CN110840984A

CN110840984A - Application of dragon's blood in preparing medicine for preventing blood brain barrier injury

Info

Publication number: CN110840984A
Application number: CN201911222060.7A
Authority: CN
Inventors: 李玉娟; 郭晶晶; 闫然然; 邓玉林; 李勇枝; 王佳平; 高建平; 梁敏
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-02-28

Abstract

The invention relates to application of dragon blood in preparing a medicine for preventing blood brain barrier injury, and belongs to the technical field of medicines. The blood brain barrier damage is blood brain barrier damage caused under microgravity environment. The dosage of the dragon blood is 1-2 g/kg/d. The medicine takes dragon's blood as the only active component, and pharmaceutic adjuvant is added to prepare the preparation. The preparation is injection, subcutaneous implant, tablet, powder, granule, capsule, oral liquid or sustained release preparation. Test results show that the dragon blood can obviously improve the phenomena of blood brain barrier cell bypass permeability increase and P-gp mediated efflux transport enhancement on blood brain barrier cell membranes caused by microgravity effect.

Description

Application of dragon's blood in preparing medicine for preventing blood brain barrier injury

Technical Field

The invention relates to application of dragon blood in preparing a medicine for preventing blood brain barrier injury, and belongs to the technical field of medicines.

Background

In recent years, people-carrying aerospace industry in China has developed rapidly. Microgravity, as a persistent factor in the aerospace environment, can cause multiple system injuries such as nerves, circulation, bones, immunity and the like of the body. Especially, the influence on the central nervous system causes the astronauts to have unfavorable symptoms such as space motion disease, sleep disorder, dyskinesia and the like, and seriously threatens the physical and mental health and flight safety of the astronauts. The Blood Brain Barrier (BBB) serves as the boundary between the blood and brain, strictly controls substance entry and exit and nutrient exchange, and is a structure that maintains the brain's internal environment and central nervous system stable.

Dragon's blood is a kind of traditional famous and precious Chinese medicine, and is resin extracted from fat-containing wood of dracaena cochinchinensis (Lour.) S.C.Chen in dracaena of Liliaceae. Modern pharmacological research proves that the dragon blood has the effects of promoting blood circulation, stopping bleeding, resisting inflammation, easing pain, resisting bacteria, resisting oxidation, repairing skin and the like. The study proves that the dragon blood can inhibit the blood viscosity, erythrocyte deformation, myocardial oxidative damage, active oxygen content in brain and the like of the rat simulating the microgravity effect, and has the protection effect on the cerebral oxidative damage and the cardiovascular system. At present, no study report on the protection of the dragon blood to blood brain barrier injury is found.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of dragon blood in preparing a medicament for preventing blood brain barrier injury, wherein the dragon blood can effectively prevent blood brain barrier injury. Further, the applicant researches and discovers that the simulated microgravity effect can damage the structure and the function of the blood brain barrier, and the dragon blood can effectively prevent the blood brain barrier from being damaged under the simulated microgravity effect.

In order to achieve the purpose, the technical scheme of the invention is as follows:

application of sanguis Draxonis in preparing medicine for preventing blood brain barrier injury is provided.

Further, the blood brain barrier injury is blood brain barrier injury caused under microgravity environment.

Furthermore, the dosage of the dragon blood is 1-2 g/kg/d.

Furthermore, the drug takes dragon's blood as the only active ingredient, and pharmaceutic adjuvants are added to prepare the preparation.

Further, the preparation is injection, subcutaneous implants, tablets, powder, granules, capsules, oral liquid or sustained release preparations.

Advantageous effects

The experimental result shows that the dragon blood remarkably improves the simulated microgravity effect to increase the blood brain barrier cell bypass permeability and enhance the P-gp mediated efflux transport on the blood brain barrier cell membrane. Specifically, the dragon blood can up-regulate the expression of intercellular abnormal connexin and maintain the complete structure of TJ and AJ on BBB, thereby improving the pathological opening of blood brain barrier cell bypass channels under the effect of simulated microgravity; the dragon blood can recover the function of simulating the microgravity effect BBB P-gp efflux transportation; dragon's blood may be assembled by inducing Rac1-Arp2/3 pathway to up-regulate peripheral actin cytoskeleton, so that actin filaments are combined and recovered with BBB connexin, and the cell-cell connection structure on BBB is stabilized to maintain complete blood brain barrier structure and normal function under simulated microgravity effect.

Drawings

FIG. 1 is an Evans blue standard curve;

FIG. 2 shows the determination of Evans blue content in the brains of rats in normal gravida;

FIG. 3 shows the measurement results of Evans blue content in the brains of rats in the simulated microgravity effect group;

FIG. 4 shows Western blot bands of each connexin between cells of the Blood Brain Barrier (BBB) of normal gravity group;

FIG. 5 shows the result of Western blot bands of each connexin between Blood Brain Barrier (BBB) cells of the simulated microgravity effector group;

FIG. 6 shows the results of the Claudin-5 semi-quantitative analysis of the intercellular connexin of BBB of the simulated microgravity effector group;

FIG. 7 is the result of semi-quantitative analysis of the extracellular connexin Occludin in the BBB cell of the simulated microgravity effect group;

FIG. 8 is a semi-quantitative analysis statistical result of BBB intercellular connexin ZO-1 of the simulated microgravity effect group;

FIG. 9 is a semi-quantitative analysis statistical result of BBB intercellular connexin VE-cadherin of the simulated microgravity effect group;

FIG. 10 shows the results of semi-quantitative analysis of BBB intercellular junction protein β -catenin of the simulated microgravity effector group;

FIG. 11 shows the Western blot banding results of P-gp protein in normal gravity group brains;

FIG. 12 shows the Western blot banding results of P-gp protein in rat brain simulating microgravity effect;

FIG. 13 shows the statistical results of semi-quantitative analysis of P-gp protein in the brains of rats in the simulated microgravity effect group;

FIG. 14 shows the results of the measurement of P-gp ATPase activity on BBB of the simulated microgravity effect group;

FIG. 15 shows the Western blot bands of rat brain Rac1, Wave2 and Arp3 proteins in the simulated microgravity effect group;

FIG. 16 shows the statistics of the semi-quantitative analysis of rat brain Rac1 protein in the simulated microgravity effect group;

FIG. 17 shows the statistics of the semi-quantitative analysis of brain Wave2 protein in rats simulating microgravity effect group;

FIG. 18 shows the statistics of the semi-quantitative analysis of rat brain Arp3 protein in the simulated microgravity effect group.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

The blood brain barrier is mainly composed of two parts, namely a passive physical barrier formed by intercellular connection physiological structures and an active barrier formed by efflux pump proteins. The invention explores the protective effect of the dragon blood to the blood brain barrier injury of the rat with the 21d simulated microgravity effect from the two aspects. The experiment adopts a rat tail suspension method to simulate microgravity effect, and the model is established and the dragon blood is administrated by intragastric administration. Through measuring the content of Evans blue in brains of 5 groups of rats and the expression of intercellular connexin, the influence of the dragon blood on the damage of the structure and the function of a 21d (medium-long term) simulated microgravity effect rat blood brain barrier cell bypass channel is discovered; detecting the expression and the efflux function of the P-gp protein and the activity of the P-gp ATP enzyme, and revealing the repair effect of the dragon blood on the structural and functional change of the P-gp efflux protein of the blood brain barrier cell membrane of the rat with the 21d simulated microgravity effect.

In the following examples:

(1) drugs and agents, as shown in table 1:

TABLE 1

(2) Animals:

the healthy SPF male SD rat (with the weight of 200 +/-20 g) is provided by the experimental animal resource center of China food and drug testing research institute, the license number SCXK- (Jing) 2009-0017, the feeding environment temperature is 20-25 ℃, the humidity is 50-60%, the free water is drunk for feeding, and the male SD rat is adaptively fed for 1 week before the experiment. Both animal feeding and experiments are carried out according to the experimental animal feeding and use regulations of Beijing university of Physician.

(3) Apparatus, as shown in table 2:

TABLE 2

(4) Preparing a reagent:

physiological saline: weighing 9g of NaCl solid powder, dissolving in a proper amount of ultrapure water, finally fixing the volume to 1L, preparing a NaCl solution with the mass fraction of 0.9%, and storing at 4 ℃.

2% of Evans blue solution by mass: accurately weighing 2g of evans blue, dissolving with normal saline, diluting to 100mL of constant volume, filtering, sterilizing, and subpackaging in sterile bottles for later use.

Trichloroacetic acid (TCA) extract with mass fraction of 50%: 50g of TCA is precisely weighed, the volume is adjusted to 100mL by using physiological saline, the filtration and the sterilization are carried out, and the TCA is preserved in a refrigerator at 4 ℃.

Rhodamine 123(Rh123) solution: rh123 of 1mg was precisely weighed and dissolved sufficiently in 100. mu.L of sterile physiological saline to prepare a Rh123 solution of 10mg/mL concentration. Rh123 solution is taken and prepared into Rh123 standard solution with 7.8125, 15.625, 31.25, 62.5, 125 and 250ng/mL series concentration by using physiological saline.

Sodium vanadate (Na)₃VO₃) Solution: precisely weighing Na₃VO₃27.5mg of the powder was dissolved in distilled water and made up to 5mL to a stock solution of 30mM sodium alum.

Dragon's blood suspension: weighing 10g of dragon blood powder, suspending with 0.5% (mass fraction) of sodium carboxymethylcellulose to 100mL, and making into 0.1g/mL dragon blood suspension.

(5) Statistical analysis

Excel, SPSS 20 and GraphPad Prism 6 software are adopted to carry out data statistical analysis, the result is expressed by Mean + -SD, single-factor variance analysis is adopted among multiple groups, and the difference is considered to have statistical significance when the probability p is less than 0.05.

Example 1: determination of blood brain barrier intercellular bypass permeability

Evans blue is a classical Blood Brain Barrier (BBB) tracer. The content of evans blue in brain is detected to be used as a quantitative index for evaluating and simulating the permeability of the intercellular bypass of the rat BBB cell with the microgravity effect.

(1) Averagely dividing 35 healthy SPF male SD rats into 5 groups, namely a 21d normal gravity group I (21d-NG-1) and a 21d normal gravity group plus dragon blood (21d-NG + DB); 21d Normal gravity two (21d-NG-2) group, 21d simulated microgravity (21d-SMG) group and 21d simulated microgravity plus dragon's blood (21d-SMG + DB) group. The 21d-NG-1 and 21d-NG-2 groups are raised under normal gravity environment, the 21d-NG + DB group is rat gavage dragon blood suspension under normal gravity, the 21d-SMG group and the 21d-SMG + DB group are raised under simulated microgravity effect, each rat in the 21d-NG + DB and 21d-SMG + DB group is fed with dragon blood suspension by gavage, the dose of the dragon blood is 1g/kg/d, and each rat in the 21d-NG-1, 21d-NG-2 group and 21d-SMG group is fed with 0.5 percent of sodium carboxymethylcellulose solution.

The method is characterized in that a classical Morey-Holton method is adopted, the tail of a rat is suspended to simulate the microgravity effect, and the method comprises the following steps: fixing a healthy SPF male SD rat by using a fixer, cleaning and disinfecting rat tails by using water and 75% ethanol in sequence, smearing tails by using saturated benzoin tincture, and fixing the tail of the rat to a pulley on the top of a tail hanging cage by using a medical adhesive tape. The forelimbs of the rat step on the bottom of the cage, the head is low, the trunk and the horizontal plane of the mouse cage form 30 degrees, the hind limbs are freely suspended without bearing a load, and the rat can freely move in the cage and drink water for ingestion.

(2) After 21 days, each rat was anesthetized with ether and injected with 4mL/kg of 2% Evans blue solution via the femoral vein. After 1h, 3% sodium pentobarbital is injected into the abdominal cavity at a rate of 1mL/kg to anaesthetize each rat, after the pain is completely disappeared, the thoracic cavity is opened to perfuse the heart with normal saline, and simultaneously the right auricle is cut open, and the operation is stopped after the effluent is colorless. The rat skull was quickly opened, the meninges were stripped and rat brain tissue was removed.

(3) Sample pretreatment

Taking a proper amount of rat brain tissue, respectively adding 4 times of 50% TCA extracting solution, grinding in an ice bath by using an electric homogenizer, and fully crushing in an ultrasonic crusher. Centrifuging at 10000r/min at 4 deg.C for 20min, collecting supernatant to obtain brain tissue Evans blue extractive solution (35 parts in total), and wrapping with tinfoil to protect from light.

(3) Preparation of standard curve and content measurement

The 2% Evans blue solution was diluted to 0.0391, 0.0781, 0.1563, 0.3125, 0.625, 1.25, 2.5, 5, 10. mu.g/mL standard solutions using 50% TCA extract. 200 μ L of each standard solution and each brain tissue Evans blue extract were added to each well of a 96-well plate. The fluorescence value of each well was measured with a multifunctional microplate reader. The instrument parameter selects excitation wavelength 620nm and emission waveThe length is 680nm, and the bandwidth is 7 nm. A standard curve is plotted with the measured fluorescence values on the vertical axis (y-axis) and the evans blue concentration on the horizontal axis (x-axis), as shown in fig. 1, with the standard curve equation being y 488.2x + 100.6. Coefficient of correlation r²0.9972, has good linear dependence. The evans blue content in each sample was calculated from the measured fluorescence values and expressed as the amount of evans blue contained in brain tissue per gram of wet weight (μ g/g).

The results of the evans blue content assay in the brain tissue of 5 groups of rats are shown in FIGS. 2-3. The brain tissue content of Evans blue in rat brain tissue of 21d-NG-1 group and 21d-NG + DB group is 0.48 + -0.11 μ g/g and 0.52 + -0.07 μ g/g respectively as shown in FIG. 2; the brain tissue content of Evans blue in rat brain tissues of 21d-NG-2 group, 21d-SMG group and 21d-SMG + DB group were 0.57 + -0.13 μ g/g, 0.72 + -0.24 μ g/g and 0.54 + -0.16 μ g/g, respectively, as shown in FIG. 3. There was no significant change in Evans blue content in brain tissue of rats in the 21d-NG + DB group compared to the 21d-NG-1 group. Compared with the 21d-NG-2 group, the brain tissue of the rats in the 21d-SMG group has 26.7 percent higher Evans blue content (p is less than 0.05). Compared with the 21d-SMG group, the brain tissue of rats in the 21d-SMG + DB group has obviously reduced content of evans blue by 24.9 percent. Preliminarily shows that the simulated microgravity effect can increase the blood brain barrier cell bypass permeability, and the dragon blood resin obviously improves the phenomenon that the simulated microgravity effect causes the blood brain barrier cell bypass permeability to increase.

Example 2: blood brain barrier intercellular connexin expression assay

(1) Same as example 1, step (1).

(2) After 21 days, the rats were anesthetized with 3% sodium pentobarbital, and after complete anesthesia, brain tissue was collected and stored in a refrigerator at-80 ℃.

(3) Protein extraction and quantification: respectively taking a proper amount of brain tissue of each rat, mixing the brain tissue in groups, weighing the brain tissue, adding a precooled strong RIPA lysate (containing holoprotease and phosphatase inhibitor) with the volume 5 times that of the lysate, and homogenizing the lysate by adopting a glass homogenizer under the ice bath condition. The homogenate is subjected to ultrasonic disruption, and then centrifuged at 12000g centrifugal force at 4 ℃ for 10min, and the supernatant is collected to obtain a total protein extract (4 parts in total), and then the total protein extract is subpackaged and stored in a refrigerator at-20 ℃ for later use.

(4) Measurement by BCA methodDetermining the protein concentration of the brain tissue protein extracting solution: accurately weighing 0.010g of Bovine Serum Albumin (BSA), dissolving in 1ml of 0.01M PBS, and performing ultrasonic treatment for 10min to fully dissolve the BSA to obtain a BSA standard substance of 10 mg/ml. Stock solutions of BSA standards were diluted sequentially with PBS to a range of concentrations of 15.625, 31.25, 62.5, 125, 250, 500, 1000, 2000 μ g/mL. 25 mu L of each of the whole protein extract and the diluted BSA standard sample is put into a 96-well plate, and 200 mu L of Coomassie brilliant blue staining solution is added. Mixing, reacting at 37 deg.C for 30min, cooling to room temperature, and measuring OD of each well with multifunctional enzyme labeling instrument₅₉₅The value is obtained. And drawing a standard curve according to the relation between the light absorption value and the concentration of the standard substance. And finally, obtaining the protein concentration of the sample to be detected according to the standard curve.

(5) Mixing the whole protein extract with 4 × Loading Buffer at volume ratio of 3:1, boiling in water bath for 10min to obtain mixed protein sample, and subpackaging at-20 deg.C for storage.

(6) Western blot

① separating gel is prepared by washing glass plate with water and ethanol, drying, placing on a gel preparation rack, preparing 12% separating gel according to the formula of separating gel shown in Table 3, pouring into the glass plate, immediately adding appropriate amount of isopropanol, sealing, pouring off isopropanol after the separating gel solidifies, and drying with filter paper.

TABLE 3

② the preparation method comprises washing glass plate with water and ethanol, drying, placing on a gel preparation rack, preparing 5% concentrated gel according to the formula shown in Table 4, filling the glass plate, immediately inserting into a 10-hole comb, and allowing the concentrated gel to solidify.

TABLE 4

③ loading and electrophoresis, loading the prepared separation gel and concentrated gel into electrophoresis electrode, pouring 1 × electrophoresis solution, removing 10-hole comb, loading the mixed protein sample of each group for 3 times, loading the sample in equal amount, performing electrophoresis in constant voltage mode with initial voltage of 80V, converting voltage of 100V to continue electrophoresis after the sample enters the separation gel, and stopping electrophoresis until the dye runs 1cm away from the gel bottom.

④ transferring membrane, after electrophoresis, performing protein transferring membrane by a Bio-Rad wet method, soaking the PVDF membrane (cutting angle mark in advance) in methanol for about 30 seconds, then soaking the PVDF membrane, sponge and filter paper in precooled transfer membrane liquid for balancing for at least 5min, opening the electric transfer clamp, sequentially placing a sponge pad, the filter paper, separation glue, the PVDF membrane, the filter paper and the sponge pad on the black negative electrode surface, removing bubbles by a roller, inserting the clamp into a transfer tank, fully pouring the transfer membrane liquid, and transferring the membrane in ice water under the conditions of constant pressure of 100V and 2 h.

⑤ sealing, after the film transfer is finished, the PVDF film is washed once by TBST buffer solution and put into 5 percent (mass fraction) of skimmed milk powder, and the obtained product is placed on a decolourization shaking table for 2 hours at room temperature.

⑥ Primary antibody incubation after blocking, the PVDF membrane was washed once with TBST, placed in TBST diluted primary antibody (Claudin-5, Occludin, VE-cadherin, ZO-1 or β -catenin), and incubated overnight at 4 ℃ with shaking.

⑦ incubation of secondary antibody after the primary antibody incubation was complete, the PVDF membrane was removed, washed 4 times with TBST, 15min each time, then the membrane was placed in the corresponding dilution of secondary antibody and incubated for 2h at room temperature with shaking.

⑧ developing, after the incubation is finished, washing the PVDF membrane with TBST for 4 times, 15min each time, preparing ECL developing solution according to the proportion of A: B ═ 1:1, then putting the PVDF membrane in a gel imager, carrying out gray scale analysis on a target band by using ImageLab software, taking GAPDH as an internal reference, and carrying out semi-quantification on the connexins Claudin-5, Occludin, ZO-1, VE-cadherin and β -catenin in a sample.

The results of Western blot bands of the respective connexins are shown in FIGS. 4-5, the results of Western blot analysis are shown in FIGS. 6-10, compared with the 21d-NG-1 group, the relative contents of Claudin-5, Occludin, ZO-1, VE-cadherin and β -catenin proteins in the 21d-NG + DB group are not obviously changed (p > 0.05), compared with the 21d-NG-2 group, the levels of Claudin-5 and VE-cadherin proteins in brain microvascular endothelial cells of rats in the 21d-SMG group are obviously reduced (p < 0.05), 18.8% and 22.8% are respectively reduced, Occludin, ZO-1 and β -catenin proteins are not obviously changed (p > 0.05), compared with the 21d-SMG group, the levels of Claudin-5 and Claudin-5 in brain in the 21d-SMG + DB group are obviously reduced, the contents of Claudin-5 and ZO-1 and β -catenin proteins in brain are obviously reduced, compared with the 21d-SMG + SMG group, the results of binding proteins in brain, the results of the binding protein in the BBB-BCB and the BBB are obviously reduced, the BBB-B are obviously reduced, the results of the binding protein in the BCB-B binding protein in the BBG + BCB, and the BBG + BCB are obviously reduced, the BCB binding protein is improved, the BCB binding protein is improved, the binding protein in the BCB, the binding protein is improved in the binding protein in the BCB, the BCB.

Example 3: blood brain barrier P-gp protein expression assay

In this example, antibodies P-gp (Abcam) were used, and protein extraction, quantification and Western blotting were carried out as described in example 2.

FIG. 11 shows the Western blot bands of P-gp protein in rat brains from groups 21d-NG-1 and NG + DB. FIG. 12 shows the Western blot bands of P-gp protein in the brains of rats in groups 21d-NG-2, SMG and SMG + DB. The band gray scale analysis was semi-quantitative and the statistical results are shown in FIG. 13. Compared with the 21d-NG-1 group, the relative content of the P-gp protein in the 21d-NG + DB group is not obviously changed (P is more than 0.05). Compared with the 21d-NG-2 group, the relative content of the P-gp protein in the rat brain of the 21d-SMG group is obviously increased by 20.4 percent (P is less than 0.05). Compared with the 21d-SMG group, the P-gp content in the brain of the rats of the 21d-SMG + DB group is obviously reduced by 29.7 percent (P is less than 0.05). Research results show that the microgravity effect simulated for a medium-long period of 21d can increase the expression level of the P-gp protein, and the dragon blood can obviously inhibit the expression of the dragon blood and restore the normal level. Regarding the regulation and control reasons, the related documents are found that many traditional Chinese medicines can inhibit the expression of MDR from the gene transcription and protein translation levels, so that the expression level of P-gp protein is reduced, and dragon blood as a multi-component compound contains more components such as flavonoid, terpenoids, steroids and steroid saponins, and the like, and can possibly regulate and control the expression of P-gp. In addition, the dragon blood has the effects of promoting blood circulation to remove blood stasis, resisting inflammation and oxidation, eliminating free radicals and the like, and can indirectly regulate and control the expression of P-gp protein by reducing excessive injury substances such as inflammatory factors, active oxygen and the like in the blood or brain of a rat simulating the microgravity effect.

Example 4: blood brain barrier P-gp substrate efflux assay

Rh123 is a classical substrate of P-gp, and the efflux function of P-gp is evaluated by measuring the accumulation and outflow of Rh123 in the brain.

(1) Same as example 1, step (1).

(2) After 21 days, rats were anesthetized with ether and injected with Rho123 solution via the femoral vein at a dose of 0.2 mg/kg. Pentobarbital sodium is injected into the abdominal cavity after 1h, and after complete anesthesia, blood is collected from the orbit and collected in an anticoagulation tube. Then the heart perfusion of the normal saline is stopped until the effluent is colorless, the brain shell meninges is stripped, and the brain tissue is collected to be tested.

(3) Sample pretreatment

Taking a proper amount of brain tissue of each rat, respectively adding 4 times of physiological saline to extract Rh123, centrifuging the brain tissue homogenate and the blood sample at a high speed of 12000g (centrifugal force) for 10min, and collecting the supernatant and storing in a dark place. Taking 100 μ L of brain tissue and blood supernatant, adding 100 μ L of normal saline, adding 300 μ L of methanol, vortex for 10s, centrifuging at 15000rpm/min for 10min, and collecting supernatant to obtain brain and blood sample solutions (28 parts each).

(4) Preparation of Rh123 Standard Curve

100 mu L of Rh123 standard solution with 7.8125, 15.625, 31.25, 62.5, 125 and 250ng/mL series of concentrations is put into a centrifuge tube, 100 mu L of blank plasma (or blank brain tissue) and 300 mu L of methanol are respectively added, vortex is carried out for 10s and mixed evenly, centrifugation is carried out for 10min at 15000rpm, and the blank plasma (or blank brain tissue) Rh123 standard curve is obtained by taking supernatant.

(5) Rh123 content detection

The sample to be tested and the supernatant of each standard (200. mu.L) were injected into a 96-well plate, and the fluorescence intensity of Rh123 in the brain tissue and plasma was measured using a multifunctional microplate reader (λ ex/λ em: 495nm/530 nm). And performing linear regression on the concentration by using the fluorescence intensity to obtain an Rh123 standard curve equation in brain tissues and blood. Calculating the Rh123 content in brain and blood of rats in each group by a standard curve method, and measuring the transport function of P-gp on the blood brain barrier by the Rh123 brain-blood ratio.

The statistical results of the brain blood distribution ratio in the brain, blood and both of the rats in the 21d-NG-2 group, the 21d-SMG group and the 21d-SMG + DB group are shown in Table 5. The brain and blood contents of the rats in the 21d-NG-2 group are 23.24 +/-1.46 NG/g and 17.46 +/-2.20 NG/mL respectively, and the distribution ratio of the brain blood is 1.37 +/-0.10; the contents of brain and blood in the rats in the SMG group are 20.71 +/-1.16 ng/g and 18.54 +/-3.90 ng/mL, and the distribution ratio of brain blood is 1.15 +/-0.18. The contents of the brain and the blood of the rats in the SMG + DB group are 24.85 +/-2.26 ng/g and 16.91 +/-5.56 ng/mL, and the distribution ratio of the brain blood is 1.44 +/-0.42. The result shows that compared with the 21d-NG-2 group, the Rh123 content in the brains of the rats of the 21d-SMG group is remarkably reduced by 10.9 percent (p is less than 0.05), the cerebral blood distribution ratio is reduced by 16.7 percent, and the Rh123 content in the blood has no statistical difference. Compared with the 21d-SMG group, the Rh123 content in the rat brain of the 21d-SMG + DB group is obviously increased by 20.2 percent (p is less than 0.05), the cerebral blood distribution ratio is increased by 25.6.3 percent, and the Rh123 content in the blood has no statistical difference. Research results indicate that the efflux transport of P-gp on rat BBB simulating microgravity effect in 21d is obviously increased, and the efflux transport function of P-gp can be obviously recovered by dragon's blood.

TABLE 5

Example 5: blood brain Barrier P-gp ATPase Activity assay

The transport activity of P-gp to the substrate is measured by measuring the amount of inorganic phosphorus produced by ATP hydrolysis, based on the fact that P-gp transports the substrate with the hydrolysis of ATP. This example measures the effect on P-gp ATPase (ATPase) activity between different groups by the difference between the amounts of inorganic phosphorus produced by each group in the presence and absence of the P-gp ATPase inhibitor vanadate. The specific operation comprises two steps: the first is enzymatic reaction and the second is phosphorus determination reaction.

(1) Same as example 1, step (1).

(2) Carrying out enzymatic reaction and phosphorus determination reaction according to the operation requirement of the ultramicro ATP enzyme activity test box:

precisely weighing a proper amount of brain tissue, adding 9 times volume of physiological saline, mechanically homogenizing under ice bath condition, 2500r/min, centrifuging for 10min, taking supernatant (namely 10% homogenate supernatant), diluting to 1% by 10 times volume of physiological saline, and simultaneously determining the tissue protein concentration by using Coomassie brilliant blue reagent. If the pre-test results are too high, the tissue homogenate of 1% is diluted to different concentrations and the pre-test is performed before the sampling concentration is determined.

The reaction system of the enzymatic reaction is shown in Table 6, and ATP enzyme in each sample in the system can combine with ATP to react to generate inorganic phosphorus. Adding Na₃VO₃Except for P-gp ATPase, other ATPases all participate in the reaction. As shown in Table 7, the formed inorganic phosphorus reacted with the phosphorus-fixing color-developing agent, and the absorbance was measured at 636 nm.

TABLE 6

TABLE 7

After the reaction, the absorbance of each tube was measured by adjusting the light path to zero with double distilled water at 636nm at 1 cm.

The enzyme activity calculation formula is as follows:

p-gp ATPase activity (U/mgprot) ═ total ATPase activity-ATPase activity (U/mgprot) of other ATPase (sodium vanadate) activities (determination OD value-control OD value)/(standard OD value-blank OD value) × phosphorus standard concentration × 7.8 × 6 ÷ concentration of sample protein to be measured (mgprot/mL)

The results of the measurement of P-gp ATPase activity on the BBB of rats in the 21d-NG-2, SMG and SMG + DB groups are shown in FIG. 14. The results of the rat brain P-gp ATPase activity assays of the 21d-NG-2 group, the 21d-SMG group and the 21d-SMG + DB group are 58.70 plus or minus 12.3U/mgprot, 68.38 plus or minus 4.3U/mgprot and 60.36 plus or minus 5.3U/mgprot respectively. Compared with the 21d-NG-2 group, the P-gp ATPase activity in the brain of the rats of the 21d-SMG group is obviously increased by about 16.5 percent. Compared with the 21d-SMG group, the P-gp ATPase activity in the brain of the rats of the 21d-SMG + DB group is obviously reduced by about 11.7 percent. The result proves that the dragon blood can obviously inhibit the activity of P-gp ATP enzyme under the effect of simulated microgravity, thereby reducing the activity of efflux function. By combining the results of the accumulation and increase of Rh123 in brain and the inhibition of P-gp efflux under the action of the dragon blood in example 4, the dragon blood is further proved to be capable of recovering the simulated microgravity effect BBB P-gp efflux transport function.

Example 6: influence of dragon blood on expression of Rac1-Arp2/3 pathway key protein in simulated microgravity effect rat brain

Differential proteomics analysis finds that 21d mimics the microgravity effect and may be based on altering the actin cytoskeleton assembly process mediated by the "Rac 1-Arp 2/3" pathway, threatening BBB structure and function. The invention measures the expression of key protein of Rac1-Arp2/3 pathway, and explores the protective mechanism of dragon's blood to blood brain barrier injury caused by 21d simulated microgravity effect.

In this example, Rac1, Wave2 and Arp2/3 antibodies were used as antibodies, and protein extraction, quantification and Western blotting were carried out as described in example 2.

Western blot bands for rat brains Rac1, Wave2 and Arp3 of the 21d-NG-2, 21d-SMG and 21d-SMG + DB groups are shown in FIG. 15. The bands were subjected to gray scale analysis and the two sets of statistics are shown in FIGS. 16-18. Compared with the 21d-NG-2 group, the relative contents of cytoskeleton related proteins Rac1, Wave2 and Arp3 in the brains of the rats of the 21d-SMG group are obviously reduced by 25.6 percent, 23.0 percent and 41.9 percent respectively (p is less than 0.05); compared with the 21d-SMG group, the contents of Rac1, Wave2 and Arp3 in the brain of the rats of the 21d-SMG + DB group are respectively increased by 42.8 percent, 13.9 percent and 47.0 percent (p is less than 0.05). The results show that the simulated microgravity effect can reduce the complex formed by the combination of Rac1, Wave2 and Arp3 protein levels, reduce the combination of Rac1 and downstream signal molecule Wave2, reduce the combination of activated Wave2 protein and Arp2/3, weaken the action of activated Arp2/3, lead to the inhibition of peripheral actin cytoskeleton polymerization mediated by Arp2/3 complex, unbalance actin cytoskeleton polymerization-depolymerization, dominate depolymerization, lead to the depolymerization of peripheral actin cytoskeleton, influence TJ and AJ proteins connected with actin filaments, lead the actin filaments to be loosely or disconnected with connexin, lead to the loosening or breaking of cell-cell connection on the blood brain barrier, increase intercellular space and further improve the permeability of the blood brain barrier. However, the dragon blood can remarkably induce Rac1, Wave2 and Arp3 protein expression in the rat brain simulating the microgravity effect, the complex formed by combining Rac1 and downstream signal molecules Wave2 is increased, Wave2 is activated, the activated Wave2 is combined with Arp2/3, Arp2/3 is further activated, and the assembly of a peripheral actin cytoskeleton is promoted. Dragon's blood may be assembled by inducing Rac1-Arp2/3 pathway to up-regulate peripheral actin cytoskeleton, so that actin filaments are combined and recovered with BBB connexin, and the cell-cell connection structure on BBB is stabilized to maintain complete blood brain barrier structure and normal function under simulated microgravity effect.

In summary, the invention includes but is not limited to the above embodiments, and any equivalent replacement or local modification made under the spirit and principle of the invention should be considered as being within the protection scope of the invention.

Claims

1. Application of sanguis Draxonis in preparing medicine for preventing blood brain barrier injury is provided.

2. The use of dragon's blood as claimed in claim 1 in the preparation of a medicament for preventing blood brain barrier injury, wherein: the blood brain barrier damage is blood brain barrier damage caused under microgravity environment.

3. The use of dragon's blood as claimed in claim 1 in the preparation of a medicament for preventing blood brain barrier injury, wherein: the dosage of the dragon blood is 1-2 g/kg/d.

4. The use of dragon's blood resin as claimed in any one of claims 1-3 in the preparation of a medicament for preventing blood brain barrier injury, wherein: the medicine takes dragon's blood as the only active component, and pharmaceutic adjuvant is added to prepare the preparation.

5. The use of dragon's blood as claimed in claim 4 in the preparation of medicament for preventing blood brain barrier injury, wherein: the preparation is injection, subcutaneous implant, tablet, powder, granule, capsule, oral liquid or sustained release preparation.