CN112516160A - Pharmaceutical composition and medical application thereof - Google Patents
Pharmaceutical composition and medical application thereof Download PDFInfo
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- CN112516160A CN112516160A CN202011241775.XA CN202011241775A CN112516160A CN 112516160 A CN112516160 A CN 112516160A CN 202011241775 A CN202011241775 A CN 202011241775A CN 112516160 A CN112516160 A CN 112516160A
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Abstract
The application discloses a medical composition and medical application thereof, wherein the medical composition comprises an effective dose of a lithium agent and a pharmaceutically acceptable carrier. Through the mode, the application can play an effective medicine role in treating ischemic stroke and blood brain barrier injury caused by the ischemic stroke.
Description
Technical Field
The application relates to the technical field of medicines, in particular to a medicinal composition and a medicinal application thereof.
Background
Ischemic stroke (also known as cerebral infarction) is one of the most lethal and disabling diseases in the human disease spectrum, and the best treatments for it are revascularization therapies including alteplase (rtPA) and surgical embolectomy, all of which have a strict time window limit, resulting in a significant proportion of patients not receiving such treatment. Among them, the time window for revascularization therapy is set mainly to avoid or reduce blood-brain barrier (BBB) damage caused by reperfusion. If the BBB is effectively preserved, the time window for revascularization therapy may be extended, thereby facilitating more people to receive revascularization therapy. However, no effective BBB protective medicine exists in clinic at present.
Among them, the main existing treatment methods for the ultra-early stage after acute ischemic stroke are alteplase rtPA venous thrombolysis and intra-arterial surgical embolectomy, which have strict time window limitations, wherein the former is 4.5 hours, and the latter is 6-8 hours (the time can be prolonged to 24 hours for a few patients). Clinical studies show that severe complications such as hemorrhage transformation and cerebral edema can occur when the time window is exceeded for vascular opening treatment, so that a considerable proportion of patients with acute cerebral infarction can not receive vascular opening treatment because the time window of vascular opening treatment is exceeded when the patients arrive at a hospital due to main and objective factors such as lack of timely medical attendance, long distance from the hospital, traffic jam on the road and the like. Also, studies have shown that rtPA itself is detrimental to the BBB, and that a significant proportion of patients, even when used within a time window, develop BBB-damaging phenotypes such as hemorrhagic transformation, cerebral edema, and the like.
Currently, no targeted BBB protective drug exists clinically. When cerebral edema or hemorrhage transformation occurs, for example, cerebral hernia is induced, dehydration treatment is performed on mild patients with 20% mannitol, hypertonic salt, diuretic and the like, and bone flap removing decompression is performed on severe patients; if the cerebral hernia is not induced, patients are generally subjected to supportive treatment such as sedation, pressure reduction, correction of water-electricity-acid balance disorder and the like.
Disclosure of Invention
The technical problem mainly solved by the application is to provide a pharmaceutical composition and medical application thereof, and the pharmaceutical composition can play an effective pharmaceutical role in treating ischemic stroke and blood brain barrier injury caused by the ischemic stroke.
In order to solve the technical problem, the application adopts a technical scheme that: provided is a pharmaceutical composition for treating ischemic stroke, comprising: the lithium preparation is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
Wherein the pharmaceutical composition is in dosage unit form.
Wherein the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmole/kg, wherein mmole/kg is the amount of the substance of the lithium agent relative to the mass of the patient.
Wherein the dose of the lithium agent in the pharmaceutical composition is 3.0 mmole/kg.
Wherein the pharmaceutical composition further comprises an effective amount of alteplase for treating ischemic stroke, and the amount of alteplase in the pharmaceutical composition is 0.9 mg/kg, wherein mg/kg is the mass of alteplase relative to the mass of the patient.
In order to solve the above technical problem, another technical solution adopted by the present application is: provides the application of a lithium agent in the preparation of a medicine for treating cerebral arterial thrombosis, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebral vascular endothelial cell and protecting the structure of a tight junction protein of the endothelial cell of a blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
In order to solve the above technical problem, the present application adopts another technical solution: there is provided a pharmaceutical composition for treating blood-brain barrier injury, the pharmaceutical composition comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of the blood brain barrier.
In order to solve the above technical problem, the present application adopts another technical solution: provides the application of a lithium agent in the preparation of a medicament for treating blood brain barrier injury, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting the structure of a tight junction protein of the endothelial cell of the blood brain barrier.
In order to solve the above technical problem, the present application adopts another technical solution: there is provided a pharmaceutical composition for treating a blood brain disease, the pharmaceutical composition comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
In order to solve the above technical problem, the present application adopts another technical solution: provides the application of a lithium agent in the preparation of a medicament for treating blood brain diseases, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of cerebrovascular endothelial cells and protecting the structure of tight junction protein of the endothelial cells of a blood brain barrier.
The beneficial effect of this application is: different from the situation of the prior art, the lithium agent in the pharmaceutical composition plays an important role in up-regulating the Wnt/beta-catenin signal pathway of the brain vascular endothelial cells and protecting the structure of the tight junction protein of the endothelial cells of the blood brain barrier, so that the blood brain barrier can be protected. Therefore, the lithium agent can play an effective pharmaceutical role in treating ischemic stroke and blood brain barrier injury caused by the ischemic stroke.
Drawings
FIG. 1 is a comparative illustration of an experiment in which coronary sections of a mouse brain were stained with TTC;
FIG. 2 is a graph showing the relative proportion of infarcted volume in the brain hemisphere of a mouse;
FIG. 3 is a schematic representation of the relative proportions of the Evans blue staining;
FIG. 4 is a graphical representation of the relative percentage of evans exudation by hemisphere weight;
FIG. 5 is a schematic view of immunofluorescence double staining for observing the exudation of endogenous IgG from blood vessels;
FIG. 6 is a bar graph of the relative density of extravasation of IgG from the blood vessel;
FIG. 7 is a bar graph of neurobehavioral scores;
FIG. 8 is a schematic diagram of immunofluorescence double staining for neuronal apoptosis;
FIG. 9 is a histogram of the number of apoptotic neurons in the infarct zone versus the density of neurons in the mouse;
FIG. 10 is a schematic diagram of immunofluorescence double staining for observing the expression of active beta-catenin on endothelial cells;
FIG. 11 is a bar graph showing the relative density of active β -catenin expressed on endothelial cells;
FIG. 12 is a schematic representation of the active and total β -catenin protein levels in the infarct zone brain tissue of each group;
FIG. 13 is a bar graph of relative density of the immunoblots of FIG. 12;
FIG. 14 is a further bar graph showing the relative density of the immunoblots of FIG. 12;
FIG. 15 is a schematic horizontal bar graph of axin2 in groups of cerebral infarcted tissues;
FIG. 16 is a bar graph showing the levels of apcdd1 mRNA in various groups of cerebral infarcted tissues;
FIG. 17 is a graph showing the effect of different concentrations of lithium chloride on the level of bEnd.3Axin2 mRNA;
FIG. 18 is a graph showing the effect of different concentrations of lithium chloride on the bEnd.3TCF/LEF TOPflash values;
FIG. 19 is a schematic diagram showing the effect of lithium chloride on a Wnt/beta-catenin pathway-deficient bEnd.3 cell line constructed by knocking out Fzd 4;
FIG. 20 is a schematic diagram showing a further effect of lithium chloride on Wnt/β -catenin pathway deficient bEnd.3 cell lines constructed by knockout of Fzd 4;
FIG. 21 is a bar graph of axin2 mRNA levels for each group.
Detailed Description
The present application will be described in detail below with reference to the drawings and embodiments.
The application provides a pharmaceutical composition for treating ischemic stroke, which comprises the following components: an effective dose of lithium for treating ischemic stroke and a pharmaceutically acceptable carrier.
Ischemic stroke is a general term for brain tissue necrosis caused by stenosis or occlusion of blood supply arteries (carotid and vertebral) of the brain and insufficient blood supply to the brain. There are four types of cerebral ischemia: transient Ischemic Attack (TIA); reversible neurological dysfunction (RIND); progressive Stroke (SIE); complete Stroke (CS). TIA did not have cerebral infarction, while RIND, SIE and CS had different degrees of cerebral infarction.
The influence range of ischemia can be divided into local cerebral ischemia and diffuse cerebral ischemia. The causes of localized cerebral ischemia are: middle cerebral artery embolism; intracranial or intracranial carotid stenosis, occlusion, or thrombosis; cerebral artery spasm. The causes of diffuse cerebral ischemia are: sudden cardiac arrest, hypotension, anemia, hypoglycemia, etc.
Among them, the best existing treatment for ischemic stroke (also known as cerebral infarction) is revascularization therapy including alteplase (rtPA) and surgical embolectomy, which all have strict time window limitations, resulting in a significant proportion of patients not receiving such treatment. The time window for revascularization therapy is set mainly to avoid or reduce blood brain barrier damage caused by reperfusion, while the alteplase rtPA itself has damage to the blood brain barrier, and even if used within the time window, part of patients still have BBB damage phenotype such as hemorrhagic transformation, cerebral edema, and the like. However, if a BBB protectant is used in combination with rtPA to reduce BBB damage, the effectiveness of the revascularization therapy will be improved, and the time window for revascularization therapy may be extended and more people will receive revascularization therapy.
The main use of lithium agents in current clinical or basic research is the prophylactic and therapeutic effect on bipolar affective disorders with alternating episodes of mania and depression. The inventor of the application finds that when the lithium agent is used for the ischemic stroke of a mouse, the Wnt/beta-catenin pathway of the cerebral vascular endothelial cell of the mouse can be up-regulated, and the BBB damage in the acute stage of the cerebral infarction of the mouse also has an obvious protective effect, so that the indication of the lithium agent is increased potentially.
In addition, although the targeting Wnt/beta-catenin pathway agonist is available at present, the agonist is still in the experimental stage. As the lithium agent is a clinical commonly used psychiatric drug, the lithium agent is expected to be directly used in clinical tests and applications.
In contrast, the inventors of the present application found in experimental studies that lithium agents have a dual protective effect on BBB function and nerve cells. And the lithium agent can improve the functional integrity of the cerebrovascular endothelial cells by up-regulating the Wnt/beta-catenin pathway of the cerebrovascular endothelial cells, and can protect the structure of the tight junction protein of the endothelial cells of the blood brain barrier, thereby realizing the protection effect on the BBB.
Wherein the lithium agent is lithium chloride (LiCl) and/or lithium carbonate (Li)2CO3). In other embodiments, the lithium agent may be any one or more of lithium compounds such as lithium hydride, lithium nitride, and lithium hydroxide, which are not limited in this application.
Wherein the pharmaceutical composition is in dosage unit form.
Wherein the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 millimoles per kilogram, the quantity of the lithium agent relative to the mass of the patient. In other embodiments, when the pharmaceutical composition is used on a specific human body, the dosage of the lithium agent in the pharmaceutical composition may also comprehensively consider the variation of the toxic and side effects of the drug or the blood concentration and other factors, and any other reasonable dosage is adopted, which is not limited by the comparison in the present application.
Wherein the dose of the lithium agent in the pharmaceutical composition is 3.0 mmole/kg. Wherein the pharmaceutical composition further comprises an effective amount of alteplase for treating ischemic stroke, and the amount of alteplase in the pharmaceutical composition is 0.9 mg/kg, wherein mg/kg is the mass of alteplase relative to the mass of the patient.
The application also provides an application of the lithium agent in preparation of a medicine for treating cerebral arterial thrombosis, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebral vascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
The present application also provides a pharmaceutical composition for treating blood-brain barrier injury, comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of the blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
Wherein the pharmaceutical composition is in dosage unit form.
Wherein the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmole/kg, which is the mass of the lithium agent relative to the patient.
Wherein the dose of the lithium agent in the pharmaceutical composition is 3.0 mmole/kg.
The application also provides an application of the lithium agent in preparation of a medicine for treating blood brain barrier injury, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of the blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
The present application also provides a pharmaceutical composition for treating a blood brain disease, the pharmaceutical composition comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
Wherein the pharmaceutical composition is in dosage unit form.
Wherein the dosage of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmole/kg, which is the mass of the lithium agent relative to the patient.
Wherein the dose of the lithium agent in the pharmaceutical composition is 3.0 mmole/kg.
The application also provides an application of the lithium agent in the preparation of a medicament for treating the blood brain diseases, wherein the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
Wherein the lithium agent is lithium chloride and/or lithium carbonate.
The lithium agents described herein may constitute the active ingredient of a pharmaceutical composition and may generally be administered in the form of oral tablets, intravenous injections or capsules, mixed with a suitable excipient or carrier selected appropriately. Dosage compositions such as tablets, capsules, pills, suppositories, and powders depend on the intended mode of administration, which may be by any acceptable route. These routes of administration include oral, intravenous (intravenous), intramuscular (intramuscular), subcutaneous (subcutaneous). One or more of these routes may be used in one patient. In one embodiment, the compounds of the present application are used as an intravenous dosage form and may be combined with a non-toxic pharmaceutically acceptable inactive carrier, such as water, glycerol, ethanol, physiological saline, and the like. Inert excipients which are commonly used as binders, disintegrants and colouring agents may also be added to the oral mixture.
If desired, the pharmaceutical compositions administered may also contain minor amounts of non-toxic substances such as pH buffers, emulsifiers, sodium acetate and the like. The dosage regimen for the compounds to be used will depend upon the species, sex, body weight, age, medical condition, route of administration and the severity of the condition being treated. The skilled practitioner can readily determine and prescribe an effective amount of the drug to treat the disease.
The term "treatment" as used herein includes one or more of curative, palliative and prophylactic treatment, depending on the disease and condition of the patient. The precise dose administered for each active compound will vary depending on a number of factors including, but not limited to, the type of patient and the type of disease state being treated, the age of the patient, and the route of administration.
For the above therapeutic uses, the dosage administered will, of course, vary with the mode of administration, the treatment desired, and the disease condition indicated. The total daily dose may be administered in a single dose or in divided doses. Sustained release compositions are also contemplated by the present application.
For example, the pharmaceutical composition may be in a form suitable for oral administration, such as one of a tablet, capsule, pill, powder, sustained release formulation, and solution; alternatively, the pharmaceutical composition may be in a form suitable for intravenous injection, such as an infusion bottle or bag. The pharmaceutical compositions may be in unit dosage form suitable for single administration of precise dosages. The pharmaceutical compositions will include conventional pharmaceutical carriers and active compounds. In addition, it may include other pharmaceutical or medical agents, carriers, adjuvants, and the like.
Suitable pharmaceutical carriers include inert diluents or fillers, water. If desired, the pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, and the like. Thus for oral administration, tablets containing various excipients, such as citric acid, may be employed along with various disintegrants (e.g., starch, alginic acid and certain complex silicates) and binding agents (e.g., sucrose, gelatin and acacia). Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often used to prepare tablets. Solid compositions of a similar type may also be used in soft and hard-filled gelatin capsules. Useful components of these compositions include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compound therein may be combined with various sweetening or flavoring agents, coloring matter or dyes, and optionally emulsifying or suspending agents and diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. For intravenous administration, it may be used together with distilled water or physiological saline.
Methods of preparing various pharmaceutical compositions having a particular amount of an active compound are well known or will be apparent to those skilled in the art.
The dosage ranges set forth herein are exemplary only, and are not intended to limit the scope or practice of the claimed compositions. For example, the dosage may be adjusted according to pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Accordingly, this application encompasses dose escalation in patients as determined by one of skill in the art. Determining an appropriate dose and regimen for administering a chemotherapeutic agent is well known in the relevant art and, once the teachings disclosed herein are provided, should be understood to be encompassed by those skilled in the art.
The pharmaceutical compositions of the present application may be prepared, packaged or sold in bulk form, in single unit dosage form, or in multiple unit dosage forms. As used herein, a "unit dose" is an individual amount of a pharmaceutical composition comprising a predetermined amount of active compound. The amount of active compound is typically equal to the dose of active compound to be administered to the subject, or a convenient fraction of such a dose, such as, for example, one-half or one-third of such a dose.
The relative amounts of the active compound, pharmaceutically acceptable carrier, and any other ingredients in the pharmaceutical compositions of the present application will vary depending upon the identity, size, and condition of the subject being treated, and further depending upon the route of administration of the composition. For example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
In addition to the active compound, the pharmaceutical compositions of the present application may further comprise one or more additional therapeutically effective compounds as discussed above.
The following detailed data illustrate that lithium agents provide effective pharmacological effects in the treatment of ischemic stroke. Among them, the lithium agent is exemplified by lithium chloride.
The model adopted by the application is a cerebral ischemia reperfusion model with mouse middle cerebral artery embolism (MCAO) for 1 hour (h) and blood vessel recanalization for 48 hours, and the intervention effect of the ultra-early use of the lithium-dose cerebral infarction is observed. The method comprises the following specific steps:
1. animal protocols and lithium agent treatments
C57BL/6 male mice, 8 to 10 weeks old and weighing 20 to 23g, were first obtained. These mice were kept in a pathogen-free animal facility for 12 hours of light and dark cycles. Mice were randomly selected for sham surgery, ischemic models or post-ischemic treatment studies. When it is subjected to surgery, it is first subjected to induction anesthesia in an induction chamber with 4% isoflurane and maintained anesthesia by mask delivery of 2% isoflurane. Throughout the procedure, the core temperature of each mouse was maintained at 37 ± 0.5 ℃ using a heating pad and the mice were treated for induced, transient Middle Cerebral Artery Occlusion (MCAO) by a modified endoluminal fibrous model. After MCAO lasted 60 minutes, reperfusion was established by retracting the fibers.
The mice had free access to food and water throughout the reperfusion period. After MCAO lasted 48 hours, the mice were evaluated for neurological function by the observer, e.g., for deficit score (20), grip test score (21), and horizontal ladder test score (22). Mice with no neurological deficit after surgery were excluded according to a pre-established exclusion plan.
Immediately after MCAO reperfusion, mice were injected intraperitoneally (ip) with lithium chloride (LiCl) at a concentration of 2%, intraperitoneal (ip) reperfusion was initiated and then re-injected at the same dose after 24 hours interval.
2. Infarct size analysis
After 48 hours of reperfusion, mice were deeply anesthetized with isoflurane. The entire brain of the mouse was removed and coronal sections (thickness 2mm) were made in a special recess. Sections were immediately immersed in 1ml of 1% 2,3, 5-triphenyltetrazolium chloride (TTC) and incubated at 37 ℃ for 10 minutes. Then, the TTC solution was replaced with 4% paraformaldehyde at room temperature for 1 hour. The sections were photographed with a digital camera and infarct size was measured by ImageJ software (java based public image processing software). To eliminate the effect of post-ischemic edema on lesion volume, infarct size was corrected as previously described (24). Wherein the infarct area (%) is calculated as [ ((volume of left hemisphere-non-infarct volume of right hemisphere)/volume of left hemisphere) 100%.
3. Evans blue leak measurement
Evans blue (a biological stain of the formula 6,6'- [ [3,3' -dimethyl (1,1 '-diphenyl) -4,4' -diyl ] bis (azo) ] bis (4-amino-5-hydroxy-1, 3-naphthalenedisulfonic acid) tetrasodium salt) was used for perfusion, and after administration of 100 μ l 2% Evans blue by retroorbital intravenous (i.v.) injection for 18-20h, mice were deeply anesthetized with isoflurane, and then perfused heart-beating heart with 20ml cold saline via left ventricle to remove intravascular dye. The brains of the mice were removed and photographed with a digital camera. The brain was then divided into ipsilateral and contralateral non-ischemic hemispheres and homogenized in 1ml of 50% trichloroacetic acid and centrifuged (10,000rpm, 20 minutes), and after diluting the above solution four times with ethanol, the concentration of evans was measured with a fluorescence reader (620nm excitation; 680nm emission) and expressed as μ g/g brain tissue.
4. Immunofluorescence and TUNEL detection staining
Frozen 7 μm thick sections were dried on adhesive microscope slides at room temperature and then rehydrated in PBS (phosphate buffered saline). Sections were blocked in 10% normal goat serum in PBS + 0.2% Triton X-100 for 1 hour at room temperature. Samples were incubated with the following primary antibodies in PBS + 5% goat serum + 0.2% Triton X-100 at 4 deg.C: hamster anti-mouse CD31 (platelet-endothelial cell adhesion molecule), donkey anti-mouse IgG (immunoglobulin G), rabbit anti-claudin-5 (for Claudin-5), rabbit anti-ZO-1 (zonulin 1), rabbit anti-collagen IV (intravenous), rabbit anti-lamin (laminin), rabbit anti-albumin rabbit anti-active beta-catenin. Excess antibody was removed by washing in PBS for 5 minutes (3 times). The samples were then incubated with the following secondary fluorescently labeled antibodies for 1 hour at room temperature: cy3 (macroscopic red light after excitation) goat anti-hamster IgG and Alexa Fluor (fluorescent dye) 488 donkey anti-rabbit IgG were diluted 1: 500% goat serum + 0.2% Triton X-100 was left at room temperature for 1 hour.
Among them, terminal deoxynucleotidyl transferase mediated staining for terminal labeling (TUNEL) of dUTP (deoxyuridine triphosphate) -digoxigenin was performed using TUNEL FITC (apoptosis detection kit). For double immunofluorescent staining, TUNEL sections were washed in PBS and incubated overnight at 4 ℃ with rabbit anti-NeuN (nuclear antigen) followed by 1h anti-rabbit IgG at room temperature with Alexa Fluor 647 donkey. Excess antibody was removed by washing in PBS for 5 minutes (3 times). Slides were fixed with DAPI (4', 6-diamidino-2-phenylindole) in a fade-resistant fixing medium and imaged with a microscope to obtain 10, 20 or 40 images. Immunofluorescence signal area or density was quantified by ImageJ (java based public image processing software) and normalized by blood vessel area (CD31 signal area) in 5 to 8 random ischemic areas per mouse.
5. Western blot analysis
Proteins were extracted from the ischemic side (about 3.30 mm) of 2mm thick coronal sections to the forebrain and analyzed by Western (Western blot) blotting. Brain tissue was collected 48h after occlusion. Brain tissue proteins were isolated and collected by RIPA (soluble protein extracted from animal tissues and animal cells) lysis buffer supplemented with protease and phosphatase inhibitors and quantified by BCA (BCA protein concentration detection) assay according to standard protocols. Equal amounts of protein lysate of each sample were separated on a 10% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Proteins were transferred to polyvinylidene fluoride membranes, which were then incubated with primary antibodies including rabbit anti-claudin-5, rabbit anti-ZO-1, rabbit anti-occludin, rabbit anti-MMP-9, rabbit anti- β -catenin, rabbit anti-phospho-JNK Thr183/Tyr185 (rabbit anti-phospho-c-Jun amino terminal kinase-threonine 286/threonine 185), rabbit anti-JNK (c-Jun amino terminal kinase), rabbit anti-phospho-Camk II Thr286 (calmodulin kinase II-threonine 286), rabbit anti-Camk II (calmodulin-dependent protein kinase II), dilution ratio 1: 1,000. Beta-actin was used as a normalized loading control. The membranes were incubated with the corresponding secondary antibodies and blots were developed using a GelView 6000M system (nucleic acid dye-6000M).
6. Real-time quantitative PCR (polymerase chain reaction)
Total RNA was extracted using Direct RNA MiniPrep (Direct-RNA-plasmid extraction kit) kit. Reverse transcription of RNA was performed by RT-qPCR (real-time fluorescent quantitative PCR) using reverse transcription Supermix (object-based macro language). RT-qPCR was performed on a system of real-time fluorescent quantitative PCR instruments using the Power SYBRi Script Green (a powerful premix of dyes with Green excitation wavelength) method. RNA expression was calculated using the comparative Ct (electron computer tomography) method normalized to actin. Data are presented using the 2- Δ Δ Ct method with respect to the calibrator.
7. Cell culture, oxygen glucose deprivation and recovery (OGD/R) and lithium treatment
Mouse brain-derived endothelial cells 3 were cultured in Dulbecco (Dulbecco's phosphate buffered saline) modified eagle's medium (DMEM, a medium containing various amino acids and glucose) containing 10% fetal bovine serum and 1% penicillin. All cultures were maintained in a CO2 incubator at 5% humidity, 37 ℃ and passaged routinely when 80-90% confluent. 15 hours after LiCl or vehicle treatment, cells were exposed to OGD/R (sugar oxygen deprived ex vivo cerebral ischemia model). Then, the culture broth was replaced with Dulbecco modified medium with LiCl or vehicle control, and the cultured cells were placed in a modular incubator containing 0.5-1% O2 and 99% N2 (nitrogen) and monitored with O2 (oxygen) analyzer. After 6 hours of OGD (oxygen sugar deprivation), the cells were returned to normal culture conditions containing LiCl to recover oxygen glucose for 3 hours.
8. Cell count kit 8(CCK-8) test
Cell viability was measured with CCK-8 and cells were seeded at a concentration of 5,000 cells/well in 96-well plates. Ten milliliters of CCK-8 solution was added to each well containing 100 milliliters of media. Cells were incubated at 37 ℃ for 2 hours. OD values (optical density values) were measured at 450nm with a MULTIPLE SCANNER GO.
9. Luciferase assay
For a typical luciferase assay, a stable b end.3 (mouse brain microvascular endothelial cell) cell line bearing TOP-flash firefly luciferase with Wnt/β -catenin signaling [ constructs with TCF/LEF (T-cytokine, lymphokine-enhancing) binding sites ] and a renilla control reporter were seeded in 96-well plates 48 hours later, cells were stimulated with 100ng/ml Wnt3a for 24 hours.
10. Transendothelial resistance (TEER) and permeability measurements
TEER was measured using Millicell-ERS-2 (millipore, germany). bEnd.3 cells were seeded on a Transwell permeable membrane (24-well cell culture insert) with a pore size of 0.4 μm and allowed to grow for 3 days. Baseline TEER was measured prior to LiCl and OGD/R treatment. All measurements were normalized by subtracting TEER values measured in a blank Transwell (invasion test) filter. After OGD/R, the effect of LiCl on the permeability of the endothelial monolayer of fluorescein isothiocyanate conjugated 70kDa FITC-dextran was evaluated. Ten microliters of FITC-dextran (5ug/ml) was added to the chamber and the mixture was incubated for 30 minutes. Permeability was assessed by measuring the fluorescence at 520nm emitted from a 100 μ l aliquot of medium taken from the chamber using a fluorescent plate reader multistkan GO.
11. Statistical analysis
Statistical analysis was performed using the Windows software package SPSS 16.0 (SPSS, usa). Data are presented as mean ± Standard Error (SE). Student's t-test was used for both group comparisons. Statistical differences were determined for all observations from different groups using one-way analysis of variance (ANOVA). If the variance is heterogeneous, the differences are further analyzed using the Least Significant Difference (LSD) T test or Dunnett T3 (New Bipolar test) test. Statistical significance was defined as P < 0.05.
Specifically, please refer to fig. 1 to 21 (in which, the original figures of fig. 1,3, 4, 8, 10, 19 and 20 are color drawings) for experimental results, fig. 1 is a comparison diagram of the experiment after staining the coronal section of the mouse brain with TTC; FIG. 2 is a graph showing the relative proportion of infarcted volume in the brain hemisphere of a mouse; FIG. 3 is a schematic representation of the relative proportions of the Evans blue staining; FIG. 4 is a graphical representation of the relative percentage of evans exudation by hemisphere weight; FIG. 5 is a schematic view of immunofluorescence double staining for observing the exudation of endogenous IgG from blood vessels; FIG. 6 is a bar graph of the relative density of extravasation of IgG from the blood vessel; FIG. 7 is a bar graph of neurobehavioral scores; FIG. 8 is a schematic diagram of immunofluorescence double staining for observing apoptosis of neurons; FIG. 9 is a histogram of the number of apoptotic neurons in the infarct zone versus the density of neurons in the mouse; FIG. 10 is a schematic diagram of immunofluorescence double staining for observing the expression of active beta-catenin on endothelial cells; FIG. 11 is a bar graph showing the relative density of active β -catenin expressed on endothelial cells; FIG. 12 is a schematic representation of the active and total β -catenin protein levels in the infarct zone brain tissue of each group; FIG. 13 is a bar graph of relative density of the immunoblots of FIG. 12; FIG. 14 is a further bar graph showing the relative density of the immunoblots of FIG. 12; FIG. 15 is a schematic horizontal bar graph of axin2 in groups of cerebral infarcted tissues; FIG. 16 is a bar graph showing the levels of apcdd1 mRNA in various groups of cerebral infarcted tissues; FIG. 17 is a graph showing the effect of different concentrations of lithium chloride on the level of bEnd.3Axin2 mRNA; FIG. 18 is a graph showing the effect of different concentrations of lithium chloride on the bEnd.3TCF/LEF TOPflash values; FIG. 19 is a schematic diagram showing the effect of lithium chloride on a Wnt/beta-catenin pathway-deficient bEnd.3 cell line constructed by knocking out Fzd 4; FIG. 20 is a schematic diagram showing a further effect of lithium chloride on Wnt/β -catenin pathway deficient bEnd.3 cell lines constructed by knockout of Fzd 4; FIG. 21 is a bar graph of axin2 mRNA levels for each group.
Wherein FIG. 1 shows the ischemic area after staining a coronal section of a mouse brain with TTC, the white area being the ischemic area; fig. 2 shows the relative proportion (%) of infarcted volume in the brain hemisphere, n-8/group; FIG. 3 shows stained areas of Evans blue (blue areas are regions of Evans blue leakage into brain tissue); FIG. 4 corresponds to the relative percentage (%) of Evensan exudation by hemisphere weight, n 6/group; FIG. 5 shows immunofluorescence double staining to observe the extravasation of endogenous IgG (green) from blood vessels (CD31, red), corresponding to a scale of 100 um; fig. 6 shows a histogram of the relative density of IgG extravasation from the blood vessel, n being 3/group; figure 7 shows a histogram of neurobehavioral scores, n-8/group. Data are mean (mean Data) ± s.e.m., P <0.05, P <0.01. As can be seen from a combination of fig. 1-7, the use of lithium chloride can reduce the volume of cerebral infarction and BBB leakage in mice, and can improve the symptoms of neurological deficit.
Wherein, fig. 8 shows immunofluorescence double staining observation of neuronal (red) apoptosis (TUNEL staining, green), scale 100 um; figure 9 shows a histogram of the number of apoptotic neurons in the infarct zone versus the density, n being 3/group. Data are mean ± s.e.m., P < 0.05. As can be seen from a combination of fig. 8-9, the use of lithium chloride reduced neuronal apoptosis in the infarcted tissue.
Wherein, fig. 10 shows the expression of active β -catenin (green) on endothelial cells (red) observed by immunofluorescence double staining, scale 100 um; FIG. 11 shows a histogram of the relative density of active β -catenin expression on endothelial cells, n being 3/group; FIG. 12 shows the active and total β -catenin protein levels for each set of infarct zone brain tissue; fig. 13 and 14 show the immunoblot relative density histograms in the "C" plot, n-3/panel; fig. 15 and 16 show histograms of axin2 and apcdd1 mRNA levels in each group of cerebral infarcted tissues, n 3/group. Ischemia/reperfusion: I/R, ischemia/reperfusion. n is 8/group. Data are mean ± s.e.m., P <0.05, P <0.01. As can be seen from a combination of fig. 10-16, the use of lithium chloride can reduce the volume of cerebral infarction and BBB leakage in mice, and can improve the symptoms of neurological deficit.
Wherein, figure 17 shows the effect of different concentrations of lithium chloride on levels of bned.3axin 2 mRNA, n 4/group; figure 18 shows the effect of different concentrations of lithium chloride on the values of bned.3tcf/LEF TOPflash, n-5/group; fig. 19 and fig. 20 show that the effect of lithium chloride observed by knocking out Fzd4 to construct a Wnt/β -catenin pathway-deficient bnd.3 cell line, and that lithium chloride can significantly improve the decreased cell resistance value after OGD/R of WT and Fzd 4-/-bnd.3 cell line, and reduce FITC-dextran leakage rate, where n is 3/group; fig. 21 shows histograms of axin2 mRNA levels for each group, n 3/group. Data are mean ± s.e.m., P <0.05, P <0.01, sugar oxygen deprivation/recovery: OGD/R, oxygen-glucose depletion and recovery; normal control: NC, normal control; wild type: WT, wild type; endothelial cell transmembrane resistance: TEER, trans-intrinsic electrical resistance. As can be seen from a combination of fig. 17-21, the use of lithium agents can up-regulate Wnt/β -catenin pathway following sugar oxygen deprivation/recovery (mimicking ischemia reperfusion injury) of mouse brain microvascular endothelial cells (bned.3) and reduce endothelial cell damage.
The experiment shows that: 1) when three doses of 1.5, 3.0 and 6.0mmol/kg (millimole/kg) of lithium chloride are used, the lithium chloride is injected subcutaneously and is administered immediately after the blood vessel is opened, and the reduction of the cerebral infarction volume of a mouse is observed and the reduction of the apoptosis of nerve cells in an infarct area is also observed at 1 time/day; 2) when the protective effect of using lithium chloride with the concentration of 3.0mmol/kg on the BBB function is observed, the BBB function tracer Evans blue (Evans blue) of the infarcted hemisphere and the leakage of mouse serum IgG protein are obviously reduced; 3) through behavioral determination, the lithium agent is found to improve neuro-behavioral impairment in early cerebral infarction of mice.
Molecular biological experiments of mouse brain tissues show that active-beta-catenin which is a marker of a Wnt/beta-catenin pathway is increased in expression on cerebrovascular endothelial cells, and the protein level of the Wnt/beta-catenin pathway in the brain tissues is also remarkably increased after lithium intervention. In addition, the marker gene apcdd1 of the Wnt/beta-catenin pathway in the infarcted brain tissue is also significantly increased after the intervention of lithium agent. In vitro experiments on cerebrovascular endothelial cells show that the lithium agent can significantly up-regulate the Wnt/beta-catenin pathway. Also, lithium agent treatment enhanced both the integrity of the cerebrovascular endothelial cell monolayer and BBB function in an in vitro glucose deprivation/recovery model (mimicking ischemia reperfusion injury in vivo) compared to control cells without lithium agent treatment.
And the lithium agent has the double functions of BBB and nerve cell protection; the BBB protection effect of the lithium agent is realized by up-regulating the Wnt/beta-catenin pathway of cerebrovascular endothelial cells and protecting the structure of the endothelial intercellular tight junction protein of the BBB. Meanwhile, experiments prove that the protective effect of the lithium agent on the BBB mainly derives from the protection of tight connection between cerebrovascular endothelial cells.
In addition, the lithium preparation is a common clinical medicine, so that the pharmacokinetics and toxic and side effects are clear, and the cost is low, so that the lithium preparation is expected to be directly applied to patients with clinical cerebral infarction.
The lithium agent has BBB protection effect and neuroprotection effect, and has higher safety compared with other medicines for targeting intervention of Wnt/beta-catenin pathway.
The lithium preparation can be used for treating other diseases causing BBB damage, such as cerebral hemorrhage, toxic encephalopathy, metabolic encephalopathy, and cerebral hemorrhage secondary to central nervous system leukemia.
In summary, the pharmaceutical composition in the application can up-regulate the Wnt/β -catenin signaling pathway of the cerebrovascular endothelial cells by using the lithium agent, and has an important effect on protecting the structure of tight junction protein of the endothelial cells of the blood brain barrier, thereby protecting the blood brain barrier. Therefore, the lithium agent can play an effective pharmaceutical role in treating ischemic stroke and blood brain barrier injury caused by the ischemic stroke.
The above description is only for the purpose of illustrating embodiments of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings of the present application or are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.
Claims (12)
1. A pharmaceutical composition for treating ischemic stroke, comprising: the lithium preparation is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
2. The pharmaceutical composition of claim 1, wherein the lithium agent is lithium chloride and/or lithium carbonate.
3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is in dosage unit form.
4. The pharmaceutical composition of claim 3, wherein the dose of the lithium agent in the pharmaceutical composition is one of 1.5-6.0 mmoles/kg, wherein mmoles/kg is the amount of the substance of the lithium agent relative to the mass of the patient.
5. The pharmaceutical composition of claim 4, wherein the dose of the lithium agent in the pharmaceutical composition is 3.0 mmoles/kg.
6. The pharmaceutical composition of claim 1, further comprising an amount of alteplase effective to treat ischemic stroke, and wherein the amount of alteplase in the pharmaceutical composition is 0.9 mg/kg, wherein mg/kg is the mass of the alteplase relative to the mass of the patient.
7. The application of a lithium agent in the preparation of a medicine for treating cerebral arterial thrombosis is characterized in that the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebral vascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
8. Use according to claim 7, characterized in that the lithium agent is lithium chloride and/or lithium carbonate.
9. A pharmaceutical composition for treating blood-brain barrier injury, comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of the blood brain barrier.
10. The application of a lithium agent in the preparation of a medicament for treating blood brain barrier injury is characterized in that the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting the structure of tight junction protein of the endothelial cell of the blood brain barrier.
11. A pharmaceutical composition for treating a cerebral-vascular disease, said pharmaceutical composition comprising: the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of a cerebrovascular endothelial cell and protecting a structure of a tight junction protein of the endothelial cell of a blood brain barrier.
12. The application of a lithium agent in the preparation of a medicament for treating blood brain diseases is characterized in that the lithium agent is used for up-regulating a Wnt/beta-catenin signal pathway of cerebrovascular endothelial cells and protecting the structure of tight junction protein of the endothelial cells of a blood brain barrier.
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| CN101404984A (en) * | 2006-03-23 | 2009-04-08 | 3M创新有限公司 | Powder filling processes |
| KR20130026762A (en) * | 2011-09-06 | 2013-03-14 | 동국대학교 산학협력단 | Pharmaceutical composition comprising statin-based drug and wnt signal transduction regulator for preventing or treating arteriosclerosis and stroke |
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| CN101404984A (en) * | 2006-03-23 | 2009-04-08 | 3M创新有限公司 | Powder filling processes |
| KR20130026762A (en) * | 2011-09-06 | 2013-03-14 | 동국대학교 산학협력단 | Pharmaceutical composition comprising statin-based drug and wnt signal transduction regulator for preventing or treating arteriosclerosis and stroke |
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