CN113244271B

CN113244271B - Application of donkey-hide gelatin in preparation of medicine for improving cerebral ischemia

Info

Publication number: CN113244271B
Application number: CN202110280809.4A
Authority: CN
Inventors: 刘海滨; 顾建军; 任汝静; 高登峰; 刘维岩; 张淹; 孙阳恩; 王春艳
Original assignee: Dong E E Jiao Co Ltd
Current assignee: Dong E E Jiao Co Ltd
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2022-12-23
Anticipated expiration: 2041-03-16
Also published as: CN113244271A

Abstract

The invention relates to application of donkey-hide gelatin in preparation of a medicament for improving cerebral ischemia, and belongs to the technical field of preparation of cerebral ischemia medicaments. The medicine prepared by the application of the invention has obvious improvement effect on cerebral ischemia, expands the application range of the donkey-hide gelatin and provides research basis for developing medicines for treating cerebral ischemia.

Description

Application of donkey-hide gelatin in preparation of medicine for improving cerebral ischemia

Technical Field

The invention relates to the technical field of preparation of cerebral ischemia medicaments, in particular to application of donkey-hide gelatin in preparation of medicaments for improving cerebral ischemia.

Background

With the increasing aging of the population, the number of patients suffering from the disease of 'Vascular Cognitive Impairment (VCI)' related to aging is increased remarkably, and the disease has serious influence on families and society. Vascular cognitive impairment is a diverse disease in which cerebrovascular pathologies predominate, with varying degrees of cognitive impairment. The clinical manifestations are decline of learning and memory function, slow performance of movement, mental disorder, and progressive worsening of disease with age.

The pathology of VCI is complex, and the condition of hemorrhage and vascular blockage in the brain of a patient often exists, so that great challenges are brought to clinical treatment. Bleeding may be exacerbated if vasodilation therapy is used; while inhibiting bleeding may accentuate the vessel occlusion. Cerebral ischemia and hemorrhage may cause infarction of brain tissue, causing severe damage to neurons and neurites, and therefore, there is an urgent need to develop new drugs that can improve cerebral blood perfusion and control disease processes.

Disclosure of Invention

The invention aims to provide application of donkey-hide gelatin in preparing a medicament for improving cerebral ischemia. The invention provides application of donkey-hide gelatin in preparing a medicament for improving cerebral ischemia, and provides a research basis for developing medicaments for improving cerebral blood perfusion and treating cerebral ischemia.

The invention provides application of donkey-hide gelatin in preparing a medicament for improving cerebral ischemia.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving the learning and memory function reduction caused by cerebral ischemia.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving brain tissue injury caused by cerebral ischemia.

Preferably, the brain tissue injury comprises inflammation or edema.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving the cerebral atrophy caused by cerebral ischemia.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving cerebral ischemia by regulating key metabolites of brain tissues.

Preferably, the brain tissue key metabolites include N-acetyl aspartic acid, creatine, choline, lactic acid and glutamic acid.

The invention also provides the application of the donkey-hide gelatin in preparing the medicine for promoting the opening and regeneration of cerebral cortex parenchyma cerebral vessels of vascular dementia caused by cerebral ischemia.

Preferably, the cerebral ischemia comprises chronic cerebral ischemia.

The invention provides application of donkey-hide gelatin in preparing a medicament for improving cerebral ischemia. The donkey-hide gelatin takes 'brain' as a target organ, takes 'vascular cognitive dysfunction' caused by chronic cerebral ischemia as a target disease for proving drug effect, and researches the function and the mechanism of the donkey-hide gelatin for improving the cerebral ischemia. Test results show that the donkey-hide gelatin can improve the decline of learning and memory functions caused by cerebral ischemia; can improve brain tissue injury caused by cerebral ischemia, including inflammation or edema; can improve brain atrophy caused by cerebral ischemia; can improve cerebral ischemia by regulating key metabolites of brain tissues; can promote the opening and regeneration of cerebral cortex parenchymal cerebral vessels of vascular dementia caused by cerebral ischemia, and can also improve cerebral cortex blood flow perfusion of the vascular dementia caused by cerebral ischemia. The application of the invention can provide drug selection for the preparation of cerebral ischemia drugs, provide scientific and rigorous experimental evidence for the donkey-hide gelatin in the aspect of treating cerebral ischemia, and lay the working foundation for the development of clinical drugs.

Drawings

Fig. 1 is a schematic diagram of a modeling method provided by the present invention, wherein a is a model building method, and B is a model image that is actually successfully built;

FIG. 2 is an elevated plus maze of experimental equipment for evaluating whether a rat is in an anxiety state, provided by the present invention;

FIG. 3 is a graph showing the results of a study conducted to evaluate whether a rat is in an anxiety state;

FIG. 4 is a diagram of an eight-arm maze study of colla Corii Asini for improving learning and memory due to cerebral ischemia;

FIG. 5 is a graph showing the results of the study of the inhibition of cerebral ischemia by E.coli T2-weighted MRI-cerebral injury edema;

FIG. 6 is a quantitative analysis chart of the study of T2-weighted MRI-brain injury edema of cerebral ischemia inhibited by colla Corii Asini provided by the present invention;

FIG. 7 is a diagram of the result of MRI study of living animals with donkey-hide gelatin to improve cerebral ischemia-encephalatrophy;

FIG. 8 shows the studies of the donkey-hide gelatin on cerebral ischemia, a key metabolite in brain tissue, in vivo animals;

fig. 9 is a two-photon laser in vivo scan in vitro graph (3D graph of cerebral blood vessel) of cerebral cortex and brain parenchyma blood vessel with colla Corii Asini provided by the invention.

Detailed Description

The invention provides application of donkey-hide gelatin in preparing a medicament for improving learning and memory function reduction caused by cerebral ischemia. In the present invention, the cerebral ischemia includes chronic cerebral ischemia. The result shows that the donkey-hide gelatin can obviously improve the anxiety state and the learning and memory functions of chronic severe cerebral ischemia (the total carotid artery constriction is 2/3, and the death rate of animals after cerebral ischemia is about 25%).

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving brain tissue injury caused by cerebral ischemia. In the present invention, the cerebral ischemia includes chronic cerebral ischemia. In the present invention, the brain tissue injury preferably comprises inflammation or edema. Test results of specific embodiments of the invention show that the donkey-hide gelatin can significantly inhibit cerebral ischemia T2-weighted MRI-cerebral injury edema through in vivo neuroimaging detection.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving the cerebral atrophy caused by cerebral ischemia. In the present invention, the cerebral ischemia includes chronic cerebral ischemia. The test results of the specific embodiment of the invention show that the donkey-hide gelatin has the function of obviously inhibiting the encephalatrophy.

The invention also provides application of the donkey-hide gelatin in preparing a medicament for improving cerebral ischemia by regulating key metabolites of cerebral tissues. In the present invention, the cerebral ischemia includes chronic cerebral ischemia. In the present invention, the key metabolites preferably include N-acetyl aspartic acid (NAA), creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid (Glu), which are the most basic substances involved in the metabolism of neurons in the brain; NAA is produced in nerve mitochondria and is involved in energy metabolism of neurons; creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid (Glu) and the like are necessary raw materials for synthesizing neurotransmitters; normally, these substances are mostly present inside nerve cells; when nerves are damaged, these substances are released into the blood. The experimental results of the examples show that after the rats are fed with the donkey-hide gelatin, the donkey-hide gelatin has obvious inhibition effect on the reduction of key metabolites.

The invention also provides the application of the donkey-hide gelatin in preparing the medicine for promoting the opening and regeneration of cerebral cortex parenchyma cerebral vessels of vascular dementia caused by cerebral ischemia. In the present invention, the cerebral ischemia includes chronic cerebral ischemia. The experimental results of the examples show that after the rats are fed with the donkey-hide gelatin, the donkey-hide gelatin has obvious effect of promoting the regeneration of blood vessels.

The application of donkey-hide gelatin in preparing a medicament for improving cerebral ischemia is further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

Materials and methods

Experimental animals: SD inbred rats, SPF grade, female. 12 weeks old. The weight is 280.0-320.0 g. Purchased from the laboratory animal center of the department of medicine of Beijing university. Animal license number: SYXK (Kyoto) 2016-0041. The animals are placed in 12h/12h light and dark alternate environment and eat freely. All animal experimental procedures were performed under the Peking laboratory animal Care regulations.

And (3) preparing a sample: powder preparation: colla Corii Asini powder, drying at room temperature, and sealing.

The main experimental apparatus:

siemens MAGNETOM Trio TimSystem 3.0t, living animal superconducting magnetic resonance MRI imaging system, siemens, germany (Siemens MR Center, gain c.ave.,2nd, hi-Tech Industrial Park, nanhan District, shenzhen, p.r. china). Used for rat brain MRI detection analysis;

leica SP8 DIVE Leica high resolution positive two-photon laser living body scanning microscope system (SP8 DIVE Deep In Vivo Explorer, company address 1700Leider Lane, buffalo Grove, IL 60089United States) for detection and analysis of living rat cerebral cortical vascular network;

3.Q Exactive ^TM HF combined quadrupole rod Orbitrap ^TM Mass spectrometry: thermo Fisher Scientific, inc. (168ThirdAvenue Waltham, MAUSA02451) for analysis of rat serum proteomics;

4. nikon inverted microscope: the kit is used for observing the detection and analysis of isolated culture blood vessels, nerves and glial cells.

5. Pathological section imaging system: all pathological sections were produced using the macoodi digital section system (moticvv12.0, motic Group co., ltd.

6. A cold microtome (Leica CM 3050Crystate, a product of Leica Microsystem Nussloch GmbH, D-69226, nussloch, germany).

7. Flow cytometers (Guava easy cell System, guava technologies, inc,25801industrial blvd, hayward, CA94545, USA);

HTS, TRANSWELL-24 film-in-0.3 micron PET film Transwell, product of Corning incorporated (2Alfredrd, kennebbunk, ME 04043, USA).

Manufacturing a model:

1. the purchased inbred line SD rat is bred in cages with 4 rats per cage. The male and female are half. The animal feeding conditions were: 22 ± 2 ℃, humidity: 45-60%, alternating light and shade for 12 hours. Free food intake. After 1 week of animal acclimation, animal model production was started, and the specific procedures were as follows.

2. Detection before molding: after one week of animal adaptive feeding, the preparation of chronic cerebral ischemia model was started. The specific experimental procedure is as follows.

Establishing a bilateral carotid artery long-term ischemia model: the 2% sodium pentobarbital 0.3ml/100g was injected intraperitoneally to anesthetize rats, and the skin (3-4 cm) was cut along the median line of the neck. Muscle is separated layer by layer in a blunt way, common carotid arteries on both sides are exposed, and the bifurcation of the internal and external carotid arteries is found towards the far end. And ligating a part 2mm away from the distal end of the common carotid artery to a bifurcation by using a silicone tube, wherein the specification is that the inner diameter is 0.5mm, the outer diameter is 2.5mm and the length is 3mm, putting the blood vessel into a sleeve from a longitudinal incision, and tightly attaching the silicone tube to the outside of the blood vessel and fixing the outside of the silicone tube by using a No. 4 suture. In the sham operation group, the silicone tube was placed beside the common carotid artery without compressing the blood vessel, and the rest treatment was identical to the model group. Intraperitoneal injection of penicillin prevents infection. After the operation is completed, the brain ischemia state is detected by MRI living body. The modeling method is shown in fig. 1, wherein a is a schematic diagram of a model building method, and B is a model image which is successfully built actually.

3. In order to identify the success of model preparation, the anxiety state of the rat was examined by elevated plus maze (Highplus maze) and the learning and memory function of the rat was examined by Eight-wall maze (Eight-armmaze) at the fourth and eighth weeks after the cerebral ischemia operation; at week four, the model rat behavioural did not show significant abnormalities, as compared to week eight.

4. Elevated cross maze: the present invention uses the rat elevated plus maze experiments (elevewed maze rats) to test the anxiety degree of rats, wherein the cruciate palace comprises two opposite open arms (open arms) of 50cm x10cm and two opposite closed arms (closed arms) of 50mx10cmx40cm, the upper part of the closed arms is open, and the center of the closed arms is provided with a parallel broad part of 10cmx10 cm. The height of the ground surface of the maze is 50cm. The rat was placed in the central open part with the head towards the closed arm. The observer recorded the number of times the rat entered the open and closed arms and the residence time in both arms (based on the arms of all four limbs out of the arm) within 5min, outside of 1m from the center of the maze. The number of arm openings and the time taken by the rats were calculated as a percentage of the total number (sum of the number of arms) and the total time (sum of the residence time in the arms), respectively.

The movement of the rat in the open arm is equivalent to the movement at the cliff. Rats with anxiety tend to move in the relatively safe Closed arm (Closed arm), while normal rats move in the open and Closed arms approximately the same frequency. The experimental facility elevated plus maze for evaluating whether rats are in anxiety state is shown in fig. 2.

The results of the experiment are shown in FIG. 3 (a graph of the results of the study to evaluate whether rats are in an anxiety state). The results show that the Model group (Model) showed significant anxiety-characterized behavior at week eight. Whereas the Sham group (Sham) showed no manifestations of anxiety.

5. Eight arm maze analysis: animals were acclimated to the experimental environment for 1 week, weighed, and fasted for 24 hours. The next day, the arms and center area of the maze were sprinkled with food particles (4-5 particles each, about 3-4 mm in diameter). Then, 4 animals were placed in the center of the maze simultaneously (doors to each arm were opened). Let it feed freely and explore for 10min. Then, randomly selecting 4 arms, and putting one food particle on each arm; each arm door is closed, and the animal is placed in the center of the maze; after 30s, the arm gate was opened, allowing the animal to move freely in the maze and ingest pellets until the animal had eaten all 4 arms of pellets. If the grains are not eaten after 10min, the experiment is terminated. The following 4 indices were recorded: 1) Working memory errors (working memory errors), i.e. the animal enters the arm which has eaten the nibbles again in the same training; 2) Reference memory errors (reference memory errors), i.e. animals enter the arm that has not been fed a grain; 3) Total number of arm entries; 4) Test time, i.e., the time it takes for the animal to finish eating all of the pellets. In addition, the computer can record the movement of the animal in the radial arm and in the central area, including the movement distance, the movement time and the like.

The results showed that cerebral ischemia showed a clear decline in learning and memory capacity at week eight.

6. Animals were grouped and after successful model creation, animals were divided into the following groups and testing was started with the drug to be tested. The specific grouping is shown in table 2:

TABLE 2 donkey-hide gelatin feeding dose

7. And (3) drug treatment: after the model was successfully made, experimental treatments were performed. The dose was determined as follows;

determining the amount of the donkey-hide gelatin powder: the human dosage is as follows: 3 g/day/person. The body weight was calculated as 70 kg. According to the conversion of the dosage of the human body and the dosage of the rat (according to the conversion standard of the international human dosage and the dosage of the rat), the dosage of the rat is about 8 times of the dosage of the human body. The clinical human dose is 3 g/day/per person ÷ 70kg (human body mean body weight) × 8.0 (human-rat conversion factor) =0.03/kg. Rats of 100g weight were fed with 0.03g per day. For example, a rat with a body weight of 300g has a dose of 0.03 × 3= 0.09g/day/300 g body weight. Dissolving colla Corii Asini powder 0.09g in distilled water 2.0 ml.

After 2 months of experimental treatment, the following assays were performed:

8. elevated plus maze: rats were tested for anxiety using the rat elevated plus size test in rats, which consisted of two 50cm x10cm opposed open arms (open arms) and two 50mx10cm x40cm opposed closed arms (closed arms) with an open top and a 10cm x10cm wide center. The ground surface of the maze is 50cm high. The rat was placed in the central open, head towards closed arm. The number of times the rat entered the open and closed arms and the residence time in both arms (based on the total arms of the four limbs out of the arms) was recorded by the observer within 5min from the center of the maze by 1 m. The number of times and the time the rat entered the open arm were calculated as a percentage of the total number of times (sum of the number of times in both arms) and the total time (sum of the residence time in both arms), respectively.

9. Eight arm maze analysis: animals were acclimated to the experimental environment for 1 week, weighed, and fasted for 24 hours. The next day, the arms and central area of the maze were dusted with food particles (4-5 particles each, about 3-4 mm in diameter). Then, 4 animals were placed in the center of the maze simultaneously (doors to each arm were opened). Let it eat freely and explore for 10min. Then, randomly selecting 4 arms, and putting one food particle on each arm; each arm door is closed, and the animal is placed in the center of the maze; after 30s, the arm gate was opened, allowing the animal to move freely in the maze and ingest pellets until the animal had eaten all 4 arms of pellets. If the grains are not eaten after 10min, the experiment is terminated. The following 4 indices were recorded: 1) Working memory errors (working memory errors), i.e. the animals re-enter the arm that has eaten the grain in the same training; 2) Reference memory errors (reference memory errors), i.e. animals enter the arm that has not been fed a grain; 3) Total number of arm entries; 4) Test time, i.e., the time it takes for the animal to finish eating all of the pellets. In addition, the computer can record the movement of the animal in the radial arm and in the central area, including the movement distance, the movement time and the like.

10. In vivo brain MRI neuroimaging analysis (SIEMENS MAGNETOM Trio Timesystem 3.0T), rats are anesthetized by intraperitoneal injection of chloral hydrate, placed in a special rat imaging coil, the body position of the rats is adjusted, and the rats and the imaging coil are moved into the central region of a magnetic field through a detection bed. The method comprises the steps of firstly collecting a positioning image of the head and the neck of a rat, then collecting a T1 weighted image and a T2 weighted image of the coronal position and the axial position of the head and the neck of the rat by using a fast spin echo sequence, carrying out TOF three-dimensional gradient echo sequence imaging on arterial blood vessels of the neck, carrying out axial T2Mapping imaging on intracranial brain tissues to obtain a T2 value of the tissues, and carrying out magnetic resonance spectrum imaging on the brain tissues by using a single-voxel point-resolution spin echo spectrum sequence to obtain information of metabolites such as N-acetyl aspartic acid, choline, creatine and the like of the brain tissues.

11. A Leica high-resolution positive two-photon laser living body scanning microscopic system (SP 8 DIVE Deep InVivo Explorer) is adopted to detect and analyze the cerebral cortex vascular network. After rats were anesthetized to open craniotomy, fluorescent dye (fluoroscein isothiocyanate-dextran, molecular weight: 59, 000-77, 000, catalogNo D70, lotNo. A Leica sp8 live two-photon laser scanning microscope system (Leica sp8 live), HC FLUOTAR L25x/1.00IMM motCORRVISIR long-distance working objective lens are used, under the condition of excitation wavelength of 920nm, the receiving range is 500-550 nm, and (1) a blood vessel network, (2) blood flow, (3) blood flow speed and (4) cerebrovascular wall permeability of a living rat can be observed for analysis.

12. Serum proteomics sample preparation: after the above experiments were completed, each animal was anesthetized by intraperitoneal injection with pentobarbital sodium solution (1.0% pentobarbital solution, 50mg/kg body weight), the blood was taken from the abdominal aorta of the rat, placed in a centrifuge tube containing heparin, centrifuged at 3000rpm for 10 minutes, and the plasma was transferred to a 1.5ml eppendorf tube and stored frozen at-80 ℃ for serum proteomics analysis.

13. Serum proteomics test analysis: q active ^TM HF combined quadrupole rod Orbitrap ^TM Mass spectrometry: thermo Fisher Scientific product (168ThiirdAvenue Waltham, MA USA02451) was used for the analysis of rat serum proteomics.

14. Study of laser confocal: then, the animals were fixed by perfusion at 4% Parafumydide, frozen sections were prepared, and immunohistochemistry and laser confocal studies were performed.

Data processing and statistics:

data are shown as Mean ± standard deviation (Mean ± s.d.) comparisons of different groups were performed using one way ANOVA with T-test statistical tests performed using Microsoft excel software.

The pathological tissue image analysis adopts Chi-Square Test Chi-Square Test Chi-Square Test, and statistical analysis is carried out by using SPASS 20.

Results of the experiment

1. Living animal research for improving learning and memory of cerebral ischemia by donkey-hide gelatin

The decline of learning and memory ability is the most obvious clinical manifestation of various cerebral ischemia diseases such as vascular dementia, etc. The invention observes whether the donkey-hide gelatin blood-enriching preparation can improve the learning and memory functions of the brain by improving the cerebral blood flow perfusion. The invention adopts eight-arm Maze (Eightarm Maze) to detect, test and treat the disease, and then learns and memorizes the ability. The results are shown in figure 4 (donkey-hide gelatin improves learning and memory eight arm maze research of blood deficiency cerebral ischemia), and donkey-hide gelatin improves learning and memory eight arm maze research of cerebral ischemia.

The results show that:

1) In sham (i.e. non-ischemic control), animals were trained to find food quickly;

2) The model group narrows the common carotid artery by 2/3, which causes the blood perfusion of brain to be obviously reduced, and the learning and memory functions of brain tissues to be obviously reduced, although no food can be found all the time after hunger treatment. The rats had food placed artificially and continued to ingest food continuously. The rat learning and memory ability is seriously declined;

3) The colla Corii Asini powder has effects of improving learning and memory, and p value is less than 0.05.

2. MRI neuroimaging research of living animal with donkey-hide gelatin for improving cerebral ischemia-brain tissue injury (inflammation and edema)

After cerebral ischemia, neurons and their surrounding glial cells are damaged and marked edema occurs. Cerebral edema is the leading cause of death of patients due to cerebral ischemia (compressing medulla oblongata), and the formation of cerebral edema can be inhibited by weakening the damage of brain tissues and cerebral vessel walls. The invention is to prove whether the donkey-hide gelatin can protect nervous tissues and cerebral vessels and inhibit the formation of cerebral edema. In detail, the result of the study on inhibition of cerebral ischemia by donkey-hide gelatin on T2-weighted MRI-cerebral injury edema is shown in figure 5.

In order to accurately and quantitatively assess the curative effect of the medicament to be tested, the invention carries out quantitative analysis on MRI, the result is shown in figure 6, and the donkey-hide gelatin inhibits cerebral ischemia T2-weighted MRI-brain injury edema research quantitative analysis diagram, p is less than 0.05.

The results show that:

1) In the sham operation group (i.e. the non-ischemia control group), no obvious brain tissue damage and edema are seen;

2) The model group narrows the common carotid artery by 2/3, which causes the cerebral blood perfusion to be obviously reduced, and the living MRI shows that the brain tissue has obvious edema, and the p value is less than 0.001;

3) After the rats are fed with the donkey-hide gelatin, the injury and edema of the rats obviously decrease, and the p value is less than 0.05.

3. Living animal MRI research for improving cerebral ischemia-encephalatrophy by donkey-hide gelatin

Clinically, chronic cerebral ischemia for a long time can cause brain tissue atrophy, such as sequela of stroke, vascular dementia and senile dementia, and one of the most prominent pathological changes is brain atrophy. The invention aims to prove whether the blood nourishing can inhibit the atrophy of a core organ (brain) caused by cerebral ischemia. The invention uses MRI to detect the maximum section area of rat brain, the result is shown in figure 7, the result of MRI research of animal living body with donkey-hide gelatin for improving cerebral ischemia-encephalatrophy, p is less than 0.05, p is less than 0.01.

The results show that:

1) Sham operated groups (i.e., non-ischemic control group), no atrophy of brain tissue was seen (compared to non-operated normal rats);

2) The model group narrows the common carotid artery by 2/3, which leads to significant reduction of cerebral blood perfusion, and living MRI shows that brain tissues have obvious atrophy, and the p values are all less than 0.001;

3) The colla Corii Asini powder has obvious effect of inhibiting brain atrophy, and p values are less than 0.05.

4. Living animal research of donkey-hide gelatin for improving cerebral ischemia-key metabolite of cerebral brain tissue

The invention analyzes key metabolites of rat brain through living MRI, thereby judging the degree of nerve injury and evaluating the effect of the medicine.

N-acetyl aspartic acid (NAA), creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid (Glu), are the most basic substances involved in brain neuron metabolism. NAA is produced in the mitochondria of neurons and is involved in energy metabolism of neurons. Creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid Glu, and the like are essential raw materials for synthesizing neurotransmitters. Normally, these substances are mostly present inside nerve cells. When nerves are damaged, these substances are released into the blood.

How to evaluate the damage of neurons in vivo is a difficult problem at home and abroad at present. The invention adopts MRI living body to detect the damage condition of the neuron, and is convenient for researching the effect of drug treatment.

The results are shown in figure 8, and figure 8 shows the study of colla Corii Asini on cerebral ischemia, a key metabolite of brain tissue, in vivo animal.

The results show that:

1) In the sham operation group (i.e., non-ischemia control group), key substances in brain cells and key substances in energy metabolism, such as N-acetyl aspartic acid (NAA), creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid (Glu) and the like, are all present in neurons, and are not decreased. Indicating that the neuron is not obviously lost;

2) The model group narrows the common carotid artery by 2/3, which causes the significant reduction of cerebral blood perfusion, and living MRI shows that the N-acetyl aspartic acid (NAA), creatine (Cr), choline (Cho), lactic acid (Lac), glutamic acid Glu and the like of the brain tissue are all significantly reduced, and the p value is less than 0.05;

3) After the rats are fed with donkey-hide gelatin, the feed has obvious inhibition effect on the reduction of key metabolites, and the p values are all less than 0.05.

5. Study of donkey-hide gelatin on live animals with cerebral ischemic (vascular dementia) -cerebral cortical vascular network

The invention considers that the blood deficiency of the traditional Chinese medicine not only comprises the reduction of blood cells, but also belongs to the category of blood deficiency of the traditional Chinese medicine. The invention considers that promoting the opening of closed blood vessels or promoting the regeneration of blood vessels is probably an important action mechanism of the donkey-hide gelatin. Therefore, the invention researches the condition that the donkey-hide gelatin and the differentiated preparation thereof open and close cerebral cortex blood vessels.

The invention adopts a come high-resolution upright two-photon laser living body scanning microscope system (SP 8 DIVE Deep InVivo Explorer) to detect and analyze the cerebral cortex vascular network. The results are shown in fig. 9, which shows two-photon laser in vivo scan of two-photon in vivo scan of brain parenchyma blood vessels of cortex of donkey-hide gelatin (3D image of brain blood vessels).

The results show that:

1) The model group narrows the common carotid artery by 2/3, which leads to the remarkable reduction of cerebral blood perfusion, and the two-photon laser living body scanning shows that the blood vessels in the brain parenchyma are degenerated and closed, the blood brain barrier of the tiny blood vessels is damaged, and the fluorescent marker is exuded. The p values are less than 0.05 through image analysis and chi-square test;

2) After the rats are fed with the donkey-hide gelatin, the donkey-hide gelatin has obvious effect of promoting the regeneration of blood vessels, and the p values are all less than 0.05.

And (3) knotting:

the donkey-hide gelatin can obviously improve the anxiety state of chronic severe cerebral ischemia (the total carotid artery is narrowed by 2/3, and the death rate of animals after cerebral ischemia is about 25 percent).

The donkey-hide gelatin can remarkably improve the learning and memory functions p value of chronic severe cerebral ischemia (the total carotid artery is narrowed by 2/3, the death rate of animals after cerebral ischemia is about 25 percent) and is less than 0.05;

through in vivo neuroimaging detection, the donkey-hide gelatin can obviously inhibit blood deficiency cerebral ischemia T2-weighted MRI-cerebral injury edema, and the p values are all less than 0.05.

Through MRI research on living animals, the donkey-hide gelatin improves brain atrophy caused by blood deficiency cerebral ischemia, and the p value is less than 0.05.

The study of living body MRI shows that the donkey-hide gelatin recovers the blood deficiency cerebral ischemia, a key metabolite of cerebral brain tissue.

The colla Corii Asini has obvious effect of promoting blood vessel regeneration, and p value is less than 0.05.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The application of the donkey-hide gelatin in preparing the medicine for improving the learning and memory function reduction caused by cerebral ischemia, wherein the cerebral ischemia is chronic cerebral ischemia.

2. Application of colla Corii Asini in preparation of medicine for improving brain tissue injury caused by cerebral ischemia, wherein the cerebral ischemia is chronic cerebral ischemia, and the brain tissue injury is edema.

3. Application of colla Corii Asini in preparation of medicine for improving brain atrophy caused by cerebral ischemia is described, wherein the cerebral ischemia is chronic cerebral ischemia.

4. The application of donkey-hide gelatin in preparing a medicament for promoting the regeneration of cerebral cortex parenchymal cerebral vessels of vascular dementia caused by cerebral ischemia, wherein the cerebral ischemia is chronic cerebral ischemia.