CN110934857B

CN110934857B - Application of skin resinol in preparing medicine for preventing or treating brain injury and pharmaceutical composition thereof

Info

Publication number: CN110934857B
Application number: CN201911266372.8A
Authority: CN
Inventors: 庞涛; 阮文臣; 河泰麟; 王云杰; 高成龙; 张陆勇
Original assignee: China Pharmaceutical University
Current assignee: Jiangsu Zhengrui Pharmaceutical Technology Co.,Ltd.
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-03-29
Anticipated expiration: 2039-12-11
Also published as: CN110934857A

Abstract

The invention discloses application of corticosterol in preparing a medicine for preventing or treating brain injury and a pharmaceutical composition thereof. The invention provides the application of the cortisol in preparing the medicine for preventing or treating the brain injury for the first time, the cortisol has obvious effect in preventing or treating the brain injury, and particularly, the brain injury caused by the ischemic stroke can be obviously relieved. The invention discovers that the skin resinol has the function of inhibiting the damage of brain microvascular endothelial cells caused by sugar oxygen deprivation; the corticoid alcohol (Medinoresinol) can obviously reduce the permeability of blood brain barrier under oxygen sugar deprivation; the skin resinol has the function of obviously reducing the cerebral infarction volume under the blockage of the middle cerebral artery, and the pharmaceutical composition containing the skin resinol can become a novel medicament for preventing and/or treating brain injury and treating various cerebrovascular diseases including ischemic stroke.

Description

Application of skin resinol in preparing medicine for preventing or treating brain injury and pharmaceutical composition thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of cortisol in preparation of a medicine for preventing or treating brain injury and a pharmaceutical composition of the medicine.

Background

The stroke is a refractory disease seriously harming human health and life safety in the world at present, and has the characteristics of high morbidity, high disability rate, high recurrence rate and high mortality, China is the country with the most serious stroke disease burden in the world, about 200 million new stroke patients are treated every year, wherein the ischemic stroke accounts for about 75-85% of the total number of the stroke patients, the treatment cost for the stroke reaches more than 100 billion yuan every year in the country, and the serious economic burden is caused to the country and families. Over the last two decades, numerous studies have shown that neuroprotective agents are effective at improving cerebral ischemic symptoms at the animal level, but have failed in clinical trials, suggesting that the treatment of ischemic stroke by neuroprotective strategies alone is far from sufficient. At present, the acute-phase treatment means of ischemic stroke mainly comprises thrombolytic treatment, recombinant tissue plasminogen activator (tPA) is given for treatment within 3-4.5 hours of the attack of patients, but only a few patients benefit clinically due to the narrow treatment time window and the intracranial hemorrhage risk of the method. Therefore, it is urgent to research a new effective treatment for cerebral arterial thrombosis.

Cerebrovascular endothelial injury and blood brain barrier damage caused by the cerebrovascular endothelial injury are important pathological mechanisms of ischemic stroke, and are important means for treating the ischemic stroke starting from protecting endothelial cells. The brain vascular endothelial cells are important components of neurovascular units and vascular neural networks, are main components of brain microvasculature and structural basis of blood-brain-barrier (BBB), and have a leading role in maintaining the integrity of BBB and brain homeostasis under normal physiological conditions. In the process of cerebral ischemia, cerebral vascular endothelial cells are firstly damaged, and firstly, the cerebral vascular endothelium is directly damaged under the ischemic condition, so that the structural and functional integrity of the endothelial cells is damaged; secondly, the ischemic condition activates the brain vascular endothelial cells to generate endothelial inflammation, and the activated endothelial cells generate and secrete a large amount of inflammatory factors, the inflammatory factors generated by the vascular endothelium comprise selectin (P-selectin, E-selectin), adhesion molecules (VCAM-1, ICAM-1) and the like, the inflammatory factors generated by the vascular endothelium promote the adhesion of the white blood cells in the peripheral blood circulation to the endothelial cells and further migrate to the ischemic area of the brain tissue, and further the inflammatory factors generated by the white blood cells aggravate the brain tissue injury. A large number of studies show that brain endothelial cell injury, endothelial cell inflammation and subsequent endothelial function injury caused by cerebral ischemia increase the permeability of cerebral vessels and the leakage of blood brain barrier, resulting in ischemic brain injury. Endothelial cell damage and the resulting inflammatory response lead to blood brain barrier destruction and dysfunction, altered cerebrovascular permeability and integrity, vasogenic cerebral edema, production of inflammatory factors or entry of large amounts of inflammatory factors into ischemic injury areas, thereby exacerbating necrosis and apoptosis of nerve cells. Therefore, starting from the protection of cerebrovascular endothelial cells, the method has become an important treatment means for preventing cerebrovascular dysfunction caused by ischemic stroke.

Pectinols (Medioresinol) (CAS number: 40957-99-1) are a novel lignan compound isolated from plants such as eucommia ulmoides oliv. At present, no relevant research reports the biological activity.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention aims to find a medicine for relieving brain microvascular endothelial cell injury caused by sugar oxygen deprivation (OGD), and the medicine plays a role in treating ischemic stroke and other heart, lung, peripheral blood vessels and other ischemic vascular diseases.

The invention provides the use of, for example, cortisol in the manufacture of a medicament for the prevention or treatment of brain injury; the skin resinol can obviously reduce the damage of mouse brain microvascular endothelial cells under a sugar oxygen deprivation (OGD) model and the release of proinflammatory cytokines; the skin resin alcohol can obviously reduce the permeability of blood brain barrier under a sugar oxygen deprivation (OGD) model; cortisol significantly reduced the cerebral infarct volume in mice under the model of middle cerebral artery occlusion (tMCAO). Therefore, the cortisol is expected to be used for treating various cerebrovascular diseases including ischemic stroke.

The invention also provides a pharmaceutical composition for preventing or treating brain injury.

The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for preventing or treating brain injury, comprising administering a therapeutically effective amount of a compound of formula (I) to a subject in need thereof.

Furthermore, the use of the cortisol in the preparation of medicaments for preventing or treating brain injuries caused by cerebrovascular diseases such as ischemic stroke, hemorrhagic stroke or high altitude cerebral edema.

Wherein the dermestol (CAS number: 40957-99-1) is a lignan compound isolated from eucommia ulmoides and other plants, and has the following structural formula:

the skin resinol is used for obviously reducing brain microvascular endothelial cell injury caused by sugar oxygen deprivation and releasing proinflammatory cytokines, and is used for preparing a medicine for treating brain injury.

Wherein, the skin resinol is used for obviously reducing the permeability of a blood brain barrier under the condition of sugar oxygen deprivation and is used for preparing the medicine for treating brain injury. In particular, cortisol significantly reduces blood brain barrier permeability in the sugar oxygen deprivation (OGD) model.

Wherein, the skin resinol is used for obviously reducing the cerebral infarction volume under the middle cerebral artery occlusion model and preparing the medicine for treating the brain injury.

The pharmaceutical composition for preventing or treating brain injury comprises the corticosterol or the pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

Further, the pharmaceutical composition is in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, creams, ointments, suppositories or patches.

The dosage of the medicine of the invention is as follows: cell assay 20 μ M; animal experiments 10 mg/kg.

The results of the experiments in the cells show that: sugar oxygen deprivation (OGD) obviously promotes the release of lactate dehydrogenase LDH and the expression of an inflammatory factor IL-1 beta in brain microvascular endothelial cells, and 20 mu M of dermolide (Medioresinol) can obviously inhibit the release of lactate dehydrogenase LDH and the expression of the inflammatory factor IL-1 beta. In an in vitro blood brain barrier model established by co-culture of primary brain microvascular endothelial cells and astrocytes, 20 mu M of cortisol can remarkably inhibit leakage of FITC-labeled dextran and decrease of transendothelial resistance caused by sugar oxygen deprivation (OGD). In animal models, 10mg/kg of dermoresinol (Medioresinol) significantly reduced the cerebral infarct volume in the middle cerebral artery occlusion (tMCAO) model mice.

The invention relates to a new functional compound discovered by random screening, and the main action mechanism of the compound is probably to protect the damage of brain microvascular endothelial cells under the OGD condition by inhibiting the expression of endothelial cell apoptosis signal molecules, thereby protecting the blood brain barrier and improving the new function of cerebral ischemia damage.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides the application of the cortisol in preparing the medicine for preventing or treating the brain injury for the first time, and the cortisol has obvious effect in preventing or treating the brain injury, such as the brain injury caused by cerebrovascular diseases such as ischemic stroke, hemorrhagic stroke or high altitude cerebral edema, and the like, and particularly has the effect of obviously relieving the brain injury caused by the ischemic stroke.

The invention discovers that the corticoid alcohol (Medioresinol) has the effects of inhibiting the damage of brain microvascular endothelial cells caused by sugar oxygen deprivation (OGD) (figure 1) and inhibiting the expression of an inflammatory factor IL-1 beta (figure 2); corticosterol (Medioresinol) significantly reduced blood brain barrier permeability in the sugar oxygen deprivation (OGD) model (fig. 3); corticosterol (Medioresinol) had the effect of significantly reducing the cerebral infarct volume in mice under the middle cerebral artery occlusion (tMCAO) model (fig. 5). The medicinal composition containing the phloroglucinol (Medioresinol) can become a novel medicament for preventing and/or treating brain injury and treating various cerebrovascular diseases including ischemic stroke.

Drawings

FIG. 1 shows the release of cerebral vascular endothelial cell (bEND3) lactate dehydrogenase LDH by sugar oxygen deprivation (OGD) by dermolide (Medioresinol), wherein^***P<0.001vs Control(CN)；^##P<0.01, ^###P<0.001vs DMSO,(Graphpad 7.0,one-way ANOVA)；

FIG. 2 is a graph showing the effect of cortisol (Medioresinol) on the expression of IL-1 β, an inflammatory factor of cerebrovascular endothelial cells (bEND3) induced by sugar oxygen deprivation (OGD), in which^***P<0.001vs Control(CN)；^##P <0.01,^###P<0.001vs DMSO,(Graphpad 7.0,one-way ANOVA)；

FIG. 3 is a graph of the effect of dermolide (Medioresinol) on the leakage of the blood brain barrier FITC-dextran established by co-culture of brain microvascular endothelial cells and astrocytes induced by sugar oxygen deprivation (OGD); wherein^*P <0.05,^**P<0.01vs OGD,(Graphpad 7.0,one-way ANOVA)；

FIG. 4 is a graph of the effect of dermolinol (Medioresinol) on the TEER resistance of the blood brain barrier established by co-culture of brain microvascular endothelial cells and astrocytes following sugar oxygen deprivation (OGD); wherein^*P<0.05, ^**P<0.01vs OGD,(Graphpad 7.0,one-way ANOVA)；

FIG. 5 is a graph of the effect of cortisol (Medioresinol) on the infarct volume in a transient middle cerebral artery occlusion (tMCAO) model mouse; wherein^***P<0.001vs Sham,^###P<0.001vs Vehicle, (Graphpad 7.0,one-way ANOVA)。

Detailed Description

The present invention will be described in further detail with reference to the attached drawings, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.

Drugs and reagents: examples the skin resinols (Medioresinol) used were purchased from Yunan West Living Biotechnology Ltd (CAS number: 40957-99-1, trade name BBP01919), and other reagents were commercially available analytical grade reagents.

The mouse brain microvascular endothelial cell line bEND3 was purchased from a cell bank of the culture Collection of the national academy of sciences; extracting primary astrocytes from the brains of mice born for 1-2 days; extracting primary brain microvascular endothelial cells from the brain of a rat born for 21 days; type I collagen was purchased from Corning, USA; male C57/BL6 mice were purchased from the Qinglongshan animal breeding farm in Jiangning district, Nanjing, license number: SYXK (threo) 2018-.

Example 1

Release of lactate dehydrogenase LDH and expression assay of inflammatory factor IL-1 β in cells:

the method comprises the steps of pretreating brain vascular endothelial cells (bEND3) with sebacinol (20 mu M), removing a culture medium after 12h, replacing a sugar-free culture medium, placing the culture medium in an anoxic culture box for continuous culture for 6h, collecting cell supernatant, detecting Lactate Dehydrogenase (LDH) released by the vascular endothelial cells (Shanghai Biyunshi biotechnology, Ltd.) by using a commercial lactate dehydrogenase detection kit, collecting adherent cells, and detecting mRNA expression of an inflammatory factor IL-1 beta in the vascular endothelial cells by RT-PCR.

(1) Establishment of an in vitro oxygen sugar deprivation (OGD) endothelial cell injury model:

the mouse brain microvascular endothelial cell line bEND3 was cultured at 1X 10⁵The density of each cell/ml is inoculated on a 24-hole plate which is pre-coated with type I collagen, after 24 hours of stabilization, the culture medium in the hole is replaced by fresh DMEM culture medium containing dermolide (the final concentration is 20 mu M), after 12 hours of action, the culture medium is discarded, and the endothelial cells are gently washed for three times by using sterile PBS. Adding DMEM sugar-free medium containing dermolide (final concentration of 20 μ M), placing in a three-gas incubator, introducing nitrogen and carbon dioxide gas, and allowing the gas in the incubator to balance to 1% O₂、94％N₂、5％CO₂The timing is started. After 6 hours of sugar-deficient and oxygen-deficient culture, the cell culture plates were removed and the DMEM medium in each well was transferred to a new 1.5ml centrifuge tube for measurement of LDH release.

(2) Determination of LDH Release amount

The release amount of LDH is detected by adopting an LDH detection kit produced by Shanghai Biyuntian biotechnology limited company. OGD damage can cause a large amount of apoptosis or necrosis, the structure of a cell membrane is damaged, LDH in cytoplasm is released into a culture medium, and the damage degree of OGD to cells can be quantitatively determined by detecting the activity of LDH in the culture medium. The culture medium in each well after OGD molding is transferred to a new 1.5ml centrifuge tube, stored at 4 ℃ and measured the same day. And (3) diluting the INT (10 x) solution to 1 x by using INT diluent, thus obtaining the INT working solution. And (3) mixing the INT working solution, the lactic acid solution and the enzyme solution in equal volumes to prepare the LDH detection working solution. Adding a sample to be detected into a transparent 96-well plate, wherein each well is 100 mu l, then respectively adding 60 mu l of LDH detection working solution into each well, fully and uniformly mixing, and placing on a horizontal shaking table to incubate for 15 minutes at room temperature in a dark place. Measuring the absorbance of each hole at 490nm by using a multifunctional microplate reader; the results of LDH release are shown in table 1 and figure 1.

(3) Real time PCR (RT-PCR) detection of expression of inflammatory factor IL-1 beta

RT-PCR method is adopted to measure mouse brain microvascular endothelial cell line bEND3 cell inflammatory factor caused by OGD by using cortisolInhibition of IL-1 β elevation. The mouse brain microvascular endothelial cell line bEND3 was cultured at 1X 10⁵The density of each cell/ml is inoculated on a 24-hole plate which is pre-coated with type I collagen, after 24 hours of stabilization, the culture medium in the hole is replaced by fresh DMEM culture medium containing dermolide (the final concentration is 20 mu M), after 12 hours of action, the culture medium is discarded, and the endothelial cells are gently washed for three times by using sterile PBS. Adding DMEM sugar-free medium containing dermolide (final concentration of 20 μ M), placing in a three-gas incubator, introducing nitrogen and carbon dioxide gas, and allowing the gas in the incubator to balance to 1% O₂、94％N₂、5％ CO₂The timing is started. After 6 hours of sugar-deficient anaerobic culture, the cell culture plate was removed, the cells were lysed sufficiently with Trizol (Nanjing Nodezam Biotech Co., Ltd.), and the samples were placed in a 1.5mL centrifuge tube without ribozyme and frozen in a freezer at-80 ℃. Melting the sample at room temperature, adding a trichloromethane solution to uniformly mix the sample with the sample, centrifuging, absorbing the upper colorless water phase, and transferring the upper colorless water phase into a new 1.5mL centrifuge tube. Adding equal volume of isopropanol, and centrifuging to obtain RNA precipitate. Then 1mL of 75% ethanol solution is added to wash the precipitate, and finally 20 μ L of ribozyme-free water is added to melt the precipitate to obtain an RNA solution. The concentration and purity of RNA in the sample are measured by using a nucleic acid quantitative analyzer, and the purity of RNA is qualified when 2.2 & gt A260/A280 & gt 1.7. The reverse transcription reaction was performed using an RNA reverse transcription kit, and the total amount of RNA to be reversed was 1. mu.g per sample. After the reversal was completed, real-time fluorescent quantitative PCR was performed to determine the relative amounts of IL-1. beta. and reference primer 18s in the bEND3 cell sample. The primers used were all synthesized by Shanghai Biotech, and the sequences of the primers were as follows:

IL-1β：Forward:5’-TCCAGGATGAGGACCCAAGC-3’

Reverse:5’-TCGTCATCATCCCACGAGTCA-3’

18s rRNA：Forward:5’-CTTTGGTCGCTCGCTCCTC-3’

Reverse:5’-CTGACCGGGTTGGTTTTGAT-3’。

the expression of IL-1. beta. mRNA in the treated bEND3 cells was determined by RT-PCR and the results of IL-1. beta. mRNA expression are shown in Table 2 and FIG. 2.

TABLE 1 Pectinols significantly reduced OGD-induced LDH release from bEND3 cells

TABLE 2 Pectinol significantly reduced the OGD-induced expression of bEND3 cytokine IL-1. beta. mRNA

The results of the experiments in the above cells show that: sugar oxygen deprivation (OGD) significantly promoted the release of lactate dehydrogenase LDH (as shown in Table 1 and FIG. 1) and the expression of inflammatory factor IL-1 beta (Table 2 and FIG. 2) in brain microvascular endothelial cells, and 20. mu.M of cortisol (Medioresinol) was able to significantly inhibit the release of lactate dehydrogenase LDH and the expression of inflammatory factor IL-1 beta.

Example 2

Measurement of blood brain barrier permeability established by co-culture of primary brain microvascular endothelial cells and astrocytes: firstly, establishing a brain microvascular endothelial cell and astrocyte co-culture model. The inside and backside of the 24-well cell culture plates and Millicell culture chambers were coated with rat tail collagen and air dried. Inoculating astrocytes on the back side of the Millicell culture chamber, culturing in an incubator for 6h until the astrocytes are completely adhered to the wall, placing the Millicell culture chamber in a 24-hole cell culture plate, inoculating brain microvascular endothelial cells on the inner side of the Millicell culture chamber, and culturing by adopting a brain microvascular endothelial cell growth culture medium. After model success, cells were treated with dermolide (20 μ M) for 12h followed by OGD for 6h, and the effect of dermolide on blood brain barrier permeability was examined by FITC-labeled dextran (40kDa) leakage assay. The specific operation is as follows: 200ml of FITC-labeled dextran with the concentration of 10mg/ml is added into the co-culture chamber, the culture plate is placed into an incubator for continuous culture, and the fluorescence intensity of the FITC-labeled dextran leaking to the outer chamber at different time points (1h,2h,3h, 4h,5h and 6h) is detected by a fluorescence microplate reader and a fluorescence leakage curve is drawn. Meanwhile, at the corresponding time point, the resistance value between the co-culture layers was measured by a transmembrane resistance meter (EVOM2) and recorded to be plotted as a resistance change curve.

(1) In vitro blood brain barrier model establishment

Inverting the Transwell chamber, and subjecting the primary astrocytes to electrophoresis at 1X 10⁵The density of each cell/ml is seeded on the lower surface of the chamber and placed in CO₂Culturing for 4 hours in a cell culture box; turning over the Ttranswell cell after the astrocytes are adhered to the wall, placing the Ttranswell cell in a 24-hole cell culture plate, adding DMEM/F12 culture medium containing 10% FBS into the cell and out of the cell, and placing the cell in CO₂Continuously culturing for 24 hours in the cell culture box; primary brain microvascular endothelial cells were plated at 1 × 10⁵The density of each cell/ml is inoculated on the upper surface of a Transwell chamber pre-coated with type I collagen, ECM culture medium is added in the chamber and outside the chamber, and the chamber is placed in CO₂Continuously culturing in a cell culture box; then measuring the transmembrane resistance of the blood brain barrier co-culture system every day, wherein the transmembrane resistance tends to be stable after about 6 days, and the method can be used for subsequent experimental operation.

(2) Oxygen-glucose depletion (OGD) damage model establishment

After an in vitro blood brain barrier model is established by co-culturing primary brain microvascular endothelial cells and astrocytes, the culture medium in and out of a Transwell cell is replaced by a fresh ECM culture medium containing dermolinol (the final concentration is 20 mu M). After 12 hours of action, the medium was discarded and the endothelial cells were gently washed three times with sterile PBS. Adding DMEM sugar-free medium containing dermolide (final concentration of 20 μ M), placing in a three-gas incubator, introducing nitrogen and carbon dioxide gas, and allowing the gas in the incubator to balance to 1% O₂、94％N₂、5％CO₂The timing is started.

(3) TEER assay

Before the OGD molding is started, at the beginning of the reoxygenation stage and at the end of the reoxygenation stage, the internal and external transmembrane resistance values of the Transwell cell of each hole are measured by using a cell resistance meter respectively, the resistance values of three different positions of each hole are measured and averaged, and the internal and external transmembrane resistance values of the cell at the moment are obtained. The TEER result is obtained by multiplying the measured transmembrane resistance value by the bottom area of the cell,in omega cm²The results are shown in FIG. 4.

(4) FITC-dextran leakage assay

At the end of the reoxygenation phase, ECM medium containing FITC-dextran dye (final concentration 1mg/ml) was added to the chamber, CO was added₂And continuing culturing in the cell culture box. After 1, 2, 3 and 6 hours, 30. mu.l of the out-chamber medium was removed and transferred to a 96-well white plate to read the fluorescence intensity values (excitation wavelength of 495nm and emission wavelength of 520 nm). FITC-dextran dye was dissolved in ECM medium to prepare FITC-dextran standards at final concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0. mu.g/ml, which were transferred to a 96-well white plate to read the fluorescence intensity values. Subtracting the fluorescence value of the blank hole from the fluorescence values of the sample hole and the standard hole, making a standard curve by using the corrected fluorescence value of the standard hole and the corresponding FITC-dextran concentration, substituting the corrected fluorescence value of the sample hole into the standard curve to obtain the FITC-dextran concentration in the sample, wherein the result is shown in figure 3.

As shown in fig. 3 and 4, 20 μ M of cortisol was able to significantly inhibit leakage of FITC-labeled dextran and decrease in transendothelial resistance caused by sugar oxygen deprivation (OGD) in an in vitro blood brain barrier model established by co-culture of primary brain microvascular endothelial cells and astrocytes.

Example 3

Establishment of a mouse middle cerebral artery occlusion (tMCAO) model and determination of cerebral infarction volume:

(1) establishment of mouse tMCAO model

The mouse tMCAO model is referred to the internal carotid artery embolization method of Longa and the like. The main operation steps are as follows: male C57/BL6 mice weighing about 25-30g are placed in an anesthesia induction box after being weighed, 3% -4% isoflurane is given for induction anesthesia, and the mice are fixed on an operation table in a supine position after being anesthetized. The mice are put on a breathing mask and 1.0-2.0% of isoflurane is given to maintain the anesthesia state. The skin of the neck of the mouse is wiped by a 75% alcohol cotton ball, the skin is cut in the center of the neck, each layer of muscle and soft tissue is separated bluntly by a bent forceps, the common carotid artery on the right side is exposed, and a nylon thread is placed for standby. The soft tissue was bluntly isolated from the common carotid artery to the mouse cephalad, exposing the external and internal carotid arteries, taking care to avoid damaging the vagus nerve. The common carotid artery and the internal carotid artery are clamped by an artery clamp, the external carotid artery is ligated, and the external carotid artery is scalded and cut by an electric coagulation pen. The method comprises the steps of cutting a beveled small opening at the free stub of the external carotid artery, inserting a silica gel line plug into the external carotid artery, then drawing the stub of the external carotid artery and the internal carotid artery into a straight line, smoothly pushing the line plug into the internal carotid artery, loosening an internal carotid artery clamp, continuously pushing the line plug to the intracranial along the internal carotid artery, waiting to feel slight resistance, and determining that the molding is successful when a laser Doppler blood flow meter detects that the right cerebral blood flow drops to 25% of the front of the molding and below the molding. The internal carotid artery was ligated to fix the tap and prevent bleeding, the muscle and skin were sutured, and the mice were placed in a 37 ℃ incubator to maintain body temperature. After 45 minutes of ischemia, the mice are anesthetized by isoflurane, the thread plug is slowly pulled out, the stump of the external carotid artery is ligated, the right cerebral blood flow is re-perfused, and the laser Doppler blood flow meter detects that the right cerebral blood flow is increased to 75% or more before molding and determines that the re-perfusion is successful. The mice in the sham operation group are operated with the same model, but the cord plug should be pulled out immediately after insertion.

(2) Dosing regimens

Weighing and dissolving 2.5mg of the resin alcohol in 0.1mL of DMSO, adding 0.1mL of 15-hydroxystearic acid polyethylene glycol ester (Kolliphor HS15) for cosolvent, finally adding 0.8mL of physiological saline to prepare an injectable solution, calculating the administration volume of each mouse according to the body weight, and carrying out tail vein injection 2h after ischemia, wherein the administration concentration is 10 mg/kg. The corresponding medicine and solvent are injected into tail vein again 24h after operation. The sham operation group and the model group were injected with the same volume of solvent in tail vein for 2h and 24h after operation, respectively.

(3) Cerebral infarction volume determination

The cerebral infarction volume after tMCAO in mice was determined by TTC staining. 48h after ischemia, after neuroethology testing was completed, the mice were euthanized quickly by removing the cervical vertebrae, brain tissue was removed, and immediately placed in an ultra-low temperature freezer at-80 ℃. After freezing for 10 minutes, the brain tissue was removed and coronal sections were made with a razor blade, and 5 slices were cut per brain. The cut brain pieces were gently placed in 2% TTC stain, protected from light, and incubated at 37 ℃ for 10 minutes. And lightly taking out brain slices from the dye liquor, and taking a picture for storage after the brain slices are arranged in order. The normal tissue of the brain slice stained by TTC is red, and the infarcted tissue is white. Infarct volume and total brain volume of brain sections were calculated using Image J software. The percent infarct volume by volume is the percent of the total volume of the brain, and the results are shown in table 3 and figure 5.

Table 3 dermolide reduces cerebral infarct volume following tMCAO injury in rats

Experimental results in animal models showed that 10mg/kg of dermoresinol (Medioresinol) significantly reduced the cerebral infarct volume in the middle cerebral artery occlusion (tMCAO) model mice (table 3 and fig. 5).

In the above embodiment, the invention evaluates the effects of protecting the integrity of the blood brain barrier and resisting ischemic stroke of the cortisol from the cellular level and the animal level respectively, and the experimental results show that: the damage of brain microvascular endothelial cells caused by oxygen sugar deprivation (OGD) is remarkably improved by the corticosterol (Medioresinol); in an in-vitro blood brain barrier model established by co-culture of primary brain microvascular endothelial cells and astrocytes, the cortisol can also remarkably inhibit leakage of FITC-labeled glucan and reduction of transendothelial resistance caused by oxygen sugar deprivation (OGD), and shows that the cortisol has a certain blood brain barrier protection effect; in animal models, 10mg/kg of dermoresinol (Medioresinol) significantly reduced the cerebral infarction volume of middle cerebral artery occlusion (tMCAO) model mice, which further indicates that dermoresinol has the potential of preventing or treating diseases related to cerebrovascular endothelial injury, including ischemic stroke, hemorrhagic stroke, high altitude cerebral edema and other cerebrovascular diseases.

Claims

1. Application of cortisol in preparing medicine for preventing or treating cerebral injury caused by ischemic stroke is provided.

2. Use according to claim 1, wherein the corticosterol has the formula:

。

3. the use of claim 1, wherein said cortisol is used for the manufacture of a medicament for the treatment of brain damage due to ischemic stroke by significantly reducing the release of pro-inflammatory cytokines and brain microvascular endothelial cell damage due to glucose deprivation.

4. Use of the cortisol according to claim 1 in the manufacture of a medicament for the treatment of brain damage caused by ischemic stroke by significantly reducing the permeability of the blood brain barrier under conditions of glucose deprivation.

5. The use according to claim 1, wherein said cortisol is used for the manufacture of a medicament for the treatment of brain damage caused by ischemic stroke by significantly reducing the volume of cerebral infarcts in a middle cerebral artery occlusion model.