CN104473924A

CN104473924A - Application of cytisine in medicines for treating cerebral arterial thrombosis

Info

Publication number: CN104473924A
Application number: CN201410733516.7A
Authority: CN
Inventors: 余建强; 王永胜; 周茹; 李玉香; 郑婕; 吴璟; 乔海琦; 刘河涛
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2014-12-04
Filing date: 2014-12-04
Publication date: 2015-04-01

Abstract

The invention discloses application of cytisine (CYT) in medicines for treating cerebral arterial thrombosis. Experiment results indicate that when the cytisine is 1mg/kg which is in a safety dosage range, the autonomic dysfunction of a mouse focal cerebral ischemia model can be remarkably improved, the volume of cerebral infarction in an ischemia area and the apoptosis rate in an ischemic core area and penumbra can be reduced, and a certain dose-pharmacological effect correlation is represented. The experiment results prove that the cytisine has the effects of preventing occurrence of local cerebral arterial thrombosis, reducing death and disability after stroke and promoting neurological function recovery.

Description

Cytisine is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine

Technical field

The present invention relates to the application of cytisine, particularly cytisine is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine.

Background technology

Apoplexy (stroke) is that a kind of onset is anxious, disease progression is fast, the cerebral blood supply insufficiency, the circulation disorder that are feature with autonomic activities obstacle, cognitive function disappearance.Its cause of disease mainly contains region brain tissue ischemia damage body circulatory disturbance (as hypotension, sudden cardiac arrest, mistake ischemic shock) that cerebral arteries luminal stenosis or obturation cause this tremulous pulse to arrange and causes transient ischemic attack.Clinical signs is transient or the sings and symptoms of the delayed ischemic neurological deficits of irreversibility, and has the features such as high incidence, high fatality rate, high disability rate, high relapse rate, leaves over the dysfunction such as hemiplegia, aphasia in living patients more.The display of statistical data that official website of Ministry of Health of the People's Republic of China provides, cerebrovascular disease in 2003 is all number two position in city and country disease death reason, is only second to malignant tumor.Wherein the mortality rate in city is 105.40/10 ten thousand, and rural area is 89.89/10 ten thousand.The case fatality rate of male no matter in city or rural area all higher than women, but in rural females disease death reason, cerebrovascular disease proportion is 21.35%, ranks the first.Regional distribution present by north orientation south wherein Fujian and Anhui sickness rate minimum.Along with rhythm and the intensity increase of the raw Working Life of society, this disease is just toward rejuvenation development, and cerebral infarction, once occur, brings great financial burden and harmful effect to sufferers themselves, family and even society.In patients with cerebral apoplexy, 80% belongs to cerebral infarction, clinically in therapeutic time window, adopt plasminogen thrombolytic (t-Pt) means in early days at present, clinical treatment still lacks effective means, and ischemia-reperfusion Secondary cases malignant lesions after thrombolytic cannot be avoided.Current foreign study personnel apply NR2B receptor antagonist and obtain a series of achievement in research, as the Yi Fenglada just in clinical trial, are eliminated because its earlier generations medicine exists cardiac toxicity.Animal prepares the discovery of central nervous system injury experimental animal model by experiment, a series of waterfall sample retardance biochemical reactions of being induced by ischemia are the key reasons causing cerebral ischemia tissue ischemia core space pathologic damage (tectology changes and cell dysfunction) and penumbra region cell tardive death and long-term action obstacle, its pathomechanism comprises toxicity of excitatory amino acid, calcium overload, oxygen free radical injury, inflammatory reaction and energy regeneration obstacle etc., further investigation apoplexy nerve injury and Neuroprotective Mechanisms, the medicine that apoplexy is effectively treated in developing has become the social problems that can not be ignored, therefore from sending out from Chinese medicine extract component drug efficacy study, research and develop desirable cerebral infarction nerve prevention very urgent with therapeutic strategy with medicine.

Cytisine CYT (Cytisine C ₁₁h ₁₄n ₂o) be a kind of goldspink flower pattern alkaloid, it is active that domestic pharmaceutical research discovery in early stage CYT has similar nicotine (Nicotine) sample, the excited respiratory system in energy reflexive ground, CYT is developed to antiarrhythmic drug clinically, for because of operation, wound or the poisoning During Reflexive Apnea that causes and shock.Recent study also finds that CYT and α 4 β 2 nAChR (this receptor is the action target spot of part stop smoking medicine) has very high affinity and combines, the effectiveness of experiment display CYT stop smoking medicine.In addition the correlational study that cytisine derivant has dopaminergic pathway in regulation and control parkinson disease disease nigrostriatum is had been reported abroad.The neuronal cell glutamate excitotoxicity damage of domestic report cytisine to isolated culture in 2013 has the effect of certain neuroprotective; but whether there is neuroprotective and possible mechanism has no report both at home and abroad at present in the damage of body cerebral ischemia toxicity of excitatory amino acid; therefore, developed into cerebral infarction medicine and be there is high potential value and social meaning.

Summary of the invention

The object of the invention is by the pharmacological research to cytisine, provide cytisine as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine.

The present invention is achieved through the following technical solutions goal of the invention:

Cytisine provided by the invention is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, and the structural formula of described cytisine is such as formula shown in (1):

Particularly, described Ischemic Stroke is the cerebral infarction of acute stage or the phase of reparation.

Further, described cytisine single application dosage is mice 0.25 ~ 1.0mg/kg, rat 0.0875mg/kg ~ 0.35mg/kg, people 0.028mg/k ~ 0.056gmg/kg; Standard body weight people 70kg single use amount 1mg ~ 4mg.

Preferably, described cytisine single application dosage is mice 0.25mg/kg.

Preferably, described cytisine single application dosage is mice 0.5mg/kg.

Preferably, described cytisine single application dosage is mice 1mg/kg.

Particularly, described cytisine single application dosage is only limitted to the dosage that do not cause blood glucose to reduce.

Particularly, the dosage form of described medicine is peroral dosage form or injection type that pharmaceutics allows.

Cytisine provided by the invention has following beneficial effect as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine:

(1) cytisine significantly can improve focal mice medium-sized artery thromboembolism model (MCAO) mouse Nerve dysfunction not falling under hypoglycemic dosage, reduces cerebral infarction volume and apoptosis rate;

(2) brain tissue impairments such as cerebral edema can be alleviated;

(3) recovery of oxidative stress lipid peroxidation and promotion oxygen free radical scavenger content can be weakened, significantly suppress the expression that Neurons Against Cerebral Ischemia organizes NR2B albumen and mRNA, promote the phosphorylation of CREB and REK and the expression of ERK, CREB mRNA thereof.

Show that cytisine has the effect of neuronal damage caused by treatment cerebral infarction, can be used for the medicine preparing cerebral infarction.

Accompanying drawing explanation

Fig. 1: cytisine is to the TTC colored graph of mice focal ischemia injury protection effect;

Fig. 2: cytisine is on the HE of CA1 pathological change impact after focal cerebral ischemic in mice

Colored graph;

Fig. 3: cytisine affects the pathological change of focal cerebral ischemic in mice damage cortical layer

HE colored graph;

Fig. 4: DNA situ end labeling (Tunel dyeing) observe cytisine on the impact of murine cerebral ischemia side Hippocampus and cortical area apoptosis rate (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.05 ^*p<0.01 wherein, Fig. 4 A is murine cerebral ischemia side hippocampus CA1 (× 200), cortical areas (× 400) apoptosis figure, Fig. 4 B be murine cerebral ischemia side hippocampus CA1, cortical area apoptosis rate cartogram);

Fig. 5: cytisine murine cerebral ischemia side cortex NR2B immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01; Compare with model group: ^*p<0.01 wherein, Fig. 5 A is that murine cerebral ischemia side cortex NR2B positive cell expresses immunofluorescence figure (× 400), and Fig. 5 B is murine cerebral ischemia side cortex NR2B positive cell expression intensity cartogram);

Fig. 6: cytisine Hippocampal CA 1, murine cerebral ischemia side NR2B immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Fig. 6 A is murine cerebral ischemia side CA1 district NR2B positive cell immunofluorescence figure (× 200, × 400), and Fig. 6 B is Hippocampal CA 1, murine cerebral ischemia side NR2B positive cell expression intensity cartogram);

Fig. 7: cytisine murine cerebral ischemia side Hippocampus dentate body (DG) district NR2B immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Fig. 7 A is murine cerebral ischemia side dentate body NR2B positive cell immunofluorescence figure (× 200, × 400), and Fig. 7 B is murine cerebral ischemia side dentate body NR2B positive cell expression intensity cartogram);

Fig. 8: cytisine cortical area, focal cerebral ischemia side p-ERK immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Fig. 8 A is cortical area, murine cerebral ischemia side p-ERK positive cell immunofluorescence figure (× 400) to x, and Fig. 8 B is murine cerebral ischemia side cortex p-ERK positive cell expression intensity cartogram);

Fig. 9: cytisine Hippocampal CA 1, murine cerebral ischemia side p-ERK immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.05, ^*p<0.01 wherein, Fig. 9 A is Hippocampal CA 1, murine cerebral ischemia side p-ERK positive cell immunofluorescence figure (× 200, × 400), and Fig. 9 B is Hippocampal CA 1, murine cerebral ischemia side p-ERK positive cell expression intensity cartogram);

Figure 10: cytisine focal cerebral ischemia side Hippocampus dentate body (DG) district p-ERK immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.05, ^*p<0.01 wherein, Figure 10 A is murine cerebral ischemia side dentate body p-ERK positive cell immunofluorescence figure (× 200, × 400), and Figure 10 B is murine cerebral ischemia side dentate body p-ERK positive cell expression intensity cartogram);

Figure 11: cytisine focal cerebral ischemia side cortical areas p-CREB immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Figure 11 A is cortical area, murine cerebral ischemia side p-CREB positive cell immunofluorescence figure (× 200, × 400), and Figure 11 B is cortical area, murine cerebral ischemia side p-CREB positive cell expression intensity cartogram);

Figure 12: cytisine (compares with sham operated rats the impact that Hippocampal CA 1, mice ischemia side p-CREB immuning positive cell is expressed ^##p<0.01, compares with model group: ^*p<0.01 wherein, Figure 12 A is Hippocampal CA 1, murine cerebral ischemia side p-CREB positive cell immunofluorescence figure (× 200), and Figure 12 B is Hippocampal CA 1, murine cerebral ischemia side p-CREB positive cell expression intensity cartogram);

Figure 13: cytisine mice ischemia side dentate body (DG) district p-CREB immuning positive cell is expressed impact (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.05, ^*p<0.01 wherein, Figure 13 A is murine cerebral ischemia side dentate body p-CREB positive cell immunofluorescence figure (× 200, × 400), and Figure 13 B is murine cerebral ischemia side dentate body p-CREB positive cell expression intensity cartogram);

Figure 14: cytisine on the impact of mice ischemia side cerebral tissue NR2B protein expression level (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Figure 14 A is that mice respectively organizes cerebrum ischemia side cerebral tissue NR2B protein expression immunity printing and dyeing histogram, and Figure 14 B is that mice respectively organizes cerebrum ischemia side cerebral tissue NR2B protein expression level cartogram);

Figure 15: cytisine on the impact of mice ischemia side cerebral tissue p-ERK, t-ERK protein expression level (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01 wherein, Figure 15 A is that mice respectively organizes cerebrum ischemia side cerebral tissue p-ERK, t-ERK protein expression immunity printing and dyeing histogram, and Figure 15 B is that mice respectively organizes cerebrum ischemia side cerebral tissue p-ERK, t-ERK protein expression level cartogram);

Figure 16: cytisine on the impact of mice ischemia side cerebral tissue p-CREB, t-CREB protein expression level (compare with sham operated rats: ^#p<0.05 ^##p<0.01, compares with model group: ^*p<0.05 ^*p<0.01 wherein, Figure 16 A is that mice respectively organizes cerebrum ischemia side cerebral tissue p-CREB, t-CREB protein expression immunity printing and dyeing histogram, and Figure 16 B is that mice respectively organizes cerebrum ischemia side cerebral tissue p-CREB, t-CREB protein expression expression cartogram);

Figure 17: cytisine on the impact of mice ischemia side cerebral tissue NR2B mrna expression level (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01

Figure 18: cytisine on the impact of mice ischemia side cerebral tissue ERK mrna expression level (compare with sham operated rats: ^##p<0.01, compares with model group: ^*p<0.01

Figure 19: cytisine on the impact of mice ischemia side cerebral tissue CREB mrna expression level (compare with sham operated rats: ^#p<0.05 ^##p<0.01, compares with model group: ^*p<0.05 ^* ^*p<0.01

In above all cells immunofluorescence figure: A: sham operated rats, B: model group, C:1mg/kg cytisine group, D:6mg/kg nimodipine group.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further detailed explanation.

Embodiment 1

Cytisine is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, and the structural formula of cytisine is such as formula shown in (1):

Wherein, Ischemic Stroke is acute stage cerebral infarction, and cytisine single application dosage is only limitted to the dosage not causing blood glucose to reduce, and cytisine single application dosage is mice 0.25mg/kg, and the dosage form of medicine is injection type.

Embodiment 2

Wherein, Ischemic Stroke is reparation phase cerebral infarction, and cytisine single application dosage is only limitted to the dosage not causing blood glucose to reduce, and cytisine single application dosage is mice 0.5mg/kg, and the dosage form of medicine is pin powder-type.

Embodiment 3

Wherein, Ischemic Stroke is reparation phase cerebral infarction, and cytisine single application dosage is only limitted to the dosage not causing blood glucose to reduce, and cytisine single application dosage is mice 1mg/kg, and the dosage form of medicine is pin powder-type.

Zoopery below further illustrates the effect of above-described embodiment 1 to 3:

One, experiment material

1.1 animal process

The 2-3 monthly age, male ICR mouse, 25-30g, purchased from Ningxia Medical University's Experimental Animal Center, animal productiong credit number: NCXK (rather) 2013-0005.Feeding conditions comprises standard feed, tap water, and room temperature remains on (24 ± 2) DEG C, humidity 50-60%, illumination time every day each 12h.Before experiment, animal is placed in experimental situation and adapts to 3 days.

1.2 experimental drugs and instrument

Cytisine (Yanchi County, ningxia pharmaceutical factory, lot number 1612009), with normal saline, mother liquid concentration 0.2mg/mL ,-20 DEG C of storages.2, 3, 5-triphenyltetrazolium chloride (2, 3, 5-triphenyltetrazoliumchloride, TTC, purchased from sigma company), SOD, CAT, MDA, GSH-px test kit (building up Bioengineering Research Institute purchased from Nanjing), Tunel cell apoptosis detection kit (In Situ Cell Death Detection Kit, purchased from Roche company), the anti-NMDAR2B polyclonal antibody of rabbit (purchased from Protientech company), rabbit anti-p44/42MAPK ERK1/2 (137F5) polyclonal antibody, the anti-Phospho-p44/42MAPK of rabbit (ERK1/2) (Thr202/Tyr204) polyclonal antibody, the anti-CREB of rabbit (48H2) polyclonal antibody, the anti-Phospho-CREB polyclonal antibody of rabbit is (all purchased from Cell Signaling Technology, CST company).Commercial goods is, fluorescence microscope (BX51, Japanese olympus company) more than NR2B, CREB, ERK mRNA, internal reference primer (purchased from invitrogen company).Electrophresis apparatus, electroporation, gel imaging instrument.PCR instrument, real-time PCR, nucleic acid concentration analyzer (equal purchased from American Bio-RAD company).

1.3MCAO Establishment of mouse model: a mice preoperative curfew eats, freely drink water, lumbar injection 3.5% chloral hydrate anesthesia mice is adopted with the dosage of 0.1ml/10g, with reference to Longa method, with 0.1mm nylon wire preparation right side middle cerebral artery occlusion (middle cerebral arteryocclusion, MCAO) model.After ischemia 2h, mice, in the brain after obstruction of artery 2h, is again used chloral hydrate anesthesia, cuts off cervical region stitching thread, and be separated and expose external carotid artery (ECA), pull out Outlet bolt, ligation ECA stump, can realize Reperfu-sion.Layer-by-layer suture muscle, body of gland and skin, use iodophor disinfection wound to protect from infection, and lies on the back to be placed in incubator and to treat that anesthesia is clear-headed.Whole operation maintains 37 DEG C to recovery from anesthesia process body temperature.Sham operated rats animal, accepts identical anesthesia and operation process, but does not carry out medium-sized artery thromboembolism.

1.4 administrations detect with sampling: ICR mice is divided into focal cerebral medium-sized artery thromboembolism re-perfusion model group (I/R) at random, cytisine various dose group.Mice 1h before operation modeling presses 0.1ml/10g body weight intraperitoneal administration.Sham operated rats gives the normal saline of equivalent with method.After mice ischemia-reperfusion 24h, carry out delayed ischemic neurological deficits scoring, cerebral infarction volume, histopathology and morphological change, molecular biology express pharmacodynamic evaluations such as changing.

Two, experimentation

(1) neurological deficit scoring

1.1 experimental techniques:

With reference to the methods of marking of Bederson, adopt mono blind method to mark to mouse Nerve functional defect, neuromotor dysfunction scoring is carried out to each group of mice, 0 point: be normal, impassivity sign; 1 point: left fore becomes bent; 2 points: deflection circle during walking; 3 points: topple over to the left during walking; 4 points: to reduce with level of consciousness without autonomous.

1.2 experimental results:

As shown in Table 1, there is the forelimb not tensible of damage offside in MCAO model group mice Post operation, turn-takes or topple on the left of during walking, even cannot autonomous, level of consciousness reduce or the significant cerebral infarction delayed ischemic neurological deficits symptom such as death.Single intraperitoneal injection cytisine is amphicheirality on the impact of function of nervous system's moving obstacle caused by mice MCAO, when dosage increasing action strengthens, (contrasts with MCAO the most by force to protective effect during 1mg/kg ^*p ﹤ 0.05 or ^*p ﹤ 0.01), weaken with the increase protective effect of dosage after being greater than this dosage or occurred certain toxic reaction, test and establish nimodipine (6mg/kg) positive drug control group.Prompting cytisine (1mg/kg, 0.5mg/kg) has the effect alleviating delayed ischemic neurological deficits caused by mice MCAO, is dosage correlation to the protective effect of cerebral infarction nerve injury.

(2) cerebral infarction volume measures

2.1 experimental techniques:

With 3.5% chloral hydrate anesthesia mice and sacrificed by decapitation, take out cerebral tissue rapidly, remove olfactory bulb, cerebellum and low brain stem, the freezing 10min of organization embedding liquid-20 DEG C.The 2mm brain section cutting 5 thickness from front pole (anteriorpole) coronalplane is continuously placed in 2%2,3, in 5-triphenyltetrazolium chloride normal saline solution, 37 DEG C of lucifuges hatch 10min, and 10% formaldehyde neutral buffered liquid (pH7.4) fixedly spends the night.Automatic microspur camera shooting, Image pro plus5.1 image analysis software calculates and statistical brain Infarction volume.

2.2 experimental results:

As can be seen from table 1 and Fig. 1, single intraperitoneal injection cytisine is two-way reaction to the increase of cerebral infarction volume caused by mice MCAO, increases cerebral infarction volume and reduces, (contrast with MCAO the most by force to protective effect during 1mg/kg with dosage ^*p ﹤ 0.05 or ^*p ﹤ 0.01), the increase protective effect after this with dosage weakens or a few nothing, tests and establishes nimodipine (6mg/kg) positive drug control group.Prompting cytisine (1mg/kg, 0.5mg/kg) all has the effect reducing mice MCAO cerebral infarction volume.

(3) cerebral edema measures

3.1 experimental techniques:

After Cerebral Ischemia-reperfusion in Mice 24h, sacrificed by decapitation mice, complete cerebral tissue is taken out on ice bag, remove olfactory bulb, cerebellum and low brain stem, get ischemia side cerebral tissue, be weighed as weight in wet base, then put into 110 DEG C of oven for baking 24h, be weighed as dry weight, brain water content (%)=(weight in wet base-dry weight)/weight in wet base × 100%.

3.2 experimental results:

As shown in Table 1, single intraperitoneal injection cytisine is two-way reaction to the increase of cerebral infarction volume caused by mice MCAO, increases degree of cerebral edema and reduces, reduce degree (compare with MCAO the most by force to edema during 1mg/kg with dosage ^*p ﹤ 0.05 or ^*p ﹤ 0.01), the increase protective effect after this with dosage weakens or a few nothing, tests and establishes nimodipine (6mg/kg) positive drug control group.Prompting cytisine (1mg/kg, 0.5mg/kg) has the effect reducing MCAO mouse brain edema.

Table 1. cytisine damages Infarction volume to focal cerebral ischemic in mice, neurological deficit is marked and the impact of brain water content

(4) biochemical indicator detects

4.1 experimental techniques:

By the fast fetching brain that breaks end after mouse anesthesia, be stored in-80 DEG C of liquid nitrogen stand-by after the brain of normal saline of short duration cleaning ischemia side on ice.The glass homogenizer using soak with sulphuric acid to cross operates on ice by adding Tissue lysates by 9:1 (V/W), and homogenate loses piece of tissue to naked eyes 20 times, centrifuging and taking supernatant repeatedly.Use bovine serum albumin to press Coomassie Brilliant Blue and detect testing sample absorbance at microplate reader 512nm place, be total protein concentration standard curve (R ²>0.99) and measure each experimental mice cerebral tissue protein concentration, biochemical indicator detection is carried out in strict accordance with test kit operating instruction.

4.2 experimental results:

As shown in Table 2, single intraperitoneal injection cytisine presents two-way reaction to the changes of biochemical indexes caused by mice MCAO, antioxidant (SOD, GSH-px, CAT) increased activity in cerebral tissue is increased with dosage, lipid peroxidase (MDA) activity reduces, and (contrasts with MCAO to the oxygen free radical scavenger activity the strongest lipid peroxide activity in cerebral tissue during 1mg/kg is the most weak ^*p ﹤ 0.05 or ^*p ﹤ 0.01), the protective effect after this with the increase antioxidant stress injury of dosage weakens or a few nothing, tests and establishes nimodipine (6mg/kg) positive drug control group.Prompting cytisine (1mg/kg, 0.5mg/kg) all has the oxidativestress damage effect alleviating MCAO Mice brain tissues.

Table 2. cytisine is to MDA content, SOD, GDH-in focal cerebral ischemic in mice damaged tissue px, CAT activity impact

(5) HE dyeing tissues observed structure pathological change

5.1 experimental techniques:

Hematoxylin-eosin dyeing (hematoxylin-eosin staining, HE staining), paraffin section toasts 20min in 65 DEG C of baking ovens, slide is soaked in successively dimethylbenzene I (10min) afterwards, dimethylbenzene II (5min), dehydrated alcohol I (1min), dehydrated alcohol II (1min), 95% ethanol (30s), 80% ethanol (30s) and 70% ethanol (30s), wash three times, 5min is soaked afterwards in haematoxylin, wash three times, 20s is soaked in the hydrochloride alcohol solution of 1%, washing, 2min is soaked in the solution of Yihong, water speed is washed, paraffin section continues to be soaked in 80% ethanol (30s) successively afterwards, 95% ethanol (30s), dehydrated alcohol I (1min), dehydrated alcohol II (3min), dimethylbenzene I (2min) and dimethylbenzene II (2min), finally use neutral gum mounting, after often group Mice brain tissues paraffin section HE has dyeed, be placed in basis of microscopic observation, often open section and get 200 × and 400 × two visuals field respectively, each visual field is got 3-6 region respectively and is carried out Taking Pictures recording.

5.2 experimental results:

As shown in Figures 2 and 3, single intraperitoneal injection cytisine presents two-way reaction to the organizational structure pathological change impact caused by mice MCAO, contrast MCAO model group increases the cortex in cerebral ischemia region with dosage and the damaging pathological change of hippocampus alleviates, cellular edema, cavity sample reduces, karyopycnosis limit collection weakens, necrocytosis and neutral grain are ingratiated with inflammatory exudation phenomenon and are reduced, cell membrane integrity and cell arrangement distributing homogeneity by force cumulative, the lightest to cerebral tissue inner cell structure pathologic damage during 1mg/kg, contrast with MCAO, the protective effect that dosage is greater than 1mg/kg or is less than 0.5mg/kg antibody Monoclonal weakens or a few nothing, test and establish nimodipine (6mg/kg) positive drug control group, prompting cytisine (1mg/kg, 0.5mg/kg) there is the effect alleviating MCAO Mice brain tissues cellularity pathologic damage.

(6) apoptosis detects

6.1 experimental techniques:

In situ terminal labeling (Tunel) method of DNA break surveys apoptosis degree in the cerebral tissue of mice ischemia side, often organize mice and get 6 paraffin sections (every sheet is with the ultra-thin cerebral tissue sample of 2-3 sheet) containing ischemia side cerebral tissue, rewarming process 45min in 65 DEG C of baking ovens.Cerebral tissue dewaxes: slide is soaked in dimethylbenzene I, dimethylbenzene II (each 10min) successively, dehydrated alcohol I, dehydrated alcohol II (each 10min), 95% ethanol (5min), 90% ethanol (5min), 80% ethanol (5min) and 70% ethanol (5min), carry out dewaxing treatment.Continue and use PBS soaking flushing 3 times, each 5mim, suck dry moisture, drip E.C. 3.4.21.64, process 30min in 37 DEG C, PBS rinses.Slide adds Tunel reactant liquor and rinses 3 times, each 5min at 37 DEG C of process 1h, PBS, then drips the fluorescein-labelled liquid of DAPI nucleus.Tunel detects apoptosis and establishes the positive and negative control simultaneously.Basis of microscopic observation, slide gets 200 × and 400 × two visuals field observe mice ischemia side cortexes and Hippocampus, each visual field is got 6 regions and is carried out observing and taking pictures.Count often to organize and often open apoptotic cell and normal cell number in the cerebral tissue of photo small mouse ischemia side, thus draw apoptosis rate.

Apoptosis rate=apoptotic cell number/(apoptotic cell number+normal cell number) × 100%.

6.2 experimental results:

As shown in Figure 4; the Apoptosis of lumbar injection cytisine to the ischemic area caused by mice MCAO presents two-way reaction; contrast MCAO model group increases with dosage; cortex and the Apoptotic cells of brain hippocampus number quantity in cerebral ischemia region reduce gradually; inner cell apoptosis minimum number is knitted to brain during 1mg/kg; protective effect is remarkable, simultaneously ischemic tissue's surrounding normal cell arrangement align mutually, fine and close, compare with MCAO ^*p ﹤ 0.05 or ^*p ﹤ 0.01, tests and establishes nimodipine (6mg/kg) positive drug control group, and prompting cytisine (1mg/kg, 0.5mg/kg) all has the apoptotic effect of reduction MCAO focal cerebral ischemia regional organization.

(7) determination of immunofluorescence method focal cerebral ischemia damage side brain tissue cell p-CREB, p-ERK, NR2B expression

7.1 experimental techniques:

Paraffin section 65 DEG C of baking oven for heating 45min, conventional dimethylbenzene dewaxing, gradient alcohol dehydration, section is immersed in the pressure cooker of the sodium citrate buffer containing 0.01M/L and is boiled 4min, naturally cools to room temperature, completes antigen hot repair multiple.Drip 3% hydrogen peroxide at room temperature and soak 10min blocking-up endogenous peroxidase activity.Lowlenthal serum closes heterogenetic antigen, and room temperature places 20min, does not rush, and drip primary antibodie 4 DEG C by certain dilution ratio and spend the night, next day takes out, and 37 DEG C of rewarming 45min, rinse 3 times with PBS, each 5min.The FITC labelling goat antirabbit two dripping 1:20 resists, in wet box, lucifuge 37 DEG C hatches 1h, PBS rinses afterwards, drip fluorescent dye Dapi process 15min mounting, often open section under Hippocampus and cortex get 6 regions 400 × and 200 × visual field fluorescence microscope, take pictures, and respectively organize the average optical density value of each associated protein in the cerebral cortex of mice ischemia side by IPP software statistics.

7.2 experimental results:

As shown in Fig. 5 to Figure 13, by the intensity that Immunofluorescence test histiocyte NR2B, p-ERK, p-CREB, immuning positive cell are expressed, cortical area, each group of focal cerebral ischemia side, Hippocampal CA 1 and DG district tissue slice all visible positive signal under fluorescence microscope is expressed, contrast sham operated rats, cytisine (1mg/kg), nimodipine (6mg/kg) administration group NR2B, p-ERK, p-CREB positive signal significantly strengthen ( ^##p ﹤ 0.01); From Fig. 5 to Fig. 7, contrast MCAO model group, cytisine (1mg/kg), its NR2B immuning positive cell of nimodipine (6mg/kg) administration group above 3 Zonal expression quantity and positive signal intensity thereof significantly decline ( ^*p ﹤ 0.01), positive signal intensity and object weaken because of nuclear translocation protein expression phenomenon; From Fig. 8 to Figure 13, contrast MCAO model group, in cytisine (1mg/kg) and nimodipine (6mg/kg) administration group, cell ERK, CREB protein phosphorylation strengthens, and karyon is expressed obviously, cortex, DG district cell p-ERK positive signal significantly strengthen ( ^##p<0.01), hippocampus cell positive signal enhancing ( ^*p ﹤ 0.05); From Figure 11 to Figure 13, contrast MCAO model group, above 3 region cell p-CREB positive signal significantly strengthen ( ^##p ﹤ 0.01); Prompting cytisine (1mg/kg) protective effect to mice MCAO cerebral ischemia may be the downstream NR2B-ERK-CREB signal path by participating in the induction of regulation and control excitatory amino acid; reduce the expression of NR2B albumen, promote ERK, CREB protein phosphorylation.

(8) Western blot detects the expression of focal cerebral ischemia damage side cerebral tissue p-CREB/t-CREB, p-ERK/t-ERK, NR2B albumen

8.1 experimental techniques:

Use triumphant base whole protein to extract test kit and extract total protein, BCA protein content detection kit measures sample total protein concentration and demarcates albumen unifies concentration.SDS-polyacrylamide gel electrophoresis, carries out wet method transferring film with by nitrocellulose filter (NC film).Transferring film terminates rear taking-up nitrocellulose filter, is inserted by film in 5% defatted milk powder confining liquid and closes 1h.Closed end hatch 5% defatted milk powder dilution primary antibodie, wash film hatch two resist.NC film is washed three times, each 10min with PBST.Drip protein chemistry luminous agent (ECL), NC film is fixed in film magazine, and press-in film exposes.Take out film, put into developer solution and each 1min of fixative solution, last clean water.Gel image analysis imaging system (Bio-Rad, Universal Hood II) scans and graphical analysis object band each on film.

8.2 experimental results:

As shown in Figure 14 to Figure 16, compare with sham operated rats, model group Cerebral Ischemia-reperfusion in Mice damage side cerebral tissue nmda receptor subunit NR2B albumen, the relative sham operated rats of p-ERK, p-CREB protein expression obviously increase ( ^##p<0.01), t-ERK, t-CREB protein expression there was no significant difference; As seen from Figure 14, compare with model group, cytisine (1mg/kg), nimodipine (6mg/kg) organize NR2B protein expression significantly reduce ( ^*p ﹤ 0.01), and the result of Figure 15 and Figure 16 display p-ERK, p-CREB protein expression significantly increase ( ^*p ﹤ 0.01), t-ERK, t-CREB protein expression there was no significant difference, the protective effect of prompting cytisine may be and pass through to regulate NR2B-ERK-CREB signal path, reduces the expression of NR2B albumen, promotes ERK, CREB protein phosphorylation.

(9) PCR real time fluorescent quantitative detects NR2B, ERK, CREB mrna expression level

9.1 experimental techniques:

Carry out design of primers according to NCBIGenbanck small mouse nmda receptor NR2 subunit (NR2B), CREB, ERK and mactinf gene order number, primer is synthesized by invitrogen company:

NR2B forward primer: 5'-TCTGAGCATCGTTACCTTGG-3'

NR2B downstream primer: 5'-CTTCTGGCACGGGACTGTAT-3'

Product length: 100bp

CREB forward primer: 5'-TAGTCCCAGCAACCAAGT-3'

CREB downstream primer: 5'-GGACGCCATAACAACTCCAG-3'

Product length: 110bp

ERK forward primer: 5'-CTCTCATTTGAGGACAGGCA-3'

ERK downstream primer: 5'-ATGTGGCATGCAGTGTAGGT-3'

Product length: 129bp

Mactinf forward primer: 5'-GAGACCTTCAACACCCCAGC-3 '

Mactinf downstream primer: 5'-ATGTCACGCACGATTTCCC-3 '

Product length: 285bp

Annealing temperature: 57.9 DEG C

Extract test kit operating instruction carry out total tissue RNA extraction in strict accordance with the love RNA that pursues progress.RNA reverse transcription is carried out with TransScript First-Strand cDNA Synthesis SuperMix test kit.In strict accordance be template with cDNA on real-time fluorescence quantitative PCR instrument according to TransStart Green qPCRSuperMix test kit operating instruction with the amplification condition optimized under carry out PCR specific amplification and fluorescent quantitation.

9.2 experimental results:

As shown in Figures 17 to 19, the result display of quantitative fluorescent PCR, compared with sham operated rats, model group NR2B mrna expression relative value obviously increases ( ^##p<0.01), ERKmRNA, CREB mrna expression relative value significantly reduce ( ^##p<0.01); Compared with model group, the administration group NR2B mrna expression relative value that cytisine (1mg/kg) and nimodipine (6mg/kg) are organized significantly reduces ( ^*p ﹤ 0.01), ERK mRNA, CREB mrna expression relative value significantly increases ( ^*p ﹤ 0.01), the protection of prompting cytisine is done may be and the expression by reducing NR2B mRNA promote that the expression of ERK mRNA, CREB mRNA is relevant.

Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, without departing from the concept of the premise of the invention, can also make some distortion and improvement, these all belong to protection scope of the present invention.

Claims

1. cytisine is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: the structural formula of described cytisine as the formula (1):

Formula (1).

2. cytisine according to claim 1 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described Ischemic Stroke is the cerebral infarction of acute stage or the phase of reparation.

3. cytisine according to claim 1 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is mice 0.25 ~ 1.0mg/kg.

4. cytisine according to claim 3 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is mice 0.25mg/kg.

5. cytisine according to claim 3 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is mice 0.5mg/kg.

6. cytisine according to claim 3 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is mice 1mg/kg.

7. cytisine according to claim 1 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is rat 0.0875/kg ~ 0.35mg/kg, people 0.014mg/kg ~ 0.056gmg/kg; Standard body weight people 70kg single use amount 1mg ~ 4mg.

8. cytisine according to claim 1 is as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, it is characterized in that: described cytisine single application dosage is only limitted to the dosage not causing blood glucose to reduce.

9. the cytisine according to any one of claim 1-8 claim, as the purposes for the treatment of ischemic cerebral apoplexy Chinese medicine, is characterized in that: the dosage form of described medicine is peroral dosage form, injection type or injectable powder type that pharmaceutics allows.