CN104857319B

CN104857319B - A kind of pharmaceutical composition and its preparation method and application for treating diabetes mellitus encephalopathy

Info

Publication number: CN104857319B
Application number: CN201510314447.0A
Authority: CN
Inventors: 王飞; 张廷模; 袁有才
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu Huashen Technology Group Co.,Ltd.
Priority date: 2015-06-09
Filing date: 2015-06-09
Publication date: 2018-08-03
Anticipated expiration: 2035-06-09
Also published as: CN104857319A

Abstract

The present invention provides a kind of pharmaceutical compositions for treating diabetes mellitus encephalopathy, it is the preparation that the raw material matched by following weight is prepared：12 18 parts of pueraria lobata, 8 12 parts of the coptis, 8 12 parts of radix scutellariae, 12 18 parts of the stem of noble dendrobium, 24 parts of bear gall, 24 parts of Radix Notoginseng.The present invention also provides the preparation method of the pharmaceutical composition and purposes.Medical treatment diabetes mellitus encephalopathy of the present invention is with obvious effects, is better than Cilostazol, especially best with middle dose group.Drug of the present invention promotes angiogenesis, improves hippocampus hypoxic-ischemic state, adjust hippocampus IGF 1 and PI3K/AKT/CREB signal path activity by protecting capilary (blood-brain barrier).

Description

A kind of pharmaceutical composition and its preparation method and application for treating diabetes mellitus encephalopathy

Technical field

The present invention relates to a kind of pharmaceutical compositions for treating diabetes mellitus encephalopathy, belong to drug field.

Background technology

Diabetes (diabetes mellitus, DM) and its complication have become the health problem of public's concern, Because recently as socio-economic development, global diabetic far beyond speed expected from people to increase sharply, according to the world It is 3.7 hundred million that diabetes alliance (IDF), which counts global diabetic's number in 2011, and developing country just accounts for the 80% of morbidity, Diabetes prevalence is even more to reach 9.7% in China 20 years old or more crowd, has become the most country of diabetic.Glycosuria The course of disease is long, complication is more, medical treatment cost is high, and extreme influence is caused to patient and society.Currently, diabetes, tumour, heart and brain blood Noninfectious disease based on pipe disease etc. has become the principal disease of global death.Carry out the prevention of diabetes and complication Research has great social effect.

Diabetes are the metabolic diseases characterized by chronic hyperglycemia, and principal pathogenetic is the reason is that not due to insulin secretion Foot or (and) caused by insulin action defect.Due to sugar, fat, protein metabolism disorder cause whole body multisystem, a variety of groups It knits, the damage of the pathologic, physiologic of organ, the organ complications such as eye, kidney, heart and brain is caused to occur.Wherein neurological complication is most normal See, such as diabetic neuropathy, ischemic cerebrovascular disease, atherosclerosis.Currently, medical research is to diabetes both at home and abroad More, the diabetes mellitus encephalopathy (Diabetic caused by diabetes central lesion is reported in peripheral neuropathy research Encephalopathy, DE) concern it is less.In recent years, people gradually recognize the importance of diabetes mellitus encephalopathy, start to put forth effort Research.

Diabetes mellitus encephalopathy is using cognition dysfunction as outstanding behaviours, simultaneously with intracerebral structure, one kind of pathophysiological change Send out disease；Also there is scholar to propose to be named as " decline of diabetes related cognitive ", do not obtain yet at present unified.Its pathogenesis, pathology Physiological change is still unclear, previously from glucose toxicity, insulin signaling obstacle, calcium homeostasis is unbalance, inflammation, oxidative stress damage Wound, vascular lesion and hypothalamus-pituitary-adrenal axis Anomaly, obtain some progress.Also have and think that diabetes increase A Er The incidence of Zi Haimo diseases, it is believed that sporadic alzheimer disease represents diabetes brain type or insulin brain is resisted, referred to as 3 types sugar Urine disease.However cerebral functional deterioration in diabetes mellitus encephalopathy, mechanism are still difficult to reach, because and not all diabetes all occur recognize work( It can damage, also only part alzheimer disease has diabetes medical history.

Diabetes mellitus encephalopathy is the chronic complicating diseases of diabetes characterized by acquired cognitive behavior defect, should belong to Chinese medicine and disappear Thirsty disease merges dementia, forgetful scope, kidney qi virtual loss, and damp phlegm, hemostasis, the mutual knot of turbid poison are the basic diseases that diabetes mellitus encephalopathy occurs Machine answers kidney tonifying, essence replenishing nutrition brains to effect a permanent cure on clinical treatment, and to take stopgap measures, treating both manifestation and root cause of disease can improve improvement blood supply promoting blood circulation and removing blood stasis Cognitive impairment in diabetic patients.

Invention content

The technical solution of the present invention is to provide a kind of pharmaceutical compositions of new treatment diabetes mellitus encephalopathy.The present invention's is another There is provided the preparation method of the pharmaceutical composition and purposes for one technical solution.

The present invention provides a kind of prevention or/and the pharmaceutical compositions for the treatment of diabetes mellitus encephalopathy, it is matched by following weight The preparation that the raw material of ratio is prepared：

12-18 parts of pueraria lobata, 8-12 parts of the coptis, 8-12 parts of radix scutellariae, 12-18 parts of the stem of noble dendrobium, 2-4 parts of bear gall, 2-4 parts of Radix Notoginseng.

It is further preferred that it is the preparation that the raw material matched by following weight is prepared：

12 parts of pueraria lobata, 6 parts of the coptis, 9 parts of radix scutellariae, 12 parts of the stem of noble dendrobium, 1.5 parts of bear gall, 3 parts of Radix Notoginseng.

Pharmaceutical composition of the present invention be by pueraria lobata, the coptis, radix scutellariae, the stem of noble dendrobium, bear gall, Radix Notoginseng primary medicinal powder, water or organic Solvent extractable matter is active constituent, and the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared is added.

Wherein, the preparation is tablet, capsule, granule, pill, oral solution.

The present invention also provides a kind of methods preparing the pharmaceutical composition, it is characterised in that：It includes following step Suddenly：

A, the bulk pharmaceutical chemicals of each weight proportion are weighed；

B, pueraria lobata, the coptis, radix scutellariae, the stem of noble dendrobium add water to cook concentration；

C, bear gall, Radix Notoginseng is taken to beat powder；The concentrate and pharmaceutically acceptable auxiliary material or auxiliary of addition b step Property ingredient is prepared into pharmaceutically common preparation.

The present invention also provides the pharmaceutical compositions in preparing Prevention or/and treating the drug of diabetes mellitus encephalopathy Purposes.

Wherein, the drug is the drug for mitigating cognition dysfunction, preventing hippocampal neuron microvascular damage.

Wherein, the drug is prevention hippocampal neuron microvascular damage, protects tight junction protein and protect blood brain Barrier safeguards the drug of brain homeostasis.

Wherein, the drug is protection hippocampus microvascular endothelial cells, promotes endothelial progenitor cells migration and differentiation, promotes sea The micro- blood in horse area is newborn and improves the drug of diabetes mellitus encephalopathy ability of learning and memory.

Wherein, the drug is to improve by adjusting hippocampus IGF-1 and PI3K/Akt/CREB signal path activity Practise the drug of memory function.

Drug of the present invention is made of pueraria lobata, radix scutellariae, the coptis, the stem of noble dendrobium, bear gall powder, Radix Notoginseng powder, full side's replenishing the vital essence and removing heat, promoting blood circulation solution Malicious dredging collateral and treat diabetes mellitus encephalopathy.Pueraria lobata, stem of noble dendrobium nourishing generate fluid are monarch drug in a prescription, the coptis, radix scutellariae bitter-cold herbs expelling heat eliminating dampness, purging intense heat in side Removing toxic substances, is altogether ministerial drug, and bear gall powder is clearing heat and detoxicating, and Radix Notoginseng powder promoting blood circulation and removing blood stasis is adjutant.

Medical treatment diabetes mellitus encephalopathy of the present invention is with obvious effects, is better than Cilostazol, especially best with middle dose group.The present invention Drug promotes angiogenesis, improves hippocampus hypoxic-ischemic state, adjust hippocampus IGF-1 by protecting capilary (blood-brain barrier) And PI3K/AKT/CREB signal path activity.

Description of the drawings

The CA 1 of Hippocampus Fig. 1 HE dyes picture (1. blank group 2. model group 3. Chinese medicine large dosage group 4. Chinese medicine middle dosage 5. 6. western medicine group (SP × 400), white arrow are spongiocyte to Chinese medicine small dose group to group, and black arrow is karyopycnosis necrosis Neuron)

Fig. 2 rat hippocampus Electronic Speculum nervus vasculairs members picture (1. blank group 2. model group 3. Chinese medicine large dosage group 4. in Chinese medicine Dosage group 5. Chinese medicine small dose group 6. western medicine group (SP × 10000))

Fig. 3 rat hippocampus Electronic Speculum neuronal synapses picture (1. blank group 2. model group 3. Chinese medicine large dosage group 4. in Chinese medicine Dosage group 5. Chinese medicine small dose group 6. western medicine group (SP × 40000))

(1. 2. 3. diabetic sciatic nerve is 4. for diabetes rat brain for normal rat for Fig. 4 azovan coerulen vascular leakages figure 5. 6. diabetic glomeruli oozes out glomerular capillary to diabetes rat liver to diabetes rat lung tissue.)

Occludin expressions figure in Fig. 5 rat hippocampus

Claudins-5 expressions figure in Fig. 6 rat hippocampus

JAM-1 expressions figure in Fig. 7 rat hippocampus

ZO-1 expressions figure in Fig. 8 rat hippocampus

Fig. 9 HIF-1 α expand solubility curve

Figure 10 GAPDH expand solubility curve

Figure 11 GAPDH expand solubility curve

Figure 12 rat peripheral blood endothelial progenitor cells are expressed and variation (A negative controls, B detection gatings, C EPC_SPositive signal Figure)

Figure 13 EPC_STrend chart after diabetes

Figure 14 EPC_SEach group compares figure after diabetes

HIF-1 α mRNA, VEGFmRNA expressions (※ P compared with blank group in Figure 15 hippocampus<0.05, ※ ※ and blank Group compares P<0.01, * compared with model group P<0.05, * * P compared with model group<0.05.)

(1. 2. 3. Chinese medicine large dosage group is 4. for model group for blank group for the expression (S-P × 400) of Figure 16 each group rat hippocampus CD34 Chinese medicine middle dose group 5. Chinese medicine small dose group 6. western medicine group)

Figure 17 PI3K amplification solubility curve by

Figure 18 AKT expand solubility curve

Figure 19 CREB expand solubility curve

Figure 20 GAPDH expand solubility curve

PI3KmRNA, AKTmRNA, CREBmRNA are expressed in Figure 21 hippocampus

Expression (S-P × 200) (1. blank group 2. model group 3. big dose of Chinese medicine of Figure 22 each groups CA 1 of Hippocampus IGF-1 Amount group 4. Chinese medicine middle dose group 5. Chinese medicine small dose group 6. western medicine group)

Comparison (※ ※ P compared with blank group of Figure 23 each groups CA 1 of Hippocampus IGF-1<0.01, * with model group ratio Compared with P<0.05, * * P compared with model group<0.05.)

Expression (S-P × 400) (1. blank group 2. model group 3. big dose of Chinese medicine of Figure 24 each groups CA 1 of Hippocampus CREB Amount group 4. Chinese medicine middle dose group 5. Chinese medicine small dose group 6. western medicine group)

Expression (S-P × 400) (※ ※ P compared with blank group of Figure 25 each groups CA 1 of Hippocampus CREB<0.01, * with Model group compares P<0.05, * * P compared with model group<0.05.)

Specific implementation mode

The preparation of 1 drug suspension oral solution of the present invention of embodiment

Take pueraria lobata 12g, coptis 6g, radix scutellariae 9g, stem of noble dendrobium 12g, bear gall powder 1.5g, Radix Notoginseng powder 3g；Jin Chai may be used in the stem of noble dendrobium The stem of noble dendrobium, pueraria lobata apply elegant jessamine, the coptis to be produced for Chongqing；

Experiment is water decoction, and pueraria lobata 12g coptis 6g radix scutellariae 9g stem of noble dendrobium 12g water boiling concentrations are the solution of 0.65g/ml, Bear gall powder, Radix Notoginseng powder is added, suspension is made.

The preparation of 2 medicinal granule of the present invention of embodiment

Pueraria lobata 12g, coptis 6g, radix scutellariae 9g, stem of noble dendrobium 12g, bear gall 1.5g, Radix Notoginseng 3g are taken, is extracted as described in Example 1, Concentration, granulation, whole grain.

The preparation of 3 medicine capsule of the present invention of embodiment

Pueraria lobata 12g, coptis 6g, radix scutellariae 9g, stem of noble dendrobium 12g, bear gall 1.5g, Radix Notoginseng 3g, are extracted as described in Example 1, dense Contracting, granulation, whole grain are encapsulated.

Beneficial effects of the present invention are proved below by way of pharmacodynamics test.

Influence of 1 drug of the present invention of test example to diabetes mellitus encephalopathy Cognition Function in Rats

One, experiment material

1. experimental animal

Healthy SPF grades of hero SD rats 120, weight 200g ± 20g, experimental animal are purchased from Xi'an Communications University experimental animal Center, quality certification book number SCXK (Shan) 2012-003. are in Medical experimental center animal house sub-cage rearing, per 10, cage.Animal house At 20-26 DEG C, relative humidity control keeps illumination in 12 hours, night to turn off the light on 45-65%, ventilation, daytime for temperature control.

2. observing drug

2.1 drugs of the present invention：It is prepared by embodiment 1, pueraria lobata, the coptis, radix scutellariae, stem of noble dendrobium decocting filtering, it is every milliliter to concentrate 0.65g containing crude drug.Crude drug is purchased from Beijing Tongrentang (Bozhou) company, bear gall powder is added before gavage, suspension is made in Radix Notoginseng powder 0.725g/l。

2.2 control drug：Cilostazol：50mg/ pieces, Zhejiang great Zhong pharmaceutical Co. Ltds.

2.3 high lipid food：10% lard, 10% sucrose, 2.5% cholesterol, 77.5% basal feed；Basal feed is by west Pacify university of communications's animal experimental center to provide, high lipid food is voluntarily prepared according to formula.

Two, experimental method

1. diabetes mellitus encephalopathy model copy

SPF grades of SD rats 120, male, animal adaptable fed 3 days under the conditions of available light, free water are ingested, Separating 15 is only used as blank control group at random, gives normal diet nursing；Remaining 105 is only used as modeling group and gives high lipid food (10% lard, 10% sucrose, 2.5% cholesterol, 77.5% basal feed) uses intraperitoneal injection STZ's after feeding 4 weeks, 4 weeks Method manufactures rat diabetes model, and fasting (can't help water) 12h, is dissolved in citrate buffer by STZ before rat modeling 1% solution is made in (0.1mmol/L, pH=4.3), and disposable celiac injection chain urine makees rhzomorph 45mg/kg.Tail vein after 3 days Blood glucose is surveyed in blood sampling, and the rat for reaching 16.7mmol/L thinks that diabetes model is successfully established, is included in experiment, not up to glycemic criteria To be considered as modeling unsuccessful, reject.Blank control group gives the citrate buffer of intraperitoneal injection equivalent.

2. grouping and treatment

Reach totally 96 of glycemic criteria Cheng Mo, is divided into STZ model groups, drug bolus group of the present invention, the present invention immediately Drug middle dosage, drug small dose group of the present invention, Cilostazol control group, every group 19.Blood glucose, blood are surveyed again within 7 days after modeling Sugar person still up to standard enters pharmaceutical intervention, blank group and model group：Pure water 1ml/100g gavages, 1 times/day.Large dosage treatment Group：Drug water decoction 1ml/100g (0.725g/ml crude drugs are equivalent to 10 times of adult's kg body weight dosage) of the present invention, in Dosage treatment group：(0.363g/ml crude drugs are equivalent to the 5 of adult's kg body weight dosage to drug water decoction 1ml/100g of the present invention Times), low dose for the treatment of group：(0.182g/ml crude drugs are equivalent to adult's kg body weight and use drug water decoction 1ml/100g of the present invention 2.5 times of dose), western medicine group：(1.65mg/ml crude drugs are equivalent to adult's kg body weight medication to Cilostazol 1ml/100g 5 times of amount) gavage, once a day, above each processing method adjusts at any time according to changes of weight, and during which all rats freely drink Water with ingest.Continuous 8 weeks.Water maze test is carried out after perfusion, judges rat intelligence situation.

3. observation index

(1) ordinary circumstance is observed：The rat state of mind, activity condition, chroma of hair, drinking-water, feed, bedding and padding etc..

(2) rat blood sugar, weight are surveyed weekly primary；

(3) insulin level measures

(3) Morris water maze tests

(4) hippocampus morphological change：Light microscopic, Electronic Speculum.

4.Morris water maze tests

Morris water mazes are developed using institute of Materia Medica,Chinese Academy of Medical Sciences to be tested, which is diameter 214cm, high 50cm, depth of water 30cm, black matrix when starting experiment, prepared Chinese ink are instilled in water, water is contaminated black.According to suitable on pool wall Clockwise is divided into west, south, east, four, north quadrant along four white marking points of equidistant label, by pond.In Chi Zhongbei quadrants Put an a diameter of 10cm, the circular platform of high 28cm in center.There are one cameras for installation right over pond, can clearly shoot To entire pond.Experiment carries out in quiet, artificial light room, and water temperature will control when testing at 20-23 DEG C, daily will be more The water in pond is changed, fasting 4 hours before testing, hair dryer dries up experimental rat after experiment.

(1) orientation navigation is tested：It needs to be carried out continuously 4 days, 4 place of entry (different daily) is selected outside platform quadrant, Animal is faced into pool wall gently into the water when experiment, cannot disturb the water surface, record rat is from water is entered to climbing up what platform was undergone Time absconds incubation period, then rat is allowed to stop 15s on platform.If can not find platform in 120s, incubation period note of absconding For 120s, and rat is placed in rest 15s on platform.4 achievements of the preclinical arithmetic equal value as this day of absconding carry out Statistical analysis, incubation period of absconding represent the Spatial learning ability of rat.

(2) space exploration is tested：After orientation navigation off-test 1 day, platform is removed, does space exploration experiment.By rat Into the water, record rat enters the pond went swimming path after water in 120s and enters target area quadrant offside quadrant where the platform Index of the number in domain as evaluation Rats With Memory achievement.

5. the acquisition of sample

Each group rat fasting 12 hours before execution are weighed, tail vein surveys blood glucose.

Rat anesthesia, anesthesia are injected intraperitoneally using 10% chloraldurate by the standard of 0.3ml/100g after surveying blood glucose, fiber crops After liquor-saturated satisfaction, dorsal position is fixed on mouse platform, and cutting off thoracic cavity from xiphoid-process exposes heart, observes the position of the left heart and right auricle of heart, Needle for injection (blood transfusion specialized transfusion system) is inserted into aorta ascendens from the left apex of the heart, while cutting off right auricle of heart, 0.9% sodium chloride of ice Injection 200ml rinse to trickle it is limpid after, 250ml is perfused in 4% paraformaldehyde, and rat body places 2 hours after hardening, Broken end takes brain, a part of rat cerebral tissue to be placed in 10% formaldehyde and fix overnight on rapid ice pan, specimens paraffin embedding slices thickness 5um, row HE is dyed.Another part rat cerebral tissue is placed in 4% paraformaldehyde and fixes overnight, and specimens paraffin embedding slices thickness 5um carries out immune group Change and measures.

After doing 0.9% chloride injection perfusion of Electronic Speculum rat, using 4% paraformaldehyde and 0.5% glutaraldehyde mixed liquor 250ml is perfused, rapid ice pan broken end takes brain, detaches hippocampus, take and accomplish 1³5 pieces of the hippocampal tissue of mm, implantation 2.5% glutaraldehyde electricity Mirror liquid inspection.

6.HE is dyed

(1) tissue paraffin block cuts the slice of 5um, drags for piece, lacing film；

(2) slice is set into oven 56 DEG C of roasting pieces 30 minutes；

(3) conventional to dewax to water：Slice is placed in dimethylbenzene I 15 minutes respectively by following procedure, dimethylbenzene II 15 minutes, Followed by enter 100%, 95%, 90%, 80%, 70% alcohol respectively each 5 minutes, remove removal xylene, distilled water flushing 5 minutes removes Remove alcohol；

(4) bush sperm contaminates 10~15 minutes, extra dye liquor on pure water rinsing slide；

(5) 0.5% acidic alcohols break up the several seconds, and flowing water rinses 3~5 minutes；

(6) Yihong liquid dyes 5 minutes, pure water rinsing 3 minutes；

(7) it is dehydrated：It is put into 70%, 80%, 90%, 95%, 100% alcohol each 5 minutes；

(8) transparent 10 minutes of dimethylbenzene, keep sample transparent；

(9) sealing：Appropriate neutral gum capping slide mounting is added dropwise；

(10) optical microphotograph microscopic observation rat hippocampus morphology and neuron distribution.

7. electron microscope specimen makes

By 1³5 pieces of the hippocampal tissue of mm, the glutaraldehyde fixer for being placed in the 2.5% of 4 DEG C (contain 0.1M phosphoric acid in fixer Buffer solution, 4% paraformaldehyde) in fix 2 hours or more, 0.1M phosphate buffers embathe 30 minutes, place into the 1% 4 of 4 DEG C Somuum oxide fixer fixes 2 hours after (containing 0.1M phosphate buffers in fixer), and 0.1M phosphate buffers embathe 10 minutes, Alcohol serial dehydration：30% alcohol 10 minutes, 50% alcohol 10 minutes, 70% alcohol 10 minutes；70% acetate ethanol uranyl Block dye is overnight；90% alcohol 10 minutes, 2 times；100% alcohol 10 minutes, 3 times repeatedly；Also Ethylene Oxide is replaced 10 minutes；Asphalt mixtures modified by epoxy resin Fat Epon812 is impregnated with, embeds, and makees half ultra-thin section 1-2 μ after polymerization, is positioned under an optical microscope after methylene blue dyeing, use is auspicious V type ultramicrotome of allusion quotation LKB- carries out ultra-thin section 50-70nm, after acetic acid uranium, lead citrate dyeing, Hitachi, Japan H-600/ It observes, take pictures under Hitachi, Japan H-7650/ Japan Electronics JEM-100SX transmission electron microscope.

Three, statistical procedures

It is handled using spss17.0 softwares, data are indicated with mean ± standard deviation (Mean ± SD), and comparison among groups are using single Analysis of variance, P<0.05 is statistically significant for difference.

Four, experimental result

1. ordinary circumstance modeling group passes through 4 weeks high fat diets, rat body weight obviously increases, most weight 350-420g it Between, obesity is formed, more drinks obviously occurs in rat after STZ is injected, mostly food, diuresis (is replaced, urinated after modeling for 3 days before bedding and padding modeling Measure showed increased, it is necessary to replace daily), it gradually becomes thin, illustrates that diabetes model is extremely successful.Modeling rat spirit is withered It wastes, hair color jaundice is slow in action, and weight loss is apparent, and rats death is more in 2 weeks after STZ injections, and the cause of death is mainly blood Sugared excessively high, the too fast rat of weight loss, rat of 2 weeks weight loss in 100g or more are easy death；STZ injects 4 Zhou Houzai The dead more phenomenon of secondary appearance, especially weight are easily dead less than the rat of 250g.In addition, flatulence caused by diabetes, Intestinal obstruction is also major reason, and when gavage is strayed into tracheae and causes 2 death.

2. influence of the drug of the present invention to rat blood sugar

3 days rat blood sugars drastically increase after modeling, with more statistically significant (P before blank group and this group of modeling< 0.01), hereafter there is different degrees of decline, but remain above 16.7mmol/L, maintain high level ever since.At the 8th week, together Normal group compares, remaining each group blood glucose still there were significant differences (P<0.01), large, medium and small with model group drug more of the present invention Dosage group and control group have different degrees of reduction trend, but only drug bolus group statistical significance (P of the present invention< 0.05).There was no significant difference between remaining group (P>0.05).1 is see the table below, table 2.

3. influence of the drug of the present invention to weight

Weight obviously increases after 4 weeks after high sugar high in fat is fed, more statistically significant with blank group, 2 after STZ injections Weight loss is apparent after week, statistically significant (P<0.01), weight tends to be steady after 2 weeks.

3.Mirris water mazes

Blank group rat finds the time of platform in pond and swimming distance is gradually shortened in 4 days training process, Model group escape latency and swimming are slow apart from longer and shortening, have with blank group comparing difference in the 5th day escape latency Conspicuousness (p<0.01), the big-and-middle small dose group rat performance of drug of the present invention is more apparent than model group more preferable, and small group has Notable difference (p<0.05), control group also has improvement trend compared with model group, but there was no significant difference.Space exploratory experiment in 6th day, The number that model group rats enter target area significantly reduces (p<0.05), illustrate that Rats With Memory ability is decreased obviously；The present invention The big-and-middle small dose group rat of drug and control group relatively have different degrees of improvement with model group, but only middle dose group has significantly Difference (p<0.05).

The comparison (x ± s) of 3 ability of learning and memory of table

※ P compared with blank group<0.05, ※ ※ P compared with blank group<0.01, * compared with model group P<0.05.

4. influence of the present invention to hippocampal formation and neuron vascular morphology

4.1HE dyeing

Blank group neurons of hippocampus CA 1 form is normal, marshalling, and cell level is apparent, and cell is rounded, and film is complete Boundary rule, nucleus is clear, kernel indigo plant dye.Model group neurons of hippocampus CA 1 is disorganized, and neural levels are reduced, god It is reduced through first number, structure owes clear, cell space shrinkage or swelling, it is seen that many karyopycnosis necrosis neurons, it is seen that spongiocyte Hyperplasia phenomenon.The above-mentioned pathological change of the large, medium and small dosage group of gegen qinlian decoction mitigates compared with model, but group difference unobvious.It is right More whole according to group neural identical permutation, structure owes clear, sees more karyopycnosis necrosis neuron, it is seen that spongiocyte, above-mentioned each disease Neo-Confucianism improves to be improved obviously compared with model group, relatively has different with Chinese medicine group (see Fig. 1).

5.2 Electronic Speculum

Blank group：Neuron volume is larger, and cell nuclear is mostly round, and Distribution of chromatin is uniform in core, and euchromatin is rich Richness, heterochromatin are less；Cytoplasm inner cell organ is abundant, and Golgi complex is flourishing, and mitochondria is round or rod-shaped, is scattered in thin In cytoplasm, a large amount of intensive rough surfaced endoplasmic reticulum (RER) stacked arrangements, free ribosome is abundant, core Zhou Changjian Golgi complexs.Capillary Vascular endothelial cell is flat, and nuclei dyeing chromaticness is fine and closely woven, core week cytoplasm it is abundant, lumen of vessels is complete, containing few in endothelial cell matter The rough surfaced endoplasmic reticulum (RER) of amount, mitochondria small volume and ridge is few, ribosomes is dispersed in cytoplasm.Cynapse is (between presynaptic membrane, cynapse Gap, postsynaptic membrane) and postsynaptic cell device it is clear in structure, myelin institutional framework is complete.

Model group：Neuron mild swelling, electron density reduce；Show slightly spacious in cytoplasm, Golgi complex is flat Capsule is slightly expanded, mitochondrial swelling, and partial mitochondrial ridge disappears, and rough surfaced endoplasmic reticulum (RER) has no significant change, free ribosome relatively on Group is reduced.Capillary endothelial cell mild swelling, electron density reduce, and cytoplasm mitochondrial swelling, lumen of vessels is irregular, Visible larger white space around it.Cynapse swelling, structure is spacious, and electron density reduces, part myelin layer structure part Loosely.

Chinese medicine large dosage group：The relatively upper group of neuron swelling degree slightly mitigates；Capillary lumen rule, around have no bright Aobvious white space；Synaptic structure still has mild swelling, the relatively upper group of synaptic versicle slightly to increase；Myelin layer structure is substantially complete.

Chinese medicine middle dose group：Eucaryotic cell structure is normal, and neuronal cell matter mitochondrial swelling slightly mitigates, myelin plate Layer structure is substantially complete.Capillary lumen rule, around have no apparent white space.

Chinese medicine small dose group：Eucaryotic cell structure is normal, and the relatively upper group of neuronal cell matter mitochondrial swelling is apparent, myelin Layer structure is loosely apparent.Capillary lumen rule, around have no apparent white space.

Western medicine group：Eucaryotic cell structure is normal.Capillary lumen rule, around have no apparent white space.(see Fig. 2, Fig. 3)

Five, conclusion

1. high fat diet composite S TZ injection diabetes rat 8 weekend ability of learning and memory is decreased obviously, there is cognitive function Obstacle.

2. cognition dysfunction occurs in diabetes mellitus encephalopathy rat, damaging or taking off occur in hippocampal neuron and capilary It loses.

3. prophylactic use drug of the present invention can mitigate diabetes mellitus encephalopathy Cognition Function in Rats obstacle, with prevention hippocampus god It is related through first microvascular damage.

The repair that 2 drug of the present invention of test example damages diabetes mellitus encephalopathy blood brain barrier of rats

One, experiment material

1. experimental animal

Healthy SPF grades of hero SD rats 120, weight 200g ± 20g, experimental animal are purchased from Xi'an Communications University experimental animal Center, the quality certification book number SCXK (Shan) 2012-003.In Medical experimental center animal house sub-cage rearing, per 10, cage.Animal house At 20-26 DEG C, relative humidity control keeps illumination in 12 hours, night to turn off the light on 45-65%, ventilation, daytime for temperature control.

2. observing drug

2.1 drugs of the present invention：It is made of pueraria lobata, the coptis, radix scutellariae, the stem of noble dendrobium, bear gall powder, Radix Notoginseng powder etc., pueraria lobata, the coptis, Huang A kind of reed mentioned in ancient books, stem of noble dendrobium decocting filtering, concentrate as every milliliter contained 0.65g.Crude drug is purchased from Beijing Tongrentang (Bozhou) company, adds before gavage Enter bear gall powder, suspension 0.725g/l is made in Radix Notoginseng powder.

Two, experimental method

1. animal model and interference method

1.1 model

SPF grades of SD rats 120, male, animal adaptable fed 3 days under the conditions of available light, free water are ingested, Separating 15 is only used as blank control group at random, gives normal diet nursing；Remaining 105 is only used as modeling group and gives high lipid food (10% lard, 10% sucrose, 2.5% cholesterol, 77.5% basal feed) uses intraperitoneal injection STZ's after feeding 4 weeks, 4 weeks Method manufactures rat diabetes model, and fasting (can't help water) 12h, is dissolved in citrate buffer by STZ before rat modeling 1% solution is made in (0.1mmol/L, pH=4.3), and disposable celiac injection chain urine makees rhzomorph 45mg/g.Tail vein is adopted after 3 days Blood surveys blood glucose, and the rat for reaching 16.7mmol/L thinks that diabetes model is successfully established, be included in experiment, not up to glycemic criteria It is unsuccessful to be considered as modeling, rejects.Blank control group gives the citrate buffer of intraperitoneal injection equivalent.

1.2. it is grouped and treats

2. collection of specimens

Collection of specimens is perfused in 2.1 azovan coerulens

3 are randomly selected from each group rat, and fiber crops are injected intraperitoneally by the standard of 0.3ml/100g only with 10% chloraldurate Liquor-saturated, after anesthesia is satisfied, dorsal position is fixed on mouse platform, notch at the groin of right side, and exposure right side iliac vein, skin test pin puncture is pressed Lml/100g weight injects 2% azovan coerulen, stands 30 minutes, cuts off thoracic cavity from xiphoid-process, observe the position of the left heart and right auricle of heart, from The left apex of the heart is inserted into needle for injection (blood transfusion specialized transfusion system) to aorta ascendens, while cutting off right auricle of heart, 0.9% sodium chloride of ice note Penetrate liquid 200ml rinse to trickle it is limpid after, broken end takes brain on rapid ice pan, and left and right brain separates, as the first of 5 times of brain volumes For surveying OD values in amide.Blank group and model group respectively take side brain to be placed in 4% paraformaldehyde and fix, serial dehydration, paraffin It is fixed to carry out light microscopic sight by embedded section thickness 5um.

2.2Westen-blot collection of specimens

Every group of remaining 6 rats dislocation is put to death, rapid broken end takes brain, rapid ice pan to detach hippocampal tissue, left and right sides It is respectively put into different cryopreservation tubes and is put into liquid nitrogen container freezing rapidly, -80 DEG C of refrigerators are moved into after acquisition.

3. azovan coerulen tissue specimen

(1) the brain paraffin mass of azovan coerulen injection embedding cuts the slice of 5um, drags for piece, lacing film；

(2) slice is set into oven 56 DEG C of roasting pieces 30 minutes；

(3) conventional to dewax to water：Slice is placed in dimethylbenzene I 15 minutes respectively by following procedure, II 15 points of dimethylbenzene

Clock followed by enters 100%, 95%, 90%, 80%, 70% alcohol each 5 minutes respectively, removes removal xylene,

Distilled water flushing 5 minutes removes alcohol；

(4) the light dyeing of Yihong liquid 5 minutes, pure water rinsing 3 minutes；

(5) it is dehydrated：It is put into 70%, 80%, 90%, 95%, 100% alcohol each 5 minutes；

(6) transparent 10 minutes of dimethylbenzene, keep sample transparent；

(7) sealing：Appropriate neutral gum capping slide mounting is added dropwise；

(8) azovan coerulen distribution situation around optical microphotograph microscopic observation rat brain blood vessel.

4. azovan coerulen assay

Left and right brain is separated, is placed in the formamide of 5 times of brain volumes, 37 DEG C of water-baths 48h, 15000r/min centrifuge 20 points Clock, takes supernatant to survey absorbance (OD) value at 630nm wavelength, and the standard items that formamide is prepared make blank control.According to standard Curve calculates EB contents (ug/mL), and brain tissue EB contents are 5 times of above-mentioned value.

5.Westen-blot is measured

(1) utilize RIPA protein lysates extraction histone (specific steps are shown in specification)；

(2) BCA kit protein quantifications (specific steps are shown in specification)；

(3) sample is denatured by boiling：By the sample dissolved by 4:It is moved into after 1 ratio and 5X sample-loading buffer mixings It in 1.5mLep pipes, is placed on foam with holes and boils 5-10min in boiling water, centrifuge：10’000rpm/10min；

(4) 10% gel slab prepared is put into electrophoresis tank, it is ensured that electrophoresis tank does not leak；

(5) 1 × Running Buffer are poured into；

(6) it is loaded with thin pipette tips, determines Loading sequence and calculates sample-adding amount, the upper limit of volume is true according to instrument model It is fixed.This experiment sample-adding is all 20ug total proteins, first plus pre-dyed Protein Marker (article No. 26616)；

(7) electrophoresis concentrate glue voltage 100V, separation gel voltage 200V, keeps low temperature as possible, prevents heat production excessively high.

(8) film is taken out under flowing water flushing, cuts corner and marks.

(9) wet turn：Filter paper, glue, film are put well successively by direction, until entire clamping plate keeps close；

(10) it is closed with the TBS containing 5% skimmed milk power, 1-4h can be closed in room temperature.This Laboratory Temperature is closed 4 hours；

(11) primary antibody is combined, overnight at 4 DEG C；

(12) it rinses, room temperature rinses 3 times, each 10min；

(13) secondary antibody, room temperature combination 2h are combined；

Goat anti-Mouse IgG,(H+L),Peroxidase Conjugated 1：10000

Goat anti-Rabbit IgG,(H+L),Peroxidase Conjugated 1：10000

(14) it rinses, room temperature rinses 3 times, each 10min.Last time is rinsed with TBS to remove tween；

(15) chemiluminescence (by specification operation), the exposing operation in darkroom.Graded exposure -5 minutes 10 seconds.

Three, statistical method

Four, result

1. tissue observation azovan coerulen leakage scenarios

The light dye picture in brain Yihong after the injection of normal rat azovan coerulen, no azovan coerulen ooze out blood vessel.Diabetes mellitus encephalopathy rat The light dye picture in brain Yihong after the injection of brain azovan coerulen, a large amount of azovan coerulens ooze out blood vessel.Diabetes rat azovan coerulen injects recoil The light dye picture in bone nerve Yihong, it is seen that azovan coerulen slightly oozes out blood vessel.Lung tissue Yihong is light after the injection of diabetes rat azovan coerulen Contaminate picture, it is seen that azovan coerulen oozes out blood vessel.The light dye picture in liver Yihong after the injection of diabetes rat azovan coerulen, it is seen that azovan coerulen oozes Go out blood vessel.The light dye picture in glomerulus Yihong after the injection of diabetes rat azovan coerulen, it is seen that azovan coerulen oozes out glomerular capillary. (see Fig. 4)

2. azovan coerulen assay in brain tissue

Model group rats brain tissue EB contents are increased compared with blank group, and difference has statistical significance (P<0.05).Through drug therapy, Each group EB contents decline (P compared with model group<0.05) no significant difference (P, but between each group>0.05).

EB content tables (n=6) in 4 brain tissue of table

※ ※ P compared with blank group<0.05, * compared with model group P<0.05, * * P compared with model group<0.01.

3.Westen-blot measures tight junction protein expression

The influence that 3.1 drugs of the present invention express occludin

Experimental study shows that the occludin expression of model group rats hippocampus is reduced compared with blank group, significant difference (P< 0.01) it, is remarkably reinforced through drug therapy each group occludin expression, wherein drug middle dose group of the present invention, western medicine group table Up to highest, with model group comparing difference significantly (P<0.01), for drug middle dose group of the present invention compared with model group, difference has statistics Learn meaning (P<0.05).Drug bolus group of the present invention, compared with model group without significant difference (P>0.05), drug of the present invention In, the statistics indifference opposite sex (P between three groups of small dose group and western medicine group>0.05).Illustrate diabetes rat blood-brain barrier disruption, Occludin lowers, and chemoprophylaxis administration of the present invention can reduce the destruction of occludin, there is protection blood-brain barrier effect, Er Qiezhong Small dose group, western medicine group effect are optimal.(see Fig. 5)

3.2 the influence that drug of the present invention expresses claudins-5

Experimental study shows that the claudins-5 expression of model group rats hippocampus significantly reduces (P compared with blank group<0.01), through medicine Object treatment each group claudins-5 expression is remarkably reinforced, and for drug bolus group of the present invention compared with blank group, difference has statistics Meaning (P<0.05), in drug of the present invention, small dose group, control group and blank group comparing difference are without conspicuousness (P>0.05).With Model group compares, and the large, medium and small dosage group of drug of the present invention, the claudins-5 expression enhancings of control group group, difference have statistics meaning Justice (P<0.01).Compared with drug bolus group of the present invention, in drug of the present invention, small dose group expression there were significant differences (P< 0.05), indifference (P in drug of the present invention, between small dose group, control group group>0.05).Illustrate that diabetes rat blood-brain barrier is broken Claudins-5 lowers when bad, and claudins-5, which is destroyed, after pharmaceutical intervention of the present invention is reduced, small group and Western medicine Xi Luota Azoles group effect is suitable, is optimal.The possible mechanism of action is to protect the tight junction protein claudins-5 of blood-brain barrier.(see Fig. 6)

The influence that 3.3 drugs of the present invention express JAM-1

This is experimental studies have found that model group rats hippocampus JAM-1 is expressed compared with blank group apparent increase (P<0.01), through drug It treats each group JAM-1 expression in various degree to lower, but the still significant difference (P of control group compared with blank group<0.01).This The large, medium and small dosage group of invention drug and model group comparing difference are notable, statistically significant (P<0.01).In drug of the present invention Dosage group JAM-1 expression is larger, small dose group is substantially reduced, significant difference, statistically significant (P<0.01).Drug of the present invention Large and small dosage comparison among groups are without significant difference (P>0.05).Control group no significant difference (P compared with model group>0.05).Explanation When diabetes 8 weeks, blood brain barrier of rats destruction is increased with JAM-1, because JAM-1 is mainly electrostatic barrier, it may be possible to diabetes Blood brain barrier of rats increases the compensatory of other transmembrane protein reductions when destroying, and drug of the present invention may pass through increase The albumen such as occludin effectively to lower the expression of JAM-1, protection blood-brain barrier effect, and middle dose group effect is optimal.(see Fig. 7)

The influence that 3.4 drugs of the present invention express ZO-1

Experiment shows that model group ZO-1 expression significantly reduces (P compared with blank group<0.01), through drug intervention groups ZO-1 tables Up to being remarkably reinforced, wherein in drug of the present invention, small dose group express highest, with model group comparing difference significantly (P<0.01), with Blank group compares no significant difference (P>0.05), without significant difference (P compared with drug bolus group of the present invention>0.05), two groups Between indifference (P>0.05).With western medicine group there were significant differences (P<0.01).Illustrate diabetes rat blood-brain barrier disruption, ZO-1 lowers, and chemoprophylaxis administration of the present invention, the ZO-1 alleviated is destroyed, and has protection blood-brain barrier effect, and small group Effect is optimal.(see Fig. 8)

Six, conclusion

1. diabetes can cause blood-brain barrier disruption, permeability to increase, it may be possible to one of the mechanism that diabetes mellitus encephalopathy occurs.

2. the low expression of tight junction protein occludin, claudins-5, ZO-1 in diabetes may be diabetes The reason of Blood Brain Barrier (BBB) permeability.

3. drug of the present invention protects blood-brain barrier by protecting tight junction protein, brain homeostasis is safeguarded, from And prevent diabetes mellitus encephalopathy.

Effect of 3 drug of the present invention of test example to diabetes mellitus encephalopathy rat Angiogenesis

One, experiment material

1. experimental animal

2. design of primers and synthesis

Primer synthesis is completed by the prosperous bio tech ltd of Beijing AudioCodes.Sequence is as follows：

Two, animal model and intervention

1. animal model and interference method

1.1 model

SPF grades of SD rats 120, male, animal adaptable fed 3 days under the conditions of available light, free water are ingested, Separating 15 is only used as blank control group at random, gives normal diet nursing；Remaining 105 is only used as modeling group and gives high lipid food (10% lard, 10% sucrose, 2.5% cholesterol, 77.5% basal feed) uses intraperitoneal injection STZ's after feeding 4 weeks, 4 weeks Method manufactures rat diabetes model, and fasting (can't help water) 12h, is dissolved in citrate buffer by STZ before rat modeling 1% solution is made in (0.1mmol/L, pH=4.3), and disposable celiac injection chain urine makees rhzomorph 45mg/g.Tail vein is adopted after 3 days Blood surveys blood glucose, and the rat for reaching 16.7mmol/L thinks that diabetes model is successfully established, be included in experiment, not up to glycemic criteria It is unsuccessful to be considered as modeling, rejects.Survey a blood glucose every 2 weeks afterwards.The citrate that blank control group gives intraperitoneal injection equivalent is slow Fliud flushing.

1.2. it is grouped and treats

2. collection of specimens

2.1 blood specimen collection

Before STZ intraperitoneal injections, tail vein blood 0.1ml is placed in ethylenediamine tetra-acetic acid (EDTA) anticoagulant tube and send loss thin Born of the same parents' instrument detects the quantity of endothelial progenitor cells.Treatment is drawn blood inspection again after 3,24,56 days.

2.2 immunohistochemistry collections of specimens

3 progress immunohistochemistry materials are randomly selected from each group rat.0.3ml/100g is pressed using 10% chloraldurate Standard intraperitoneal injection of anesthesia, after anesthesia is satisfied, dorsal position is fixed on mouse platform, and cutting off thoracic cavity from xiphoid-process exposes heart, is seen The position for examining the left heart and right auricle of heart is inserted into needle for injection (blood transfusion specialized transfusion system) to aorta ascendens from the left apex of the heart, is cut simultaneously Open right auricle of heart, 0.9% sodium chloride injection 200ml of ice rinse to trickle it is limpid after, 150ml is perfused in 4% paraformaldehyde, greatly Mouse body is placed 2 hours after hardening, and broken end takes brain on rapid ice pan, is placed in 4% paraformaldehyde and is fixed overnight, serial dehydration, stone Wax embedded section thickness 5um divides left and right brain, carries out immunohistochemistry measurement.

2.3 PCR collections of specimens

Three, experimental method

(1) FCM analysis

1. being completed being detected in the blood preparation 24 hours of acquisition；

2. using Two Colour Fluorescence label FCM analysis method, the double fluorescencepositive cells of observation rat peripheral blood (CD34+, VEGFR+2) variation of ratio judges rat peripheral blood EPCs quantity：By 1 day before modeling, 3 days, 4 weeks, 8 week point samplings.

3. every rat takes ethylenediamine tetra-acetic acid (EDTA) anticoagulated whole blood 50uL；Measure the anti-rat of pipe plus FEI-C labels Anti- each 20ul of rat CD34 monoclonal antibodies of VEGFR2 monoclonal antibodies and PE labels；

4. room temperature, which is protected from light, is incubated 15min；

5. often pipe plus 500uL erythrocyte cracked liquids, room temperature, which is protected from light, is incubated 20min；

6. each pipe plus lmL FCM analysis buffer solutions, 1200r/min centrifuge 5min, abandon supernatant；

7. the phthalate buffer that phosphorates (PBS) 300uL is resuspended, upper machine testing.

8. being excited with 488nm argon lasers, 10000 cells are counted every time, and double fluorescence are analyzed with Cellquest softwares Positive cell proportion.

(2) PCR is detected

1.RNA is extracted

Preparation before 1.1 RNA extractions：

1) before use, all glasswares (including graduated cylinder, beaker, homogenizer, surface plate etc.) use washing lotion (mixing Acid) impregnate, done at a high temperature of then arising from 200 DEG C with materials instrument (knife is cut, tweezer, lunch box, kidney basin etc.) one it is 4-6 hours roasting, It is cool spare to room temperature.

2) plastic ware (including EP pipes, cryopreservation tube and pipette tips etc.) uses 0.1%DEPC water to impregnate, high pressure steam sterilization Then 30min is placed on 40 DEG C of ovens and is dried for standby.

3) solution all in RNA extraction process is all prepared with autoclaved 0.1%DEPC.

4) disposable breathing mask, cap, gloves are worn during whole operation, diligent hand-off set in experiment.

1.2 RNA are extracted：

1) clip 50-100mg tissues are put into homogenate tube, 1ml TriPure Isolation Reagent are added, after homogenate Pour into 1.5ml centrifuge tubes, 15-25 DEG C of incubation at room temperature 5min.

2) chloroform 0.2ml is added, mixing 15s is placed at room temperature for 2-15min,

3) 4 DEG C, 12000g, 15min is centrifuged.

4) it draws upper phase and is transferred to another EP pipes, isopropanol 0.5ml is added, manually violent concussion 15s, 15-25 DEG C 5-10min is placed at room temperature for,

5) it 4 DEG C, 12000g, centrifuges 10min, abandons supernatant (RNA is sunken to tube bottom, so paying attention to preventing precipitation from pouring out).

6) 75% ethyl alcohol (1ml ethyl alcohol/1mlTRIzol) is added to wash, mildly shakes centrifuge tube, suspend precipitation.

7) 4 DEG C, 7500g, 5min is centrifuged.

8) supernatant is abandoned, 10min or so is air-dried.

9) plus DEPC water (20-50ul) dissolving precipitates.

10) RNA extracted measures OD values and quantifies.

The OD values of RNA are measured with trace measurement instrument.It is returned to zero using DEPC water as blank.2ul RNA samples are added, read 260/280 ratio, i.e. OD values, and record RNA concentration (ug/ul).

2. reverse transcription reaction

Using TaKaR companies PrimeRT reagent Kit Perfect Real Time(DRR037A) Reverse transcription reagent box is loaded on ice according to the following steps.

Mild mixing simultaneously centrifuges.

Reverse transcription reaction condition is as follows：

1) 37 DEG C of 15min (reverse transcription reaction)

2) 85 DEG C of 5s ((reverse transcriptase inactivation reaction)

Reaction terminates, and is immediately placed in cooled on ice, is measured on NanoDrop ND-1000spectrophotometer CDNA contents and purity, synthesized cDNA can be directly used for PCR or saved backup in -20 DEG C.

3.Real-time PCR reactions

Using the SYBY Premix Ex Taq of TaKaRa Biotechnology companies^TMⅡ(Perfect Real Time (DRR081A) kit sequentially adds following reagent, structure PCR reaction systems

After the centrifugation of lid lid mixing, it is placed in BIO-RAD companies IO5^TM Multicolor Real-Time PCR It is expanded by following reaction conditions in Detection System fluorescence quantitative PCR instruments.

PCR reaction conditions are as follows：

Stage 1：Dissociation Stage:

Stage 2：

3.2 solubility curves are analyzed

PCR after reaction, by PCR reaction solution temperature from 60 DEG C to 95 DEG C slowly heatings, monitors SYBR Green I in real time Fluorescence signal intensity variation, obtain melt curve analysis, with confirm PCR reaction specificity.(see Fig. 9, Figure 10, Figure 11)

Amplification curve diagram reflects HIF-1 α, VEGF, GAPDH exponent phase in this experiment in Fig. 9, Figure 10, Figure 11 Slope is big, is expanded for high efficiency, collimation is preferable between curve and curve, close, the quantitative accuracy of each reaction tube amplification efficiency With it is reproducible, the low concentration exponent phase is apparent, is less prone to erroneous judgement, high sensitivity；Melting curve is in single peak value curve, It shows that HIF-1 α, VEGF, GAPDH amplified production are pure, shows that real-time quantitative PCR-CT values are accurate.Fang Jinhang mRNA relative expressions The calculating of amount and compare.

3.3 sample rna relative amounts use 2^-⊿⊿CTMethod calculates

^⊿CT=CT^{Target gene}One CT^{Reference gene}

^⊿⊿CT=^⊿CT^{Test _ ⊿}CT^Control

Real-time qPCR are the variations using fluorescence signal, detect each be circulated throughout in pcr amplification reaction in real time The amplified production brightness change of journey show that CT values and standard curve carry out starting template quantitative analysis.

(3) immunohistochemistry

1. embedded wax stone to be pressed to the serial section of Coronal row about 5um thickness；

2. use the processed glass slide of poly-D-lysine, 59 DEG C of fishing piece postposition oven 60 minutes, so that slice is close glutinous Attached anti-flake；

3. slice dewaxes through dimethylbenzene to water；

4.30%H₂O₂1 is pressed with distilled water:10 ratios mix, and room temperature 10 minutes is with inactivating endogenous enzyme, distilled water flushing 3 It is secondary；

5. antigen is answered in hot repair:Slice is immersed into 0.0lM citrate buffers (PH6.0), electric furnace breaks after being heated to boiling Electricity, after being spaced 10min, 2 times repeatedly, PBS (PH7.2-7.6) is washed 2 times after cooling；

6. 5%BSA confining liquids are added dropwise, room temperature 20min gets rid of surplus liquid, does not wash；

7. dropwise addition is diluted to 1:100 primary antibody (rabbit igg, respectively IGF-1/CREB), 37 DEG C 1 hour, PBS is washed 2 minutes × 3 times；

8. biotinylated secondary antibody rabbit anti-mouse igg is added dropwise, 20-37 DEG C of 20min, PBS are washed 2 minutes × 3 times；

9. reagent SABC is added dropwise, 20-37 DEG C of 20min, PBS are washed 5 minutes × 4 times；

10.DAB develops the color:Using DAB colour reagents box (AR1022), 1ml distilled water is taken, in reagent adding box

A, B, C reagent each 1 drips, and slice is added to after mixing.Color development at room temperature controls the reaction time under mirror, distills water washing.

11. haematoxylin is added dropwise slightly to redye, alcohol serial dehydration, dimethylbenzene is transparent, resinene mounting, is seen under microscope It examines.

Four, statistical analysis

It is handled using spss17.0 softwares, data are indicated with mean scholar standard deviation (Mean scholar SD), and comparison among groups are using single Analysis of variance, P<0.05 is statistically significant for difference.

Five, result

1. drug of the present invention is to diabetes rat EPC_SThe influence of proliferation

This experiment carries out endothelial progenitor cells analysis in peripheral blood, the VEGFR marked with FITC using using flow cytometer⁺² With the dual anti-label endothelial progenitor cells of CD34 of PE labels, as a result indicated with the EPCs numbers in every 10000 monocytes.Figure 12 is Negative control sets the 1st cell door according to forward angle light scatter (FSC) and side scatter (SSC), the circle menu in scatter plot Nucleus region (Figure 13)；According to VEGFR⁺²FITC and CD34PE sets the 2nd cell door, draws a circle to approve CD34 and VEGFR⁺²Double positives Cell mass region, such as figure right upper quadrant, that is, EPC (Figure 14).

Experimental result shows that rat surveys peripheral blood EPCs after injection STZ is 3 days and counts display, compared with Normal group, EPC, which is counted, to be reduced, the statistically significant (P of difference<0.05).The quantity of more each treatment group's peripheral blood EPCs after STZ is injected 4 weeks Obviously increase earlier above, with the more significant increase of model group, wherein in drug of the present invention, small dose group have compared with model group it is aobvious Write sex differernce (P<0.05).Drug bolus group of the present invention also has increase trend, but not statistically significant (P>0.05).Control group The quantity of EPCs obviously increases (P earlier above<0.05), in drug of the present invention, small dose group is compared with model group there was no significant difference (P> 0.05)。

There is increase trend before the quantity of more each treatment group's peripheral blood EPCs was compared with 4 weeks after STZ is injected 8 weeks, but without statistics Meaning.Each comparison among groups, model group EPC countings compared with blank group still reduce, the statistically significant (P of difference<0.05).Respectively control The quantity for the treatment of group peripheral blood EPCs obviously increases earlier above, with the more significant increase of model group, wherein in drug of the present invention, Small dose group difference (P significant compared with model group<0.05).Drug bolus group of the present invention also has increase trend, but without statistics Learn meaning (P>0.05).The quantity of control group EPCs obviously increases (P earlier above<0.05), in drug of the present invention, small dose group compared with Model group there was no significant difference (P>0.05).(see Figure 12, Figure 13, Figure 14)

2. influence of the drug of the present invention to diabetes mellitus encephalopathy rat hippocampus HIF-1 α mRNA, VEGFmRNA

Relative expression quantities and blank group obvious increase (P of this experiment PCR detections discovery HIF-1 α mRNA in model group <0.05), weaken compared with model group through each treatment group HIF-1 α mRNA relative expression quantities of prevention administration, wherein drug of the present invention is small Dosage group significance (P compared with model group<0.05).Remaining each group HIF-1 α mRNA relative expression quantities compared with model group Weaken, not statistically significant (P>0.05).The large, medium and small dosage group of drug of the present invention and western medicine group group difference are without statistics Learn meaning (P>0.05).

Relative expression quantities and blank group obvious increase (P of the VEGFmRNA in model group<0.01), each through prevention administration Treatment group's VEGFmRNA relative expression quantities weaken compared with model group, and difference is statistically significant (P<0.01).Medicine of the present invention The large, medium and small dosage group of object and the not statistically significant (P of western medicine group group difference>0.05).See Figure 15

HIF-1 α mRNA, VEGFmRNA expression (2 in 5 hippocampus of table^{- ⊿ ⊿ CT,} N=6)

The influence that 3 drugs of the present invention express diabetes mellitus encephalopathy rat hippocampus CD34

This experiment immunization histochemical staining brown yellow granule is that CD34 expression is positive, blank group rat hippocampus CD34 stained positives For cell distribution around capilary, distribution density is uniform.Model group dyeing CD34 positive cells significantly reduce, and distribution density is apparent Reduce (P<0.05).Drug each group of the present invention and western medicine group CD34 positive cells relatively increase with model group, distribution density Increase.In drug wherein of the present invention, small dose group and model group comparing difference significance (P<0.01), drug of the present invention is big Dosage group and western medicine group and model group comparing difference significantly (P<0.05).

MVD is measured using technical method under light microscope, and vascular counts are using the positive after immunohistochemistry CD34 labels Cell judges inner cell, determines capilary, is dyed to pale brown or sepia, the clear endothelium of boundary or endothelial cell cluster and makees For a capilary.Vascular distribution highest zone is selected under 100 times of light microscopics, observation vascular morphology is capilary under 400 times, 5 visuals field are selected under 400 times of light microscopics, count capilary number, take its average value, calculate MVD values (a unit area).(see figure 16)

6 rat hippocampus capilary distribution density MVD of table (N=6)

※ P compared with blank group<0.05, ※ ※ P compared with blank group<0.01, * compared with model group P<0.05, * * The P compared with model group<0.05_。

Conclusion

1. newly plus gegen qinlian decoction can promote diabetes cognition dysfunction endothelial progenitor cells from rat bone marrow proliferation, transfer ability, Participate in reparation and the new life of blood vessel.

2. newly adding gegen qinlian decoction that can increase the density of diabetes cognition dysfunction rat hippocampus capilary, blood is promoted Pipe is newborn.

3. newly plus gegen qinlian decoction may be by protecting hippocampus microvascular endothelial cells, promoting endothelial progenitor cells migration and differentiation And promotes the micro- blood of hippocampus newborn and improve diabetes cognition dysfunction learning and memory in rats ability.

Influence of 4 drug of the present invention of test example to diabetes mellitus encephalopathy rat IGF1/PI3K/AKT/CREB signal paths

One, experiment material

1. experimental animal

2. design of primers and synthesis

5. observing drug

5.1 drugs of the present invention：It is made of pueraria lobata, the coptis, radix scutellariae, the stem of noble dendrobium, bear gall powder, Radix Notoginseng powder etc., pueraria lobata, the coptis, Huang A kind of reed mentioned in ancient books, stem of noble dendrobium decocting filtering, concentrate as every milliliter contained 0.65g.Crude drug is purchased from Beijing Tongrentang (Bozhou) company, adds before gavage Enter bear gall powder, suspension 0.725g/ml is made in Radix Notoginseng powder.

5.2 control drug：Cilostazol：50mg/ pieces, Zhejiang great Zhong pharmaceutical Co. Ltds.

5.3 high lipid food：10% lard, 10% sucrose, 2.5% cholesterol, 77.5% basal feed；Basal feed is by west Pacify university of communications's animal experimental center to provide, high lipid food is voluntarily prepared according to formula.

Two, animal model and intervention

1. animal model and interference method

1.1 model

1.2. it is grouped and treats

2. collection of specimens

2.1 immunohistochemistry collections of specimens

6 progress immunohistochemistry materials are randomly selected from each group rat.0.3ml/100g is pressed using 10% chloraldurate Standard intraperitoneal injection of anesthesia, after anesthesia is satisfied, dorsal position is fixed on mouse platform, and cutting off thoracic cavity from xiphoid-process exposes heart, is seen The position for examining the left heart and right auricle of heart is inserted into needle for injection (blood transfusion specialized transfusion system) to aorta ascendens from the left apex of the heart, is cut simultaneously Open right auricle of heart, 0.9% sodium chloride injection 200ml of ice rinse to trickle it is limpid after, 150ml is perfused in 4% paraformaldehyde, greatly Mouse body is placed 2 hours after hardening, and broken end takes brain on rapid ice pan, is placed in 4% paraformaldehyde and is fixed overnight, serial dehydration, stone Wax embedded section thickness 5um carries out immunohistochemistry measurement.

2.2 PCR collections of specimens

Three, experimental method

(1) immunohistochemistry

3. slice dewaxes through dimethylbenzene to water；

(2) PCR is detected

1.RNA is extracted

Preparation before 1.1 RNA extractions：

1.2 RNA are extracted：

3) 4 DEG C, 12000g, 15min is centrifuged.

7) 4 DEG C, 7500g, 5min is centrifuged.

8) supernatant is abandoned, 10min or so is air-dried.

9) plus DEPC water (20-50ul) dissolving precipitates.

10) RNA extracted measures OD values and quantifies.The OD values of RNA are measured with trace measurement instrument.Using DEPC water as blank Zeroing.2ul RNA samples are added, read 260/280 ratio, i.e. OD values, and record RNA concentration (ug/ul).

2. reverse transcription reaction

Using TaKaR companies PrimeRT reagent Kit Perfect Real Time(DRR037A) Reverse transcription reagent box is loaded on ice according to the following steps.Table 7

Mild mixing simultaneously centrifuges.

Reverse transcription reaction condition is as follows：

1) 37 DEG C of 15min (reverse transcription reaction)

2) 85 DEG C of 5s ((reverse transcriptase inactivation reaction)

3.Real-time PCR reactions

3.1 use the SYBY Premix Ex Taq of TaKaRa Biotechnology companies^TMⅡ(Perfect Real Time (DRR081A) kit sequentially adds following reagent, structure PCR reaction systems

PCR reaction conditions are as follows：

Stage 1：Dissociation Stage:

Stage 2：

3.2 solubility curves are analyzed

PCR after reaction, by PCR reaction solution temperature from 60 DEG C to 95 DEG C slowly heatings, monitors SYBR Green I in real time Fluorescence signal intensity variation, obtain melt curve analysis, with confirm PCR reaction specificity.(see Figure 17, Figure 18, Figure 19, Figure 20)

Amplification curve diagram reflects that PI3K, AKT, CREB, GAPDH are bent in this experiment in Figure 17, Figure 18, Figure 19, Figure 20 Linear index phase slope is big, is expanded for high efficiency, and collimation is preferable between curve and curve, and each reaction tube amplification efficiency is close, quantitative Accuracy with it is reproducible, the low concentration exponent phase is apparent, is less prone to erroneous judgement, high sensitivity；Melting curve in unimodal, Show that PI3K, AKT, CREB, GAPDH amplified production are pure, product meets design length requirement, shows real-time quantitative PCR-CT values Accurately.

3.3 sample rna relative amounts use 2^-⊿⊿CTMethod calculates

^⊿CT=CT^{Target gene}One CT^{Reference gene}

^⊿⊿CT=^⊿CT^{Test _ ⊿}CT^Control

Four, statistical analysis

Five, result

1. the influence that drug of the present invention expresses diabetes mellitus encephalopathy hippocampus PI3K/AKT/CREB mRNA

This experimental study shows that model group rats hippocampal tissue PI3KmRNA, Akt mRNA, CREBmRNA expression is more empty White group is substantially reduced (P<0.0l), show that feeding composite S TZ intraperitoneal injections duplication T2DM rat models with high sugar high in fat exists It coincide with document report in terms of PI3KmRNA, Akt mRNA, the expression of CREBmRNA relative quantifications.Each group and model group ratio is administered Compared with hippocampal tissue PI3KmRNA, Akt mRNA, CREBmRNA gene relative expression quantity pole significantly rises (P<0.01).Wherein, PI3K gene expressions no significant difference (P between the large, medium and small dosage group of drug of the present invention and western medicine group>0.05)；Akt Each treatment comparison among groups in terms of mRNA expression, drug bolus group of the present invention is low compared with other groups, relatively has with small dose group extremely aobvious Write difference (P<0.01), with middle dose group, western medicine group comparing difference significantly (P<0.05) in, small dose group and Western medicine pair According to group difference without conspicuousness (P>0.05)；Each treatment comparison among groups, drug bolus group of the present invention in terms of CREBmRNA expression It is low compared with other groups, between middle dose group, western medicine group compared with have pole significant difference (P<0.01), with small dose group comparing difference Significantly (P<0.05), the significant (P of difference between small dose group and middle dosage and western medicine group<0.05).Show medicine of the present invention In object, small dose group and western medicine group activation T2DM Rat hippocampus PI3K/Akt/CREB signal path related genes Expressional function is preferable.It the results are shown in Table 8.(see Figure 21)

PI3KmRNA, AKTmRNA, CREBmRNA expression (2 in 8 hippocampus of table^{- ⊿ ⊿ CT,} )

※ P compared with blank group<0.05, ※ ※ P compared with blank group<0.01, * compared with model group P<0.05, * * The P compared with model group<0.05,

Δ P compared with large dosage of group<0.05, Δ Δ P compared with large dosage of group<0.01, # small dose group P<0.05.

Influence of 2 drugs of the present invention to diabetes mellitus encephalopathy hippocampus IGF-1, CREB

IGF-1 immunohistochemical stainings positive cell is in brown color, blank group hippocampus god in hippocampal neuron in this experiment It is positive through apparent intensive brown color is presented in member；The different dye cell of model group brown color significantly reduces (P<0.01)；Medicine of the present invention After object and the treatment of Western medicine comparison medicine, the different dye cell of each group yellow has different degrees of increase, but drug middle dose group of the present invention Increase apparent (P compared with model group<0.01), small dose group, control group and the statistically significant (P of model group comparing difference<0.05). (see Figure 22, Figure 23)

CREB immunohistochemical stainings positive cell is in karyon brown yellow granule, blank group in hippocampal neuron in this experiment The apparent intensive different dye cell of brown yellow granule is presented in hippocampal neuron；Model group cell arrangement is loose, and the different dye of brown color is thin Born of the same parents significantly reduce (P<0.01)；After drug and Western medicine comparison medicine treatment of the present invention, the different dye cell of each group yellow has in various degree Increase, but drug middle dose group of the present invention increases apparent (P compared with model group<0.01), small dose group, control group and model group ratio (P statistically significant compared with difference<0.05).(Figure 24, Figure 25)

Conclusion

1. diabetes cognitive disorder rat hippocampus IGF-1 and PI3K/Akt/CREB signal path obstacle is damaged with cognitive function Evil is related.

2. newly plus gegen qinlian decoction is by increasing hippocampus IGF-1 expression and adjusting PI3K/Akt/CREB signal path activity Improve learning and memory function.

Above-mentioned experiment feeds composite S TZ intraperitoneal injections in 4 weeks using high-sugar-fat-diet can be successfully, reproduced diabetes model, should Model hyperglycemia after diabetes are formed persistently exists, and learning and memory in rats ability is decreased obviously at 8 weeks, can reflect diabetes brain Cerebral lesion situation when sick.Diabetes chronic hyperglycemia early stage i.e. damage neural blood vessel unit, causes blood-brain barrier disruption, peripheral blood Middle endothelial progenitor cells quantity is reduced, and microvascular lesions deformation, quantity are reduced, and capilary reparation and nascent capacity decline, hippocampus god Through first necrosis, vacuolation, glial cell invasion etc., with diabetes mellitus encephalopathy Hippocampal Injury positive correlation.Drug of the present invention can improve Diabetes rat ability of learning and memory, mechanism and protection blood-brain barrier, promote capilary repair newborn and activation IGF-1 and PI3K/AKT/CREB signal paths protect hippocampal neuron related in turn.

Claims

1. a kind of pharmaceutical composition for treating diabetes mellitus encephalopathy, it is characterised in that：It is the bulk pharmaceutical chemicals system matched by following weight Preparation made of standby：

2. pharmaceutical composition according to claim 1, it is characterised in that：It is by pueraria lobata, the coptis, radix scutellariae, the stem of noble dendrobium, bear Courage, Radix Notoginseng primary medicinal powder, water or extractive with organic solvent are active constituent, and pharmaceutically acceptable auxiliary material is added and is prepared Preparation.

3. pharmaceutical composition according to claim 2, it is characterised in that：The preparation is tablet, capsule, particle Agent, pill, oral solution.

4. a kind of method preparing the pharmaceutical composition described in claim 1-3 any one, it is characterised in that：It includes as follows Step：

A, the bulk pharmaceutical chemicals of each weight proportion are weighed；

C, bear gall, Radix Notoginseng is taken to beat powder；The concentrate and pharmaceutically acceptable auxiliary material that b step is added are prepared into pharmacy Upper common preparation.

5. the pharmaceutical composition described in claim 1-3 any one is preparing the drug for preventing or/and treating diabetes mellitus encephalopathy In purposes.

6. purposes according to claim 5, it is characterised in that：The drug is to mitigate cognition dysfunction, prevention sea The drug of horse neuron microvascular damage.

7. purposes according to claim 5, it is characterised in that：The drug is prevention hippocampal neuron capilary damage Evil protects tight junction protein and protects blood-brain barrier, safeguards the drug of brain homeostasis.

8. purposes according to claim 5, it is characterised in that：The drug be protection hippocampus microvascular endothelial cells, Promote endothelial progenitor cells migration and differentiation, promotes hippocampus Angiogenesis and improve the medicine of diabetes mellitus encephalopathy ability of learning and memory Object.

9. purposes according to claim 5, it is characterised in that：The drug be by adjust hippocampus IGF-1 and PI3K/Akt/CREB signal path activity improves the drug of learning and memory function.