inhibitor gefitinib inhibits astragaloside VI for the proliferation function of neural stem cell

Nerve ball in 2~5 generations is dispelled to single cell suspension, 96 orifice plates are inoculated in the density of 500 cells in every hole In, use the proliferated culture medium culture that growth factor is added.First group of Radix Astragali soap for being separately added into 10nmol/L, 100nmol/L Glycosides VI (HQ22), control group (ctrl) are not added with.Another group of EGF inhibitor gefitinib for adding 10nmol/L.Processing 7 days Count the diameter of the nerve ball number formed under different disposal under the microscope afterwards, as a result as shown in figure 21.The result of statistics is as schemed Shown in 22.After gefitinib is added in statistical result showed, astragaloside VI treated neural stem cell bulb diameters reduce.EGF Inhibitor gefitinib significantly suppresses the rush neural stem cell self-renewing effect of astragaloside VI.

BrdU (cell proliferation markers) incorporation methods are further used to determine the proliferative capacity of neural stem cell, as a result as schemed Shown in 23, wherein Figure 23 (a) figures indicate to be not added with after the cell BrdU that astragaloside VI is handled is dyed as a result, (b) figure indicates to add It is after the cell BrdU dyeing of the astragaloside VI processing of 10nmol/L as a result, (c) figure indicates plus the astragaloside of 100nmol/L It is after the cell BrdU dyeing of VI processing as a result, (d) figure indicate plus the EGF inhibitor gefitinib processing of 10nmol/L it is thin It is after born of the same parents BrdU dyeing as a result, (e) figure indicates plus the EGF inhibitor of the astragaloside VI and 10nmol/L of 10nmol/L It is after the cell BrdU dyeing of gefitinib processing as a result, (f) figure indicates plus the astragaloside VI and 10nmol/ of 100nmol/L Result after the cell BrdU dyeing of the EGF inhibitor gefitinib processing of L.The statistical result of BrdU positive cells such as Figure 24 It is shown.As a result show that gefitinib inhibits the rush nerve stem cell proliferation of astragaloside VI to act on, after gefitinib is added, Astragaloside VI treated neural stem cell BrdU positive cell numbers are reduced.

Embodiment 7~9 the result shows that astragaloside has the function of skins Porcine HGF, and pass through molecule mould When intending data and show that the binding force of astragaloside and EGF receptor is strong, and using EGF receptor antagonist, astragaloside promotes nerve The effect of stem cell regenerating is suppressed.It is in close relations with EGF after illustrating that astragaloside enters in vivo.If by astragaloside and EGF Receptor forms pharmaceutical composition, and astragaloside can effectively facilitate neural stem cell regeneration, and synergistic effect is generated with EGF receptor, The combination of astragaloside and EGF receptor is improved, neural stem cell regeneration is further promoted.

Embodiment 10

Astragaloside VI has repaired the learning ability being damaged after apoplexy and spatial memory capacity

It is used for assessing the learning and m emaory of rat by water maze laboratory.Memory capability is the important of hippocampus nerve Function, we tested with this assess astragaloside VI (HQ22) processing after, ischemia-reperfusion rat learning and m emaory is extensive Multiple situation.Experiment is as follows：Experimental rat was sent into operating room in 30 minutes in advance, non-toxic prepared Chinese ink is poured into a diameter of 150cm Prototype water tower in, water tower is divided into 4 quadrants, a cylindrical bar is being placed at tower edge 20cm, table top floods Not in water, to ensure that rat cannot see that platform surface.Every rat of continuous 5 days training records exists from 4 different quadrants The duration of platform is searched out in 60 seconds.If rat cannot search out platform in 60 seconds, it is recorded as 60 seconds.When rat sends out After existing platform, then rat is stopped to the operation for carrying out next again in 15 seconds on platform.Between the experiment of each quadrant of every rat It must be over 4 minutes every the time.In the 6th day revocation platform, every rat of observational record was from different quadrant set off in search platforms Moving line, and front platform where the quadrant residence time, the data obtained by it is single it is blind in a manner of united by an other laboratory technician Meter.Platform passes through that preclinical statistical result is as shown in figure 25, and the time letter of land regions is found after astragaloside VI intervenes It is short.Target quadrant residence time than statistical result it is as shown in figure 26, after astragaloside VI intervenes, in staying for target quadrant Time dramatically increases.The statistical result of escape latency is as shown in figure 27, after astragaloside VI intervenes, finds the time of platform It is remarkably decreased, illustrates that learning ability significantly improves.

The above result shows that neural stem cell regeneration can be effectively facilitated with astragaloside, and it is damaged after repairing rat stroke Learning ability and spatial memory capacity, can be used as promote the regenerated drug of neural stem cell.

Several embodiments of the invention above described embodiment only expresses, 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 the protection of the present invention Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.