CN108567920B - Traditional Chinese medicine composition for treating acute ischemic stroke - Google Patents

Abstract

The invention relates to a traditional Chinese medicine composition for treating acute ischemic stroke, which consists of effective components and medically acceptable excipient, wherein the effective components are prepared from the following raw material medicines in percentage by weight: 10.4-11.5% of rheum officinale, 10.4-11.5% of divaricate saposhnikovia root, 26.0-28.7% of cape jasmine fruit, 1.6-1.7% of calculus bovis factitius, 10.4-11.5% of tabasheer, 10.4-11.5% of magnolia officinalis, 10.4-11.5% of red paeony root and 15.6-17.2% of giant knotweed rhizome. The traditional Chinese medicine composition can be tablets, granules or capsules. Clinical verification shows that the traditional Chinese medicine composition has obvious curative effect on treating acute ischemic stroke.

Description

Traditional Chinese medicine composition for treating acute ischemic stroke

Technical Field

The present invention relates to medical formulations, in particular to pharmaceutical formulations of undefined structure from medicinal plants.

Background

Acute ischemic stroke is a common and high-incidence cerebrovascular disease in clinic, has the characteristics of acute morbidity, fast progress, high disability rate and high disease death rate, is the first acknowledged human health killer in the medical field, and is one of the major threats to the health of urban and rural residents in China. With the continuous progress of treatment means, the fatality rate of cerebral apoplexy is reduced, but the disability rate is gradually increased. According to the traditional Chinese medicine, the triple energizer is related to cerebral vessels, cerebral spirit and the like, the triple energizer congestion plays an important role in the pathological evolution of the stroke, and the curative effect of treating the acute ischemic stroke through a method of dredging the triple energizer is obvious clinically.

At present, the first effective treatment scheme for treating acute cerebral apoplexy at home and abroad is to use alteplase to carry out intravenous thrombolysis, the treatment effect is proved by experiments of national institute of neuropathy and apoplexy (NINDS), but the treatment scheme is limited in practical clinical application, because the method for treating acute cerebral apoplexy has strict indications, contraindications and time window, the proportion of patients treated by actual intravenous thrombolysis is very low, and is as low as 1.23% in China. The key link of reducing the fatality rate and the disability rate lies in the intervention treatment in the early stage of diseases, so that the fatality rate and the disability rate are reduced and the life quality of patients is improved by reasonably adopting traditional Chinese medicine intervention treatment through the treatment based on syndrome differentiation of traditional Chinese medicine, and the traditional Chinese medicine composition which has small toxic and side effects, is convenient to take for a long time and is specially used for treating the acute ischemic stroke is urgently found.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the traditional Chinese medicine composition for treating acute ischemic stroke, which is suitable for treating acute ischemic stroke and has obvious effect.

The technical scheme for solving the problems is as follows:

a traditional Chinese medicine composition for treating acute ischemic stroke comprises effective components and medically acceptable excipient, wherein the effective components are prepared from the following raw material medicines in percentage by weight: 10.4-11.5% of rheum officinale, 10.4-11.5% of divaricate saposhnikovia root, 26.0-28.7% of cape jasmine fruit, 1.6-1.7% of calculus bovis factitius, 10.4-11.5% of tabasheer, 10.4-11.5% of magnolia officinalis, 10.4-11.5% of red paeony root and 15.6-17.2% of giant knotweed rhizome.

The traditional Chinese medicine composition comprises the following raw materials, by the optimal ratio, 10.9% of rheum officinale, 10.9% of divaricate saposhnikovia root, 27.5% of gardenia, 1.6% of calculus bovis factitius, 10.9% of tabasheer, 10.9% of magnolia officinalis, 10.9% of red paeony root and 16.4% of giant knotweed rhizome.

The active ingredients in the scheme are prepared by the following method:

(1) pulverizing artificial calculus bovis into fine powder, and sieving;

(2) extracting the rest raw materials except calculus bovis factitius with water under reflux for 4 hr, filtering, collecting filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.0-1.2 at 60 deg.C, vacuum drying, and pulverizing into fine powder;

(3) and (3) mixing the fine powder prepared in the step (1) and the fine powder prepared in the step (2) to obtain the effective components.

The traditional Chinese medicine composition is a common oral preparation which is a tablet, a granule or a capsule prepared from the effective components and medically acceptable excipient according to a conventional method.

Based on the main pathogenesis of acute ischemic stroke, the inventor takes the general idea of 'dredging the triple energizer, relaxing the bowels, reducing phlegm and inducing resuscitation' as the prescription according to the traditional Chinese medicine theory. In the formula, the divaricate saposhnikovia root, the rhubarb and the gardenia are monarch drugs which respectively play roles in ventilating the lung, relaxing the bowels and promoting urination and can ventilate the triple warmer; the ministerial drugs of the artificial bezoar and the tabasheer clear heat and have the effects of reducing phlegm and inducing resuscitation; the adjuvant and guiding drugs comprise rhizoma Polygoni Cuspidati, radix Paeoniae Rubra, cortex Magnolia officinalis, and has effects of clearing hollow viscera, promoting blood circulation, and clearing hollow viscera. The whole formula has the effects of dredging the triple warmer, clearing hollow viscera, eliminating phlegm and inducing resuscitation. The monarch drug, the ministerial drug, the adjuvant drug and the conductant drug in the prescription respectively play their own roles, and the effect of treating the acute ischemic stroke is obvious.

The effect of the traditional Chinese medicine composition for treating acute ischemic stroke is proved by the following statistical results of clinical experiments and animal experiments.

Drawings

FIG. 1 is a diagram of the result of the MACO model evaluated by the laser speckle blood flow imaging system.

FIG. 2 is a graph showing the statistical results of the effect of the Chinese medicinal composition on the neurobehavioral scores of MCAO rats.

FIG. 3 is a result chart of the effect of the traditional Chinese medicine composition on the cerebral infarction area of MCAO rats.

FIG. 4 is a graph showing the effect of the Chinese medicinal composition on the brain morphology of MCAO rats (HE staining, x 200).

FIG. 5 is a graph showing the effect of the Chinese medicinal composition of the present invention on the morphology of MCAO rat brain tissue (Nissl staining, x 200).

FIG. 6 is a bar graph showing the results of the effect of the Chinese medicinal composition of the present invention on serum LPO, MDA and NO levels and SOD activity in MACO rats.

FIG. 7 is a bar graph showing the results of the effect of the Chinese medicinal composition of the present invention on the serum IL-1. beta. and TNF-. alpha.content of MACO rats.

FIG. 8 is a bar graph showing the effect of the Chinese medicinal composition of the present invention on the expression of MACO rat brain tissue-associated proteins.

FIG. 9 is a graph showing the results of the effect of the Chinese medicinal composition of the present invention on the number of apoptotic cells in brain tissue of MACO rats (Tunel staining, X200).

FIG. 10 is a graph showing the results of the effect of the Chinese medicinal composition of the present invention on the expression of MACO rat brain tissue-associated proteins (Bax, Cyt-C, LC3B) (immunofluorescence,. times.200).

The meanings of the reference symbols in fig. 2 above are: the Model group (Model), the invention medicament low dose group (TST-L), the invention medicament medium dose group (TST-M), the invention medicament high dose group (TST-H) and the Angong Niuhuang pill group (ANW) have statistical significance (P <0.05) compared with the Sham operation group (Sham), and the # indicates that the invention medicament low dose group (TST-L), the invention medicament medium dose group (TST-M), the invention medicament high dose group (TST-H) and the Angong Niuhuang pill group (ANW) have statistical significance (P <0.05) compared with the Model group (Model).

The meanings of the reference symbols in fig. 3 and fig. 6-9 are: indicates that the Model group (Model) has statistical significance (P <0.05) compared with the Sham group (Sham), and indicates that the # indicates that the Model group (Model) has statistical significance (P <0.05) compared with the Model group (Model), the # indicates that the Model group (Model) has statistical significance (TST-L), the TST-M, the TST-H, and the ANGONGNIUHUANG pill group (ANW).

(I) clinical experiments

1 object and method

1.1 general data

60 patients are from hospitalized patients with ICU encephalopathy in Dongguan city, 2016-8-2018, and are proved to be cerebral infarction or cerebral hemorrhage by skull CT/MRI. The treatment groups and the control group are randomly divided according to the treatment sequence. The treatment groups consisted of 30 cases, 19 men and 11 women; the control group had 30 cases, 20 men and 10 women. There was no statistical difference in the general data of age, sex, etiology, course and complications (P > 0.05).

1.2 diagnostic criteria

The western medicine diagnosis standard refers to the diagnosis standard of Chinese guidelines for acute ischemic stroke (2014). The Chinese medicine diagnosis standard refers to the Chinese viscera blockage syndrome standard of Chinese traditional medicine diagnosis and curative effect evaluation standard (trial) formulated by the cooperation of Chinese medicine administration in 1996 with brain disease and acute disease.

1.3 inclusion criteria

The following conditions are also satisfied: those who meet the diagnosis standards of traditional Chinese medicine and Western medicine for cerebral apoplexy; the syndrome diagnosis is consistent with the patients with viscera blockage in the stroke; the onset time is within 7 days; the age is between 35 and 80 years old; consciousness impairment score (Glasgow score) <13 points; the family members of the patients voluntarily sign the informed consent form.

1.4 exclusion criteria

One of the following conditions is satisfied: those that do not meet inclusion criteria; diagnosing a transient cerebral ischemia initiator; complicated with serious primary diseases such as blood, digestive, urinary system, or mental disorder.

1.5 case shedding criteria

One of the following conditions is satisfied: treatment of death cases during the observation period; those who were discharged halfway (giving up treatment, discharging or transferring to an outside hospital for continued treatment); the clinical observation was terminated for various reasons.

1.6 grouping method:

according to the random principle, a random number table is consulted to divide the treatment group and the control group into two groups for observation, and 30 cases are observed in each group. The sex and age of the patients in the two groups are compared, statistical analysis is carried out, and the difference between the two groups has no statistical significance.

1.7 methods of treatment

Because acute ischemic stroke has high fatality rate and disability rate, the life health and safety of patients must be kept in the first place in the process of clinical efficacy verification. The disease is generally in the acute stage within the initial 2 weeks after onset and in the convalescent stage after 2 weeks, so that two groups of patients must be treated routinely simultaneously in the acute stage 2 weeks after onset. The specific treatment modalities for the control and treatment groups were as follows:

basic treatment: refer to Chinese guidelines for acute ischemic stroke (2014), and perform basic treatment on the treated group and the control group respectively. The main treatment measures are as follows: basic nursing, vital sign maintenance (body temperature, blood pressure, heartbeat, pulse and the like), prevention and treatment of complications such as gastrointestinal hemorrhage and infection, control of blood pressure, blood fat and blood sugar, monitoring of the amount of entering and exiting, maintenance of water electrolyte balance, symptomatic treatment of cerebral edema and cranial hypertension, anti-platelet aggregation, fiber reduction, anticoagulation and the like.

Treatment groups: nasal feeding (after patient clearance, oral administration may be changed) on the basis of basal therapy the following granules described in example 1, 15g each time, dissolved in 100ml of warm boiled water 2 times a day. If the frequency of defecating exceeds 4 times per day, the dosage is reduced by half, and the smooth flow of fu-qi is kept (defecating is performed 1-3 times per day). 7 days is a treatment course, and the treatment course is 1 treatment course.

Control group: nasal feeding (after patient's mental clearance, oral administration can be changed) AngongNiuhuang pill (a pharmaceutical industry Co., Ltd., national standard Z44020047 in Baiyunshan, Guangzhou) on the basis of basic treatment, 3g each time, dissolving in 100ml of warm boiled water, 1 time per day. 7 days is a treatment course, and the treatment course is 1 treatment course.

1.8 Observation items

Vital signs (body temperature, blood pressure, etc.), glasgow coma scale score at admission; the refreshing time, the GCS scale scoring condition before treatment, 3 days after treatment and 7 days; observing the occurrence of routine blood, biochemical indexes, bedside electrocardiogram and adverse reactions before and after treatment.

1.9 therapeutic efficacy criteria

Patients were scored and compared using the Glasgow coma rating scale before treatment, 3 days after treatment, and 7 days after treatment, respectively. The clinical cure is as follows: the score of the consciousness disturbance reaches more than 13 points; the effect is shown: improvement in consciousness impairment score by more than 5 points; the method has the following advantages: improvement in consciousness impairment score by more than 2 points; and (4) invalidation: the disturbance of consciousness score is unchanged or even aggravated.

2.0 statistical treatment

All statistical data are analyzed by SPSS 13.0 statistical software, and the measured data are expressed by means of mean plus or minus standard deviation and are tested by t; the counting data is checked by chi-square. The difference is statistically significant with P < 0.05.

2 results

2.1 comparison of conscious time of two groups of patients

The difference between the mean conscious time of the patients in the treatment group and the patients in the control group is statistically significant (P is less than 0.05), which is shown in Table 1.

TABLE 1 mean conscious time comparison of two groups of patients: (

Sky)

Note: the clear time comparison of the treatment group and the control group shows that the difference is significant (P < 0.05).

2.2 Pre-and post-treatment GCS score comparison between two groups of patients

The comparative differences between the two groups before treatment were not statistically significant (p > 0.05). 3 days after treatment, the GCS scores of the two groups are improved to different degrees, and the difference has significance (P is less than 0.05) compared with the GCS scores of the same group before treatment; comparison of differences between the two groups 3 days after treatment was not statistically significant (P > 0.05). After 7 days of treatment, the treated group has significant difference compared with the control group, and has statistical significance (P <0.05), which is shown in Table 2.

TABLE 2 comparison of GCS scores before and after treatment in two groups: (

Minute)

Note: comparing the two groups 3 days after treatment with the groups before treatment, wherein P is less than 0.05; ② compared with the control group after 3 days of treatment, P is more than 0.05; ③ 7 days after the treatment, the P is less than 0.05 compared with the control group.

2.3 clinical efficacy comparison of two groups of patients

The difference between the total effective rate of the treatment group and the control group has statistical significance (P <0.05), and is shown in Table 3.

TABLE 3 comparison of post-treatment clinical efficacy of the two groups (n,%)

Note: the comparison of the total effective rate of the treatment group and the control group shows that the significant difference exists (P is less than 0.05).

2.4 safety analysis

There were no deaths in both groups during the observation period of the study, and no adverse events occurred. The safety indexes of the two groups of medicines have no obvious influence on 1 st and 7 th days after the medicine is put into the group, and the two groups of medicines are safe and have no damage to organs such as liver, kidney, heart and the like.

2.5 conclusion

The Angong Niuhuang Wan is a Chinese patent medicine which is most negative and popular in clinical critical illness treatment and has wide application in the traditional Chinese medicine, is a traditional prescription for treating stroke amenorrhea syndrome, and has the effects of relieving fever, tranquilizing, relieving cerebral edema, promoting the repair of an injured nervous system, improving cerebral circulation, increasing cerebral blood flow and the like. The research finds that the traditional Chinese medicine composition or the bezoar chest functioning pill can improve the consciousness state of patients with the viscera closed diseases in the acute stage of the apoplexy by matching with the basic treatment. Compared with the control group, the treatment group has the advantages of shortening the waking time of the patients and improving the GCS score of the patients 7 days after treatment.

(II) animal experiments

1 Experimental materials and instruments

1.1 Experimental drugs

The treatment group medicine is granules described in the embodiment 1 of the invention;

the control group of drugs was AngongNiuhuang Wan (Beijing Tongrentang science and technology development, Inc. batch No. 16017026).

1.2 Experimental reagents

Rat line plug 250-280 g: specification: 250-280g/0.37mm 0.28mm/5cm (Shenzhen Riwold Co.); sodium pentobarbital (Merck, Germany, batch No. 110320); specification of 2% lidocaine hydrochloride: 5ml (Hubei Tian pharmaceutical Co., Ltd., lot number: 31604283); general tissue fixative (neutral) (Wuhan Severe Biotech, Inc., lot number 174801); 2,3, 5-triphenyltetrazolium chloride (Amresco, USA, with a batch number of 20170701); BCA protein assay kit (Biyuntian Bio Inc., lot number 022616160511); malonic acid (MDA) kit (tokyo founded bio-inc, lot number 20180103); SOD reagent box (Nanjing Biochemical company, batch number: 20180103); lipid Peroxide (LPO) kit (Nanjing Biotech, lot number 20180103); a kit for detecting the content of Nitric Oxide (NO) (Beijing Soilebao Tech Co., Ltd., batch No. 20180224); rat IL-1 beta ELISA kit (Abbkine, product No. KET9001-2, batch No. ATQSF 2601); rat TNF-alpha ELISA kit (Abbkine, product No. KET9007-2, batch No. ATQSE 2101); tunel kit (Roche, cat # 11684817910); Anti-Bcl-2antibody (abcam, cat # Ab 32124); Anti-LC3B antibody (abcam, cat # Ab 192890); Anti-Baxantibody (abcam, cat # Ab 32503); anti-active caspase-3antibody (abcam, cat # Ab 32042); Anti-Cytochrome C antibody (abcam, cat # Ab 33504); Anti-Nrf2antibody (abcam, cat # Ab 31163); Anti-Heme Oxygenase 1 antibody (abcam, cat # Ab 13248); Anti-GAPDH Rabbit Polyclonal antibody (Kangjiu Shiji Co., Cat: CW0101M, lot 30118); anti-beta-actin Mouse Monoclonal antibody (Kangjiu century Co., Cat: CW0096M, Lot 10209); IRDye 800CW Goat anti-Rabbit (second antibody) (LI-COR Co., Ltd., product No. 926-32211, lot No. C70301-05); IRDye 800CW Goat anti-Mouse (second antibody) (LI-COR Co., Ltd., cat # 926-32210, lot # C70301-02); goat serum for blocking (Kangjie Shiji Co., Cat: CW0130S, lot 30245); anti-fluorescence attenuation packaged tablets (Beijing Soilebao Tech Co., Ltd., product number: S2110).

1.3 Experimental instruments

(1) Dewatering machine: the model is as follows: STP 120; the manufacturer: sammer Feishel technologies, Inc.;

(2) a tissue embedding machine: the model is as follows: HistoStar; the manufacturer: sammer Feishel technologies, Inc.;

(3) paraffin slicer: the model is as follows: HM 340E; the manufacturer: sammer Feishel technologies, Inc.;

(4) inverted fluorescence microscopy: the model is as follows: a BDS 300; the manufacturer: chongqing ott optical instruments ltd;

(5) sample rapid grinding appearance: the model is as follows: JXFSPRP-32; the manufacturer: shanghai Jing Shi Kogyo development Co., Ltd;

(6) an enzyme-labeling instrument: the model is as follows: multiskan G01510; the manufacturer: sammer Feishel technologies, Inc.;

(7) two-color infrared laser imaging system: the model is as follows: odyssey SA; the manufacturer: gene Take Ltd;

(8) horizontal shaking table: the model is as follows: WD-9045B, manufacturer: six instrument factories in Beijing;

(9) GXG electronic balance: the model is as follows: JJ 3000; the manufacturer: double jie test instrument factory, normal-maturing city;

(10) analytical balance: the model is as follows: an EL 204; the manufacturer: shanghai Merle-Torledo instruments, Inc.;

(11) a centrifuge: the model is as follows: D3024R; the manufacturer: sailyker, USA;

(12) transferring the power supply of the electrophoresis apparatus: the model is as follows: DYY-7C; the manufacturer: beijing, six Biotechnology Ltd;

(13) double vertical electrophoresis apparatus: the model is as follows: DYCZ-25D; the manufacturer: beijing, six Biotechnology Ltd;

(14) glass plate support: the model is as follows: WD-2106A; the manufacturer: beijing is a Biotechnology Ltd.

1.4 consumables

Syringes, shavers, scalpels, iodophors, cotton swabs, medical gauze, fixing plates, cotton threads, suture needles, surgical scissors, tweezers, sodium citrate anticoagulation tubes, vacuum blood collection tubes and the like.

2 method of experiment

2.1 Experimental animals

2.1.1 variety, Source and weight

SPF male rats aged 8-10 weeks and having a body weight of about 250-270g were purchased from the Experimental animal center of Guangzhou university of traditional Chinese medicine, and the animals were given license numbers: SCXK (yue) 2013-: SCXK (Yue) 2013-. Adaptive feeding for 1 week before experiment. The practical operations of feeding, gavage and the like of the experimental rat are performed according to international guidelines.

2.1.2 animal receiving, quarantine and quarantine duration markers

(1) Receiving: after the animals are purchased, the receiving personnel check and accept, and after the animals are qualified, the animals are transported to an animal room of a pharmacy building A401, 4-5 animals are placed in each cage, and normal feed and drinking water are given.

(2) Quarantine content and results: the fur is glossy and has no depilation phenomenon; eyes, nose and ears are normal and no secretion exists; no trauma and inflammation on each part of the body; normal behavior, gait and autonomic activity; the bowels are normal; normal food intake and water drinking. During the quarantine observation period, the indications of the animals such as appearance signs, behavior activity, stool character and food intake are observed, and unqualified animals are not found.

(3) Site and time of quarantine observation: animals were received for 3 days of quarantine at pharmacy building a401, university of traditional Chinese medicine, guangzhou.

2.1.3 animal feeding and rearing conditions

(1) A breeding environment: the animals are raised in stainless steel cages and are raised in experimental animal houses, the environmental temperature range of the animal houses is 22-25 ℃, the relative humidity is 50-60%, the light and shade alternate for 12 hours, the working illumination is 200-300 Lux, and the animal illumination is 15-20 Lux. The food box is labeled, and the label content comprises: experiment name, gender, person in charge of subject, date of receipt, and the like.

(2) Feeding conditions are as follows: feeding rats according to the principle of 'timing and quantifying', taking a proper amount of pellet feed, distributing the pellet feed into each cage of rat hamper, and feeding the rats twice a day. The rat feed was provided by the laboratory animal center of the university of traditional Chinese medicine, Guangzhou. The drinking water is pure water and is changed every day.

2.2 dosage and method of administration

The drug was gavaged 6 hours after surgery for 1 time, followed by continuous gavage for 7 days, 1 time per day. The model group and the sham operation group are given pure water, the AngongNiuhuang pill group is given 0.3g/kg, and the low, medium and high dose groups of the treatment group are respectively given 0.6g/kg, 0.3g/kg and 0.15g/kg of the granules of the invention example 1. The intragastric volume is 10 ml/kg.

TABLE 4 dosage form for animals

2.3 Experimental groups

Healthy male SD rats are adopted, drinking water is normally taken, after adaptive feeding is carried out for 1 week, cerebral ischemia reperfusion operation is carried out, and after the operation is successful, the operation is randomly divided into 6 groups: sham (Sham), Model (Model), inventive Low dose (TST-L), inventive high dose (TST-H), Angong Niuhuang pill (ANW). The sham operation group was the same as the operation group except that no wire plug was inserted. Each group had 15 animals, and if death occurred during the experiment, rats were supplemented.

2.4 preparation of the model

Male SD rat is fasted and not forbidden to water before operation one night, is injected with 1% pentobarbital sodium for anesthesia, is fixed by a rat plate in a supine position, the neck is depilated by a hair shaver, the neck is disinfected by a 75% alcohol cotton ball, a 1-2cm opening is cut in the center of the neck, the muscle gap between sternocleidomastoid muscle, sternum and hyoid muscle is separated bluntly, the common carotid artery and vagus nerve are exposed at the left internal cervical triangle, then internal and external carotid arteries are separated bluntly along the common carotid artery, the external carotid artery is ligated to the proximal end of the external carotid artery by an operation thread after dissociating the external carotid artery, the internal carotid artery and the common carotid artery are respectively clamped by a micro-artery clamp, an operation thread is prepared under the internal carotid artery, an incision is cut at the position of the common carotid artery by a micro-ophthalmic scissors, a thread plug is inserted into the common carotid artery along the incision, a loose knot is formed at the incision, and the artery clamp at the internal artery is loosened, slowly inserting the thread plug into the internal carotid artery from the common carotid artery upwards, finally entering the intracranial section of the internal carotid artery, reaching the initial part of the middle cerebral artery, inserting the thread plug into the intracranial section of the internal carotid artery to a depth about 18 +/-1 mm away from the bifurcation of the internal carotid artery and the external carotid artery, blocking all blood supply sources of the middle cerebral artery on one side, then fastening a slipknot at the incision of the common carotid artery, loosening a common carotid artery clamp, suturing layer by layer, and exposing the other end of the thread plug outside the skin. After 1.5 hours, the plug was slowly pulled out until resistance indicated that the plug tip reached the carotid artery incision, cerebral blood reperfusion was complete, and the plug exposed outside the skin was cut off. Postoperative penicillin antiphlogistic treatment is carried out, and 0.25% lidocaine hydrochloride analgesic treatment is given at the same time. The sham operation group was the same as the operation group except that no wire plug was inserted.

2.5 Observation index

2.5.1 laser speckle data acquisition

After the operation is finished, one animal of the sham operation group and one animal of the model group are randomly extracted, and the rat experiment is subjected to gas anesthesia in the whole process by using isoflurane mixed with compressed air (oxygen and nitrogen). At the beginning of the experiment, anesthesia was induced with 5% isoflurane and then continued with 2% isoflurane, and the procedure was performed under this anesthesia and the laser speckle imaging raw data was collected. The rat is stably fixed on a stereotaxic apparatus, the hair of the head of the rat is removed by a hair shaver, the scalp and the fascia are cut off along the sagittal suture from the center of the head, connective tissues such as periosteum and the like on the surface of the skull are cleaned by an operating knife, and then a high-speed dental drill with a drill bit with the diameter of about 1.6mm is used for grinding the skull area between the coronal suture and the herringbone suture of the rat until the cortical blood vessels are clearly visible. In the process of polishing the skull, physiological saline is used for cooling in due time, so that the thermal injury to the brain tissue of the rat caused by too fast polishing is prevented. After polishing, the round stainless steel base of the imaging device is arranged in the thinned rat skull region.

In the experiment, the skull is only ground, the skull is not removed, the integrity of dura mater and cortical tissue is maintained, the change of intracranial pressure caused by the loss of the skull can not be caused, cerebral edema and the like, so that the stable long-term observation of the experimental rat is ensured, and errors possibly caused by heart beating and respiration are reduced.

2.5.2 general index

Carefully observing and recording the mental state, fur change, activity condition, drinking water and food intake of each group of rats every day; and the body weight of each rat was measured on days 1, 3 and 7 after the operation.

2.5.3 neurobehavioral Scoring

Rats were scored neuro-behaviorally on days 1, 3, and 7 after molding, respectively. And (4) removing the rats in the plug-in surgery group with the total score of less than 3 on the first day after the surgery, and filling the rats. Neurobehavioral scoring criteria: firstly, a rat tail is provided to observe the flexion condition of forelimbs, if the two forelimbs symmetrically extend to the ground, the flexion is counted as 1 minute, the flexion of elbows is counted as 2 minutes, the internal rotation of shoulders is counted as 3 minutes if the contralateral forelimbs of the operation appear, and the flexion of the wrists and the internal rotation of the shoulders are counted as 4 minutes if the wrists penetrate and flex; secondly, the animal is placed on a flat ground, the animal is pushed to the two shoulders to move towards the opposite sides respectively, the resistance is checked, if the resistance on the two sides is equal and powerful, the resistance is counted as 0min, if the resistance is descended when the animal is pushed to the opposite sides of the operation, the resistance is divided into three degrees of light, medium and heavy according to the descending degree, and the resistance is counted as 1 min, 2 min and 3min respectively. Thirdly, placing the two forelimbs of the animal on a metal mesh, and observing the muscle tension of the two forelimbs. The muscle tension of the double forelimbs is equal and strong, and the score is 0, and the score is 1, 2 and 3 according to the difference of the tension reduction degree of the contralateral limbs after the operation. Fourthly, the animal turns round without stopping turning to one side, and the score is 1. The higher the score, the more severe the behavioral disturbance of the animal.

2.5.4 TTC staining

The animals are anesthetized and sacrificed, the head is quickly cut off, the skull is stripped, the brain is taken out in an ice bath environment, the cerebellum, the olfactory bulb, the lower brain stem and the like are removed, the animals are placed into a refrigerator at the temperature of minus 20 ℃ for freezing for 20min, and after the animals are taken out, coronal sections are made by an operation blade at intervals of 2mm from the frontal pole, and 4 animals are made continuously. Immediately placing the section in 2% TTC for staining, incubating for 20min at 37 ℃ in a dark condition, placing the section in 4% formic acid for fixing for 1h after staining, taking a picture by a digital camera, inputting the picture into a computer, measuring the area of ischemic brain tissue on the picture by an Image-pro plus6.0 Image analysis system (the ischemic part is not stained, and the ischemic part is stained red), and calculating the area of an infarcted part.

2.5.5 pathological staining (HE staining and Nissen staining)

And (3) fixing a coronal section tissue block of 2mm before and after bilateral cerebral vision cross on 4% of poly formic acid after ice bath decapitation, slicing after paraffin embedding, and observing and evaluating pathological changes of brain tissues of rats in each group under a light microscope after HE staining. And (3) Nie dyeing: the sections were stained with 1% toluidine blue solution, then color-separated with 95% alcohol, followed by gradient dehydration with alcohol, xylene was transparent, and the nissl body morphology, quantity and distribution changes were visualized on a light microscope after mounting.

2.5.6 Biochemical index detection

The traditional Chinese medicine composition has the following effects on the oxidative stress condition of cerebral ischemia reperfusion of rats: the method comprises the following steps of (1) determining relevant indexes of oxidative stress in serum by a colorimetric method: lipid Peroxide (LPO) content, superoxide dismutase (SOD) activity, Malondialdehyde (MDA) content, and Nitric Oxide (NO) content. The corresponding operation is carried out according to the instruction steps on the kit of LPO, SOD, MDA and NO, and the content of the LPO, SOD, MDA and NO in the serum of each group of rats is measured and recorded.

2.5.7 ELISA method for detecting IL-1 beta and TNF-alpha of inflammatory factors

And (3) measuring the contents of IL-1 beta and TNF-alpha in serum by using an ELISA kit, and prompting operation and measurement according to instructions.

2.5.6 immunoblotting method for detecting protein expression

Detecting the expression of apoptosis-related proteins Bax, Bcl-2 and Cyt-C, detecting the expression of autophagy-related protein LC-3 and detecting the expression of oxidative stress-related proteins Nrf2 and HO-1.

(1) Extracting protein from rat brain tissue and determining its concentration

Protein extraction: first, approximately 100mg of rat brain tissue was weighed into a 2mL EP tube, and the brain tissue mass was cut as much as possible. Then adding 1mL of RIPA lysate (strong) and placing on ice, then crushing the brain tissue for 3 times by a homogenizer, each time for 10 seconds, placing on ice to fully crack the brain tissue, after 30min, centrifuging at 12000rpm at 4 ℃ for 10 min; the centrifuged supernatant was dispensed into 0.5mL centrifuge tubes and stored in a freezer at-80 ℃.

Determination of brain tissue protein concentration: according to the BCA kit specification, the specific steps are as follows:

preparing 0.5mg/mL standard protein diluent: precisely sucking 20 mu L of 25mg/mL protein standard solution, and adding 980 mu L of diluent to obtain the final product.

② precisely pipetting a sample of an appropriate volume into a 96-well plate, and then adding a diluent to 20. mu.L. Separately, 20, 16, 12, 8, 4, 2, 1, 0. mu.L of a 0.5mg/mL standard protein dilution was pipetted precisely into a 96-well plate, and the dilution was added to 20. mu.L.

Preparation of BCA working solution is determined according to the number of samples, and BCA-A, BCA-B solution is prepared according to the proportion of 50: 1.

And fourthly, adding 200 mu L of BCA working solution into the plate hole, fully and uniformly mixing, and then placing for 30min at 37 ℃. The protein concentration was determined at a wavelength of 562 nm.

(2) Preparation of SDS-PAGE gel electrophoresis

Filling glue: and cleaning and drying the glass plate, keeping the two layers to be flat and aligned, and fixing the glass plate on an electrophoresis glue pouring device to prepare glue pouring. The appropriate gel concentration was chosen to measure the molecular weight of the protein of interest, and the procedure was followed according to the instruction manual to prepare 12% or 15% separation gel, fill about 6ml of separation gel and seal with ultra pure water, when the refracted ray appeared between the separation gel and water, it was indicated that the coagulation was complete. After about 5min, the supernatant water was decanted and blotted clean with filter paper. 5% concentrated gel was added to the separation gel and immediately inserted in parallel with a comb. The glue is properly supplemented on two sides in the process of gelling and solidifying, so that the volume reduction caused by gelling and solidifying is avoided. After the concentrated gel is solidified, the comb is pulled out along the vertical direction, so that the gel sample adding hole is prevented from being damaged.

Sample treatment and loading: 5 Xreduction loading buffer and deionized water were added to 30. mu.g of total protein sample solution to a total volume of 20. mu.L, followed by shaking and mixing. The resulting mixture was incubated at 95 ℃ for 10min to denature the protein, and then centrifuged. Sequentially, 20. mu.L of the denatured protein sample was pipetted by a pipette and slowly added to the loading well, taking care to avoid the generation of air bubbles.

③ electrophoresis: and after the sample loading is finished, preparing electrophoresis, setting the electrophoresis voltage to be 80mV, running for 20min, adjusting the voltage to be 120mV when the bromophenol blue in the sample enters the separation gel after being pressed into a straight line, and stopping electrophoresis when the bromophenol blue moves to a position about 1cm away from the bottom of the glass plate.

Fourthly, transferring the film: opening the clamp of the transfer module, setting the black side at the bottom, then installing a sandwich layer, and placing the sponge pad → filter paper → glue → PVDF film (needing to be activated in methanol for 5min in advance) → filter paper → the sponge pad in sequence, and paying attention to avoid bubbles in the whole placing process. And then rotating for about 1h under the voltage condition of 300mA, wherein the specific time is determined according to the molecular weight, and the whole film rotating process is carried out in an ice bath environment.

Sealing: after completing the membrane transfer, the membrane was washed in TBS quickly, and the PVDF membrane was removed and clipped off in the upper right corner to distinguish the orientation. The membrane shearing is mainly carried out according to the molecular weight of a marker and the molecular weight of protein to be measured. The sheared membrane was immersed in a TBST solution containing 5% skimmed milk powder and blocked at room temperature for 2 h. (the membrane cannot make any contact with Tween-20 before blocking is finished, otherwise the background is too high)

Sixthly, primary antibody incubation and secondary antibody incubation: the blocked PVDF membrane was rinsed gently with TBST, then diluted with primary antibody (antibody dilutions were made according to the instructions), incubated overnight (16h) in a horizontal shaker at 4 ℃ and recovered after the primary antibody was completed, and then washed with TBST 4 times for 5min each. And (3) after the membrane is washed, adding a secondary antibody diluent (in a dark place) marked by an infrared dye, incubating the mixture on a horizontal shaking table for 1h (in a dark place) at room temperature, and after the secondary antibody incubation is finished, washing the membrane for 4 times by TBST (tert-butyl-tert-butyl ether) for 5min each time.

And color development: tween-20 was removed by washing with PBS and scanning was performed using an Odyssey CLx near-infrared two-color fluorescence imaging system.

2.5.7Tunel method for detecting cell apoptosis

(1) Paraffin section dewaxing to water: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing. (properly lengthening dewaxing time in winter)

(2) Repairing: after the section is slightly dried, a circle is drawn around the tissue by a organizing pen (liquid is prevented from flowing away), proteinase K working solution (proteinase K storage solution is diluted by PBS 1:9) is dripped into the circle to cover the tissue, and the tissue is incubated for 30min at 37 ℃. The slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

(3) Membrane breaking: after the section is slightly dried, the membrane-breaking working solution is dripped into the ring to cover the tissue, the incubation is carried out for 20min at normal temperature, and the slide is placed in PBS (pH7.4) and is shaken and washed on a decoloration shaking table for 3 times, 5min each time.

(4) Addition of reagents 1 and 2: taking a proper amount of reagent 1(TdT) and reagent 2(dUTP) in the tunel reagent box according to the number of the slices and the size of the tissues, mixing the mixed reagent 1(TdT) and the reagent 2(dUTP) according to a ratio of 2:29 (the reagents 1 and 2 are prepared as before), adding the mixed reagent into the ring to cover the tissues, horizontally placing the slices in a wet box, incubating at a constant temperature of 37 ℃ for 2 hours, and adding a small amount of water into the wet box to keep.

(5) Blocking endogenous peroxidase: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each. The sections were incubated in 3% hydrogen peroxide solution in methanol (hydrogen peroxide: methanol ═ 1:9) for 15min at room temperature in the dark, and the slides were washed 3 times in PBS (pH7.4) on a destaining shaker for 5min each time.

(6) Adding a reagent 3: after the sections were spin-dried slightly, each section was covered with an appropriate amount of reagent 3 (coverer-POD), and the sections were placed flat in a wet box and incubated in an incubator at 37 ℃ for 30 min. Slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each.

(7) DAB color development: after the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive nucleus is brownish yellow, and the section is washed by tap water to stop color development.

(8) Counterstaining cell nuclei: harris hematoxylin is counterstained for about 3min, washed with tap water, 1% hydrochloric acid alcohol is differentiated for several seconds, washed with tap water, returned to blue by ammonia water and washed with running water.

(9) Dewatering and sealing: placing the slices in 75% alcohol for 6 min-85% alcohol for 6 min-anhydrous alcohol I for 6 min-anhydrous alcohol II for 6 min-xylene I for 5min, dehydrating, taking out the slices from xylene, air drying, and sealing with neutral gum.

Tunel result interpretation: hematoxylin staining cell nucleus is blue, and DAB shows positive apoptosis cell nucleus is brown yellow. Each section in each group was randomly picked up at least 6 200-fold fields in the ischemic penumbra area for photographing. When taking a picture, the tissues are filled in the whole visual field as much as possible, and the background light of each picture is ensured to be consistent. The Tunel method detects cell apoptosis, and the cell nucleus of the cell is solidified and condensed into a tan, round, crescent or irregular shape. And selecting the same brownish yellow cell nucleus as a unified standard for judging positive cells of all photos by using Image-pro plus6.0 software, selecting the same blue cell nucleus as a total cell, and analyzing each photo to obtain the number of the positive cells and the total cell number of each photo.

2.5.8 immunofluorescence method

(1) Dewaxing: the slices are placed on a slice rack and washed by a series of dimethylbenzene, absolute ethyl alcohol and water to remove paraffin, and the tissues are convenient to combine with the antibody after being rehydrated;

(2) antigen retrieval: the slices were boiled in 10mM citrate buffered saline (pH 6.0), placed under sub-boiling conditions for 10min, and rinsed with a stream of cold water for 30 min.

(3) Washing: soaking and washing the glass slide with PBS for 3 times, 3min each time, and sucking the PBS with absorbent paper;

(4) and (3) circling: a circle is drawn around the tissue to prevent liquid from losing;

(5) membrane permeation: 0.5% Triton-X-100 (in PBS) for 20min at room temperature (antigen expressed on cell membrane omitted this step);

(6) washing: soaking and washing the glass slide with PBS for 3 times, 3min each time, and sucking the PBS with absorbent paper;

(7) and (3) sealing: adding normal goat serum dropwise, and sealing at room temperature for 1 h;

(8) primary antibody incubation: blotting the sealing solution with absorbent paper, adding primary antibody (diluted with 5% goat serum) dropwise, placing in a wet box, and incubating overnight at 4 deg.C (16 hr);

(9) washing: PBST (0.1% Tween-20) is soaked and washed for 3 times, 5min each time, and the PBST is blotted by absorbent paper;

(11) and (3) secondary antibody incubation: single dyeing: fluorescent secondary antibodies (diluted in 5% goat serum) were added dropwise and incubated in a wet box at 37 ℃ for 1 h.

(12) Washing: PBST is soaked and washed for 3 times, each time is 5min, and the PBST is sucked and dried by absorbent paper;

(13) counterstaining the nucleus: dripping DAPI (10ug/ml) and incubating for 5min in dark to stain the core of the specimen;

(14) washing: PBST is soaked and washed for 4 times, each time is 5min, and the PBST is sucked and dried by absorbent paper;

(15) dripping 1 drop of anti-fluorescence quenching agent on the slice, sticking the cover glass on the glass slide, and observing and collecting images under the confocal condition of laser. Scanning parameters are as follows: excitation light wavelength 488nm, emission light wavelength 518nm (green) for detection, 40 times of objective lens, spot scanning mode, Zoom of 1.0

2.6 data processing

All experimental data results were analyzed using SPSS 24.0 statistical software, and all data were averaged ± SD

Two independent samples were compared by t-test, differences between groups were compared by one-way analysis of variance (ANOVA), and pairwise comparisons between groups were performed by LSD test method. Results P<0.05 has statistical significance. GraphpadPrism6.0 is selected for software drawing.

3 results of the experiment

3.1 laser speckle blood flow imaging System evaluation MACO model

FIG. 1 shows that the Model group successfully blocks the local blood flow on the right side of the brain of the rat, and the local blood flow value is obviously reduced.

3.2 animal status and body weight

The animal status of each group before operation is normal, and the weight is basically not different. Within one week after surgery, Sham group rats had good mental status, normal hair color, normal diet, sensitive response, and normal weight gain. Model group rats had depressed mental state, dark hair color, decreased response sensitivity, and decreased body weight. The rats in each treatment group of TST and ANW group were better compared with the Model group, and the animals had better status and increased body weight to different extents.

TABLE 5 Effect of the Chinese medicinal composition of the present invention on the body weight of MACO rats

3.3 Effect of the Chinese medicinal composition of the present invention on the neurobehavioral Scoring of MCAO rats

After 1 day of surgery, the neurobehavioral scores of the Model, TST and ANW groups were significantly increased (P <0.05) compared to the Sham group. At one week post-surgery, neurobehavioral scores in TST-M, TST-H and ANW groups were significantly reduced compared to Model groups (P < 0.05). The neurobehavioral scores in the TST-M, TST-H group were not statistically different from those in the ANW group. The results are shown in FIG. 2.

3.4 Effect of the Chinese medicinal composition on the cerebral infarction area of MCAO rat

No ischemic infarct was observed in the brain tissue of Sham group rats. Compared with Sham rats, the ischemic infarction focus of Model rats is obvious, and the cerebral ischemic area is remarkably increased (P < 0.05). Compared with Model group rats, the ischemic area of rats in TST-H group and ANW group is significantly reduced (P < 0.05). The comparison of the ischemic areas of rats in the TST-H group and the ANW group has no statistical difference. The results are shown in FIG. 3.

3.5 Effect of the Chinese medicinal composition of the invention on the brain tissue morphology of MCAO rat

HE staining results show that neuron cells in cerebral cortex, hippocampus and ischemic penumbra areas of the rats in the Sham group have no obvious abnormality and no obvious ischemic damage, the cell morphology is normal, the cytoplasm is full, the cells are arranged orderly, and the cell nucleus is clear and visible. The rats of the Model group can see ischemic necrosis of the left cerebral cortex, large-area softening range, nervous tissue necrosis liquefaction, neuronal nucleus dissolution and disappearance and massive microglial cell infiltration; the local pyramidal cell nucleus in the CA1 area of the hippocampus is deeply stained and concentrated, and a few pyramidal cells are necrotic; the local cone cells in the CA3 area are reduced and disappear to form cavities, and a large amount of glia cell hyperplasia and infiltration can be seen around the cavities; the DG region is mostly subjected to retraction and deep staining by neurons or pyramidal cells, is triangular or irregular, and has unobvious nucleolus; the arrangement of nerve fibers of the bilateral corpus callosum is loose and disordered. The pathological changes of brain tissues of the TST-H group and the ANW group are obviously reduced compared with the Model group, the neuron soma visible in the cortex is reduced and is often triangular, the nucleus is deeply dyed and condensed, and the nucleolus and the chromatin are unclear; there were no obvious abnormalities in the hippocampus. The necrosis of nerve cells is improved to different degrees, the number of surviving cells is increased, the arrangement of the cells is more orderly, and the cytoplasm is more full. The results are shown in FIG. 4.

Nissl staining results show that densely arranged blue-stained neuron cells can be seen in the slices of the Sham group rats, the number of the blue-stained neuron cells is large, 4-5 layers exist, the nit bodies are rich, and the cell outlines are clear. On the other hand, the section of the Model group rat shows that the nerve cell layer becomes thin, the density is reduced, most nerve cell bodies swell, deform and necrose, even the neuron is disintegrated, and the Neisseria is greatly reduced. Compared with the Model group, pathological changes of brain tissues of the TST-H group and the ANW group are obviously reduced, the number of survival cells is increased, cell layers are arranged neatly, and only a small number of cone cells are reduced. The results are shown in FIG. 5.

3.6 Effect of the Chinese medicinal composition on serum LPO, MDA, NO level and SOD activity of MACO rats

Serum LPO and MDA of rats in each group and NO level and SOD activity are measured, and experimental results show that compared with the Sham group, the serum LPO and MDA level of rats in the Model group are remarkably increased (P <0.05), and the NO level and SOD content of rats in the Model group are remarkably reduced (P < 0.05). Compared with a Model group, the rat serum LPO of the high-dose group of the traditional Chinese medicine composition is remarkably reduced (P is less than 0.05), the MDA level of the low-dose group and the bezoar chest functioning pill group of the traditional Chinese medicine composition is remarkably reduced (P is less than 0.05), the NO level of the high-dose group and the bezoar chest functioning pill group of the traditional Chinese medicine composition is remarkably increased (P is less than 0.05), and the SOD activity of each dose group and the SOD of the chest functioning pill group of the traditional Chinese medicine composition is remarkably increased (P is less than 0.05). The results are shown in FIG. 6.

3.7 content influence of inflammatory factors IL-1 beta and TNF-alpha in rat serum

FIG. 7 shows that the serum IL-1 β and TNF- α levels in Model group are significantly higher than those in Sham group, and the difference is statistically significant (P <0.05), while the serum IL-1 β and TNF- α levels in rats in each administration group are significantly reduced compared with the Model group, and the difference is statistically significant (P <0.05), and the difference between each dosage group of the Chinese medicinal composition of the present invention and the bezoar chest functioning pill group is not statistically significant.

3.8 Effect on Bax, Bcl-2, Cyt-C, Nrf2, HO-1, LC-3B protein expression in rat brain tissue

The immunoblotting method detects the expression of Bax, Bcl-2, Cyt-C, Nrf2, HO-1 and LC-3B proteins in rat brain tissues, and the result is shown in figure 8.

In the apoptosis-regulating proteins Bax, Bcl-2 and Cyt-C, the expression of Cyt-C, Bax in brain tissues of a Model group is obviously increased compared with that of a Sham group (P <0.05), the expression of Bcl-2 of the Model group is obviously reduced compared with that of the Sham group (P <0.05), the expression of Cyt-C, Bax of an ANW group and a TST-H group is obviously reduced compared with that of the Model group (P <0.05), and the expression of Bcl-2 is obviously increased (P < 0.05).

In oxidative stress related proteins Nrf2 and HO-1, the expression level of the Model group and the Sham group is obviously increased by more than 1 time (P <0.05), while the expression level of the ANW group and the TST-H group is obviously reduced by one time (P <0.05) compared with the Model group.

The autophagy-related protein LC-3B Model group is obviously reduced compared with the Sham group (P <0.05), and the expression level of autophagy-related protein LC-3B in the ANW group and the TST-H group is obviously increased (P < 0.05).

3.9 Tunel staining

TUNEL staining was used to detect neuronal apoptosis. As shown in FIG. 9, the number of TUNEL positive neurons in the Model group was significantly greater than that in the Sham group (P <0.05), and the number of TUNEL positive neurons in each administration group was significantly reduced (P <0.05) compared with the Model group, especially in the TST-H group and ANW group, and the difference between the Chinese medicinal composition of the present invention and the Angongniuhuang pill group was not statistically significant, which indicates that TST can reduce the number of apoptotic neurons after cerebral ischemia reperfusion injury.

3.10 immunofluorescence

Bax protein is expressed predominantly in the cytoplasm and mitochondrial membrane and is labeled green fluorescence in this experiment. As shown in FIG. 10A, only very small amounts of immunofluorescence expression, and occasional positive cells were seen in the Sham group sections. The Model group can obviously increase the intensity of immunofluorescence and the number of positive cells. Compared with the model group, the immunofluorescence intensity of each administration group is reduced, the number of positive cells is partially reduced, and particularly the TST-H group and the ANW group are obviously reduced.

Cyt-C protein is expressed predominantly in the mitochondrial matrix and is labeled green fluorescence in this experiment. As shown in FIG. 10B, only very small immunofluorescence expression was seen with the Sham group sections, with occasional positive cells. The Model group can obviously increase the intensity of immunofluorescence and the number of positive cells. Compared with the model group, the administration group has the advantages that the immunofluorescence intensity is reduced, the number of positive cells is partially reduced, and particularly TST-H is obviously reduced.

The LC-3B protein is expressed predominantly in the cytoplasmic, endomembrane system and is labeled green fluorescence in this experiment. As shown in FIG. 10C, small amounts of immunofluorescence expression and occasional positive cells were seen in the Sham sections. The Model group showed no fluorescence expression, and the number of positive cells was very small. Compared with the model group, the administration groups have increased immunofluorescence intensity and increased positive cell number, especially the TST-H group and ANW group are obviously increased.

4 conclusion

The experiment successfully establishes a rat model of cerebral ischemia-reperfusion injury, and explores the effect of the traditional Chinese medicine composition on treating acute ischemic stroke. The experimental result shows that the traditional Chinese medicine composition can effectively reduce the damage of oxidative stress to nerve cells, inhibit apoptosis and improve autophagy activity, thereby promoting the recovery of cerebral ischemia damage and providing the protection of neuron cells.

Detailed Description

Example 1 (granules)

Prescription: 100g of rhubarb, 100g of divaricate saposhnikovia root, 250g of gardenia, 15g of calculus bovis factitius, 100g of concretio silicea bambusae, 100g of magnolia officinalis, 100g of red paeony root, 150g of giant knotweed rhizome and 915g of total.

The preparation method comprises the following steps: pulverizing artificial calculus bovis into fine powder, and sieving. Then adding water into the other seven medicines, carrying out hot reflux extraction for 4 hours, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.0-1.2 at the temperature of 60 ℃, then placing the extract in an oven, carrying out vacuum drying at the temperature of 75 ℃ to obtain dry paste, and crushing the dry paste into fine powder. And finally, mixing the obtained calculus bovis factitius fine powder and the dry paste fine powder, adding a proper amount of dextrin and starch to 450g, adding water to prepare granules, and then drying, granulating and subpackaging into 15 g/bag.

Example 2 (granules)

Prescription: taking 103g of rhubarb, 100g of divaricate saposhnikovia root, 240g of cape jasmine fruit, 15g of calculus bovis factitius, 96g of concretio silicea bambusae, 100g of magnolia officinalis, 103g of red paeony root, 158g of giant knotweed rhizome and 915g of total.

The preparation method comprises the following steps: pulverizing artificial calculus bovis into fine powder, and sieving. Then adding water into the other seven medicines, carrying out hot reflux extraction for 4 hours, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.0-1.2 at the temperature of 60 ℃, then placing the extract in an oven, carrying out vacuum drying at the temperature of 75 ℃ to obtain dry paste, and crushing the dry paste into fine powder. And finally, mixing the obtained calculus bovis factitius fine powder and the dry paste fine powder, adding a proper amount of dextrin and starch to 450g, adding water to prepare granules, and then drying, granulating and subpackaging into 15 g/bag.

Example 3 (granules)

Prescription: taking 102g of rhubarb, 102g of divaricate saposhnikovia root, 262g of gardenia, 16g of calculus bovis factitius, 100g of concretio silicea bambusae, 95g of magnolia officinalis, 95g of red paeony root, 143g of giant knotweed rhizome and 915g in total.

The preparation method comprises the following steps: pulverizing artificial calculus bovis into fine powder, and sieving. Then adding water into the other seven medicines, carrying out hot reflux extraction for 4 hours, filtering, collecting filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.0-1.2 at the temperature of 60 ℃, then placing the extract in an oven, carrying out vacuum drying at the temperature of 75 ℃ to obtain dry paste, and crushing the dry paste into fine powder. And finally, mixing the obtained calculus bovis factitius fine powder and the dry paste fine powder, adding a proper amount of dextrin and starch to 450g, adding water to prepare granules, and then drying, granulating and subpackaging into 15 g/bag.

Example 4 (Capsule)

The drug granules prepared by the method of example 1 were filled into gelatin capsules to prepare capsules.

Example 5 (tablet)

The pharmaceutical granules prepared according to the method of example 1 were compressed into tablets by a conventional method.

Claims (4)

1. A traditional Chinese medicine composition for treating acute ischemic stroke comprises effective components and medically acceptable excipient, wherein the effective components are prepared from the following raw material medicines in percentage by weight:

10.4-11.5% of rheum officinale, 10.4-11.5% of divaricate saposhnikovia root, 26.0-28.7% of cape jasmine fruit, 1.6-1.7% of calculus bovis factitius, 10.4-11.5% of tabasheer, 10.4-11.5% of magnolia officinalis, 10.4-11.5% of red paeony root and 15.6-17.2% of giant knotweed rhizome.

2. The traditional Chinese medicine composition for treating acute ischemic stroke as claimed in claim 1, wherein the effective components are prepared from the following raw material medicines in percentage by weight:

10.9 percent of rhubarb, 10.9 percent of divaricate saposhnikovia root, 27.5 percent of gardenia, 1.6 percent of calculus bovis factitius, 10.9 percent of tabasheer, 10.9 percent of officinal magnolia bark, 10.9 percent of red paeony root and 16.4 percent of giant knotweed rhizome.

3. The traditional Chinese medicine composition for treating acute ischemic stroke according to claim 1, wherein the traditional Chinese medicine composition is a tablet, a granule or a capsule.

4. The traditional Chinese medicine composition for treating acute ischemic stroke as claimed in claim 3, which is prepared by the following method:

(1) pulverizing artificial calculus bovis into fine powder, and sieving;

(2) extracting the rest raw materials except calculus bovis factitius with water under reflux for 4 hr, filtering, collecting filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.0-1.2 at 60 deg.C, vacuum drying, and pulverizing into fine powder;

(3) and (3) mixing the fine powder prepared in the step (1) and the fine powder prepared in the step (2), adding a pharmaceutically acceptable excipient, and preparing tablets, granules or capsules according to a conventional preparation method.

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