Application of pilose antler-derived substance in preparation of medicine for treating cerebral ischemia injury

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a pilose antler-derived substance in preparation of a medicine for treating cerebral ischemia injury. The cornu cervi pantotrichum-derived substance comprises a cornu cervi pantotrichum extract AVE and a cornu cervi pantotrichum stem cell paracrine factor AnSC-CM. The AnSC-CM and AVE provided by the invention can reduce the water content of injured brain tissues of rats, reduce the brain injury area of the rats and repair central nervous injury of the rats, and the effect of the AnSC-CM is superior to that of the AVE.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of a substance derived from antler velvet in preparation of a medicine for treating cerebral ischemic injury. BACKGROUND

[0002] Acute ischemic stroke (AIS) is the most common type of stroke, which is caused by interruption of cerebral blood flow, leading to ischemia and hypoxia of the corresponding brain tissue, thus producing symptoms of nerve function damage, such as hemiplegia, aphasia, hemineurosis, headache, dizziness and the like, and even life-threatening in severe cases. It is the most common cause of death and disability worldwide. The current main treatment measure is to quickly restore blood supply to the ischemic area of the brain tissue to reduce nerve function damage, but it cannot promote the endogenous brain repair function, and patients still have some nerve function disorders in the chronic stage, such as balance and gait disorders, motor function disorders, sensory function disorders and the like. Therefore, it is necessary to explore a new way to relieve nerve damage caused by acute ischemic stroke. SUMMARY

[0003] The application provides application of a substance derived from antler velvet in preparation of a medicine for treating cerebral ischemic injury. The two substances derived from antler velvet, i.e., antler velvet extract (AVE) and antler conditional medium (AnSC-CM), can reduce the water content of the damaged brain tissue of a rat, reduce the brain damage area of the rat and repair the central nerve damage of the rat.

[0004] The application provides application of a substance derived from antler velvet in preparation of a medicine for treating cerebral ischemic injury. The two substances derived from antler velvet, i.e., antler velvet extract (AVE) and antler conditional medium (AnSC-CM), can reduce the water content of the damaged brain tissue of a rat, reduce the brain damage area of the rat and repair the central nerve damage of the rat.

[0005] Further, the preparation process of the antler velvet extract is as follows: fresh antler velvet slices are added to pure water overnight for extraction, centrifugal filtration is performed to obtain an antler velvet extract solution, and freeze-drying is performed to obtain the antler velvet extract.

[0006] Further, the preparation process of the antler conditional medium is as follows: antler stem cells are separated from the mesenchymal layer and the horn handle periosteum of the antler, the antler stem cells are cultured using complete culture medium, when the confluence degree is more than 90%, the antler stem cells are subcultured by adding complete culture medium, when the confluence degree is 70% to 80%, the complete culture medium is replaced by complete culture medium without fetal bovine serum and the culture is performed for 46 to 50 hours, the supernatant is collected, cell debris is removed by centrifugation, the concentrated solution is obtained by ultrafiltration and centrifugal concentration, and the concentrated solution is freeze-dried to obtain the antler conditional medium.

[0007] Further, the medicine is used for repairing central nerve injury of rats.

[0008] Further, the medicine is used for recovering nerve function of rats.

[0009] Further, the medicine is used for reducing water content of damaged brain tissue of rats.

[0010] Further, the medicine is used for reducing brain tissue injury area of rats.

[0011] Further, the medicine further comprises a pharmaceutically acceptable auxiliary material; the auxiliary material is a combination of any one or more of physiological saline, mannitol, glucose, trehalose and sucrose.

[0012] Further, the medicine is an injection preparation.

[0013] Further, the injection preparation is prepared by mixing the deer antler-derived substance and physiological saline according to a mass-volume ratio of 0.5-1:1.

[0014] Further, the medicine further comprises a pharmaceutically acceptable auxiliary material: mannitol, glucose, trehalose, sucrose.

[0015] Further, the deer antler-derived substance is added into physiological saline for rehydration according to a mass-volume ratio of 0.5 mg-1 mg:1 mL.

[0016] Compared with the prior art, the present application has the beneficial effects that: Deer antler is the only mammalian organ that can periodically regenerate completely, and the two deer antler-derived substances AVE and AnSC-CM provided by the present application can significantly promote nerve regeneration and repair damaged brain tissue.

[0017] The present application compares the repair effects of AnSC-CM and AVE on brain tissue injury of rats, AnSC-CM injection is the AnSC-CM group, and AVE injection is the AVE group, and the results show that: injection of AnSC-CM and AVE can significantly reduce the water content of brain tissue of rats (P<0.05), reduce edema in the ischemic area, thereby improving the brain microenvironment and reducing the risk of secondary injury. The brain injury area of the AnSC-CM group is significantly smaller than that of the control group (P<0.01), and the balance beam experiment and nerve function score confirm that the motor coordination recovery is faster.

[0018] The present application compares the protective effects of AnSC-CM group and AVE group on neurons, and the results show that: the neurons in the AnSC-CM group are arranged more closely, the cell membrane integrity is better, the nuclear membrane is clear, and the number of necrotic cells is significantly reduced (P<0.001 compared with the Control group), which proves that AnSC-CM has better neuroprotective effect. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort.

[0020] Figure 1 Balance beam test score comparison for each group of rats.

[0021] Figure 2 Neurological function score for each group of rats.

[0022] Figure 3 Injury side brain tissue water content for each group of rats.

[0023] Figure 4 HE staining of brain tissue for each group of rats.

[0024] Figure 5 TTC staining of brain tissue sections for each group of rats. In the figure, A is the TTC staining of brain tissue sections of rats in the Normal group, in which the six samples from top to bottom are parallel samples; B is the TTC staining of brain tissue sections of rats in the normal saline control group, in which the six samples from top to bottom are parallel samples; C is the TTC staining of brain tissue sections of rats in the Pilose Antler Extract group, in which the six samples from top to bottom are parallel samples; D is the TTC staining of brain tissue sections of rats in the Pilose Antler Stem Cell Paracrine Factor group, in which the six samples from top to bottom are parallel samples. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application will be described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0026] Example 1: Application of Pilose Antler-derived substances in the preparation of cerebral ischemic injury drugs and health products.

[0027] I. Test materials and instruments The materials and instruments used in the test are shown in Table 1 and Table 2.

[0028] Table 1 Test materials Table 2 Experimental instruments II. Experimental method 1. Preparation of Cervus Cornu Pantotrichum extract Fresh Cervus Cornu Pantotrichum slices were weighed at 100 g, washed twice with pure water, crushed, and extracted at 4°C overnight by stirring at a ratio of fresh Cervus Cornu Pantotrichum: pure water = 1:5. The extraction was repeated three times, and then extracted at 4°C overnight by stirring at a ratio of extracted Cervus Cornu Pantotrichum: pure water = 1:3 twice. The filtrates were combined, centrifuged at 4000 rpm for 10 min, and the supernatant was filtered through filter paper to obtain the Cervus Cornu Pantotrichum extract. The extract was immediately transferred to a sterile culture dish and frozen overnight in a -80°C ultra-low temperature freezer. Then, the extract was placed in a vacuum freeze dryer (-45°C, power 1700 W, vacuum degree 3 pa) for freeze-drying. The Cervus Cornu Pantotrichum extract freeze-dried powder was obtained. When used, the powder was rehydrated with physiological saline at a mass-volume ratio of 1 mg:1 mL, and filtered through a 0.45 µm filter membrane to remove bacteria.

[0029] 2. Preparation of Cervus Cornu Pantotrichum stem cell paracrine factor Cervus Cornu Pantotrichum stem cells were isolated from the mesenchymal layer and horn handle periosteum of Cervus Nippon, and the cells were cultured after being obtained. The culture system was complete culture medium, which was DMEM basic culture medium, 10% fetal bovine serum, and 1% penicillin / streptomycin. The culture conditions were 37°C and 5% CO2. When the Cervus Cornu Pantotrichum stem cells were cultured to more than 90% confluence, they were washed once with PBS, digested with trypsin for 2 min, and terminated with complete culture medium. The cells were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The cells were resuspended and subcultured into a new culture bottle with complete culture medium. When the cells reached 75% confluence, the complete culture medium was removed, the cells were washed twice with PBS, and the cells were cultured in conditioned medium without fetal bovine serum (DMEM basic culture medium, 1% penicillin / streptomycin). After 48 h, the cell culture supernatant was collected, centrifuged at 1000 rpm for 5 min to remove the detached cells and debris, and the Cervus Cornu Pantotrichum stem cell paracrine factor was obtained. The Cervus Cornu Pantotrichum stem cell paracrine factor was ultrafiltrated and concentrated at 5000 rpm for 30 min at 4°C using a 3KD ultrafiltration tube. The factor was immediately frozen overnight in a -80°C ultra-low temperature freezer after being transferred to a sterile culture dish, and then dried in a vacuum freeze dryer (-45°C, power 1700 W, vacuum degree 3 pa). The Cervus Cornu Pantotrichum stem cell paracrine factor (AnSC-CM) freeze-dried powder was obtained. When used, the powder was rehydrated with physiological saline at a mass-volume ratio of 0.5 mg:1 mL, and filtered through a 0.45 µm filter membrane to remove bacteria.

[0030] 3. Grouping of rats and model construction Forty rats were randomly divided into four groups: normal group (Normal), saline control group (Control), velvet extract group (AVE), and velvet stem cell paracrine factor group (AnSC-CM), with 10 rats in each group. Ischemic stroke injury models were prepared in rats in each group. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (30 mg / kg), and the skin was prepared and conventionally disinfected with iodophor. The rats were then fixed on a mouse plate, and the skin in the middle of the neck was incised and bluntly separated until the common carotid artery was completely exposed. The common carotid artery, external carotid artery, and internal carotid artery were separated. A live knot was tied at the proximal end of the common carotid artery and the proximal end of the external carotid artery with surgical thread, the distal end of the external carotid artery was ligated, the internal carotid artery was clamped, a V-shaped small opening was cut in the live knot and the ligation site of the external carotid artery with ophthalmic scissors, the thread plug was inserted into the internal carotid artery with forceps, the external carotid artery was completely cut and the internal carotid artery was opened, the thread plug was continuously inserted to avoid entering the opthalmic artery, and the insertion was stopped when there was slight resistance. The thread plug was fixed, the surgical thread of the common carotid artery was untied, the skin of the rat neck was sutured, and iodophor was disinfected. The Normal group did not insert the thread plug, and the steps were the same as those of the other groups. The success criteria for modeling were that the rats showed neurological impairment.

[0031] After the modeling was successful, the AVE group and the AnSC-CM group were injected with velvet extract (50 μg / rat) and velvet stem cell secreted factor (20 μg / rat) through the tail vein, the Control group was injected with saline through the tail vein, and the Normal group was not given drug treatment.

[0032] 4. Balance beam walking experiment score The motor integration and coordination ability was measured at 1 d, 2 d, 3 d, 4 d, and 5 d after modeling. A balance beam with a length of 100 cm and a width of 2.5 cm was placed 20 cm above the ground, and the rats were trained to walk smoothly on the balance beam one week before the operation. The score was evaluated according to the Feeney 5-grade scoring standard, as shown in Table 3.

[0033] Table 3. Balance beam walking experiment score 5. Neurological deficit score The neurological deficit score of the rats was evaluated 5 days after the operation. The Zeo Longa 5-point evaluation method was used to evaluate the neurological function of the rats, as shown in Table 4.

[0034] Table 4. Neurological function score standard 6. Brain tissue water content determination At 5 days after operation, 3 rats in each group were randomly selected, and the brain was taken after decapitation. The cerebellum of the rat was removed, and the brain tissue was gently separated along the midline with forceps. The weight of the ischemic injury side brain tissue was weighed as wet weight, and it was placed in a constant temperature oven for 24 h. The weight was measured as dry weight. The water content of the brain tissue of each group of rats was calculated by dividing the wet weight by the dry weight.

[0035] 7. Analysis of brain tissue neuron pathological changes At 5 days after operation, 3 rats in each group were randomly selected, and anesthetized by intraperitoneal injection of chloral hydrate. After anesthesia, the rat chest was opened, the right auricle was opened, and 200 ml PBS was slowly perfused from the left heart apex, and then 100 ml 4% paraformaldehyde was perfused until the rat limbs were white and the lung was completely white. The rat was immediately sacrificed, the brain was taken by decapitation, and was placed in 4% paraformaldehyde for fixation. After 48 h, dehydration was performed by ethanol gradient, and embedding was performed after slicing. HE staining was performed. The specific method is as follows: xylene dewaxing for 14 min, 100% ethanol immersion for 10 min, 95% ethanol immersion for 6 min, 80% ethanol immersion for 2 min, 70% ethanol immersion for 2 min, distilled water washing for 3 min, staining with hematoxylin for 5 min, 1% hydrochloric acid ethanol solution for 15 s, tap water for 5 min, 70% ethanol for 5 min, 80% ethanol for 5 min, and immersion in eosin staining solution for 3 min, 95% ethanol for 2 min, 100% ethanol for 6 min, xylene transparency for 10 min, and neutral gum sealing. Then, the pathological changes of rat brain tissue neurons were observed under a microscope.

[0036] 8. Analysis of brain tissue injury area At 5 days after operation, 3 rats in each group were randomly selected, and the brain was taken immediately after decapitation. The rat brain was cut into six equal parts, soaked in TTC staining solution, and placed in a constant temperature incubator in the dark. To avoid uneven staining, turn over every 5 min. After 30 min of staining, the TTC staining solution was recovered, the brain tissue slices were dried with filter paper, and the brain tissue injury area was calculated by taking a photograph and using Image J.

[0037] 9. Statistical analysis The data differences were analyzed and plotted by using Graph Prism 9, and T-test or One-way ANOVA was used for testing. “*” represents P<0.05; “**” represents P<0.01; “***” represents P<0.001; and “****” represents P<0.0001.

[0038] III. Test results 1. Comparison of balance beam walking experiment scores The results are as follows: Figure 1As shown, there was no statistically significant difference in the balance beam test score of the Control group, AVE group and AnSC-CM group at 1 day after modeling (P>0.5), and the scores of the three groups were higher than that of the Normal group (P<0.05). At 3 days, the score of the AnSC-CM group was significantly reduced, lower than that of the Control group (P<0.01) and lower than that of the AVE group (P>0.5), and the score of the AVE group was slightly reduced, lower than that of the Control group (P>0.5). At 5 days, the score of the AnSC-CM group was continuously reduced, lower than that of the Control group (P<0.005) and lower than that of the AVE group (P>0.5), and the score of the AVE group was lower than that of the Control group (P<0.05), and the score of the Normal group had no obvious reduction. The balance beam walking test score curve of the AVE group and the AnSC-CM group showed a downward trend, and the curve of the Control group had no obvious change. According to the Feeney 5-grade score standard, the recovery effect of the rats was inversely proportional to the upward curve. The results showed that the rats in the AnSC-CM group had good recovery effect, the rats in the AVE group gradually recovered with the increase of days, and the rats in the Control group had no obvious recovery trend. Therefore, the AnSC-CM group and the AVE group had a treatment effect on the central nervous injury of the rats, and the treatment effect of the AnSC-CM group was more significant,

[0039] 2. Comparison of neurological deficit scores As shown in the results, Figure 2 At 5 days after modeling, the neurological function score of the AnSC-CM group was significantly lower than that of the AVE group and the Control group (P<0.01), but higher than that of the Normal group. The neurological function score of the AVE group was higher than that of the AnSC-MC group and the Normal group, and lower than that of the Control group (P<0.05). The neurological function score of the Control group was the lowest. According to the Zeo Longa 5-point evaluation method, it can be obtained that the AnSC-CM group and the AVE group showed a significant recovery trend with the increase of days after injury, and the Control group had no obvious recovery effect, and the effect of the AnSC-CM group was more significant than that of the AVE group.

[0040] 3. Comparison of water content in brain tissue on the injury side As shown in the results, Figure 3As shown, the water content of all three groups was higher than that of the Normal group, with the AnSC-CM group having a lower water content than the other two groups, and the AVE group having a lower water content than the Control group. The higher the water content of the injured-side brain tissue, the more severe the brain damage in the rat. With the exception of the Normal group, the AnSC-CM group had the lowest brain tissue water content and the most significant recovery effect, followed by the AVE group. The Control group had the highest brain tissue water content on the injured side, the most severe brain edema, and no significant recovery effect. Therefore, the AnSC-CM and AVE groups demonstrated a favorable therapeutic effect in the rat central nervous system injury repair experiment, with the AnSC-CM group showing a more pronounced therapeutic effect than the AVE group.

[0041] 4. Comparison of neuronal pathological changes in brain tissue The results are as follows Figure 4 As shown, the neurons in the brain tissue of the rats in the Normal group were arranged neatly and tightly, with uniform cell size, clear neural structure, intact cell membrane and clear nuclear membrane, and uniform cytoplasm infection; the rats in the Control group had a large number of necrotic neurons, a decrease in neuronal cells, ruptured cell membrane, disappearance of nuclear membrane, and lighter cytoplasm staining; the degree of neuronal damage in the rats in the AVE group and AnSC-CM group was significantly improved compared with the Control group, with only a small number of necrotic neurons and ruptured nuclear membrane, and the cytoplasm staining gradually became uniform, and the degree of neuronal damage in the AnSC-CM group was milder than that in the AVE group.

[0042] 5. Comparison of brain tissue damage area The results are as follows Figure 5 As shown, brain sections in the Normal group were brightly colored and showed no obvious signs of injury. The injury area in the AnSC-CM group was significantly smaller than that in the AVE and Control groups, as confirmed by calculation of the injury area using Image J. The injury area in the AVE group was smaller than that in the Control group and larger than that in the AnSC-CM group. The injury in the Control group was the most severe, with no trend toward improvement. TTC staining visually demonstrated that the AnSC-CM group had a favorable recovery effect on central nervous system injury in rats. The AVE group also had a therapeutic effect, but the degree of recovery was slightly lower than that in the AnSC-CM group.

[0043] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0044] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. The use of velvet antler-derived substances in the preparation of a drug for treating cerebral ischemia injury, characterized in that: The antler-derived substance is antler extract or antler stem cell paracrine factor; The velvet antler extract is obtained by using velvet antler as raw material through water extraction and then freeze-drying; The antler stem cell paracrine factor is obtained by using the antler tip mesenchymal layer or horn stalk periosteum as raw materials through isolating the antler stem cells, culturing, passage, collecting the supernatant, ultrafiltration concentration and then freeze-drying.

2. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemia injury, characterized in that: The preparation process of the antler antler extract is as follows: fresh antler antler slices are added with pure water for overnight extraction, antler antler extract is obtained by centrifugal filtration, and antler antler extract is obtained after freeze-drying.

3. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemic injury, characterized in that: The preparation process of the antler stem cell paracrine factor comprises the following steps: isolating antler stem cells from the mesenchymal layer of the antler tip or the periosteum of the antler stalk, culturing the obtained antler stem cells using a complete culture medium, adding the complete culture medium for subculture when the confluence reaches 90% or more, replacing the culture medium with a complete culture medium without fetal bovine serum when the confluence reaches 70% to 80%, culturing for 46 to 50 hours, collecting the supernatant, centrifuging to remove cell debris, concentrating by ultrafiltration and centrifugation to obtain a concentrate, and freeze-drying the concentrate to obtain the antler stem cell paracrine factor.

4. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemic injury, characterized in that: The medicine is used for repairing central nervous system damage in rats.

5. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemic injury, characterized in that: The medicine is used for restoring the nerve function of rats.

6. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemic injury, characterized in that: The drug is used to reduce the water content of damaged brain tissue.

7. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemia injury, characterized in that: The medicine is used for reducing the damaged area of ​​rat brain tissue.

8. The use of the velvet antler-derived substance according to claim 1 in preparing a drug for treating cerebral ischemic injury, characterized in that: The drug also includes pharmaceutically acceptable excipients; The auxiliary material is any one or more combinations of physiological saline, mannitol, glucose, trehalose and sucrose.

9. The use of the velvet antler-derived substance according to claim 8 in preparing a drug for treating cerebral ischemic injury, characterized in that: The medicine is an injection preparation.

10. The use of the velvet antler-derived substance according to claim 9 in preparing a drug for treating cerebral ischemia injury, characterized in that: The injection preparation is prepared by mixing the antler-derived substance and physiological saline in a mass-to-volume ratio of 0.5-1:1.

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