CN112656883B - Traditional Chinese medicine composition for treating ischemic cerebrovascular disease and application thereof - Google Patents
Traditional Chinese medicine composition for treating ischemic cerebrovascular disease and application thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for promoting blood circulation and application thereof, belonging to the field of medicines. The invention aims to provide an effective medicament for treating diseases caused by cerebral ischemia injury. The invention provides a traditional Chinese medicine composition for promoting blood circulation, which comprises the following components in parts by weight: cornu Bubali, Ningpo Yam rhizome, Hirudo, Lumbricus, Notoginseng radix, radix rehmanniae Preparata, radix Angelicae sinensis, Saviae Miltiorrhizae radix, rhizoma corydalis Decumbentis, radix Anthrisci and resina Toxicodendri, pulverizing, sieving, decocting to obtain extract, oven drying, and tabletting. The blood circulation promoting composition provided by the invention can improve animal nerve behavior abnormality caused by cerebral ischemia, reduce cerebral infarction area of cerebral ischemia rats, improve neuron deletion condition of cerebral cortex ischemia semi-dark zone, improve cerebral edema condition, reduce permeability of blood brain barrier EB, improve neuron shrinkage, reduce synapse number, damage of capillary vessel basement membrane and other conditions, has a brain tissue protection effect on cerebral ischemia, and can be used as a specific medicine for treating ischemic cerebrovascular disease.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a blood-activating traditional Chinese medicine composition and application thereof.
Background
Ischemic cerebrovascular disease (ICVD) refers to the necrosis or softening of local brain tissue that occurs in the event of an interruption or insufficiency of blood supply, which is usually manifested as certain symptoms of neurological deficit. ICVD is a common disease with high mortality and disability rate, and is mostly found in middle-aged and elderly people. The pathogenesis of the disease is not completely clear, so the treatment medicine is deficient. The disease not only reduces the living standard of the middle-aged and the elderly, but also increases the economic burden of society and families to a certain extent.
In recent years, new achievements are obtained in the research of the treatment of cerebral ischemia injury, mainly including the elimination of free radicals, the improvement of antioxidant enzyme activity, anti-inflammation and Ca2+Antagonizing and inhibiting excitatory amino acid toxicity, resisting apoptosis, etc. However, the acute phase mainly involves thrombolysis and symptomatic treatment, and the thrombolysis therapy is limited in time (namely, thrombolysis is adopted within 6 hours of onset of disease), so that the development of a drug capable of quickly restoring blood supply in a cerebral ischemic region is of great significance. The ischemic cerebrovascular disease has the characteristics of high morbidity, high mortality and high disability rate, seriously harms human health, has the main treatment methods of ultra-early thrombolysis, symptomatic treatment and supportive treatment, and still lacks of specific medicines for treating the disease at present.
Disclosure of Invention
The invention aims to provide an effective medicament for treating diseases caused by cerebral ischemic injury, and provides a blood-activating traditional Chinese medicine composition for solving the problem of how to effectively treat the diseases caused by the cerebral ischemic injury, wherein the composition is prepared from the following components: 10-50 g of buffalo horn, 10-50 g of Ningpo yam rhizome, 3-10 g of leech, 3-10 g of earthworm, 5-30 g of pseudo-ginseng, 5-15 g of prepared rehmannia root, 10-50 g of Chinese angelica, 5-15 g of red sage root, 10-50 g of corydalis amabilis, 10-50 g of anthriscus sylvestris and 10-50 g of dried lacquer.
Further defined, the composition is made from the following components: 10-30g of buffalo horn, 10-20g of Ningpo yam rhizome, 3-5 g of leech, 3-5 g of earthworm, 5-10 g of pseudo-ginseng, 5-10 g of prepared rehmannia root, 10-20g of Chinese angelica, 5-10 g of red sage root, 10-20g of corydalis amabilis, 10-20g of anthriscus sylvestris and 10-20g of dried lacquer.
Further defined, the composition is made from the following components: 30-50 g of buffalo horn, 20-50 g of Ningpo yam rhizome, 5-10 g of leech, 5-10 g of earthworm, 10-30g of pseudo-ginseng, 10-15 g of prepared rehmannia root, 20-50 g of Chinese angelica, 10-15 g of red sage root, 20-50 g of corydalis amabilis, 20-50 g of anthriscus sylvestris and 20-50 g of dried lacquer.
Further limited, the dosage form of the blood-activating traditional Chinese medicine composition is tablets, powder, capsules or granules.
The invention also provides application of the blood-activating traditional Chinese medicine composition in preparing a medicine for treating ischemic cerebrovascular diseases.
Has the advantages that: the important combination of the blood circulation activation provided by the invention is proved by experimental research that the traditional Chinese medicine composition can improve the animal nerve behavior abnormality caused by cerebral ischemia to a certain extent, the cerebral infarction area of a cerebral ischemic rat can be reduced by a high and medium dose group of the traditional Chinese medicine composition, the arrangement of nerve cells of the high dose group and a nimodipine group in the traditional Chinese medicine composition is relatively regular, the swelling of nerve cells is weakened, the nimodipine bodies are increased, the neuron deletion condition of a cerebral cortex ischemic penumbra area is improved, the cerebral edema condition after the intervention of the high and medium doses of the traditional Chinese medicine composition is improved, the permeation quantity of blood brain barrier EB can be reduced after the administration of the high and medium dose group of the traditional Chinese medicine composition, and the situations of neuron shrinkage, reduction of synapse quantity, damage of capillary vessel basement membrane and the like can be improved by the high and medium dose traditional Chinese medicine composition. The traditional Chinese medicine composition for promoting blood circulation provided by the invention has a brain tissue protection effect on cerebral ischemic rats, and can be used as a specific medicine for treating ischemic cerebrovascular diseases.
Drawings
FIG. 1 is a graph of the cerebral infarction area of rats in each group, wherein A is a blank group, B is a sham operation group, C is a model group, D is nimodipine group, E is a low dosage group of a Chinese medicinal composition, F is a medium dosage group of the Chinese medicinal composition, and G is a high dosage group of the Chinese medicinal composition;
FIG. 2 is a transmission electron microscope photograph of nerve cells, wherein A is a blank group (x 10000), B is a sham operation group (x 10000), C is a model group (x 10000), D is a nimodipine group (x 10000), E is a high dose group (x 10000) of a Chinese medicinal composition, F is a medium dose group (x 10000) of the Chinese medicinal composition, and G is a low dose group (x 10000) of the Chinese medicinal composition;
FIG. 3 is a transmission electron microscope image of blood brain barrier, wherein A is blank group (x 10000), B is sham operation group (x 10000), C is model group (x 10000), D is nimodipine group (x 10000), E is high dosage group (x 10000) of Chinese medicinal composition, F is medium dosage group (x 10000) of Chinese medicinal composition, and G is low dosage group (x 10000) of Chinese medicinal composition;
FIG. 4 is a transmission electron micrograph of synaptic barriers, wherein A is a blank group (x 10000), B is a sham operation group (x 10000), C is a model group (x 10000), D is a nimodipine group (x 10000), E is a high dose group (x 10000) of a Chinese medicinal composition, F is a medium dose group (x 10000) of the Chinese medicinal composition, and G is a low dose group (x 10000) of the Chinese medicinal composition;
FIG. 5 is a graph of the results of staining HE of cortical neurons of rat brain in each group, wherein A is a blank group (X10000), B is a sham operation group (X10000), C is a model group (X10000), D is a nimodipine group (X10000), E is a high dose group (X10000) of a Chinese medicinal composition, F is a medium dose group (X10000) of a Chinese medicinal composition, and G is a low dose group (X10000) of a Chinese medicinal composition;
FIG. 6 is a graph showing the results of the rate of apoptosis of nerve cells in the cerebral cortex region of rats in each group; a is blank group, B is false operation group, C is model group, D is nimodipine group, E is Chinese medicinal composition high dose group, F is Chinese medicinal composition medium dose group, G is Chinese medicinal composition low dose group; h is an inhibitor group.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
1. Experimental animals: healthy adult SD male rats weighing between 180-220 g. SD rat is selected from Liaoning province experimental animal resource center. Rats were placed in the animal room for approximately 7 days of acclimatization prior to the experiment, during which time the humidity in the animal room was kept constant at 50%, the temperature was kept at 25 ℃ and the rats were free to eat and drink water. The whole animal experiment operation process strictly executes the relevant standards set by animal experiment ethics Committee of Chinese academy of science.
2. Experimental reagent: penicillin sodium powder is purchased from Harbin triple pharmaceutical industry, chloral hydrate is purchased from Fochen chemical reagent factory of Tianjin city, 2,3, 5-triphenyltetrazolium chloride is purchased from Shanghai blue season technology, Evans blue is purchased from Beijing Booltoda, formamide is purchased from Tianjin Tianli chemical reagent, paraformaldehyde is purchased from Guangzhou Kangguo biology, PBS is purchased from Shanghai Saimei Heshi, 2.5% glutaraldehyde is purchased from Beijing Raylen biotechnology, osmium acid is purchased from Beijing Raylen biotechnology, ethanol solution is purchased from Shanghai such as Ji Biotech, acetone solution is purchased from Shanghai Hanyao chemical industry, epoxy resin is purchased from Beijing Hakkaide Kangshi biological technology, uran acetate is purchased from Qingdao Jiekang Kango biological technology, lead citrate is purchased from Beihu Xin Xintai Gaitai chemical technology, xylene is purchased from Shanghai Hanhai chemical industry, solid paraffin is purchased from Shenhai Longtu wax, chemical industry, Taiyang acetate is purchased from Taiyang chemical industry, ammonia was purchased from Jiangxi Jiangjiang ammonia technology and neutral gum was purchased from Beijing Kull chemical. Nimodipine: nimodipine tablets (batch number: BJ36637, specification: 30mg) from Bayer company are selected.
Example 1.
A Chinese medicinal composition for promoting blood circulation: 30g of buffalo horn, 20g of Ningpo yam rhizome, 5g of leech, 5g of earthworm, 10g of pseudo-ginseng, 10g of prepared rehmannia root, 20g of Chinese angelica, 10g of red sage root, 20g of corydalis amabilis, 20g of anthriscus sylvestris and 20g of dried lacquer; the traditional Chinese medicinal materials all meet the national drug quality identification standard.
The preparation method comprises the following steps:
(1) pulverizing cornu Bubali, Hirudo and Lumbricus, and sieving with 80 mesh sieve;
(2) decocting rhizoma Discoreae Nipponicae, Notoginseng radix, radix rehmanniae Preparata, radix Angelicae sinensis, Saviae Miltiorrhizae radix, rhizoma corydalis Decumbentis, Anthriscus and resina Toxicodendri in water, filtering, mixing filtrates, and concentrating the filtrate to obtain extract;
(3) and (3) adding the mixture sieved in the step (1) into the extract in the step (2), drying, crushing and tabletting to obtain the blood-activating traditional Chinese medicine composition.
The method for decocting the traditional Chinese medicines comprises the following steps: soaking the medicines in cold water for 30min to completely soak the medicines in the water, then decocting with water, adding water to a position about 2 cm above the medicine surface, boiling the water with strong fire, then decocting with slow fire for about 30min, decocting one medicine twice, mixing the liquid medicines of the two times, carrying out evaporation concentration treatment, and finally controlling the concentration of the decoction to be 2.95 g/mL. The dosage of rats (rat middle dose is equal to the crude drug dosage multiplied by 0.018 multiplied by 5) is calculated by referring to the conversion formula of the equivalent dose of human and animals, and then the dosage of each group is obtained according to the ratio relation among different groups (low dose: middle dose: high dose is equal to 1:2: 4).
Example 2.
A Chinese medicinal composition for promoting blood circulation: 10g of buffalo horn, 10g of Ningpo Yam rhizome, 3g of leech, 3g of earthworm, 5g of pseudo-ginseng, 5g of prepared rehmannia root, 10g of Chinese angelica, 5g of red sage root, 10g of corydalis amabilis, 10g of anthriscus sylvestris and 10g of dried lacquer. The preparation method and the decoction method of the Chinese traditional medicine are the same as the embodiment 1.
Example 3.
A Chinese medicinal composition for promoting blood circulation: 50g of buffalo horn, 50g of Ningpo Yam rhizome, 10g of leech, 10g of earthworm, 30g of pseudo-ginseng, 15g of prepared rehmannia root, 50g of Chinese angelica, 15g of red sage root, 50g of corydalis amabilis, 50g of anthriscus sylvestris and 50g of dried lacquer. The preparation method and the decoction method of the Chinese traditional medicine are the same as the embodiment 1.
The test effect was verified using the following experiment: the effect was verified by taking example 1 as an example.
In the experiment, SD rats are divided into 7 groups, namely a model group, a blank group, a pseudo-operation group and a nimodipine group, in the study, and the traditional Chinese medicine composition group (high, medium and low dose groups) in the example 1 comprises 18 rats in each group.
The sham operation group: 1mL of distilled water is given, the injection is carried out for two weeks before the model building, once a day, and the preparation of the model of the pseudocerebral ischemia animal is carried out half an hour after the last injection.
Model group: 1mL of distilled water is given, the injection is performed for two weeks before the molding, the injection is performed once a day, and the animal model of cerebral ischemia is manufactured half an hour after the last injection.
Blank group: 1mL of distilled water is given, and the injection is carried out for two weeks before the modeling, once a day, and the modeling of cerebral ischemia is not carried out.
Nimodipine group: the administration dose is 20mg/kg, the concentration is 2mg/mL by diluting with distilled water, the administration is performed by intragastric administration for two weeks before the model building, once a day, and the animal model preparation of cerebral ischemia is performed half an hour after the last intragastric administration.
2. Preparation and evaluation of whole brain cerebral ischemia animal model
The preparation of the whole brain cerebral ischemia model is completed by adopting a two-vessel ligation method (2-vessel occlusion,2-VO), and the rats are fasted for 12 hours before molding and are free to drink water, and the detailed steps are as follows:
(1) all rats were first weighed, and the dose administered per rat was calculated according to the standard of 0.3mL/100g of 10% chloral hydrate, followed by intraperitoneal anesthetic injection.
(2) After the anesthesia operation is completed, the rat is placed in a supine position, hair in the center of the neck of the rat is removed, skin disinfection is carried out by using iodophor, then the skin is cut off along the center of the neck of the rat in a longitudinal direction, the length of the cut-off is about 20mm, each layer of subcutaneous tissue of the rat is subjected to blunt separation, and then the left carotid artery beside the trachea and the vagus nerve parallel to the carotid artery are found.
(3) The vagus nerve and common carotid artery were isolated by selecting an appropriate tool (glass needle) and then double ligation was performed using a No. 4 surgical thread. The treatment of the right common carotid artery was performed in the same manner.
(4) The operation is gentle, and the use of metal instruments to touch and excessively pull the vagus nerve is avoided as much as possible. After the operation, the skin is sutured intermittently, the heat preservation is carried out, and the anal temperature of the rat is maintained at 37.5 +/-0.5 ℃.
The TTC staining method is adopted to test the molding effect, if white infarction focuses with different ranges appear on the cerebral cortex of each group of rats after cerebral ischemia molding, the molding success is proved, and the specific operation method is shown in the following determination of cerebral infarction area.
3. Animal neurological deficit scoring
A Longa method is adopted to carry out nerve function scoring 24 hours after a rat is anesthetized and awake, a single blind method is selected for scoring, namely, an observer lifts the rat tail to be about 1 foot away from the ground under the condition that experimental groups are not understood, the stretching conditions of two forelimbs of the rat are observed, the rat is placed on the ground to observe the walking condition, simultaneously, the recording of the symptom of nerve function deficiency is carried out, and the score of the method is in positive correlation with the nerve behavior injury.
The scoring criteria were as follows:
0 minute: the rats normally move and do not show any symptom of nerve function loss;
1 minute: when the tail of the rat is lifted, lesion contralateral forelimb is difficult to be completely stretched;
and 2, dividing: when the rat crawls, the rat rotates towards the opposite side of the focus;
and 3, dividing: when the rat walks, the rat can topple towards the opposite side of the focus;
and 4, dividing: the rat has no autonomous activity and is accompanied with certain disturbance of consciousness;
4. cerebral infarction area determination
(1) After 24 hours of cerebral tissue ischemia molding treatment, 6 rats in each group are anesthetized and then the cerebral tissue is directly taken out. Because the brain tissue is soft, care must be taken when taking the brain to ensure the integrity of the brain tissue.
(2) The brain tissue is quickly transferred to a refrigerator with the temperature of 20 ℃ below zero and placed for 20min, so that the slicing treatment at the later stage is convenient.
(3) Placing the ventral side of the brain tissue in a brain groove in a downward direction, and carrying out coronal section processing along the frontal pole to the occipital pole direction to ensure that each section is about 2mm thick, and slicing 5-6 in total.
(4) Transferring the brain slice to a container filled with 2% TTC in advance, covering the container with tinfoil paper, transferring to a water bath kettle at 37 ℃, incubating for 15min under a dark condition, and turning over the brain slice according to conditions in the dyeing process to promote the brain slice to be in uniform contact with a dyeing solution.
(5) And taking out the brain slices after dyeing is finished, then washing the brain slices with normal saline, and transferring the brain slices into 4% paraformaldehyde solution for fixing for 24 hours after dyeing is finished. The digital camera can observe the cerebral infarction area after shooting.
(6) The cerebral infarction area was measured using Image processing software (Image Pro Plus), and the cerebral infarction area rate (total cerebral infarction area/total brain area × 100%) was calculated.
5. Hematoxylin-eosin staining (HE)
(1) Perfusion and fixation for material taking: firstly, 10% chloral hydrate is selected for experimental rats, and the abdominal cavity anesthesia injection treatment is carried out according to the dosage of 0.3mL/100 g. After the steps are finished, the chest cavity is opened, the heart is exposed, the right auricle is cut, a perfusion needle is inserted into the root part of the aorta from the apex of the heart, the normal saline is injected quickly, and the perfusion is stopped when the whitening of the liver is found. After completion of perfusion, the whole brain of the test rat was quickly removed and fixed with 4% paraformaldehyde fixing solution.
(2) After the brain tissue is fixedly treated by the fixing liquid for 24 hours, preparation of pathological sections is started, and the pathological sections are continuously washed by tap water for about 3 hours to remove paraformaldehyde solution contained in the tissue as far as possible.
(3) Ethanol ascending dehydration: after the steps are finished, transferring the target tissue into a dehydrator for dehydration treatment, wherein the dehydrating agent and the dehydrating sequence are respectively 75% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I and 100% ethanol II.
(4) Transparent xylene, wax dipping and embedding. And transferring the target tissue block into xylene for transparent treatment for about 30min, then respectively infiltrating the target tissue in soft wax I, soft wax II, hard wax I and hard wax II for 2h, and finally embedding the tissue in the wax block by using an embedding machine.
(5) Brain tissue was sectioned with a rotary microtome, each section was 4 μm thick, transferred to warm water at 42 ℃ and spread, and mounted on a glass slide coated with polylysine. After the completion, the mixture is transferred into an electric heating constant temperature drying oven for drying, and the parameters are controlled at 37 ℃ for 24 hours.
(6) Dyeing: the sections to be stained were dried in an electric oven (T ═ 60 ℃) for 30min, after which the sections were subjected to a conventional dewaxing treatment, with hematoxylin, tap water, 1% hydrochloric acid ethanol and eosin for 10min, several seconds and 10min, respectively. After the completion, ethanol dehydration treatment, xylene transparency and neutral gum sealing treatment are carried out.
(7) The physiological morphology and the histopathological conditions of the rat brain cortex were observed and photographed under an optical microscope.
6. Determination of water content in brain tissue
(1) After 24h of cerebral tissue ischemia molding treatment, 6 rats in each group were anesthetized (0.3mL/100g of 10% chloral hydrate) and then the cerebral tissue was directly taken out to measure the water content.
(2) The brain tissue was transferred to a prepared tin foil (weighed) and weighed immediately, wherein the difference between the weights of the two was the brain wet weight of the rat.
(3) Wrapping brain tissue with tinfoil, transferring to a 100 ℃ oven, treating for 24h, taking out to room temperature, and weighing twice, wherein the weight difference is the dry weight of the rat brain.
(4) Calculating the water content of the brain tissue: the water content of brain tissue is (wet weight of brain tissue-dry weight of brain tissue)/wet weight x 100%.
7. Evans Blue (EB) staining for testing Blood Brain Barrier (BBB) integrity
(1) After 24 hours of molding by the 2-VO method, 5mL/kg of 2% EB dye solution is injected from the left femoral vein of the rat. The rats are anesthetized after 3 hours, then physiological saline is perfused into the left ventricle, the volume is about 200-300 mL, and then the head is cut off to take the brain.
(2) Weighing rat left cerebral hemisphere, soaking brain tissue in formamide container in the ratio of 1mL/100mg, and incubating in water bath at 60 deg.C for 2 days.
(3) Controlling the parameters r of the centrifuge to be 1000r/min, centrifuging once when T is 5min, taking the supernatant after the centrifugation, placing the supernatant into a microplate reader, controlling the lambda to be 632nm to measure the absorbance value of the supernatant, and repeating the measurement for three times for each sample.
(4) Drawing an EB standard curve: precisely weighing 4mgEB, adding into 25mL of physiological saline weighed in advance to obtain 160 mug/mL solution, then respectively diluting to 0.125, 0.25, 0.5, 1, 2, 4 and 8 mug/mL solution, uniformly placing in a 60 ℃ water bath, carrying out photophobic incubation treatment for 2d, measuring the absorbance value (lambda is 632nm) by using a microplate reader, and drawing an EB standard curve.
(5) The EB concentration of the sample can be obtained by substituting the OD value measured by the sample into a standard curve, and then the EB content in the brain tissue can be obtained by substituting the EB concentration into the following formula. The specific formula is as follows:
EB content (μ g/g) ═ EB concentration (μ g/mL) × volume of formamide solution soaked brain tissue (mL)/wet weight of brain tissue.
8. Transmission Electron Microscope (TEM) for observing brain tissue ultrastructure
(1) Material taking: each group of rats was 24h after moldingPerfusing 200-300 mL of physiological saline through the left ventricle, cutting off the head to take out the brain after completion, and then transferring the cerebral cortex onto ice to prepare the brain with the thickness of 1mm3Small pieces of size.
(2) Fixing: placing the mixture in 2.5% glutaraldehyde solution at 4 deg.C for continuously fixing for not less than 2 hr, and washing with 0.1M neutral phosphate buffer solution for 3 times (15 min per time). Fixation with 1% osmic acid was then performed for 2h, and washing with the above phosphate was repeated.
(3) And (3) dehydrating: and (3) performing step-by-step dehydration treatment by using gradient ethanol, then performing replacement treatment by using propylene oxide, and transferring to a constant-temperature incubator for polymerization treatment after Epon812 epoxy resin is soaked and embedded.
(4) And (3) selecting an ultrathin slicer to slice the embedded block, ensuring the thickness of the embedded block to be about 70nm, then selecting lead citrate and uranyl acetate to carry out double dyeing treatment, and after the double dyeing treatment is finished, moving the embedded block to a TEM (transmission electron microscope) for observation and photographing treatment.
In three experiments of flow cytometry, enzyme-linked immunosorbent assay and immunohistochemistry, SD rats are divided into 8 groups, namely a blank group, a pseudo-operation group, a nimodipine group, Chinese medicinal composition groups, an inhibitor group (SB203580 is a p38 MAPK inhibitor), and the like, wherein each group comprises 6 rats. The dosage of the Chinese medicinal composition in high, medium and low dosage groups is 53.12g/kg, 26.56g/kg and 13.28g/kg respectively; the administration dosage of the nimodipine is 20 mg/kg; the model group, the sham operation group and the blank group were subjected to intragastric gavage with distilled water of equal volume. Continuous gavage is required for 14 days before the upper groups are modeled, and SB203580(5mg/kg) is injected into the abdominal cavity 30min before cerebral ischemia in the inhibitor group.
The experimental results are as follows:
first, 1. animal neurological deficit scoring results
After 24h of modeling, the rats in the sham operation group and the blank group do not have abnormal behaviors in the climbing and tail lifting processes, the gait of the rats is stable, the muscle force is normal, and the nerve function score is 0; the rats in the model group all have certain behavioral disturbance, and the rats are shown to bend forward from one forelimb when lifting the tail and walk to one side of the turning circle when crawling.
Results as shown in table 1, the neurological score of the model group rats was higher than that of the sham operation group (P < 0.01); the nerve function scores of rats in the high and medium dose groups and the nimodipine group of the traditional Chinese medicine composition are lower than those in the model group (P < 0.05). The experimental result shows that the high and medium dosage group of the traditional Chinese medicine composition has a certain effect on improving the neurobehavioral abnormality caused by cerebral ischemia.
TABLE 1 comparison of neurological scores in groups of rats
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^ 0; p<0.05 is expressed as ^.
2. Determination of cerebral infarction area
Firstly, pale infarcted foci with different ranges appear in each ischemic group, which indicates that the model is successfully made; no infarct focus exists in the sham operation group and the blank group; the pallor infarction area of the brain slice of the model group is large, and the boundary between the focal area and normal brain tissue is obvious;
as shown in table 2 and fig. 1, the cerebral infarction areas of the rats in the nimodipine group, the medium-dose group of the traditional Chinese medicine composition and the high-dose group of the traditional Chinese medicine composition are significantly reduced, and have significant difference (P <0.01) compared with the model group, wherein the cerebral infarction area reduction is most significant in the high-dose group of the traditional Chinese medicine composition; the cerebral infarction of rats in the low-dose group of the traditional Chinese medicine composition is also reduced, but the traditional Chinese medicine composition has no statistical significance compared with the model group (P > 0.05).
TABLE 2 comparison of cerebral infarction area rates of rats in each group
Note:in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^.
3. Measurement of water content in brain tissue
The results are shown in Table 3, the water content of the brain tissue of the rats in the sham operation group and the blank group has no obvious difference (P is more than 0.05), and the water content of the brain tissue of the rats in the model group is obviously increased compared with that of the rats in the former group (P is less than 0.01); the brain tissue water content of the traditional Chinese medicine composition middle dose group, the traditional Chinese medicine composition high dose group and the nimodipine group is obviously reduced, and the difference between the traditional Chinese medicine composition middle dose group and the nimodipine group is found to have statistical significance after being compared with the model group. The results prove that the nimodipine group, the medium-dose group and the high-dose group of the traditional Chinese medicine composition can reduce the water content of the brain tissue of a rat after ischemia, wherein the water content of the brain tissue of the high-dose group of the traditional Chinese medicine composition is reduced most obviously.
TABLE 3 comparison of brain tissue water content of various groups of rats
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^ 0; p<0.05 is expressed as ^.
EB staining test of blood brain Barrier integrity results
The results are shown in table 4, the brain tissue EB content of the sham operation group and the blank group is not obviously different (P > 0.05); compared with a sham operation group, the EB infiltration amount of the rats in the model group is remarkably increased (P < 0.01); the rat EB infiltration amount of the traditional Chinese medicine composition middle dose group, the traditional Chinese medicine composition high dose group and the nimodipine group is obviously reduced compared with that of the model group (P is less than 0.01); wherein, the EB infiltration amount of the high-dose group and the nimodipine group of the traditional Chinese medicine composition is obviously reduced. Experimental results prove that the medium and high dose groups of the traditional Chinese medicine composition have a certain effect on improving the integrity of the blood brain barrier.
TABLE 4 comparison of EB content in brain tissue of rats of each group
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^.
5. Result of observing ultra-microstructure of neurovascular unit by transmission electron microscope
The blood brain barrier, the cerebral cortex neuron cells and the nerve synapses are mainly observed in more detail. The results are shown in fig. 2, fig. 3 and fig. 4, the arrangement of nerve cells in the sham operation group and the blank group is regular, the gap is normal, the cells are mostly circular, the nuclear membrane is clearly visible, the structures of organelles such as mitochondria, golgi bodies and the like are normal, the ribosome is abundant, the number and the shape of synaptic vesicles are normal, the double-layer structure of the capillary basement membrane is normal, the periphery is free from edema and inflammatory cell infiltration, and the tight connection is normal. And the model group neurons are swollen, the chromatin of the neurons is condensed, the nuclear membrane is fused, the karyotype is abnormal, the quantity of ribosome and synaptic vesicle is reduced, the double-layer structure of the capillary vessel basement membrane is fuzzy, the surrounding astrocytes are severely swollen, and the tight connection structure is damaged. The experimental results show that the high and medium dosage of the traditional Chinese medicine composition can improve the conditions of neuron shrinkage, reduction of synapse number, damage of capillary basement membrane and the like.
Results of HE staining
The results are shown in fig. 5, the brain cortical neuronal cells of the rats in the sham operation group and the blank group are densely and orderly arranged, the cell morphology is normal, the cell membrane is complete, the nucleolus can be clearly observed, and the nucleosome is rich. The arrangement of the neuron cells of the model group is sparse, the structural hierarchy is disordered, the intercellular spaces of the neuron cells are widened, part of the neuron cells are also degenerated and necrotized, the boundary between nucleus and cytoplasm is fuzzy and the phenomenon of nuclear fixation and shrinkage occurs, the cell volume is reduced, a small part of nucleus is lightly stained and vacuolated, and even more, nucleolus disappears, and the phenomenon of obvious reduction of the Nissner corpuscle occurs. Compared with the model group, the arrangement of nerve cells in the traditional Chinese medicine composition is more orderly in the high-dose group and the nimodipine group, the swelling of the nerve cells is reduced, the nimodipine bodies are obviously increased, and the neuron deletion condition of the cerebral cortex ischemia penumbra area is obviously improved.
The results are summarized: (1) the experiment is carried out on neuroethology evaluation according to a Longa method, and the result proves that the traditional Chinese medicine composition can improve the animal neuroethological abnormality caused by cerebral ischemia to a certain extent.
(2) The TTC staining method is adopted to verify whether the model preparation is successful, and infarction focuses with different ranges are found in the cerebral cortex of an ischemic rat as a result, which indicates that the model building is successful; the percent of infarct part in the total brain slice area after TTC staining is analyzed and calculated by adopting IMAGE IMAGE software, and the result shows that the traditional Chinese medicine combination high and medium dose groups can reduce the cerebral infarct area of the cerebral ischemic rat.
(3) HE staining results show that the arrangement of nerve cells in the traditional Chinese medicine composition, the high-dose group and the nimodipine group is relatively regular, the swelling of nerve cells is weakened, the nimodipine bodies are increased, and the condition of neuron deletion in ischemic penumbra areas of cerebral cortex is improved.
(4) The water content of the brain tissue is measured by a dry-wet weight method, and the calculation result shows that the cerebral edema condition is improved after the intervention of high and medium doses of the traditional Chinese medicine composition.
(5) The permeability of BBB is detected by observing the content of EB in brain tissue, and the result shows that the permeability of EB in blood brain barrier can be reduced after the traditional Chinese medicine composition is administrated in a high-dosage and medium-dosage group.
(6) The electron microscope results show that the administration of the Chinese medicinal composition with high and medium dosage can improve the conditions of neuron shrinkage, reduction of synapse number, damage of capillary vessel basement membrane and the like.
II, 1, enzyme linked immunosorbent assay (ELISA) for measuring the expression of inflammatory factors
The influence of the traditional Chinese medicine composition on a serum inflammatory medium TNF-alpha of a rat with cerebral ischemia: ELISA detection results are shown in Table 5, and TNF-alpha expression is not significantly different between the sham operation group and the blank group (P > 0.05); the TNF-alpha level of the model group is obviously higher than that of the blank group, the pseudo-operation group, the nimodipine group and the high and medium dosage group of the traditional Chinese medicine composition; the nimodipine group decreased most significantly compared to the model group (P < 0.01); in addition, significant reductions in TNF-. alpha.levels were also observed in SB203580 when compared to the model group. The experimental results suggest that both the SB203580 group and the high and medium dosage groups of the traditional Chinese medicine composition can reduce the expression level of TNF-alpha.
The influence of the traditional Chinese medicine composition on the serum inflammatory mediator IL-6 of the cerebral ischemia rat is as follows: the detection results of the ELISA method are shown in Table 5, the traditional Chinese medicine composition has influence on serum IL-6 of rats with cerebral ischemia, and the result shows that the IL-6 expression has no obvious difference (P is more than 0.05) between a sham operation group and a blank group; compared with the sham operation group, the IL-6 in the model group is obviously increased (P < 0.01); after comparison with a model group, the expression level of IL-6 in a high-dosage group and a medium-dosage group of the nimodipine and the traditional Chinese medicine composition is reduced, and the reduction of the high-dosage group of the traditional Chinese medicine composition is most obvious (P is less than 0.01); the IL-6 level was also significantly decreased in the SB203580 group compared to the model group (P < 0.01). The experimental result indicates that the traditional Chinese medicine composition with high and medium dosage and SB203580 can reduce the level of IL-6 in serum of rats.
TABLE 5 Effect of Chinese medicinal composition on TNF-alpha and IL-6 in serum of rats with cerebral ischemia: (
n＝6)
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^.
The influence of the traditional Chinese medicine composition on the serum inflammatory mediator IL-1 beta of the cerebral ischemia rat is as follows: the results of detection by ELISA are shown in Table 6, there is no significant difference in IL-1. beta. expression between sham surgery and blank (P > 0.05); compared with the sham operation, the IL-1 beta level of the model group is increased remarkably (P < 0.01); the IL-1 beta level in the traditional Chinese medicine composition is reduced in the dosage group and the high dosage group (P is less than 0.01); the IL-1 beta level of the SB203580 group is also obviously reduced compared with the model group (P < 0.01); the IL-1 beta level of the traditional Chinese medicine composition in the middle and high dose groups and the SB203580 group is reduced, which shows that the three administration groups have certain effect on reducing the IL-1 beta level in the brain tissue of the rat.
The influence of the traditional Chinese medicine composition on the serum inflammatory mediator IL-2 of the cerebral ischemia rat is as follows: the ELISA detection results are shown in Table 6, and the IL-2 expression is not obviously different between the blank group and the sham operation group (P is greater than 0.05); the serum inflammatory medium IL-2 level of the model group is higher than that of the sham operation group (P < 0.01); compared with the model group, the IL-2 level of the traditional Chinese medicine composition in the high and medium dose groups and the nimodipine group is obviously reduced (P is less than 0.01); the IL-2 level was also significantly reduced in SB203580 compared to the model group (P < 0.01); the results show that the traditional Chinese medicine composition middle-dose group, the high-dose group and the SB203580 group can reduce the expression level of the serum inflammatory medium IL-2 of rats.
TABLE 6 Effect of Chinese medicinal composition on serum IL-1 beta and IL-2 of rats with cerebral ischemia: (
n＝6)
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^.
2. Flow Cytometry (FCM) for detecting apoptosis rate
The detection results are shown in fig. 6, and show that the apoptosis rates of the neuron cells in each group are respectively 3.28% in the blank group, 3.64% in the pseudo-operation group, 15.57% in the model group, 5.5% in the nimodipine group, 6.4% in the high-dose group in the traditional Chinese medicine composition, 6.58% in the medium-dose group in the traditional Chinese medicine composition, 12.54% in the low-dose group in the traditional Chinese medicine composition and 3.89% in the inhibitor group. No obvious difference exists between the sham operation group and the blank group; compared with the model group, the high and medium dose groups of the traditional Chinese medicine composition, the SB203580 and the nimodipine group of the traditional Chinese medicine composition have obviously reduced apoptosis rate. The experimental results suggest that the traditional Chinese medicine composition of the high and medium dosage groups and the SB203580 group can obviously reduce the apoptosis induced by cerebral ischemia.
3. Immunohistochemistry (IHC) method for detecting expression of apoptosis and blood brain barrier damage related protein
The detection results of the apoptosis-related factors are shown in table 7, and the sham operation group has no significant difference from the blank component (P > 0.05); compared with a sham operation group, the number of positive cells of Caspase-3 and Bax in the model group is increased, the number of positive cells of Bcl-2 is reduced, and the difference has statistical significance (P is less than 0.01); compared with the model group, the number of positive cells of Caspase-3 and Bax in the high and medium dose groups and the SB203580 group of the traditional Chinese medicine composition is obviously reduced, the number of positive cells of Bcl-2 is obviously increased, the difference has statistical significance, and the high and medium dose groups and the SB203580 group of the traditional Chinese medicine composition are prompted to reduce the expression of apoptosis-related proteins.
TABLE 7 Effect of Chinese medicinal compositions on the expression of Bcl-2, Bax and Caspase-3 in the cortical areas of brain: (
n＝6)
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^ 0; p<0.05 is expressed as ^.
The result of COX-2 detection is shown in Table 8, there is no clear difference in COX-2 expression between the blank group and the sham group (P > 0.05); compared with the sham operation group, the COX-2 positive cell number of the model group is increased (P < 0.01); compared with the model group, the number of positive cells of COX-2 in each dose group of the traditional Chinese medicine composition and the SB203580 group is obviously reduced, the difference has statistical significance, and the traditional Chinese medicine composition and the SB203580 have the effect of protecting brain tissues by down-regulating the expression of the COX-2.
The ICAM assay results are shown in table 8, there was no significant difference between sham and placebo groups (P > 0.05); compared with a sham operation group, the ICAM expression of the model group is obviously up-regulated (P is less than 0.01), and compared with the model group, the ICAM expression of the traditional Chinese medicine composition in the high and medium dose groups and the SB203580 group is obviously down-regulated, and the difference has statistical significance, and experiments suggest that the ICAM expression of the traditional Chinese medicine composition in the high and medium dose groups and the SB203580 can reduce BBB damage caused by inflammatory reaction by down-regulating the ICAM expression.
VEGF detection results are shown in Table 8, and there is no significant difference between VEGF positive cell numbers in the sham operation group and the blank group (P > 0.05); compared with a sham operation group, the VEGF positive cell value of the rat in the model group is obviously increased (P < 0.01); compared with the model group, the number of VEGF positive cells in the nimodipine group, the traditional Chinese medicine composition dose groups and the SB203580 group is obviously reduced, and the difference has statistical significance, which suggests that the traditional Chinese medicine composition and the SB203580 group can reduce the permeability of the blood brain barrier by reducing the expression of VEGF.
TABLE 8 Effect of Chinese medicinal compositions on COX-2, ICAM, VEGF expression in the cerebral cortex region: (
n＝6)
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a In comparison with the model group, P<0.01 is expressed as ^ 0; p<0.05 is expressed as ^.
The matrix metalloproteinase detection results are shown in table 9, and there is no significant difference between the sham operation group and the blank group (P > 0.05); compared with a sham operation group, the expression of MMP-2 and MMP-9 in a model group is obviously up-regulated (P is less than 0.01), and compared with a nimodipine group, a traditional Chinese medicine composition high and medium dose group and an SB203580 group, namely the expression of MMP-2 and MMP-9 is obviously down-regulated, the difference has statistical significance, and the result indicates that the traditional Chinese medicine composition high and medium dose group and the SB203580 can reduce BBB damage caused by degradation of basement membrane proteins by down-regulating the expression of MMP-2 and MMP-9.
TABLE 9 Effect of Chinese medicinal compositions on MMP-2 and MMP-9 expression in the cerebral cortex region: (
n＝6)
Note: in comparison with the sham group, P<0.01 is expressed as**(ii) a Comparison with model group P<0.01 is expressed as ^ 0; p<0.05 is expressed as ^.
Claims (6)
1. The traditional Chinese medicine composition for treating the ischemic cerebrovascular disease is characterized by being prepared from the following components: 10-50 g of buffalo horn, 10-50 g of Ningpo yam rhizome, 3-10 g of leech, 3-10 g of earthworm, 5-30 g of pseudo-ginseng, 5-15 g of prepared rehmannia root, 10-50 g of Chinese angelica, 5-15 g of red sage root, 10-50 g of corydalis amabilis, 10-50 g of anthriscus sylvestris and 10-50 g of dried lacquer.
2. The traditional Chinese medicine composition for treating ischemic cerebrovascular disease as claimed in claim 1, which is prepared from the following components: 30g of buffalo horn, 20g of Ningpo Yam rhizome, 5g of leech, 5g of earthworm, 10g of pseudo-ginseng, 10g of prepared rehmannia root, 20g of Chinese angelica, 10g of red sage root, 20g of corydalis amabilis, 20g of anthriscus sylvestris and 20g of dried lacquer.
3. The traditional Chinese medicine composition for treating ischemic cerebrovascular disease as claimed in claim 1, which is prepared from the following components: 10g of buffalo horn, 10g of Ningpo Yam rhizome, 3g of leech, 3g of earthworm, 5g of pseudo-ginseng, 5g of prepared rehmannia root, 10g of Chinese angelica, 5g of red sage root, 10g of corydalis amabilis, 10g of anthriscus sylvestris and 10g of dried lacquer.
4. The traditional Chinese medicine composition for treating ischemic cerebrovascular disease as claimed in claim 1, which is prepared from the following components: 50g of buffalo horn, 50g of Ningpo Yam rhizome, 10g of leech, 10g of earthworm, 30g of pseudo-ginseng, 15g of prepared rehmannia root, 50g of Chinese angelica, 15g of red sage root, 50g of corydalis amabilis, 50g of anthriscus sylvestris and 50g of dried lacquer.
5. The traditional Chinese medicine composition for treating ischemic cerebrovascular disease as claimed in any one of claims 1 to 4, wherein the dosage form of the traditional Chinese medicine composition for treating ischemic cerebrovascular disease is tablet, powder, capsule or granule.
6. Use of the Chinese medicinal composition for treating ischemic cerebrovascular disease as claimed in any one of claims 1 to 4 in the preparation of a medicament for treating ischemic cerebrovascular disease.
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| CN101966226A (en) * | 2010-10-08 | 2011-02-09 | 张继成 | Chinese medicinal composition for treating heart cerebrovascular diseases |
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| CN101966226A (en) * | 2010-10-08 | 2011-02-09 | 张继成 | Chinese medicinal composition for treating heart cerebrovascular diseases |
| CN102225100A (en) * | 2011-06-04 | 2011-10-26 | 李现华 | Chinese medicinal formulation for treating cardiovascular and cerebrovascular diseases and preparation process of products thereof |
| CN104027525A (en) * | 2013-03-06 | 2014-09-10 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Drug for treatment of ischemic heart disease, preparation method and application thereof |
| CN103405501A (en) * | 2013-08-12 | 2013-11-27 | 赵磊石 | Preparation method of three-component blood-activating and stasis-dissolving capsules |
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