CN114668767A - Application of beta-sitosterol in treating cerebral arterial thrombosis - Google Patents
Application of beta-sitosterol in treating cerebral arterial thrombosis Download PDFInfo
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- CN114668767A CN114668767A CN202210357380.9A CN202210357380A CN114668767A CN 114668767 A CN114668767 A CN 114668767A CN 202210357380 A CN202210357380 A CN 202210357380A CN 114668767 A CN114668767 A CN 114668767A
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- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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Abstract
The invention relates to application of beta-sitosterol in treating ischemic stroke, which utilizes a primary neuron hypoxia/reoxygenation model of a mouse and a middle cerebral artery occlusion model of the mouse to find that the beta-sitosterol can inhibit the accumulation of cholesterol in nerve cells, effectively regulate the cholesterol transport function of cholesterol transport protein NPC1L1 on cell membranes, and can obviously improve the cerebral infarction degree and nerve function damage of middle cerebral artery occlusion model animals after intraperitoneal injection. The invention provides a new drug effect substance for treating ischemic stroke, which provides a reliable basis for the independent application or combined application of beta-sitosterol in medicine.
Description
Technical Field
The invention relates to the field of medicines, in particular to application of beta-sitosterol in treating cerebral arterial thrombosis.
Background
Ischemic stroke is an acute cerebrovascular disease, has the characteristics of high morbidity, high mortality and high disability rate, and brings huge burden to family economy and social medical treatment of patients. At present, the adoption of thrombolytic drugs such as alteplase and urokinase for revascularization is considered as a standard strategy for treating ischemic stroke, but the clinical application of the thrombolytic drugs is greatly limited due to the short treatment time window and the risk of easy hemorrhage. Therefore, the intensive search for new potential therapeutic targets has become a major research direction in this field.
Ischemic stroke is a complex pathophysiological process, and the occurrence mechanism of the ischemic stroke is very complex, and comprises energy failure, excitotoxicity, neuroinflammation, apoptosis, oxidative stress and the like. These links are causal and influence each other, forming a vicious circle, and finally leading to irreversible necrosis of neuron cells. Among them, neuronal apoptosis is considered to be a central link and a key factor causing brain damage.
Homeostasis disorders of cholesterol and cholesterol metabolites in cells are a major cause of neuronal apoptosis. The NPC1L1 gene is a newly discovered site closely related to cholesterol absorption in recent years, is an important factor for maintaining the dynamic balance of cholesterol in organisms, and is an action target of a novel lipid-lowering drug ezetimibe.
The beta-sitosterol is one of phytosterol components, belongs to a tetracyclic triterpenoid compound, and is widely present in various vegetable oils, plant seeds, fruits and vegetables. The beta-sitosterol has good pharmacological action in the aspects of oxidation resistance, hyperlipidemia resistance, inflammation resistance, immunoregulation, tumor resistance, central nervous system and the like. At present, no report related to the treatment of ischemic stroke by beta-sitosterol is found. Therefore, the beta-sitosterol is introduced into the medicine for treating the cerebral arterial thrombosis (single or multi-medicine combined treatment), and has great practical significance.
Disclosure of Invention
The invention provides application of beta-sitosterol in treating and/or preventing cerebral ischemic stroke. In particular to the application in preparing the medicine for treating and/or preventing cerebral arterial thrombosis.
Another embodiment of the invention provides an application of beta-sitosterol in inhibiting excessive accumulation of cholesterol in nerve cells. It inhibits excessive accumulation of cholesterol in nerve cells by effectively binding to NPC1L1 protein.
Another embodiment of the present invention provides a use of beta-sitosterol for inhibiting apoptosis caused by endoplasmic reticulum stress of ischemic stroke nerve cells.
Another embodiment of the present invention provides the use of beta-sitosterol in the treatment and/or prevention of cerebral infarction due to ischemic stroke.
Another embodiment of the present invention provides the use of beta-sitosterol in the treatment and/or prevention of ischemic stroke neurological impairment.
The present invention provides a drug for treating and/or preventing ischemic stroke, which is characterized by comprising beta-sitosterol as an active ingredient. Optionally in combination with other drugs for treating and/or preventing ischemic stroke. The other medicines for treating and/or preventing ischemic stroke are preferably Chinese medicines, plant medicine extracts, effective parts or effective components, NPC1L1 small molecule compounds, nucleic acids such as RNAi, polypeptides or protein medicines.
The administration mode of the beta-sitosterol comprises intraperitoneal injection, subcutaneous injection and/or oral administration. The dosage to be administered is preferably 2-100mg/kg, preferably 10-50 mg/kg.
The invention has the advantages that: the application of beta-sitosterol in treating ischemic stroke is disclosed, and aims to introduce the beta-sitosterol into the treatment of the ischemic stroke, intervene OGD/R primary neuron cells by adopting the beta-sitosterol, find that the beta-sitosterol can obviously inhibit the accumulation of cholesterol in the neuron cells, and can obviously inhibit the apoptosis of the neuron of an animal model with the ischemic stroke after the intraperitoneal injection of an animal model mouse, improve the nerve behavior of the model animal and the like. The invention provides a new drug effect substance for treating ischemic stroke, which provides a reliable basis for the new application of beta-sitosterol in medicine.
Drawings
FIG. 1 is a graph showing the effect of β -sitosterol in inhibiting the accumulation of cholesterol in neuronal cells by acting directly on the NPC1L1 target; a, a graph A: cholesterol staining in primary neuronal cells of different treatment groups; and B: testing the expression level of NPC1L1 in cortical brain tissue of a mouse; and (C) figure: molecular docking diagram of beta-sitosterol and NPC1L 1.
FIG. 2 is a graph of protein expression of the genes for inhibition of MCAO/R-induced endoplasmic reticulum stress/neuronal apoptosis by beta-sitosterol.
FIG. 3 is a graph of the improvement of cerebral infarction and neurobehavioral improvement of animal model mice by beta-sitosterol; FIG. A: TTC staining patterns of experimental mouse brain sections; and B: a neuro-functional score plot of the test mice; and (C) figure: the brain water content of the brain of the mouse is tested.
Detailed Description
The following is a more detailed description of the present invention in connection with specific preferred embodiments and it is not intended that the practice of the invention be limited to these descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Example 1: effect of beta-sitosterol on intracellular cholesterol transport and transport receptor NPC1L1
1. Experimental methods
(1) Primary culture of neurons: pregnant mice (E14-15D) were sacrificed by overanesthesia outside the super clean bench, placed in an anatomical plate, sprayed with 75% alcohol for sterilization, and the abdomen was quickly dissected open to remove fetal beads, which were placed in a glass dish containing a pre-cooled (ice bath) D-Hank's balance solution. Individual embryos were separated under sterile conditions and the brain shells were peeled off to rapidly remove the bilateral brains (ice-on-ice procedures). Dissecting under microscope to remove blood vessel membrane, collecting cerebral cortex tissue, placing in pre-cooled D-Hank's solution, cutting into pieces of 1-2mm3, transferring to 0.25% trypsin solution, and digesting at 37 deg.C for 20 min. The digestion was stopped by rinsing the tissue 3 times with DMEM solution containing 20% fetal bovine serum (containing 0.5mM glutamine, 100U/mL penicillin, 100U/mL streptomycin). Repeatedly blowing and beating the mixture into turbid liquid (with gentle action and no bubbles) by using a glass pipette polished by fire until no obvious tissue blocks are visible, sieving the turbid liquid with a 200-mesh sieve to prepare single cell suspension, and counting the number of cells on a cell counting plate and under a microscope. DMEM medium was used to dilute and resuspend the single cell suspension to the appropriate density, and seeded on cell culture plates previously coated with polylysine (PLL, 25. mu.g/mL). The cell culture plates were transferred to a CO2 incubator for culturing, and after 24h the Neurobasal medium (containing 2% B27, 0.5mM glutamine, 100U/mL penicillin, 100U/mL streptomycin) was changed at full volume, and thereafter every 2d half the volume was changed, and the cell growth was observed under an inverted microscope before each change. Depending on the cell growth, cultures 7-14d can be used for the experiments.
(2) Establishment of a model of sugar oxygen deprivation (OGD/R) cells: normal culture of primary cortical neuronal cells 7-14d (depending on cell growth). On an ultra-clean bench, the normal cortical neuron cell culture medium is aspirated, PBS liquid is repeatedly washed for 3 times, then glucose-free DMEM culture medium is added, 5% CO2, 95% N2 and 37 ℃ are subjected to anoxic incubation for 2h, then sugar-free culture liquid is aspirated, PBS liquid is washed for 3 times, Neruobasal (containing 2% B27, 100U/mL penicillin and 100U/mL streptomycin) culture medium is added, and the culture medium is placed into an incubator to be perfused (reoxygenation and sugar-regaining) for culture (reoxygenation condition: 37 ℃, saturation humidity and 5% CO2) After 24h, it was used in the subsequent experiments.
(3) Intracellular cholesterol staining experiment: plating primary neuron cells into 96-well plates at a density of 2-3 × 104Cells/well, cells were cultured to appropriate conditions in a 37 ℃ incubator containing 5% CO 2. Cells were washed with 100 μ l detection buffer; adding 50-100 μ l of fixing solution, and incubating for 10 min; cells were washed 2-3 times with 100. mu.l assay buffer. The staining solution was diluted 1:100 with detection buffer before use, then 100. mu.l/well was added to a 96-well plate and incubated in the dark at 37 ℃ for 30-60 minutes. The staining solution was carefully removed with a pipette and the cells were gently washed 2-3 times with 100. mu.l/well of detection buffer. Images were acquired using a fluorescence microscope (Ex/Em ═ 340-380/385-470 nm).
(4) Establishment of MCAO/R animal model: after the mice for experiment are successfully anesthetized, the mice are fixed on an operating table in a supine position, skin preparation and disinfection are carried out conventionally, a central incision of the neck is taken, glandular tissues and fascia of the neck are separated in a blunt manner, and the common carotid artery, the external carotid artery and the internal carotid artery are exposed and separated and ligated. A V-shaped small opening is cut at the stump of the external carotid artery by an ophthalmic scissors (the common carotid artery and the internal carotid artery are clamped by a arteriole clamp before the opening is cut). A pre-treated (heparin-soaked) 8-0 nylon suture plug 15mm long was carefully inserted from the external carotid artery; after removing the arteriolar clamp of the internal carotid artery, inserting a treated nylon wire plug until just entering the intracranial anterior cerebral artery (the wire plug is inserted into the internal carotid artery by 10 +/-0.5 mm, on the basis of meeting slight resistance), and blocking the opening of the middle cerebral artery; removing the wire plug after 2h of ischemia to make the head end return to the external carotid artery, so that the blood flow of the common carotid artery can be re-infused into the middle cerebral artery; in the operation process, transcranial Doppler ultrasound is used for detecting the middle cerebral artery blood flow (the probe is fixed 2mm behind bregma and is 2mm aside from the midline), and 70-80% of the blood flow which is reduced to the position before the operation is used as a mark for successfully preparing the MCAO model; after ligation and sterilization, suturing subcutaneous tissues and skin layer by layer, and putting the animal back into a cage after the animal is anesthetized and revived; the manufacturing steps of the pseudo-operation model are the same as the previous steps, but no thread plug is inserted after the blood vessel is separated, and the subcutaneous tissue and the skin are sutured after ligation and sterilization.
(5) Western blot detection of Total protein levels: the treated tissue was homogenized 3 times (25Hz, 3 s/time) on ice with an ultrasonic cell disruptor by adding RIPA lysate (containing protease inhibitor and PMSF). Centrifugation was carried out at 12000rpm for 20min at 4 ℃ and the supernatant collected and placed on ice. Taking a certain amount of supernatant for quantifying the BCA kit, adding 1/4 volumes of 5 × loading buffer into the residual supernatant, boiling for 10min, and waiting for loading. Separating the sample by 12% SDS-PAGE gel electrophoresis, 80V, 2h, setting the gel running time according to specific conditions; then 100V membrane transfer for 100min, then 5% skimmed milk powder is used for sealing for 2-4h, primary antibody is added, and incubation is carried out overnight at 4 ℃. After 3 rinses of TBST (10mM Tris,150mM NaCl, and 0.1% Tween-20), goat/rabbit secondary antibody was added and incubated for 2h at room temperature. After rinsing 3 times with TBST, chemiluminescence was obtained.
2. Results of the experiment
As shown in FIG. 1, the intracellular cholesterol staining test results show that the OGD/R model has significantly increased intracellular cholesterol; beta-sitosterol (10 mu M) can obviously inhibit cholesterol accumulation in cells caused by an OGD/R model, and meanwhile, the beta-sitosterol has no influence on the cholesterol in normally cultured primary neuron cells. Further Western blot experiment results show that the beta-sitosterol can effectively reverse the abnormal increase of the NPC1L1 protein expression in mouse cortex tissues caused by the MCAO model. Similarly, the results of molecular docking experiments show that the beta-sitosterol molecules can be effectively combined with NPC1L1 protein. These results indicate that the cholesterol accumulation in nerve cells of mice with ischemic stroke, and the beta-sitosterol can inhibit the excessive accumulation of the cholesterol in the nerve cells by directly acting on the cholesterol transport protein NPC1L 1.
Example 2: effect of beta-sitosterol on apoptotic genes in neurons
1. The experimental method comprises the following steps:
(1) western blot detection of Total protein levels: same as above
2. Results of the experiment
As shown in fig. 2, compared with the normal group, the protein expression levels of the apoptotic genes caspase3, caspase 9, Bax in the cortical tissue of the MCAO model mouse were significantly increased; meanwhile, the endoplasmic reticulum stress marker proteins GRP78/Bip and caspase12 are detected to be obviously highly expressed; and the β -sitosterol treated group reversed this effect. This indicates that beta-sitosterol can inhibit apoptosis caused by endoplasmic reticulum stress of ischemic stroke nerve cells.
Example 3: influence of beta-sitosterol on animal model mouse nerve function behavior and cerebral infarction
1. The experimental method comprises the following steps:
(1) measuring cerebral infarction area by triphenyltetrazolium chloride (TTC) staining: each group of animals is subjected to 24h reperfusion, 10% chloral hydrate intraperitoneal injection, head breaking and brain taking after anesthesia, quick freezing for 15min in a refrigerator at the temperature of-20 ℃, and cutting 5 coronal slices with the thickness of 2 mm. Care was taken when slicing, as brain tissue easily sticks to the blade, which was first immersed in a 2% TTC solution to prevent loss. The cut brain slices are quickly placed into TTC solution for dyeing, a cover glass is pressed on the brain slices to flatten the brain tissues, the brain tissues are incubated for 30min in a thermostat at 7 ℃ in the dark, 4% paraformaldehyde is fixed, and the change of the color is continuously observed. When the surface of the brain piece begins to turn pink, the brain piece is reversed. The dyed surface has active brain tissue; the infarcted portion was unable to be stained due to mitochondrial inactivation and remained white. When the color of the surface of the brain slice changes from light red to dark red, the reaction is ended, and the TTC solution is washed away by PBS. And calculating the volume percentage of cerebral infarction by using image analysis software after the digital camera takes a picture. The percent by volume (%) of cerebral infarction is left cerebral infarction area x slice thickness/total volume of right brain tissue x 100%.
(2) Detecting the water content of the brain: after the mice of each experimental group are dosed and modeled, the mice are subjected to excessive anesthesia, brains are taken after the mice are directly decapitated, olfactory bulbs, cerebellum and low brainstem are removed, left and right hemispheres are separated, the wet weight is immediately weighed, then the left and right hemispheres are placed into an electric oven at the temperature of 110 ℃ for 24 hours to be baked to constant weight, and then the dry weight of brain tissues is quickly weighed. The brain water content (%) - (wet-dry weight)/wet weight × 100% was calculated.
2. The experimental results are as follows:
as shown in fig. 3, the MCAO model apparently caused a large infarct in the brain of the experimental mice. Compared with the model group, the cerebral infarction area of the mice of the beta-sitosterol treatment group is obviously reduced; meanwhile, the injection of the beta-sitosterol in the abdominal cavity can obviously improve the nerve function damage and the cerebral edema of the model mouse. These results indicate that beta-sitosterol can significantly improve cerebral infarction degree and nerve function damage of mice with ischemic stroke.
In conclusion, the beta-sitosterol directly acts on the cholesterol transport protein NPC1L1, so that the amount of cholesterol in nerve cells of ischemic stroke is reduced, the nerve cell apoptosis caused by accumulation of cholesterol is inhibited, and the cerebral infarction and cerebral edema after the ischemic stroke are reduced, thereby playing a role in neuroprotection. The beta-sitosterol is reasonably developed to be prepared into a proper dosage form, and a new medicament is expected to be provided for clinically treating the ischemic stroke.
Claims (10)
1. Application of beta-sitosterol in preparing a medicament for treating and/or preventing cerebral arterial thrombosis.
2. Application of beta-sitosterol in preparing medicine for inhibiting excessive accumulation of cholesterol in nerve cells.
3. The use as claimed in claim 2, characterized in that the excessive accumulation of cholesterol in nerve cells is inhibited by effective binding to the NPC1L1 protein.
4. Application of beta-sitosterol in preparing a medicament for inhibiting apoptosis caused by endoplasmic reticulum stress of ischemic stroke nerve cells.
5. Application of beta-sitosterol in preparing a medicament for treating and/or preventing cerebral infarction caused by ischemic stroke.
6. Application of beta-sitosterol in preparing a medicament for treating and/or preventing ischemic stroke nerve function damage.
7. Use according to any one of claims 1 to 6, characterized in that cholesterol transporters on the cell membrane are used as drug targets for the treatment of ischemic stroke.
8. The use as claimed in claim 7, characterized in that the specific regulatory molecules of the beta-sitosterol target comprise cholesterol and NPC1L 1.
9. Use according to any one of claims 1 to 6, characterized in that the β -sitosterol is optionally used in combination with other drugs for the treatment and/or prevention of ischemic stroke.
10. Use according to any one of claims 1 to 9, characterized in that the administration mode of β -sitosterol is selected from the group consisting of intraperitoneal injection, subcutaneous injection and/or oral administration. The administration dosage is selected from 2-100mg/kg, preferably 10-50 mg/kg.
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| 李媛媛 等: "基于网络药理学和分子对接探讨漏芦防治缺血性脑卒中的作用机制", 中西医结合心脑血管病杂志, vol. 18, no. 22, pages 3735 - 3744 * |
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