Use of anisodine hydrobromide

Abstract

Provided is use of anisodine hydrobromide in the preparation of a therapeutic drug for stroke patients after tPA thrombolysis, which can improve various clinical indexes of the patient after thrombolysis for ischemic stroke, repair blood-brain barrier permeability, inhibit leukocyte infiltration, regulate the expression levels of related proteins, etc.

Description

Use of hydrobromide anisodine

[0001] The present application claims priority to the Chinese patent application No. 202411235620.3 filed on September 04, 2024, and entitled "Use of hydrobromide anisodine", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of medicine, in particular to the use of hydrobromide anisodine. BACKGROUND

[0003] Stroke, also known as apoplexy, is a group of diseases caused by abnormal cerebral blood flow due to cerebral vascular obstruction or rupture, resulting in brain tissue damage. Stroke not only has a high mortality rate, but also has a high disability rate, which brings a heavy burden to individuals, families and society. Therefore, stroke has become a severe challenge to health and health care in China and even the world.

[0004] Stroke is divided into ischemic stroke and hemorrhagic stroke, and ischemic stroke is the most common type of stroke, accounting for 60%-80% of the total number of strokes. Tissue plasminogen activator (tPA) is the only drug included in the guidelines for the treatment of ischemic stroke. However, the biggest problem of using tPA intravenous thrombolysis is the risk of secondary cerebral vasogenic edema and hemorrhage. At present, there is still a lack of effective drugs in clinical practice that can block the cerebral edema and hemorrhage caused by tPA thrombolysis. Therefore, it is of great significance to find a method that can reduce the risk of cerebral edema and hemorrhage caused by tPA for the treatment of ischemic stroke.

[0005] Blood brain barrier (BBB) disruption is a key pathological link of tPA-induced brain edema and hemorrhage. Tight junctions, adherens junctions and basement membrane between brain vascular endothelial cells play an important role in maintaining the integrity of BBB. tPA can convert plasminogen to activated plasmin, which can dissolve fibrin-rich thrombus and open the blocked cerebral vessels. In addition to thrombolytic effect, plasmin can also degrade tight junctions, adherens junctions and basement membrane between vascular endothelial cells, leading to BBB damage. During stroke, the basement membrane is degraded by various protease systems, and matrix metalloproteinases (MMPs) are important components of these systems. Low density lipoprotein receptor-related protein 1 (LRP-1) in brain endothelial cells is up-regulated after ischemia, and tPA can bind to LRP-1 to promote the secretion of MMPs by endothelial cells. At the same time, tPA-activated plasmin can activate MMPs. MMPs can cause BBB damage by degrading junction proteins between endothelial cells and basement membrane. In addition, tPA-induced reperfusion leads to ischemia-reperfusion injury. Peroxides produced by ischemia-reperfusion activate the Src kinase system to induce high expression of Caveolin-1, a plasma membrane microvesicle protein, increasing vascular permeability. After ischemia-reperfusion, the level of MMPs also increases significantly, further damaging the integrity of the BBB and increasing the risk of brain edema and hemorrhage. The complex mechanism behind tPA-induced BBB damage makes it difficult for drugs targeting a single target to effectively alleviate brain edema and hemorrhage, and therefore no drugs have been found to truly inhibit tPA-induced cerebral vascular edema and hemorrhage.

[0006] Anisodine is an alkaloid isolated from Anisodus tanguticus. On the one hand, anisodine can block the binding of acetylcholine to muscarinic acetylcholine receptors, exerting an anticholinergic effect. On the other hand, anisodine can act as an agonist of α1-adrenergic receptors, exerting its biological activity. This dual mechanism helps to relieve vasospasm, improve microcirculation, and has effects such as antitremor, antispasmodic, and antiasthmatic. In clinical practice, anisodine is used to treat vascular headache, retinal vasospasm, ischemic optic neuritis, acute paralysis caused by cerebrovascular disease, central dysfunction caused by carbon monoxide poisoning, tremor, paralysis, bronchial asthma, motion sickness, and organophosphate pesticide poisoning, etc.

[0007] The clinical main use of the anisodine hydrobromide injection is to treat ischemic optic neuropathy, especially non-arteritic anterior ischemic optic neuropathy (NAION). The pathogenesis of this disease is mainly that the short posterior ciliary arteries supplying the optic disc are transiently perfused or low perfusion, resulting in insufficient blood supply. Previous studies have shown that anisodine hydrobromide can penetrate the blood-brain barrier and the blood-ocular barrier, directly reach the fundus lesion, relieve vasospasm, correct pathological microcirculation, improve blood perfusion, and promote vision and visual field recovery. However, there is no anisodine hydrobromide for treating stroke after tPA thrombolysis. SUMMARY

[0008] Therefore, the present application aims to provide the use of anisodine hydrobromide.

[0009] The present application provides the use of anisodine hydrobromide in the preparation of a drug for repairing the blood-brain barrier permeability of a stroke patient after tPA thrombolysis.

[0010] In the present application, the repairing of the blood-brain barrier permeability of a stroke patient after tPA thrombolysis includes improving vascular leakage, improving cerebral blood flow and / or reducing brain edema.

[0011] The present application also provides the use of anisodine hydrobromide in the preparation of a drug for inhibiting leukocyte infiltration in the brain tissue of a stroke patient after tPA thrombolysis.

[0012] Further, the present application also provides the use of anisodine hydrobromide in the preparation of a drug for regulating stroke-related proteins in the brain tissue after tPA thrombolysis.

[0013] In the present application, the related proteins include:

[0014] tight junction proteins ZO-1, JAM-1 and / or Occludin;

[0015] adhesion junction proteins VE cadherin, alpha-catenin and / or beta-catenin;

[0016] basal membrane proteins Collagen IV and / or Laminin;

[0017] metalloproteinases MMP-2 and / or MMP-9;

[0018] energy metabolism-related proteins ATP 5D, ATP alpha and / or ATP 5 beta;

[0019] plasma membrane microvesicle-related proteins Caveolin-1 and / or P-Src.

[0020] In the present application, the regulation includes at least one of the following I) to III):

[0021] I) increasing the expression level of ATP5D, Collagen IV, Laminin, ZO-1, JAM-1 and / or Occludin protein;

[0022] II) decreasing the expression level of Caveolin-1, MMP-9, VE-cadherin, alpha-catenin and / or beta-catenin;

[0023] III) decreasing the phosphorylation level of Src.

[0024] Further, the application also provides a use of the zanthesine hydrobromide in the preparation of a medicament for preventing and treating stroke after tPA thrombolysis.

[0025] In the application, the prevention and treatment include reducing the cerebral infarction area, improving cerebral blood flow, improving cerebral hemorrhage, improving neurobehavior and / or increasing survival rate.

[0026] In the application, the tPA is alteplase.

[0027] In the embodiment of the application, the administration dose of the zanthesine hydrobromide is 0.6-2.4 mg / kg.

[0028] The application finds that the use of zanthesine hydrobromide after the use of tPA has the effect of improving various clinical indexes after thrombolysis of ischemic stroke. For example, it can repair the blood-brain barrier permeability, inhibit leukocyte infiltration, regulate the expression level of related proteins, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1A shows a representative diagram of cerebral blood flow perfusion in mice, n=10;

[0030] Figure 1B shows a statistical diagram of cerebral blood flow perfusion in mice at 28.5 h, *P<0.05 vs Sham group, n=10;

[0031] Figure 2A shows a representative diagram of cerebral infarction area by TTC staining method, n=10;

[0032] Figure 2B shows a statistical diagram of cerebral infarction area / total area, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0033] Figure 3A shows a representative diagram of Evans blue exudation, n=10;

[0034] Figure 3B shows a statistical diagram of Evans blue exudation, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0035] Figure 4 shows a brain tissue water content histogram, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0036] Figure 5A shows a brain hemorrhage volume representative graph;

[0037] Figure 5B shows a brain hemoglobin content histogram, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0038] Figure 6 shows a statistical result of mouse seven-day survival rate, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=15;

[0039] Figure 7A shows a statistical result of mouse mNSS score, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0040] Figure 7B shows a statistical result of mouse NES score, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group; n=10;

[0041] Figure 8A shows a mouse brain microvessel FITC-labeled dextran leakage representative graph;

[0042] Figure 8B shows a mouse brain microvessel albumin leakage representative graph, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=10;

[0043] Figure 9A shows a mouse brain microcirculation leukocyte adhesion representative graph;

[0044] Figure 9B shows a mouse brain microcirculation leukocyte adhesion histogram, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=10;

[0045] Figure 10A shows a mouse brain tissue Tunnel protein expression;

[0046] Figure 10B shows a mouse brain tissue hippocampal CA1, DG region Nissl staining;

[0047] Figure 11A shows the relative expression amount of ZO-1, JAM-1, Occludin and Claudin-5 proteins, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0048] Figure 11B shows the morphological structure of Occludin between vascular endothelial cells;

[0049] Figure 12A shows the relative expression of VE-Cadherin, a-catenin and β-catenin proteins, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0050] Figure 12B shows the morphological structure of VE-Cadherin between vascular endothelial cells;

[0051] Figure 13A shows the relative expression of Collagen IV and Laminin proteins, *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0052] Figure 13B shows the morphological structure of Collagen IV and Laminin;

[0053] Figure 14 shows the relative expression of MMP-2 and MMP-9 proteins; *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0054] Figure 15 shows the relative expression of ATP5D, ATPa and ATPβ proteins; *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0055] Figure 16 shows the relative expression of Caveolin-1, P-Src and Src proteins; *P<0.05 vs Sham group; #P<0.05 vs Thrombus+tPA group, n=6;

[0056] Figure 17 shows the co-localization of MPO and MMP-9 in mouse brain tissue;

[0057] Figure 18 shows the technical route of the present application. DETAILED DESCRIPTION

[0058] The present application provides the use of zhusanye hydrobromide, those skilled in the art can learn from the content of this paper, and appropriately improve the process parameters. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application of this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0059] The test materials used in the present application are all ordinary market products and can be purchased in the market. The present application will be further described below in combination with examples:

[0060] Examples

[0061] 1. Materials and methods

[0062] 1.1 Animals

[0063] Clean grade C57BL / 6N male mice, weighing 22±2 g, were provided by Beijing Weishanglideshi Experimental Animal Technology Co., Ltd. with license number: SCXK(Jing)2023-0004. The animals were raised at a temperature controlled at 23±2℃, relative humidity of 45±5%, 12 hours light / dark alternation, free access to food and water. The animals were fasted for 12 hours before the experiment and had free access to water. The experimental operation procedures were performed in accordance with the guidelines of the Animal Research Committee of Peking University.

[0064] 1.2 Main reagents

[0065] Suanliuksai hydrobromide injection was provided by Chengdu First Pharmaceutical Co., Ltd. (Chengdu, China) with specifications: concentration of 0.5 mg / ml, 1 ml per bottle; alteplase (tPA) was purchased from Boehringer Ingelheim Company (Biberach, Germany); sodium pentobarbital was purchased from Sigma-Aldrich Company (MO, USA); Evans blue was purchased from Sigma Aldrich Company (MO, USA); RIPA tissue lysis buffer was purchased from CST Company (Vermont, USA), hemoglobin colorimetric assay kit was purchased from Sigma-Aldrich Company (MO, USA), ferric chloride (FeCl3) was purchased from Fukun Chemical (Tianjing, China); PBS buffer powder was purchased from Zhongshanjinqiao Company (Beijing, China); triphenylphosphonium chloride (TTC) was purchased from Sigma Aldrich Company (MO, USA).

[0066] 1.3 Establishment of thrombolytic model after ischemic stroke in mice

[0067] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (2%, 45 mg / kg, Sigma Aldrich, MO, USA) according to their body weight, and the skin of the neck was disinfected. A 1-cm-long incision was made along the midline of the neck. The right common carotid artery was gently separated with an ophthalmic forceps under a stereomicroscope, and a waterproof pad was placed under the artery to separate the carotid artery from the neck tissue. The right carotid artery was wrapped with a filter paper strip soaked with FeCl3(20%, Fuchen Chemical, Tianjin, China) for 5 min. Subsequently, the filter paper was removed, and the residual FeCl3on the neck was washed with a dropper filled with normal saline at 37°C. After 10 min, the thrombus in the carotid artery was pushed into the intracranial circulation with an ophthalmic forceps to make it fall off. The waterproof pad was removed, and the skin of the neck was sutured. After the operation, the mice were placed on a hot plate until they woke up. In the sham group, the filter paper wrapped around the carotid artery was soaked with normal saline, and the other operations were the same as those in the model group. At 4.5 h after the operation, the cerebral surface blood perfusion of the mice was detected using a laser Doppler flowmeter (moor FLPI-2, Moor Instruments, Devon, UK). Mice that met the inclusion criteria were randomly divided into groups. The left femoral vein was cannulated, and recombinant tissue plasminogen activator (rtPA, alteplase, 10 mg / kg, Boehringer Ingelheim, Biberach, Germany) or the same volume of solvent was intravenously infused. Ten percent of the dose was slowly injected in the first 5 min, and the remaining 90% was continuously pumped intravenously at a rate of 0.1 mL / h. The total administration time of tPA or solvent was about 1 h. After the administration was completed, the mice were placed on a hot plate until they woke up, and then they were placed in a mouse cage for single-cage feeding. At 5.5 h, 6.5 h, and 8.5 h after the operation, the right femoral vein was cannulated, and hexylreconin hydrobromide injection was intravenously infused at a dose determined according to the grouping, and the total administration time was about 1 h.

[0068] 1.4 Experimental grouping

[0069] The mice were divided into 12 groups according to the random principle, as follows:

[0070] (1) Control group (Sham): normal saline was used instead of FeCl3during modeling, and the other operations were the same as those in the model group. At 4.5 h after the operation, tPA or the same volume of solvent was intravenously infused through the left femoral vein. The hexylreconin hydrobromide injection infused through the right femoral vein was replaced with the same volume of normal saline;

[0071] (2) Thrombus model for 4.5 h without tPA thrombolysis group (Thrombus): FeCl3modeling, tPA or the same volume of solvent was intravenously infused through the left femoral vein at 4.5 h after the operation, and the hexylreconin hydrobromide injection infused through the right femoral vein was replaced with the same volume of normal saline;

[0072] (3) Thrombus model 4.5 hours after tPA thrombolysis group (Thrombus + tPA): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and the right femoral vein infusion of ZLJ hydrobromide injection was replaced with an equal volume of normal saline;

[0073] (4) Thrombus model 4.5 hours after tPA thrombolysis and intravenous infusion of 0.6 mg / kg ZLJ hydrobromide injection 5.5 hours after surgery (Thrombus + tPA + 0.6 mg / kg ZLJ): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and 5.5 hours after surgery ZLJ hydrobromide injection was infused through the right femoral vein according to 0.6 mg / kg mouse body weight;

[0074] (5) Thrombus model 4.5 hours after tPA thrombolysis and intravenous infusion of 1.2 mg / kg ZLJ hydrobromide injection 5.5 hours after surgery (Thrombus + tPA + 1.2 mg / kg ZLJ): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and 5.5 hours after surgery ZLJ hydrobromide injection was infused through the right femoral vein according to 1.2 mg / kg mouse body weight;

[0075] (6) Thrombus model 4.5 hours after tPA thrombolysis and intravenous infusion of 2.4 mg / kg ZLJ hydrobromide injection 5.5 hours after surgery (Thrombus + tPA + 2.4 mg / kg ZLJ): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and 5.5 hours after surgery ZLJ hydrobromide injection was infused through the right femoral vein according to 2.4 mg / kg mouse body weight;

[0076] (7) Thrombus model 4.5 hours after tPA thrombolysis and intravenous infusion of 0.6 mg / kg ZLJ hydrobromide injection 6.5 hours after surgery (Thrombus + tPA + 0.6 mg / kg ZLJ): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and 6.5 hours after surgery ZLJ hydrobromide injection was infused through the right femoral vein according to 0.6 mg / kg mouse body weight;

[0077] (8) Thrombus model 4.5 hours after tPA thrombolysis and intravenous infusion of 1.2 mg / kg ZLJ hydrobromide injection 6.5 hours after surgery (Thrombus + tPA + 1.2 mg / kg ZLJ): FeCl3 modeling, 4.5 hours after tPA infusion through the left femoral vein, and 6.5 hours after surgery ZLJ hydrobromide injection was infused through the right femoral vein according to 1.2 mg / kg mouse body weight;

[0078] (9) Thrombus + tPA + 2.4 mg / kg ZLJ: FeCl3model, tPA was infused through the left femoral vein at 4.5 hours after operation, and ZLJ injection was infused through the right femoral vein at 6.5 hours after operation according to 2.4 mg / kg mouse weight;

[0079] (10) Thrombus + tPA + 0.6 mg / kg ZLJ: FeCl3model, tPA was infused through the left femoral vein at 4.5 hours after operation, and ZLJ injection was infused through the right femoral vein at 8.5 hours after operation according to 0.6 mg / kg mouse weight;

[0080] (11) Thrombus + tPA + 1.2 mg / kg ZLJ: FeCl3model, tPA was infused through the left femoral vein at 4.5 hours after operation, and ZLJ injection was infused through the right femoral vein at 8.5 hours after operation according to 1.2 mg / kg mouse weight;

[0081] (12) Thrombus + tPA + 2.4 mg / kg ZLJ: FeCl3model, tPA was infused through the left femoral vein at 4.5 hours after operation, and ZLJ injection was infused through the right femoral vein at 8.5 hours after operation according to 2.4 mg / kg mouse weight;

[0082] 1.5 Cerebral infarction detection

[0083] Brain infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining. At 28.5 hours postoperatively, mice were deeply anesthetized and perfused with pre-chilled normal saline through the left ventricle, until the right atrium drained clear. Brains were quickly removed and cut into 5 slices of 2 mm thickness. The slices were placed in prepared TTC staining solution (2%, Sigma Aldrich, MO, USA) at 37°C for 15 min in the dark. The stained slices were then photographed under a stereomicroscope with a camera attached. After TTC staining, the non-infarcted area was stained red, while the infarcted area remained white. Image analysis software Image J was used to calculate the infarct volume.

[0084] 1.6 Evans Blue Staining

[0085] Evans Blue staining was used to evaluate the permeability of the blood-brain barrier in mice. At 25.5 hours postoperatively, mice were injected with Evans Blue dye (2%, 4 mL / kg, Sigma Aldrich, MO, USA) through the femoral vein. After 3 hours, the mice were perfused with normal saline and the brains were removed. The brains were cut into 5 slices of 2 mm thickness and photographed under a stereomicroscope. The brain tissue was then cut into small pieces and weighed, and 50% trichloroacetic acid solution (1 mL / 100 mg) was added. The mixture was incubated at 4°C for 24 hours. After ultrasonic homogenization, the supernatant was centrifuged. A 1% Evans Blue standard solution was prepared, and a standard curve was created by diluting it in proportion. The absorbance was measured at a wavelength of 620 nm using a microplate reader (Synergy 2, BioTek, VT, USA). The concentration of Evans Blue in the sample was calculated based on the standard curve.

[0086] 1.7 Brain Water Content Analysis

[0087] The brainstem wet-to-dry weight method was used to assess the degree of brain edema. At 28.5 hours postoperatively, mice were deeply anesthetized and decapitated, and the brain tissue was quickly removed. After weighing, the brain was placed in an oven at 60°C for 72 hours. The dry weight was measured, and the brain water content was calculated according to the following formula: (wet weight - dry weight) / wet weight x 100%.

[0088] 1.8 Evaluation of Brain Hemorrhage

[0089] Brain hemorrhage was evaluated by hemoglobin assay. Mice were deeply anesthetized at 28.5 hours post-surgery and perfused with normal saline through the heart. Five 2-mm-thick brain slices were taken and photographed under a stereomicroscope. Brain tissue was minced and placed in centrifuge tubes, weighed, and phosphate buffer saline (PBS, 8 mL / g) and heparin (0.16 mg / mL) were added. The samples were homogenized by sonication and centrifuged to obtain the supernatant. Hemoglobin content was measured according to the hemoglobin colorimetric assay kit (Sigma-Aldrich, MAK115, MO, USA).

[0090] 1.9 Neurobehavioral score

[0091] Two classical neurobehavioral score scales were used to evaluate the neurological deficits of mice: the modified neurological severity scores (mNSS) and the neurological evaluation scale (NES). Neurobehavioral scores were performed at 28.5 hours post-surgery. The detailed evaluation contents were as follows (see Table 1 and Table 2).

[0092] Table 1. mNSS score details

[0093] Table 2. NES score details

[0094] 1.10 Survival rate analysis

[0095] Another group of mice was allowed to recover from anesthesia and to have normal diet and water. The survival of mice was recorded every 24 hours for 7 days. The cumulative survival rate of mice at 7 days post-surgery was calculated using GraphPad Prism software.

[0096] 1.11 Brain microvascular dextran leakage

[0097] The integrity of the BBB was also evaluated by measuring the leakage of fluorescein isothiocyanate (FITC)-labeled dextran in the in vivo microcirculation apparatus. After anesthesia, the mouse's brain was fixed and the skull skin was cut open. The skull was thinned using a skull drill (NE129, NSK, Tokyo, Japan) until only a soft bone cortex remained. At 28.5 hours post-surgery, the mouse was injected with FITC fluorescent solution (FD70, 50 mg / kg, Sigma Aldrich, MO, USA) through the femoral vein. After 30 minutes, the fluorescent images of the brain surface microvessels were taken using an upright fluorescent microscope (BX51WI, Olympus, Tokyo, Japan) with an excitation wavelength of 420-490 nm and an emission wavelength of 520 nm. The fluorescent intensities inside (V) and outside (I) the capillary vessels were calculated using the image analysis software Image J, and the plasma dextran leakage was represented as I / V.

[0098] 1.12 Leukocyte adhesion to brain microvessels

[0099] The in vivo microcirculation apparatus was used to analyze the leukocyte adhesion to brain microvessels. After anesthesia, the mouse's brain was fixed and the skull skin was cut open. The skull was thinned using a skull drill. At 28.5 hours post-surgery, the mouse was injected with Rhodamine 6G solution (1.5 mg / kg, Sigma Aldrich, MO, USA) through the femoral vein. After 30 minutes, the fluorescent images of the brain surface microvessels were taken using an upright fluorescent microscope (BX51WI, Olympus, Tokyo, Japan) at a wavelength of 543 nm. Leukocytes adhering to the vessel wall for more than 10 seconds were considered as stable adherent leukocytes. The number of adherent leukocytes per mouse was counted based on the photographed images.

[0100] 1.13 Nissl staining

[0101] The fixed brain tissue was removed from 4% paraformaldehyde and dehydrated in 10% to 20% and 30% sucrose gradients for 48 hours each. The brain tissue was removed from the sucrose, embedded in OCT (optimal cutting temperature) and 10 μιη thick frozen sections were prepared. After drying for two days, the sections were stained. The OCT embedding agent was washed off with distilled water for 5 min x 2 times. Nissl staining was performed in a 37°C incubator for 10 min, and washed with distilled water for 3 min x 3 times. Differentiation and dehydration were performed in 70% to 80%, 95%, and 100% ethanol gradients for 20 s each, and in xylene for 5 min x 2 times. The sections were mounted with neutral balsam and dried before being observed and photographed under a light microscope.

[0102] 1.14 Immunofluorescence staining

[0103] After the sections are dried for two days, immunofluorescence staining can be performed. OCT is washed away with distilled water, and sodium citrate high-temperature antigen repair is performed for 10 minutes, after which the sections are naturally cooled for 90 minutes. PBST is used to permeabilize the membranes at 37°C for 45 minutes, and PBS is used to wash the sections for 5 minutes twice. Hydrogen peroxide is used to block the sections at room temperature for 40 minutes, and PBS is used to wash the sections for 5 minutes three times. Sheep serum is used to block the sections at room temperature for 15 minutes, after which the serum blocking solution is removed. The primary antibody is added, and the sections are incubated in a refrigerator overnight. The sections are removed from the refrigerator and allowed to warm for 60 minutes, and PBS is used to wash the sections for 5 minutes three times. The fluorescent secondary antibody (1:100) is added, and the sections are incubated in the dark at 37°C for 120 minutes. PBS is used to wash the sections for 10 minutes three times, and excess secondary antibody is washed away. Hoechst 33342 (1:50) is used to stain the cell nuclei, and the sections are incubated in the dark at room temperature for 15 minutes, after which PBS is used to wash the sections for 5 minutes three times. Glycerol is used to mount the sections, and nail polish is applied around the coverslips to prevent movement. After the sections are dried for 24 hours, they are observed and photographed under a laser confocal microscope (TCS SP5, Leica, Bensheim, Germany).

[0104] 1.15 Western Blotting

[0105] (1) Sample collection: At 28.5 hours after surgery, the mice are deeply anesthetized, and the brain tissue is quickly removed after sacrifice. The right cerebral cortex is separated on ice and placed in a cryogenic tube. The cryogenic tube is placed in a liquid nitrogen tank and then transferred to a -80°C refrigerator for storage.

[0106] (2) Protein quantification: The tissue is removed from the -80°C refrigerator, thawed on ice, cut into small pieces, and weighed (100 mg is appropriate), and placed in a centrifuge tube. RIPA lysis buffer (1 mL / 100 mg) is added according to the weight of the tissue, and protease inhibitors are added to the RIPA. The tissue in the centrifuge tube is homogenized by ultrasonication until the protein solution becomes transparent. The protein lysate is centrifuged at 4°C for 25 minutes at 12000 rpm, and the supernatant is removed. The sample is diluted with pure water, and 10 μl is used for quantification. The protein standard is diluted by a factor of two to create a standard curve. The protein concentration of the sample to be tested is detected according to the BCA protein quantification kit (Pulile, Beijing, China) instructions. After quantification, sample buffer is added to the protein lysate, and the centrifuge tube is boiled for 15 minutes. The boiled protein sample is aliquoted and stored at -80°C for testing.

[0107] (3) Gel making: Choose clean glass plate and fix the gel making frame. Determine the concentration of gel according to the molecular weight of the protein to be detected (10-30kD: 12%; 30-100kD: 10%; 100-200kD: 8%). First, prepare the separation gel by adding ddH2O, 30% Acr-Bis, TrisHCl-SDS (pH 8.8), 10% ammonium persulfate (APS), and TEMED in sequence, and mix well. After adding the separation gel, press the film with isopropanol, and then wait for about 30 min for solidification. Then, prepare the concentrated gel by adding ddH2O, 30% Acr-Bis, TrisHCl-SDS (pH 6.8), 10% APS, and TEMED in sequence, and mix well. Pour out the isopropanol, add the concentrated gel, insert the comb, and wait for about 40 min for solidification. Take the prepared gel plate off the frame and prepare for electrophoresis.

[0108] (4) Electrophoresis: Fix the gel plate on the electrophoresis frame, and pour the electrophoresis liquid into the electrophoresis tank. Take the sample out of the -80°C refrigerator, and cook for 5 min using the electric stove. Centrifuge for 15 s using the mini centrifuge. Pull out the comb evenly and force, and load 5-20 μl. Set the initial voltage to 80 V, and after the rainbow Maker appears, switch the voltage to 120 V until the blue band reaches the bottom of the gel plate, which takes about 90 min.

[0109] (5) Membrane transfer: Cut the polyvinylidene fluoride (PVDF) membrane, and cut it to the size of 8.5x5 cm. Before the start of the electric transfer, soak the PVDF membrane in methanol, and take out the gel and soak it in the electric transfer liquid. Make a "sandwich" for membrane transfer: the lowermost layer is a black plate, and then in sequence are a sponge, filter paper, gel, PVDF membrane, filter paper, and sponge, and close the clamp. Pour the pre-cooled electric transfer liquid into the electric transfer tank in advance, and put the "sandwich" into the electric transfer tank. Set the electric current for membrane transfer to 240 mA, and it takes about 120 min. After completion, take out the PVDF membrane and rinse it in TBST for 10 min. Put the PVDF membrane into 5% milk powder, and seal it in the shaker at room temperature for 60 min. Wash it in TBST for 5 min x 3 times.

[0110] (6) Incubation of antibody: Dilute the primary antibody to the required concentration with 1% BSA, and put the PVDF membrane into the antibody dilution liquid, and shake overnight at 4°C. Take out the PVDF membrane, and wash the membrane in TBST for 10 min x 3 times. Dilute the secondary antibody with 3% skimmed milk powder to a concentration of 1:5000. Put the PVDF membrane into the secondary antibody dilution liquid, and incubate it in the shaker at room temperature for 90 min. Take out the PVDF membrane, and wash the membrane in TBST for 10 min x 3 times.

[0111] (7) Exposure and analysis: The prepared luminescent reagent was dropped on the front of the PVDF membrane, and kept at room temperature for 2 min. The PVDF membrane was placed in a transparent plastic film and exposed to the machine. The relative intensity of each band was normalized by its corresponding internal control protein, and semi-quantitative analysis was performed using image analysis software Image J.

[0112] 1.16 Statistical method

[0113] All experimental results were expressed as mean ± standard error (mean ± SEM), analyzed by GraphPad Prism software, and plotted. One-way ANOVA was used for comparison between groups, and Tukey's honestly was used for further pairwise comparison between groups. p < 0.05 was considered statistically significant.

[0114] 2. Experimental results

[0115] 2.1 Effect of anisodine hydrobromide on cerebral blood flow in mice after tPA thrombolysis

[0116] The laser Doppler blood flowmeter was used to detect the blood perfusion of the mouse brain surface (see Figure 1). The cerebral blood flow of the mice in the thrombus group (Thrombus) decreased significantly 4.5 h after the operation (the inclusion criteria was a decrease of 60%-90%). Compared with the Thrombus group, the cerebral blood flow of the mice in the Thrombus + tPA group, the Thrombus + tPA + 0.6 mg / kg anisodine hydrobromide group, the Thrombus + tPA + 1.2 mg / kg anisodine hydrobromide group, and the Thrombus + tPA + 2.4 mg / kg anisodine hydrobromide group was significantly improved 28.5 h after the operation, but there was no significant difference in the improvement of blood flow between each group.

[0117] 2.2 Anisodine hydrobromide improved the infarct area after tPA thrombolysis

[0118] Figure 2A is the image of TTC brain infarction staining of fresh brain tissue sections taken from each group of c57 mice at 28.5h after modeling. Compared with the control (Sham) group, the right brain modeling thrombus area of the mice in the thrombus (Thrombus) group has a large area of obvious infarction area (TTC staining result is white). There is no obvious beneficial effect on cerebral infarction after thrombolysis with tPA at 4.5h (Thrombus + tPA). Figure 2B is the statistical result of TTC staining of each 10 mice in each treatment group. Compared with the Thrombus + tPA (Thrombus + tPA) group, the brain infarction area of the Thrombus + tPA + 1.2mg / kg hydrobromide anisodine group administered at 5.5h, 8.5h and the Thrombus + tPA + 2.4mg / kg hydrobromide anisodine group administered at 5.5h, 6.5h, 8.5h is significantly reduced.

[0119] 2.3 Hydrobromide anisodine reduces Evans blue extravasation after tPA thrombolysis

[0120] The residual of Evans blue dye can reflect the leakage of blood vessels. Figure 3A is the natural light image of fresh brain tissue sections taken from each group of mice at 25.5h after modeling and intravenous injection of Evans blue, and 28.5h after perfusion. Compared with the Sham group, the right brain modeling area of the mice in the Thrombus group has obvious leakage of Evans blue outside the blood vessels. There is no obvious beneficial effect on Evans blue leakage after thrombolysis with tPA at 4.5h (Thrombus + tPA). Compared with the Thrombus + tPA group, the brain Evans blue leakage of the Thrombus + tPA + 1.2mg / kg hydrobromide anisodine group administered at 5.5h, 6.5h and the Thrombus + tPA + 2.4mg / kg hydrobromide anisodine group administered at 5.5h, 6.5h, 8.5h is significantly improved. Figure 3B is the statistical result of the corresponding Evans blue leakage.

[0121] 2.4 Hydrobromide anisodine reduces brain edema after tPA thrombolysis

[0122] The brain water content detected by dry and wet weight ratio is an indicator reflecting the brain edema after modeling. Figure 4 shows the statistical result of brain edema after tPA thrombolysis and hydrobromide anisodine administration. Compared with the Thrombus + tPA group, the brain edema of the Thrombus + tPA + 2.4mg / kg hydrobromide anisodine group administered at 5.5h, 6.5h is significantly reduced.

[0123] 2.5 Hydrobromide anisodine improves brain hemorrhage in mice after tPA thrombolysis

[0124] The brain tissue homogenate hemoglobin content measured after the mouse systemic blood vessels PBS perfusion can reflect the mouse brain microvessel hemorrhage. Figure 5A shows the brain hemorrhage representative graph of the camphorphanoline hydrobromide administration after tPA thrombolysis, and Figure 5B is the corresponding brain tissue hemoglobin content statistical results. Compared with the Thrombus + tPA group, the brain hemorrhage of the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 5.5 h, 6.5 h, and 8.5 h was significantly improved.

[0125] 2.6 Camphorphanoline hydrobromide improves the 7-day survival rate of mice after tPA thrombolysis

[0126] Figure 6 shows the effect of camphorphanoline hydrobromide on the 7-day survival rate of mice after thrombus re-thrombolysis. Compared with the Thrombus + tPA group, the survival rate of mice in the Thrombus + tPA + 1.2 mg / kg camphorphanoline hydrobromide group administered at 5.5 h and 6.5 h and the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 5.5 h and 6.5 h was significantly increased.

[0127] 2.7 Camphorphanoline hydrobromide improves the neurological behavior score of mice after tPA thrombolysis

[0128] Figure 7A shows the statistical results of the mNSS score of mice administered with camphorphanoline hydrobromide after tPA thrombolysis. Compared with the Thrombus + tPA group, the mNSS score of mice in the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 5.5 h, 6.5 h, and 8.5 h was significantly reduced. Figure 7B shows the statistical results of the NES score of mice. Compared with the Thrombus + tPA group, the NES score of mice in the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 6.5 h was significantly increased. In summary, camphorphanoline hydrobromide improves the neurological behavior score of mice after tPA thrombolysis.

[0129] 2.8 Effect of camphorphanoline hydrobromide on dextran leakage of brain microvessels of stroke model mice after tPA thrombolysis

[0130] In summary of the above brain blood flow, cerebral infarction area, brain hemorrhage volume, and seven-day survival rate results, we found that the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 5.5 h and 6.5 h had better improvement effect on various indicators of stroke model mice after tPA thrombolysis, so we selected the Thrombus + tPA + 2.4 mg / kg camphorphanoline hydrobromide group administered at 5.5 h and 6.5 h for further experiments.

[0131] We used FITC-labeled dextran to evaluate the permeability of the BBB in mice (see Figure 8). Compared with the Sham group, the dextran leakage in the Thrombus group was significantly increased. Compared with the Thrombus group, the leakage of dextran was further exacerbated after the addition of tPA. Compared with the Thrombus + tPA group, the addition of hydrobromide anisodine effectively alleviated the leakage of dextran exacerbated by tPA.

[0132] 2.9 Effect of hydrobromide anisodine on leukocyte adhesion to brain microvessels in a mouse model of stroke after thrombolysis with tPA

[0133] Leukocytes were labeled with rhodamine 6G, and leukocyte adhesion to brain microvessels was observed in vivo. As shown in Figure 9, leukocyte adhesion was less in the Sham group. Compared with the Sham group, the number of adherent leukocytes was significantly increased after thrombosis. Compared with the Thrombus group, the number of adherent leukocytes was further increased in the Thrombus + tPA group. Compared with the Thrombus + tPA group, the addition of hydrobromide anisodine significantly alleviated leukocyte adhesion.

[0134] 2.10 Hydrobromide anisodine improved brain tissue apoptosis in a mouse model of stroke after thrombolysis with tPA

[0135] Based on the results of the microcirculation experiments, we determined that the Thrombus + tPA + 2.4 mg / kg hydrobromide anisodine group administered at 6.5 h had the best experimental effect, and subsequent experiments were performed. We used a Tunnel kit to detect cell apoptosis in mice in each group (see Figure 10A). Compared with the Sham group, cell apoptosis was significantly increased in the Thrombus group and the Thrombus + tPA group. Compared with the Thrombus + tPA group, cell apoptosis was significantly improved in the Thrombus + tPA + 2.4 mg / kg hydrobromide anisodine group administered at 6.5 h.

[0136] We also used Nissl staining to observe the neurons in the CA1 and DG regions of the hippocampus in mice in each group (see Figure 10B). Compared with the Sham group, the number of Nissl particles in the neurons was significantly reduced, and cell vacuolization was significantly increased in the Thrombus group and the Thrombus + tPA group. Compared with the Thrombus + tPA group, the number of Nissl particles in the neurons was increased, and cell vacuolization was significantly improved in the Thrombus + tPA + 2.4 mg / kg hydrobromide anisodine group administered at 6.5 h, indicating that hydrobromide anisodine improved the damage to the neurons in the brain tissue of a mouse model of stroke after thrombolysis with tPA.

[0137] 2.11 Effect of hydrobromide anisodine on tight junction proteins ZO-1 and JAM in the brain tissue of a mouse model of stroke after thrombolysis with tPA

[0138] 1. Effect on Occludin and Claudin-5 expression

[0139] The expression levels of tight junction proteins zonula occludens-1 (ZO-1), junctional adhesion molecule 1 (JAM-1), Occludin and Claudin-5 were detected by Western blot (see Figure 11A). Compared with the Sham group, the expression of ZO-1, JAM-1 and Occludin proteins in the Thrombus + tPA group was significantly reduced. Compared with the Thrombus + tPA group, the expression of ZO-1, JAM-1 and Occludin proteins in the Thrombus + tPA + 2.4 mg / kg hydrobromide anisodine group administered at 6.5 h was significantly increased.

[0140] We also observed the morphology of the tight junction protein Occludin using immunofluorescence. As can be seen from the figure, the expression of Occludin in the Sham group was smooth and continuous, but the expression of Occludin in the Thrombus group was relatively less and in a broken shape, and after the addition of tPA, Occludin was distributed in a punctate manner and the morphology was destroyed. Compared with the Thrombus + tPA group, the punctate discontinuous morphology of Occludin was improved after 6.5 h of 2.4 mg / kg hydrobromide anisodine (see Figure 11B).

[0141] 2.12 Effect of hydrobromide anisodine on VE-cadherin in the brain tissue of a stroke model mouse after tPA thrombolysis

[0142] Effect on VE-cadherin, α-catenin and β-catenin expression

[0143] The expression levels of the adhering junction proteins VE-cadherin, α-catenin and β-catenin were detected by Western blot (see Figure 12A). Compared with the Sham group, the expression of VE-cadherin, α-catenin and β-catenin in the Thrombus + tPA group was significantly reduced. Compared with the Thrombus + tPA group, the expression of VE-cadherin and α-catenin proteins in the Thrombus + tPA + 2.4 mg / kg hydrobromide anisodine group administered at 6.5 h was significantly increased.

[0144] We also observed the morphology of adherens junction protein VE-cadherin by immunofluorescence. As shown in Figure 12B, VE-cadherin expression was smooth and continuous in the Sham group, while VE-cadherin expression was relatively reduced and discontinuous in the Thrombus group. After the addition of tPA, VE-cadherin was distributed in a punctate pattern, and expression was reduced. Compared with the Thrombus + tPA group, the addition of 2.4 mg / kg of hydrobromide anisodine at 6.5 h improved the morphology of VE-cadherin, which was relatively more continuous (see Figure 12B).

[0145] 2.13 Effect of hydrobromide anisodine on the expression of basement membrane proteins Collagen IV and Laminin in the brain tissue of a stroke model mouse after tPA thrombolysis

[0146] The expression levels of basement membrane proteins Collagen IV and Laminin were detected by Western blotting (see Figure 13A). Compared with the Sham group, the expression of Collagen IV and Laminin proteins was significantly reduced in the Thrombus + tPA group. Compared with the Thrombus + tPA group, the expression levels of Collagen IV and Laminin proteins were upregulated in the group administered 2.4 mg / kg of hydrobromide anisodine at 6.5 h.

[0147] The morphology of basement membrane proteins Collagen IV and Laminin was observed by immunofluorescence. As shown in Figure 13B, Collagen IV and Laminin expression was complete and continuous in the Sham group, while Collagen IV and Laminin expression was relatively reduced and appeared to be broken in the Thrombus group. After the addition of tPA, Collagen IV and Laminin were more severely broken. Compared with the Thrombus + tPA group, the morphology of Collagen IV and Laminin was more continuous after the addition of 2.4 mg / kg of hydrobromide anisodine at 6.5 h.

[0148] 2.14 Effect of hydrobromide anisodine on the expression of matrix metalloproteinases MMP-2 and MMP-9 in the brain tissue of a stroke model mouse after tPA thrombolysis

[0149] The expression levels of matrix metalloproteinases MMP-2 and MMP-9 were detected by Western blotting (see Figure 14). Compared with the Sham group, the expression of MMP-9 was significantly increased in the Thrombus + tPA group. Compared with the Thrombus + tPA group, the expression of MMP-9 was significantly downregulated in the group administered 2.4 mg / kg of hydrobromide anisodine at 6.5 h. There was no significant difference in the expression of MMP-2 between the groups.

[0150] 2.15 The effect of anisodine hydrobromide on the expression of ATP 5D, ATPa and ATP5B proteins related to brain energy metabolism in the stroke model mice after tPA thrombolysis

[0151] The expression levels of ATP 5D, ATPa and ATP5B proteins related to energy metabolism were detected by Western blotting (see Figure 15). Compared with the Thrombus group, the expression of ATP5D protein in the Thrombus + tPA group was significantly down-regulated. Compared with the Thrombus + tPA group, the expression of ATP5D in the anisodine hydrobromide group administered at 2.4 mg / kg at 6.5 h was significantly up-regulated. There was no significant difference in the expression of ATPa and ATP5B proteins among the groups.

[0152] 2.16 The effect of anisodine hydrobromide on the expression of Caveolin-1 and P-Src related to brain microvesicle in the stroke model mice after tPA thrombolysis

[0153] The expression of Caveolin-1 and the phosphorylation level of Src related to brain microvesicle were detected by Western blotting (see Figure 16). Compared with the Sham group, the expression of Caveolin-1 and the phosphorylation level of Src in the Thrombus + tPA group were significantly higher than those in the Sham group. Compared with the Thrombus + tPA group, the expression of Caveolin-1 and the phosphorylation level of Src in the anisodine hydrobromide group administered at 2.4 mg / kg at 6.5 h were significantly reduced. There was no significant change in the expression of P-Caveolin-1 and Src proteins.

[0154] 2.17 The effect of anisodine hydrobromide on the leukocyte infiltration in the brain tissue of the stroke model mice after tPA thrombolysis

[0155] The infiltration of leukocytes labeled by MPO was detected by immunofluorescence. As shown in Figure 17, there was little leukocyte infiltration in the Sham group. The thrombus formation increased the leukocyte infiltration. The addition of tPA after the thrombus formation further aggravated the leukocyte infiltration. Compared with the Thrombus + tPA group, the leukocyte infiltration was significantly reduced after the addition of anisodine hydrobromide at 2.4 mg / kg at 6.5 h.

[0156] In this study, the cell source of MMP-9 was also identified by double labeling immunofluorescence. As shown in Figure 17, there was obvious co-localization between MMP-9 and leukocytes labeled by MPO. It was indicated that the MMP-9 induced by tPA was mainly released by leukocytes.

[0157] 3. Conclusion

[0158] Administration of 2.4 mg / kg of anisodamine hydrobromide at 6.5 h can improve cerebral blood flow, cerebral infarction, neurobehavioral impairment and survival rate decreased by cerebral thrombosis tPA thrombolysis.

[0159] Administration of 2.4 mg / kg of anisodamine hydrobromide at 6.5 h can inhibit cerebral edema caused by cerebral thrombosis tPA thrombolysis, which may be related to its alleviation of blood-brain barrier damage, inhibition of microvesicular protein expression, improvement of energy metabolism disorder, and inhibition of endothelial cell gap junction damage.

[0160] Administration of 2.4 mg / kg of anisodamine hydrobromide at 6.5 h can inhibit cerebral hemorrhage caused by cerebral thrombosis tPA thrombolysis, which may be related to its inhibition of vascular endothelial basement membrane damage and reduction of leukocyte-derived MMP-9.

[0161] The above is only a preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make several improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of anisodine hydrobromide in the preparation of a medicament for repairing the permeability of the blood-brain barrier in a stroke patient after tPA thrombolysis.

2. Use according to claim 1, characterized in that, The repairing the permeability of the blood-brain barrier in a stroke patient after tPA thrombolysis includes improving vascular leakage, improving cerebral blood flow and / or reducing brain edema.

3. Use of anisodine hydrobromide in the preparation of a medicament for inhibiting leukocyte infiltration in brain tissue after tPA thrombolysis.

4. Use of anisodine hydrobromide in the preparation of a medicament for regulating stroke-related proteins in brain tissue after tPA thrombolysis.

5. Use according to claim 4, characterized in that, The related proteins include: tight junction proteins ZO-1, JAM-1 and / or Occludin; adhesion junction proteins VE-cadherin, α-catenin and / or β-catenin; basal membrane proteins Collagen IV and / or Laminin; metalloproteinases MMP-2 and / or MMP-9; energy metabolism-related proteins ATP 5D, ATPα and / or ATP5β; plasma membrane microvesicle-related proteins Caveolin-1 and / or P-Src.

6. Use according to claim 4 or 5, characterized in that, The regulation includes at least one of the following I) to III): I) increasing the expression level of ATP5D, Collagen IV, Laminin, ZO-1, JAM-1 and / or Occludin protein; II) reducing the expression level of Caveolin-1, MMP-9, VE-cadherin, α-catenin and / or β-catenin; III) reducing the phosphorylation level of Src.

7. Use of anisodine hydrobromide in the preparation of a medicament for preventing and treating stroke after tPA thrombolysis.

8. Use according to claim 7, characterized in that, The prevention and treatment includes reducing the area of cerebral infarction, improving cerebral blood flow, improving cerebral hemorrhage, improving neurobehavior and / or improving survival rate.

9. Use according to any one of claims 1 to 8, characterized in that, The tPA is alteplase.

10. Use according to any one of claims 1 to 8, characterized in that, The dose of anisodine hydrobromide is 0.6-2.4 mg / kg.

Images