CN108354961A - Purposes of the Shorthorned Epimedium P.E after preparing treatment radiation in the drug of Cranial nerve injury as birth trauma and cognitive disorder - Google Patents
Purposes of the Shorthorned Epimedium P.E after preparing treatment radiation in the drug of Cranial nerve injury as birth trauma and cognitive disorder Download PDFInfo
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Abstract
The invention discloses purposes of the Shorthorned Epimedium P.E after preparing treatment radiation in the drug of Cranial nerve injury as birth trauma and cognitive disorder；Learning and memory of little mouse is decreased obviously after radiation, after being handled using Shorthorned Epimedium P.E, mouse learns, memory capability be improved significantly, and with the GAP-associated protein GAP enzyme PV of nerve cell hyperplasia, Ki67, DCX, CB expression enhancings, show Herba Epimedii truly has the effect for preventing from radiating to Cranial nerve injury as birth trauma, the damage of neuron can lead to the cognitive impairment of animal, Herba Epimedii is as traditional Chinese medicine for clinical treatment especially dept. of radiology, and core it is unexpected situations such as have good preventive effect, it is particularly suitable for radiotherapy group, prevent and treatment radiotherapy group is due to radiation-induced failure of memory, the cognitive disorders such as learning ability decrease, with great promotional value.
Description
Technical field
The invention belongs to pharmaceutical technology fields, more particularly to Shorthorned Epimedium P.E Cranial nerve injury as birth trauma after preparing treatment radiation
And the purposes in the drug of cognitive disorder.
Background technology
Herba Epimedii is Berberidaceae plant Herba Epimedii Epimedium brevicornum Maxim., Epimedium sagittatum
Epimedium sagiyyatum (Sieb.et Zucc.) Maxim, E. Pubescens Epimedium pubescens Maxim,
E. wushanense T. S. Ying Epimedium wushanense T.S.Ying or korean epimedium herb Epimedium koreanumNakai's
Dry aerial parts.Ben Xia, autumn cauline leaf are tapped when in great numbers, are removed thick stalk and impurity, are dried or dry in the shade.Herba Epimedii is that China passes
The kidney-nourishing tcm drug of system,《Compendium of Materia Medica》On the effects that recording " strengthening the essence gas, mends waist and knee, cardiac stimulant power at hard muscles and bones ", 2010 editions《In
State's pharmacopeia》On the effects that recording " kidney-replenishing, strengthening the bones and muscles, wind-damp dispelling ".Herba Epimedii mainly contains flavones, polysaccharide, lignanoid, life
The chemical compositions such as alkaloids.Modern pharmacological research also indicate that Herba Epimedii have good filling liver kidney, strengthening the bones and muscles, wind-damp dispelling, it is anti-oxidant,
Anti-aging improves immunity, inhibits various pharmacological activity such as tumour and protection cardio-cerebrovascular.
Current radiation has become a serious public health problem, with the development of science and technology, offering convenience to the mankind
While, also bring very important radiation hazradial bundle.Radiation is widely present among nature and people's life, and research finds small
To household electrical appliances such as mobile phone, electric fan, TV, computer, micro-wave ovens, arriving communication tower, satellite communication, nuclear power plant's leakage etc. greatly can produce
Raw various forms, different degrees of radiation.So the mankind are likely to also easily be exposed among different degrees of radiation, nothing
By being low dose of or the radiation of high dose can all cause different degrees of influence to people's body.Research finds that electromagnetic radiation is made
It is extremely wide with the harm to human body, the nervous system disease, angiocarpy, enterogastric diseases can be caused, after influencing reproduction, harm
The problems such as generation, reduction body immunity and eye eyesight.Research finds that radiation may cause serious consequence to the development of brain,
It influences to include backward microcephaly, intelligence and mental development, behaviouristics change, spasm and epilepsy, brain tumor and aging
Deng.According to dose of radiation and time difference, the processes such as neuron proliferation, migration, differentiation and death are may interfere with, so as to cause
The cognition dysfunction of its paramophia and development later stage.Also there are some researches prove when ionising radiation dosage be 1Gy when can influence
The survival of neural progenitor cell substantially reduces the cell number of differentiation in hippocampus, increases DNA damage, promotes cell-cycle arrest, and lure
Oxidative stress is led, finally shows that hippocampus shows as cognition dysfunction.Therefore, research and development toxicity is low, the high treatment brain of curative effect
The drug of neurotrosis and its caused cognitive disorder, repair Cranial nerve injury as birth trauma and treat by radiation-induced failure of memory,
The cognitive disorders such as learning ability reduction, receive always the attention of medicine sector scholar, although having been reported Herba Epimedii has anti-spoke
The effect penetrated, but Herba Epimedii to the protection of irradiated mice cranial nerve and the protection of radiation-induced cognitive disorder still without phase
Close report.
Currently, report of the Herba Epimedii in terms of radioresistance is broadly divided into following several respects：
Documents 1：Application No. is " CN200910073159.5 ", entitled " one kind having radioresistance, anti-fatigue effect
Preparation and preparation method thereof ", disclose it is a kind of with radioresistance, anti-fatigue effect preparation according to weight part ratio by 3~
15 parts of wilsonii, 3~20 parts of Radix Astragali, 1~6 part of Herba Epimedii and 0.003~0.006 part of beta carotene are made.It should
Preparation is curative for effect, toxic side effect is low, it is of low cost, easy to carry, be easy to preserve, convenient for taking, dosage it is accurate, to be spoke
The prevention of the clinical treatment and radiation injury penetrated after damage provides more, better drug candidate.The present invention is taken to mouse
Preparation, then observe mouse 60Co gamma-rays irradiate 30 days after survival rate, observation result show mouse survival rate ratio
The mouse survival rate of negative control group is higher by 30%~50%, while the preparation of the present invention has apparent guarantor to hemopoietic function of bone marrow
Protective function, and improve by mice spleen T lymphocyte transformation rates are shone, preparation of the invention can make mice spleen T lymphocyte transformations
Rate reaches 15%~30%, it follows that the preparation of the present invention has radiation resistance；The preparation of the present invention can be from inside to outside
Radiation is actively resisted in ground, but the patent does not refer to protective effect of the Herba Epimedii to irradiated mice cranial nerve, and anti-spoke
Penetrate with this application involves " neurotrosis and its caused cognitive disorder " be entirely different concept：The magnetic radiation referred to from above
Effect is extremely wide to the harm of human body, can cause the nervous system disease, angiocarpy, enterogastric diseases, after influencing reproduction, harm
In generation, reduces body immunity and eye eyesight；Serious consequence is caused to the development of brain, influence includes microcephaly, intelligence
Power and mental development are backward, behaviouristics changes, spasm and epilepsy, brain tumor and aging etc.；Interference neuron proliferation moves
The processes such as shifting, differentiation and death, so as to cause its paramophia and the cognition dysfunction of development later stage；It is thin to influence neural ancestral
The survival of born of the same parents substantially reduces the cell number of differentiation in hippocampus, increases DNA damages, promotes cell-cycle arrest, and induced oxidation is answered
Swash, finally shows that hippocampus shows as cognition dysfunction.And this application only refers to the survival rate of mouse, mouse beer spoiled bacteria
Thought on conversion ratio with radiation resistance, and do not refer to that radiation resistance is that Herba Epimedii is brought in the whole text, only refers to " this hair
Herba Epimedii described in bright, containing there are many have compared with the active chemical composition of Johnson ＆ Johnson's reason, mainly including flavone compound, biology
Alkali, polysaccharide, lignanoid and some essential trace elements etc. have enhancing body's immunity, reinforce gonad function, increase heart and brain
Vascular flow amount, promotes hematopoiesis function and bone metabolism, and tool anti-aging is antitumor and other effects.”
Documents 2：Application No. is " CN201110373487.4 ", entitled " a kind of anti-radiation Chinese medicine and its system
Preparation Method ", it is 2 by weight that said preparation, which is by the extract powder and microcrystalline cellulose of Chinese medicine composition,:It is obtained after 1 mixing
Capsule, the Chinese medicine composition are composed of the following components by weight：160-200 parts of Radix Astragali, 110-130 parts of psoralea corylifolia,
80-100 parts of Radix Angelicae Sinensis, 160-200 parts of Herba Epimedii, 135-165 parts of madder, 110-130 parts of the fruit of glossy privet, 110-130 parts of Radix Paeoniae Alba,
110-130 parts of radix rehmanniae preparata, 80-100 parts of Rhizoma Chuanxiong, 215-265 parts of Chinese yam, 35-45 parts of honey-fried licorice root；The extract powder is the Chinese medicine
The xeraphium of the ethanol solution extract of composition；Said preparation radioresistance is curative for effect, and toxic side effect is low, and preparation method is simple,
It is of low cost, it is easy to carry, it is easy to preserve, convenient for taking, dosage is accurate, in terms of preventing and treating radiation injury has good
Development prospect.But the patent does not refer to protective effect of the Herba Epimedii to irradiated mice cranial nerve.And the Shen
Please in " said preparation can increase the Thymus and Spleen index of 60Co γ radiation murines, protect immune organ；It can improve
60Co γ radiation murines serum and the superoxide dismutase activity in liver, reduce mda content, improve the antioxygen of body
Change ability；60Co γ radiation murine peripheral white blood cells can be increased, hematopoiesis function is improved；60Co γ radiation murines can be reduced
Bone marrow cell micronucleus rate, inhibit gamma-ray the causes chromosome aberrations of 60Co to act on；In addition, the Chinese medicine preparation toxic side effect is low,
Preparation method is simple, of low cost, easy to carry, is easy to preserve, and convenient for taking, dosage is accurate；Therefore, in prevention and treatment spoke
Penetrating has good development prospect in terms of damage, can be used for that immunity of organisms caused by radiotherapy is low, hematopoiesis function is different
Radiation injuries are often waited, can provide for the officers and men being operated under radiation environment and other related personnel and special procure drug, or
Under the conditions of high-tech modern war and the personal protection of public contingent even caused by radiation provides substance reserve." only refer to this
Preparation has anti-spoke in terms of the influence to immune function, the influence to oxidation resistance, upper three of the influence to hematopoiesis function
The effect of penetrating, and said preparation includes multiple formulations ingredient, and do not refer to that radiation resistance is that Herba Epimedii is brought in the whole text.
Therefore, research and development toxicity is low, Cranial nerve injury as birth trauma and failure of memory, learning ability after the high treatment radiation of curative effect
The drug of the cognitive disorders such as reduction is extremely urgent.
Invention content
The purpose of the present invention is to provide Shorthorned Epimedium P.E prepare treatment radiation after Cranial nerve injury as birth trauma and cognitive disorder
Drug in purposes.
The present invention is the study found that Shorthorned Epimedium P.E can be used for repairing radiation-induced Cranial nerve injury as birth trauma, and Herba Epimedii carries
Object is taken to can be used for treating radiation-induced such as failure of memory and learning ability decrease cognitive disorder.
Beneficial effects of the present invention are as follows：
Present invention finds the new applications of Cranial nerve injury as birth trauma and cognitive disorder after being used to treat radiation of Herba Epimedii：
(1) radiation patterns group mouse facial expression burnout, weight loss, this shows that radiation causes a degree of mouse to damage
Wound.Shorthorned Epimedium P.E group, the weight of Amifostine positive controls are apparently higher than radiation patterns group, Shorthorned Epimedium P.E group, ammonia
The mouse weight of phosphorus spit of fland positive controls is apparently higher than radiation patterns group, shows that Shorthorned Epimedium P.E, Amifostine have played intervention
With the important function of adjusting；
(2) spacious field experiment shows that the spontaneous activity of radiation patterns group and exploratory behavior are suppressed, and administration group (excessive sheep
Leaves of pulse plants extract group, Amifostine positive controls) then obviously performance is active, and illustrate that Herba Epimedii, Amifostine have and preferably treats spoke
Penetrate the effect of anxiety, depression caused by damage；
(3) water maze laboratory shows that radiation patterns group mouse is less in platform quadrant residence time, spanning platform number, says
Bright Radiation on Mouse Cranial nerve injury as birth trauma, decrease of memory, and Shorthorned Epimedium P.E group, Amifostine positive controls, platform as
The residence time of limit and spanning platform number obviously increase, and show that Shorthorned Epimedium P.E, Amifostine can effectively repair cranial nerve
Damage, the memory of mouse, learning ability be improved significantly；
(4) expression of Ki67, DCX, CB, PV in immunohistochemical experiment detection hippocampus of mice, experiment show to radiate
The expression of Ki67, DCX, CB, PV in mouse in hippocampus is caused to reduce, and Shorthorned Epimedium P.E group, Amifostine positive controls
The expression of Ki67, DCX, CB, PV in mouse in hippocampus increase, and the damage of neuron can lead to the cognitive impairment of animal, excessive sheep
The leaves of pulse plants truly has the effect for preventing from radiating to Cranial nerve injury as birth trauma.
(5) Herba Epimedii as traditional Chinese medicine for clinical treatment especially dept. of radiology and unexpected core situations such as have very
Good preventive effect is particularly suitable for radiotherapy group, prevent and treatment radiotherapy group due to radiation-induced failure of memory,
The cognitive disorders such as learning ability decrease, have great promotional value.
Description of the drawings
Fig. 1：Influence of the Shorthorned Epimedium P.E to the weight of the mouse of radiation injury；
Fig. 2：Spacious field experimental result；
Fig. 3：Water maze laboratory result；
Fig. 4：Immunohistochemistry detects the PV values in the hippocampus of mouse；Wherein, the expression of (A) PV positive neurons, (B) PV god
Statistical data through member expression, p ＜ 0.05；
Fig. 5：Immunohistochemistry detects the Ki67 values in the hippocampus of mouse；Wherein, the expression of (A) Ki67 positive neurons, (B)
The statistical data of Ki67 neuron expressions, p ＜ 0.05；
Fig. 6：Immunohistochemistry detects the DCX values in the hippocampus of mouse；Wherein, the expression of (A) DCX positive neurons, (B)
The statistical data of DCX positive neurons expression；
Fig. 7：Immunohistochemistry detects the CB values in the hippocampus of mouse；Wherein, the expression of (A) CB positive neurons, (B) CB god
Statistical data through member expression, p ＜ 0.05；
In the above figure, control indicates normal group, and irradiation indicates that radiation patterns group, Epimedium indicate excessive
Sheep leaves of pulse plants extract group, amifostine indicate Amifostine positive controls.
Specific implementation mode
The preparation of 1 Shorthorned Epimedium P.E of embodiment
The drying herb of epimedium herb is taken, is crushed, 70% ethyl alcohol is added according to 1: 20 ratio impregnates before decoction
0.5h is decocted 2 times, filtration, is merged 3 times and is decocted gained filtrate, is concentrated into thick paste, dry, 4 DEG C save backup.The present invention's is excessive
The sheep leaves of pulse plants refers in particular to Berberidaceae plant Herba Epimedii.
The treatment of 2 Shorthorned Epimedium P.E of embodiment is by cognitive disorders such as radiation-induced failure of memory, learning ability reductions
1, preparing experiment animal
8 SPF grades of week old male Balb/c mouse, weight (24 ± 2) g are purchased from Hubei Province Animal Experimental Study center,
Production licence number：SCXK (Hubei Province) 2015-0018.Sub-cage rearing is in Medical College of Yangtze University's Experimental Animal Center.Adaptability is fed
It supports one week, per 10 group rearings of cage.
Animal is divided into two big groups：Normal group 10,30 groups of radiation patterns group.It is disposably complete that radiation patterns group gives X lines
Body irradiates, and is disposably shone up to company's elekta precise medical computerized linear accelerator X-Rays whole bodies using Sweden's medical courses in general
It penetrates, exposure dose 5.5Gy, absorbed dose of radiation 550mu/min, the visual field is 15cm × 15cm, ource-skin Distance 100cm.Model success
Afterwards, it is randomly divided into 3 groups, every group 10, i.e. radiation patterns group, Amifostine (lot number：J0311A, Dalian U.S. logical sequence technology are limited
Company) positive controls, it is administered in 2 hours after the success of Herba Epimedii (Hubei Ji Kang pharmaceutcal corporation, Ltds) extract group model, it is excessive
Sheep leaves of pulse plants extract group carries out gavage daily by 5g/kg, and Amifostine positive controls press the abdominal cavities 30min before radiation 100mg/kg
Amifostine is injected, corresponding dosage physiological saline is given once daily in remaining every group of animal.After 4 weeks, Behaviors survey is carried out, behaviouristics is real
Test includes mainly spacious field experiment and water maze laboratory.
2, the 1st after pre-irradiation and irradiation, 2,3,4 weeks record weight.
As a result influence of the Shorthorned Epimedium P.E as shown in Fig. 1 to irradiated mice weight, compared with normal group, radiation
Model group body weight is decreased obviously, and has been less than normal group (P ＜ 0.05)；Compared with radiation patterns group, the irradiation of Shorthorned Epimedium P.E group
2 weeks weight obviously increase (P ＜ 0.05) afterwards, and weight dramatically increases (P ＜ 0.05) after Amifostine positive controls 1 week.It is testing
In, radiation patterns group mouse facial expression burnout, weight loss, this shows that radiation causes a degree of mouse to damage.Excessive sheep
Leaves of pulse plants extract group, Amifostine positive controls mouse weight be apparently higher than radiation patterns group, show Shorthorned Epimedium P.E, ammonia phosphorus
Spit of fland has played the important function intervened and adjusted.
3, spacious field is tested
Spacious field experiment (open field test) is also known as Open field test, is that evaluation experimental animal is autonomous in strange environment
A kind of method of behavior, exploratory behavior and tensity.With experimental animal among novel environment the occurrence frequency of certain behaviors and
Duration etc., independent behaviour and exploratory behavior of the reaction experiment animal in foreign environment react its anxiety with urine and stool number
Degree.Open-field test is a kind of classical performance testing method measuring mouse mood and psychological condition, and test animal in new ring
The most common method of exploratory behaviour in border, and reflect the classical way of mouse emotional reactions.It can be used for testing in animal
The excitement of pivot nervous system or depressive state.Animal, which enters a strange environmental protection consciousness reaction, to be remarkably reinforced, animal
It would generally avoid staying in open environment.Tank wall is considered as the activity of region security, so the residence time of middle section is long
It is short can the quantitatively alert level of reaction experiment animal, anxiety state and the adaptability to environment.
Experimental provision uses SuperMaze animal behaviors record analysis system (the glad Soft Inform ation Science and Technology Ltd. in Shanghai),
Spacious field experimental box length and width each 50cm, high 30cm, under casing are divided into 16.It tests and is carried out under quiet environment.By it is described just
Normal group, radiation patterns group, Amifostine positive controls, Shorthorned Epimedium P.E group mouse be respectively put into case inner bottom surface center, together
Shi Jinhang is imaged and timing.Observation stops camera shooting after a certain period of time, records the total move distance of mouse in 5min.Clean square chest inner wall
And bottom surface, in order to avoid the next test result of information (stool, urine of such as animal, smell) influence that last time animal is remaining.Animal is replaced,
Continue to test.
According to the parameter of computer software design difference observable, as in the unit interval animal in the center lattice residence time,
The grid number that a certain limbs are crossed is horizontal score, and hind leg standing number is vertical score, modifies number, urine and stool number；Movement
Speed, move distance, the time of having a rest, along side move distance, central move distance etc..
Table 1
Shown in attached drawing 2 and table 1, total displacement distance of radiation patterns group is significantly lower than normal group (P ＜ 0.05)；Herba Epimedii carries
The total displacement distance of object group is taken to be apparently higher than radiation patterns group (P ＜ 0.05), and the total displacement distance of Shorthorned Epimedium P.E group is obviously high
In Amifostine positive controls (P ＜ 0.05)；The middle section residence time of radiation patterns group is obviously considerably longer than normal group；With
Radiation patterns group is compared, and Shorthorned Epimedium P.E group, Amifostine positive controls center residence time significantly reduce (P ＜
0.05)。
Spacious field tests the change for observing spontaneous activity in mice, after normal mouse enters spacious field, first along babinet edge
Walking, observes surrounding enviroment, and then attempts to enter central area.Specifically, radiation patterns group mouse stops in central area
The time is stayed to increase, that is felt depressed, depression or anxiety cause body to ring since radiation injury causes instinctive activity limited
The reaction of border and cognitive ability declines, and incubation period is long, slow motion, and the residence time is long；Observe Shorthorned Epimedium P.E group, Amifostine sun
Property control group mice central area shorter residence time, shows that the anxiety of mouse can be effectively relieved in Shorthorned Epimedium P.E, Amifostine
State.Compared with normal group, radiation patterns group moves total length significantly lower than Shorthorned Epimedium P.E group, Amifostine sun in 5min
Property control group, and the autonomic activities ability and exploring ability of radiation patterns group are substantially reduced.These results indicate that Herba Epimedii carries
Take object, Amifostine positive controls that there is the effect of anxiety, depression caused by treatment radiation injury.Show radiation patterns group
Spontaneous activity and exploratory behavior are suppressed, and administration group (Shorthorned Epimedium P.E group, Amifostine positive controls) is then apparent
Performance is active, illustrates that Herba Epimedii, Amifostine have the function of centainly treating anxiety, depression caused by radiation injury.
4, water maze laboratory
Morris water mazes are one of the important experiments of judgment experiment animal learning memory capability, by the behavior of animal come
The intelligence situation for judging animal, achievees the purpose that research.Although mouse is born swimming top-notch player, they, which but detest, is in
State in water, while it is the activity extremely to consume one's strength to swim for mouse, they understand stopping in the searching water of instinct
Cease place.The behavior for finding recreating facility is related to a complicated Memory Process, including collects regard related with space orientation
Feel information, then these information handled, arranged, remembered, reinforce and then further taken out, it is therefore an objective to can successfully navigate by water and
The platform hidden in water is found, is finally escaped from water.Rodent moves about naturally, finds the space bit of pool wall by the light of nature
It sets and goes to roost, therefore normal mouse can reduce incubation period, increase the original platform quadrant residence time, increase spanning platform number.
Selection peace and quiet, half-light, isoperibol are tested, and experimental provision is recorded and analyzed using SuperMaze animal behaviors
System (the glad Soft Inform ation Science and Technology Ltd. in Shanghai).It will fill with clear water to predetermined altitude, add suitable in experimental provision before experiment
Food coloring (nontoxic) is measured, water is made to become opaque black liquor, heater heats water temperature to 22 ± 2 DEG C.Device surrounding is outstanding
Different graphic is hung, orientation, platform are placed in fourth quadrant center to help animal to determine, are located at the water surface or less about 1.5cm, entirely in fact
Position of platform remains unchanged during testing.Training period successively from 4 place of entry by animal towards pool wall into the water, record animal
Platform required time, i.e. escape latency are found and climbed up, is put back in cage after stopping 10s.If platform is not found in 60s, by
Experimenter is led to platform, and is put back in cage after stopping 10s on platform, and next direction training is carried out after about 30 minutes.It is fixed
Position navigation experiment is for evaluating animal learning and memory capability.It first 5 days, will be moved from the first, second, third and fourth quadrant place of entry successively
Into the water to pool wall, training 4 times daily record animal escape latency in water to object plane respectively.Space search experiment is used for
Evaluate memory of the animal to position of platform, i.e. memory retention.Remove platform within 6th day, in platform opposite, quadrant, which is faced the wall and meditated, is put into,
Respectively in original platform quadrant residence time, spanning platform number in record animal 60s.Normal group, radiation patterns group, Amifostine
Shown in D figures in positive controls, the escape latency such as attached drawing 3 of Shorthorned Epimedium P.E group, the platform quadrant residence time (s) is such as
In attached drawing 3 shown in C figures, shown in the B figures in spanning platform area frequency such as attached drawing 3, the movement locus of mouse such as attached drawing 3 in four groups
In A figures shown in.
Table 2
Shown in D figures in attached drawing 3, compared with normal group, it is known that the incubation period of radiation patterns group mouse is longer, attached drawing 3 and
Table 2 can be compared with normal group, it is known that radiation patterns group mouse is less in platform quadrant residence time, spanning platform number, says
Bright Radiation on Mouse Cranial nerve injury as birth trauma, decrease of memory.Compared with radiation patterns group, Shorthorned Epimedium P.E group, Amifostine are positive
Control group is obviously increased in the residence time of platform quadrant and spanning platform number, shows Shorthorned Epimedium P.E, Amifostine energy
Cranial nerve injury as birth trauma is effectively repaired, mouse memory is improved.
In conclusion spacious field experiment and water maze laboratory the result shows that：Learning and memory of little mouse is decreased obviously after radiation；It utilizes
Shorthorned Epimedium P.E processing after, ability of learning and memory in mice be improved significantly.
3 immunohistochemical experiments of embodiment-verification Shorthorned Epimedium P.E treats Cranial nerve injury as birth trauma
Radiation has serious influence to brain development, such as behavior change and failure of memory, learning ability weaken cognition
Obstacle.Proliferation, migration, differentiation and the dead ventricular zone (subventricular for occurring mainly in telocoele of neuron
Zone SVZ) and dentate fascia subgranular zone (subgranular zone, SGZ), hippocampal neuron is the basis of spatial memory, sea
The Spatial information processing of horse is crucial, these all to the storage of memory and are arranged extremely important.The hippocampal neuron being proliferated
It is very sensitive to radiating, so we select four labels on hippocampal neuron, i.e. PV, Ki67, DCX and CB.
It 6 weeks after irradiation, with 1% amobarbital by mouse anesthesia, is irrigated from mouse heart, utilizes 4% neutral first
Aldehyde is fixed, and excision cerebellum retains the brain tissue containing hippocampus, is fixed overnight after being put into 4% neutral formalin progress, is transferred to later
30% sucrose solution (sucrose+0.1M phosphate buffers (PB, PH：7.4) it) preserves.It is cut along median sagittal plane, the right side is taken to stay
A left side, sagittal are placed in freezing microtome (Leica 1950, Germany), brain tissue are cut to 50 μm every brain piece, is put into up
Among 24 orifice plates equipped with (1.5ml) 0.1M PB liquid, per hole 6-8 pieces, for marking PV, Ki67, DCX
(Doublecortin), CB observes the variation of Mice brain tissues.
It after cutting brain piece, is cleaned 3 times, each 5min with 0.1M PB, the H2O2 concussions for now matching 3% are incubated 10min, use
After PBST (PBS+0.1%TritonX-100) is cleaned 3 times, 2% donkey (being matched with DCX) and sheep is added (with PV, Ki67, CB
With) closing serum, after two hours be added source be sheep primary antibody DCX (Santa Cruz Biotechnology Inc., CA,
USA) and source be rabbit primary antibody Ki67 ((Gene Tex, USA) and PV, CB (Swant, USA).After 4 DEG C are incubated overnight, removal
Primary antibody is cleaned 3 times with PBST, and the anti-sheep of donkey (matching DCX) secondary antibody and goat-anti rabbit (matching PV, Ki67, CB) secondary antibody, concussion is added
It is incubated 1h, secondary antibody is removed, is cleaned 3 times with PBST, biotin-avidin compound (ABC) (Vector is added
Laboratories Inc., Burlingame, CA, USA) concussion incubation 0.5h, DAB horseradish mistakes are added after cleaning 3 times with PBST
Oxide enzymatic reagent 10min colour developings ((Vector Laboratories Inc., Burlingame, CA, USA).Piece is dragged for, waits for it
After natural drying, with resinene mounting.
Statistical disposition：It is analyzed using 17.0 software packages of SPSS, data are indicated with " x ± s ", and multiple-group analysis uses single factor test
Variance analysis is that difference is statistically significant with P ＜ 0.05.
As shown in Fig. 4, Shorthorned Epimedium P.E improves radiation-induced PV values.(A) expression of PV positive neurons, (B)
The statistical data of PV neuron expressions.P ＜ 0.05.In attached drawing 4, after 5.5gy irradiations, newly-generated granular cell or neuron are thin
Born of the same parents substantially reduce.(P ＜ 0.05), Shorthorned Epimedium P.E, Amifostine promote the increase of mouse ovarian stratum granulosum or nerve after radiating
Member regeneration (p ＜ 0.05)；Photovoltaic (PV) is the specific nerve that a kind of calbindin exists only in primate and rodent
Cell subsets.PV positive intrerneurons are the GABA neurons of hypermetabolism and high electrical activity, are the main of central nervous system
Marker plays an important role in controlling neuron activity.Neuron can pass through selectively with the neuronal synapse of surrounding
Inhibit the activity of these neurons to adjust their excitability and activity.PV positive neurons in hippocampus are easily by various damages
The influence of factor, such as ionising radiation.The damage of neuron can lead to the cognitive impairment of animal.In an experiment, radiation patterns group PV
Expression is reduced, and shows that Radiation on Mouse hippocampal neuron causes to damage.Shorthorned Epimedium P.E group, Amifostine positive controls, light
The expression of volt (PV) obviously increases, and shows the protective effect of Shorthorned Epimedium P.E, Amifostine to hippocampus PV positive neurons.
As shown in Fig. 5, it is horizontal to improve radiation-induced Ki67 for Shorthorned Epimedium P.E.(A) table of Ki67 positive neurons
It reaches, the statistical data of (B) Ki67 neuron expressions, p ＜ 0.05.In attached drawing 5, after 5.5gy irradiations, newly-generated granular cell
Or neuronal cell substantially reduces.(P ＜ 0.05), Shorthorned Epimedium P.E, Amifostine promote mouse granulosa cells layer after radiating
Increase the regeneration (P ＜ 0.05) with neuron；Ki67 is a kind of high molecular weight protein, is located in nucleus, with cell mitogen
It is related, only expressed in the nucleus of cycle proliferation, and do not expressed in other cytoplasm.It is the important mark of cell Proliferation
Will, and be highly sensitive cell proliferation marker.In an experiment, the Ki67 expression of radiation murine is reduced, and illustrates radiation injury
The proliferation of hippocampal cell.The expression of Shorthorned Epimedium P.E group, Amifostine positive controls Ki67 dramatically increases, and shows that Herba Epimedii carries
Take object, Amifostine that there is protective effect to hippocampal neuron Ki67.
As shown in Fig. 6, it is horizontal to improve irradiation DCX for Shorthorned Epimedium P.E.(A) expression of DCX positive neurons, (B) DCX
The statistical data of positive neuron expression.* P ＜ 0.05.In attached drawing 6, after 5.5gy irradiations, newly-generated granular cell or nerve
First cell substantially reduces.(P ＜ 0.05), Shorthorned Epimedium P.E, Amifostine promote radiation after mouse granulosa cells layer increase and
The regeneration (P ＜ 0.05) of neuron.DCX (double cortisols, Doublecortion) is a kind of microtubule associated protein, main to express
In neural precursor, and it is closely related with the differentiation of neuron, regeneration and migration, in neuronal migration, DCX control startings
The polymerization of process, stablize cytoskeleton, new neuron it is final position in its important function, pregnant mouse, newborn rat and at
Show in year mouse brain DCX in mature neuron without expression, DCX has expression in stem cell, but in neuronal precursor
The expression in stage is higher, therefore markers of the DCX as prematurity neuron, and therefore, DCX is moved as research neuron
Shifting, differentiation and regenerated marker.Irradiating the DCX expression of model group mouse reduces, and shows that radiation can inhibit hippocampus of mice dentation
Return the proliferation of granular cell.The expression of Shorthorned Epimedium P.E group, Amifostine positive controls DCX obviously increases, and shows excessive sheep
Leaves of pulse plants extract, Amifostine have protective effect to hippocampus DCX positive neurons.
As shown in Fig. 7, it is horizontal to improve radiation-induced CB for Shorthorned Epimedium P.E.(A) expression of CB positive neurons,
(B) statistical data of CB neuron expressions, p ＜ 0.05.The result shows that after 5.5gy irradiations, newly-generated granular cell or nerve
First cell substantially reduces.(P ＜ 0.05), Shorthorned Epimedium P.E, Amifostine promote radiation after mouse granulosa cells layer increase and
The regeneration (P ＜ 0.05) of neuron.CB is calbindin, it is the specific mark object of Studies On Neuronal.Its expression with
The activity of nerve cell is directly proportional with function, and the effect and memory of hippocampal neuron are closely related.Experiment shows mouse after radiation
CB expression reduce, show that Radiation on Mouse hippocampal neuron causes to damage.Shorthorned Epimedium P.E group, Amifostine positive control
Group, CB expression dramatically increase, and show that Shorthorned Epimedium P.E, Amifostine can reduce intracellular Ca2+Overload reduces Apoptosis
With necrosis neuron, to protect the radiation injury of brain cell.
In conclusion the attached drawing of immunization experiment 4-7's the result shows that, mouse cranial nerve major injury after radiation；Using excessive
After sheep leaves of pulse plants extract-treated, mouse cranial nerve is obviously repaired, and shows that Shorthorned Epimedium P.E energy antagonism x-ray bombardment causes
Granulosa cells or neuron reduction, and difference is significant.Conclusion Shorthorned Epimedium P.E can protect mouse cranial nerve to exempt from
Raying damages, and enhances with GAP-associated protein GAP enzyme PV, Ki67, DCX (Doublecortin) of nerve cell hyperplasia, CB expression.
Integrated embodiment 2-3 is it is found that learning and memory of little mouse is decreased obviously after radiation；After being handled using Shorthorned Epimedium P.E,
Ability of learning and memory in mice be improved significantly, and with GAP-associated protein GAP enzyme PV, Ki67, DCX of nerve cell hyperplasia
(Doublecortin), CB expression enhancing.Herba Epimedii anticipates for clinical treatment especially dept. of radiology and core as traditional Chinese medicine
Situations such as outer, has good preventive effect, is particularly suitable for radiotherapy group, prevents and treatment radiotherapy group is since radiation is drawn
The cognitive disorders such as the failure of memory, the learning ability decrease that rise, have great promotional value.The Shorthorned Epimedium P.E is being made
For Chinese medicine preparation when active constituent when, add a little auxiliary materials or carrier, can be made into capsule, tablet, oral agents, microcapsule formulation, note
Penetrate agent, suppository, spray or ointment.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art on the basis of the present invention made by equivalent substitute or transformation, in the present invention
Protection domain within.Protection scope of the present invention is subject to claims.
Claims (6)
1. purposes of the Shorthorned Epimedium P.E after preparing treatment radiation in the drug of Cranial nerve injury as birth trauma and cognitive disorder.
2. Shorthorned Epimedium P.E as described in claim 1 drug of Cranial nerve injury as birth trauma and cognitive disorder after preparing treatment radiation
In purposes, which is characterized in that the Shorthorned Epimedium P.E includes icariin.
3. Shorthorned Epimedium P.E as claimed in claim 2 drug of Cranial nerve injury as birth trauma and cognitive disorder after preparing treatment radiation
In purposes, which is characterized in that the Shorthorned Epimedium P.E further includes Hyperoside, Epimedin B, epimedin C and inositol.
4. Shorthorned Epimedium P.E as described in claim 1 drug of Cranial nerve injury as birth trauma and cognitive disorder after preparing treatment radiation
In purposes, which is characterized in that the extraction step of the Shorthorned Epimedium P.E is：Epimedium herb is taken, is crushed, ethyl alcohol leaching is added
It after bubble, decocts, filtering, gained filtrate is concentrated into thick paste, dry, saves backup.
5. Shorthorned Epimedium P.E as claimed in claim 4 drug of Cranial nerve injury as birth trauma and cognitive disorder after preparing treatment radiation
In purposes, which is characterized in that take the drying herb of the epimedium herb, crush, according to 1: 20 ratio be added 70% second
Alcohol impregnates 0.5h.
6. Shorthorned Epimedium P.E as claimed in claim 4 drug of Cranial nerve injury as birth trauma and cognitive disorder after preparing treatment radiation
In purposes, which is characterized in that decoct 3 times, filtering, merge 3 times and decoct gained filtrates and be concentrated into thick paste, dry, 4 DEG C of preservations are standby
With.
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| US20080299231A1 (en) * | 2000-11-22 | 2008-12-04 | Xia Yongchao | Herbal composition for treatment of neurodegeneration and neuronal dysfunction |
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| US20080299231A1 (en) * | 2000-11-22 | 2008-12-04 | Xia Yongchao | Herbal composition for treatment of neurodegeneration and neuronal dysfunction |
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