CN117618442A - New use of Rho kinase inhibitor Fasudil in inhibiting hallucination - Google Patents
New use of Rho kinase inhibitor Fasudil in inhibiting hallucination Download PDFInfo
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- CN117618442A CN117618442A CN202310310115.XA CN202310310115A CN117618442A CN 117618442 A CN117618442 A CN 117618442A CN 202310310115 A CN202310310115 A CN 202310310115A CN 117618442 A CN117618442 A CN 117618442A
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- hallucination
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Abstract
The invention discloses a new application of Rho kinase inhibitor Fasudil in inhibiting the illusion effect, the Fasudil can specifically inhibit the illusion effect, and has good development for inhibiting 5HT 2A The prospect of the drug for inhibiting the hallucination induced by the receptor-mediated hallucination provides a theoretical basis for inhibiting the hallucination induced by the hallucination agent in the clinical treatment of mental related diseases such as depression, and the clinical safety of Fabauil has been approved, so that the research and development cost of the drug in the research and development of inhibiting the hallucination can be saved.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a novel application of Rho kinase inhibitor Fasudil in inhibiting hallucination.
Background
The existing clinical antidepressant drugs mainly adopt a monoamine strategy, and although the drug can effectively treat depression, most of the clinical antidepressant drugs have serious defects of delayed onset (2-6 weeks), low effectiveness (50% -70%), lack of cognition improvement, even damage to cognition, sexual dysfunction and suicide tendency and the like, so that the research and development of novel fast, efficient and low-toxicity antidepressant drugs has important significance. In recent years, classical hallucinogens have made remarkable progress in the treatment of mental diseases such as depression, anxiety, post-traumatic stress disorder, and the like, and in particular have positive roles in promoting neural plasticity, regulating the immune state of the body, and regulating neurotransmitter release.
Classical hallucinogens are a class of psychoactive substances acting on 5-HT receptors, primarily by activating 5-HT 2A Receptor-induced sensory and emotional changes and hallucination effects, including 2, 5-dimethoxy-4-methamphetamine (DOM), xyloside (Psilocybin), xyloside (Psilocin), 2, 5-dimethoxy-4-iodoamphetamine (DOI), lysergic acid diethylamine (LSD), N-dimethyl primary amine (DMT), classical hallucinogens such as DMT, LSD, mescoline, and xyloside were listed as the first class of psychotropic drugs in the catalogue of psychotropic drugs published 2013 in China. Currently, the European drug administration has approved a phase III clinical study of the treatment of major depressive disorder (major depressive disorder, MDD) with the xyloside, which shows the advantages of quick onset and durable effect in antidepressant treatment. DMT shows good antidepressant and anxiolytic effects in clinical studies. In addition, LSD also shows the remarkable therapeutic effect on depression, drug dependence and anxiety in animal experimental study, and it can be seen that the classical hallucinogen is very expected to replace the traditional antidepressant drug and is widely applied in the aspect of clinically treating mental related diseases such as depression.
However, the side effect of the hallucination phenomenon induced by the classical hallucinogens greatly limits the application of the hallucinogens in clinical treatment of mental related diseases such as depression, and therefore, how to inhibit or alleviate the generation of the hallucination effect on the basis of ensuring the treatment effect of the classical hallucinogens is an important problem to be solved in the field at present.
Disclosure of Invention
Aiming at the technical problems in the background technology, the invention provides a novel application of Rho kinase inhibitor Fasudil in inhibiting the illusion. The invention discovers for the first time that Nogo-A/RhoA signal channel antagonists have remarkable potential for resisting the hallucinogen effect caused by the hallucinogen, and animal experiments prove that the Rho kinase inhibitor Fasudil can specifically inhibit the hallucinogen effect.
The invention adopts the following technical scheme to realize the purposes:
in a first aspect, the present invention provides the use of a Rho kinase inhibitor in the manufacture of a medicament for specifically inhibiting the effect of hallucinations.
Further, the Rho kinase inhibitor is Fasudil.
Further, the hallucination is 5HT 2A Receptor mediated hallucination.
Further, the medicament also comprises one or more inert, non-toxic excipients which are pharmaceutically acceptable.
Further, the excipient comprises carrier, solvent, emulsifier, dispersant, wetting agent, adhesive, stabilizer, colorant, and perfume.
Further, the medicine is injection, capsule, granule, drop, freeze-dried product, granule or tablet.
Further, the medicine is prepared into injection, capsule, medicinal granules, drops, freeze-dried substances, granules or tablets by adding medicinal auxiliary components into Fasudil with the mass fraction of more than 80%.
In the present invention, the term "Rho kinase (ROCK) inhibitor" refers to a class of agents capable of inhibiting Rho kinase. Rho kinase is a downstream acting substrate of Rho protein, is one of the main kinases which are discovered in recent years and are involved in cell movement, and has important regulation effects on cell division, shrinkage, adhesion, migration, secretion and other activities. It has close relation with the occurrence and development of cardiovascular diseases such as vasospasm, arteriosclerosis, ischemia/reperfusion injury, heart failure, myocardial infarction, hypertension, angina pectoris and the like, and Rho kinase has become an important target point for the research and development of new drugs related to cardiovascular diseases.
In a specific embodiment of the present invention, the Rho kinase inhibitor is Fasudil, also called Fasudil, purchased from Shanghai Tao Shu biotechnology limited company, with the product number of T3060, and Fasudil is a novel drug with wide pharmacological actions, and the main application is to improve and prevent cerebral vasospasm after subarachnoid hemorrhage and cerebral ischemia symptoms caused by cerebral vasospasm. The Fasudil has a molecular structure of 5-isoquinoline sulfonamide derivative, expands blood vessels by increasing the activity of myosin light chain phosphatase, reduces the tension of endothelial cells, improves brain tissue microcirculation, does not generate and aggravate cerebral blood theft, can antagonize inflammatory factors, protects nerves from apoptosis, promotes nerve regeneration, and has certain curative effects of Fasudil hydrochloride on promoting the recovery of nerve functions, relieving clinical symptoms and reducing disability rate.
In the present invention, the term "hallucination" refers to 5HT 2A Receptor-mediated hallucination, the hallucination induced by activation of the 5-HT2A receptor, is a classical hallucinogen acting on the 5-HT receptor. The classical hallucinogens include, but are not limited to: 2, 5-dimethoxy-4-methamphetamine (DOM), xyloside (Psilocin), 2, 5-dimethoxy-4-iodoamphetamine (DOI), lysergic acid diethylamine (LSD), N-Dimethyltryptamine (DMT). In addition, the illusion effect is not limited to the illusion effect induced by the classical illusion agent, and the pathological illusion effect induced by other illusion agents is within the protection scope of the classical illusion agent.
In a specific embodiment of the present invention, the influence of Fasudil according to the present invention on the hallucinogen-induced hallucination is representatively studied using a classical hallucinogen DOM or Psilocin-induced head-flick mouse model as an example, and the hallucinogen is not limited to the specific hallucinogens listed in the present invention, and any hallucinogen capable of causing a hallucination is within the scope of the present invention. In a specific embodiment of the present invention, the hallucinogen is preferably a compound that acts on 5-HT receptors by activating 5-HT 2A The receptor induces classical hallucinogens that produce hallucination.
Further, the head-flick mouse model adopted by the invention is an animal model for researching fantasy behaviors most commonly used in the field at present. The head-flick response (HTR) is a rapid left-right rotation of the head, in the administration of 5-hydroxy to rats and miceTryptamine energy hallucinogens or other 5-HT 2A The appearance of agonists followed by a head-flick response is widely used as 5-HT 2A Behavior determination of receptor activation. There is a strong positive correlation between the effect of the mouse's head-flick response and the human-induced effects, i.e. the head-flick response is indicative of the creation of hallucinogenic behaviour.
Further, whereas the medicament described in the first aspect of the invention may act systemically and/or locally, on the basis of which it may be administered in a suitable manner, for example by oral, parenteral, pulmonary or nasal route, the specific form of the medicament described in the first aspect of the invention may be administered in a manner suitable for such routes of administration.
The form of administration suitable for oral administration is capable of acting rapidly and/or releasing the medicament described in the first aspect of the invention in an improved manner and comprises tablets, e.g. in crystalline and/or amorphous and/or dissolved form (uncoated or coated tablets, e.g. with a coating against gastric juice or delayed dissolution or insolubilization, according to the characteristics of the medicament itself described in the first aspect of the invention), tablets, or films, films/lyophilisates, capsules (e.g. hard or soft capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions which break down rapidly in the mouth.
The form of administration of parenteral administration may avoid the step of absorption (e.g., intravenous, intra-arterial, intracardiac, intraspinal, intra-lumbar, intra-articular) or involve absorption simultaneously (e.g., intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal). Suitable administration forms for parenteral administration are in particular preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Suitable for another route of administration are, for example, pharmaceutical forms for inhalation, for example powder inhalation or nebulizer inhalation, or pharmaceutical forms which can be taken nasally, drops, solutions or sprays.
The medicament according to the first aspect of the invention may be converted to said administration form. This can be carried out in a manner known per se by mixing with inert, nontoxic, pharmacologically suitable excipients. These excipients include, inter alia, carriers (e.g., microcrystalline cellulose, lactose, mannitol, starch), solvents (e.g., liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (e.g., sodium lauryl sulfate, polyoxysorbitan oleate, propylene glycol), binders (e.g., polyvinylpyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants, such as ascorbic acid), colorants (e.g., inorganic pigments, iron oxides) and masking flavors and odors.
In addition, the invention also provides application of the Rho kinase activator in preparing medicines for treating mental diseases such as analgesia, addiction, autoimmune diseases, depression and anxiety improvement, alzheimer disease and related dementia, post-traumatic stress disorder, autism spectrum disorder and the like. In a specific embodiment of the present invention, the Rho kinase activator is preferably LPA.
In a second aspect, the invention provides a pharmaceutical composition for specifically inhibiting the hallucinations.
Further, the pharmaceutical composition comprises an effective amount of a Rho kinase inhibitor.
Further, the Rho kinase inhibitor is Fasudil.
Further, the pharmaceutical composition further comprises one or more inert, non-toxic excipients that are pharmaceutically acceptable.
In the present invention, the term "effective amount" refers to an amount that has a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a pharmaceutically or pharmaceutically effective amount refers to the amount of drug required to produce a desired therapeutic effect, which can be reflected by the results of a clinical trial, a model animal study, and/or an in vitro study. The pharmaceutically effective amount depends on several factors, including, but not limited to, the subject's characteristic factors (e.g., height, weight, sex, age, and history of administration), the severity of the disease.
Further, the pharmaceutical composition may also comprise other therapeutic agents for inhibiting or alleviating the effects of hallucinations.
Further, such other therapeutic agents for inhibiting or alleviating the effects of hallucinations include, but are not limited to: any presently disclosed agent useful for inhibiting or alleviating a hallucination, and/or any presently disclosed agent useful for aiding in inhibiting or alleviating a hallucination, are also within the scope of the present invention, as are pharmaceutical compositions comprising the combination of the agent and the Rho kinase inhibitor Faudil of the present invention, which may be used simultaneously or sequentially (the agent is used first followed by the Rho kinase inhibitor Faudil of the present invention, or the agent is used first followed by the Rho kinase inhibitor Faudil of the present invention), and in addition, the agent and the Rho kinase inhibitor Faudil of the present invention may be administered in the same or different modes of administration, including, but not limited to: intravenous, subcutaneous, intraperitoneal, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, intranasal, or any combination thereof.
Further, suitable modes of administration of the pharmaceutical compositions include any of a variety of methods and delivery systems known to those of skill in the art to physically introduce the pharmaceutical compositions of the present invention into a subject, including, but not limited to: oral administration, parenteral administration, administration by inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration or administration by an implanted reservoir. In particular embodiments of the invention, administration by injection is preferred, including by bolus injection or continuous infusion. In the case of pharmaceutical compositions for administration by injection, they may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and they may contain formulating agents such as suspending, preserving, stabilizing and/or dispersing agents.
Furthermore, the present invention provides a method for suppressing or alleviating the effects of hallucinations, the method comprising the steps of: administering to a subject in need thereof an effective amount of the Rho kinase inhibitor Fasudil or a pharmaceutical composition described in the second aspect of this invention.
In a specific embodiment of the present invention, the hallucination is preferably 5HT 2A Receptor-mediated hallucination, the hallucination induced by activation of the 5-HT2A receptor, is a classical hallucinogen acting on the 5-HT receptor.
Drawings
FIG. 1 is a schematic diagram of classical hallucinogens DOM and Psilocin, non-hallucinogen 5-HT 2A Results of changes in Nogo-A and RhoA protein expression levels in the cerebral cortex following intraperitoneal administration of the receptor agonists Listinide and TBG to mice;
FIG. 2 is a schematic diagram of classical hallucinogens DOM and Psilocin, non-hallucinogen 5-HT 2A Results of changes in Nogo-A and RhoA protein phosphorylation in the cerebral cortex following intraperitoneal administration of the receptor agonists Listinide and TBG;
fig. 3 is a graph of the effect of Fasudil on classical hallucinogen DOM-induced mouse head-flick behavior, where each group of n=8-10, data expressed as mean ± SEM, P <0.05, P <0.01, P <0.001, P <0.0001, data analyzed using One way ANOVA and Dunnett's test;
fig. 4 is a graph of the effect of Fasudil on classical hallucinogen pseudocin-induced mice head-flick behavior, where each group of n=8-10, data expressed as mean ± SEM, P <0.05, P <0.01, P <0.001, P <0.0001, data analyzed using One way ANOVA and Dunnett's test;
fig. 5 is a graph of the effect of the P75 NTR inhibitor TAT-Pep5 on classical hallucinogen DOM-induced mouse head flick behavior, where each group n=6, data expressed as mean ± SEM, P <0.05, P <0.01, P <0.001, P <0.0001, data analyzed using One way ANOVA and Dunnett's test.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
EXAMPLE 1Western Blot detection of Change 1 in Nogo-A, rhoA after the action of a hallucinogen, experimental Material
The experimental reagents, antibodies and instruments used in this example are shown in tables 1 to 3, respectively.
TABLE 1 Experimental reagents
Table 2 experimental antibodies
Table 3 laboratory apparatus
The main reagent configuration in this example:
10 x electrophoresis liquid running buffer: electrophoresis liquid: double distilled water is prepared according to the following proportion of 1:9 configuration.
10 Xtransfer buffer: and (3) converting the electrokinetic liquid: methanol: double distilled water is prepared according to the following proportion of 1:2:7 configuration.
10 XTBST: TBST: double distilled water is prepared according to the following proportion of 1:9 configuration.
5% skim milk powder: 2g of skim milk powder was taken and 40mL of 1 XTBE was added for complete dissolution.
5% BSA: 2g of BSA was taken and 40mL of 1 XTBE was added thereto to dissolve the BSA sufficiently.
Ammonium persulfate: 1.00g of ammonium persulfate was dissolved in 10mL of double distilled water.
2. Detection of Nogo-A, rhoA changes after the action of a hallucinogen by Western Blot
The sample used in this example was classical hallucinogen DOM (1 mg/kg) And Psilocin (0.5 mg/kg) and non-pseudogenic 5-HT 2A Protein extracts of cerebral cortex 10min after intraperitoneal administration of the receptor agonist Liquiride (T61065-Shanghai Tao Shu Biotechnology Co., ltd.) (0.1 mg/kg) and TBG (20 mg/kg) (St. Bei Fu (Beijing) Biotechnology Co., ltd., C57, male, 18-22g,6-8 weeks). The variation of the expression levels of Nogo-A and RhoA proteins in the above samples was detected by Western Blot. The specific Western Blot experiment method is as follows:
(1) Glue making
(1) Selecting a glass plate with a smooth lower edge, cleaning the glass plate and the comb, and then flushing and drying by using distilled water.
(2) The thick glass plate and the thin glass plate are aligned and then put into a clamp to be clamped, and are vertically clamped on a frame. The lower edges of the two glass plates are aligned during operation so as to avoid glue leakage.
(3) Preparing the required separating gel according to the polyacrylamide gel formula, adding TEMED, and immediately shaking to obtain the final product. The 10% split gum formulation is shown in table 4 below and the 5% concentrate gum formulation is shown in table 5 below.
Table 4 10% release gum formulation
Table 5 5% concentrated gum formulation
(2) Filling glue and loading
(1) During glue filling, a gun is used for adding along one side of the glass plate, and the glue surface is lifted to be about 15mm away from the upper edge of the short plate. Then adding a layer of water, and setting the gel surface after liquid sealing more quickly. The glue filling can be started faster, and the glue surface is slowed down when reaching the required height. The glue seal is slow and uniform from left to right, otherwise the glue will be punched out.
(2) When there is a fold line between the water and the gel (about 20min at room temperature), this indicates that the gel has set. And waiting for 3min to solidify the gel sufficiently, pouring out the upper water and sucking the upper water with filter paper.
(3) 5% concentrated gel is prepared according to the polyacrylamide gel formula, and the gel can be filled after being immediately shaken up after being added with TEMED. The remaining space is filled with the concentrated glue from one side and then the comb is inserted into the concentrated glue. The comb is inserted so that one side of the comb is firstly inserted, then the other side of the comb is slowly inserted, and finally whether the comb is horizontal or not is checked.
(4) After gelation, the gel was rinsed with distilled water and placed in an electrophoresis tank. The thin glass plate is on the inside and the thick glass plate is on the outside. If only one glue is run, a plastic plate replacing the glass plate is placed on the other side of the electrophoresis tank.
(5) And (3) filling the inner tank with new electrophoresis liquid, and then preparing for loading, wherein the electrophoresis liquid in the inner tank at least needs to be over the inner side glass plate, and the electrophoresis liquid with the height of about 3cm is added in the outer tank and then over the lower edge of the glass plate. The two sides of the comb are respectively pinched by two hands, the comb is pulled out slightly vertically upwards, the l mL pipetting gun is used for blowing and flushing the sample adding hole, the sample is sucked by the pipetting device, and the gun tip is inserted into the gap between the two plates above the sample adding hole, so that the sample is slowly added. The wells that were not loaded were filled with 1×loading buffer.
(3) Electrophoresis
(1) And (3) electrophoresis is carried out by selecting a constant voltage of 80V, and the voltage can be adjusted to 120V to increase the speed after the sample enters the separation gel. Electrophoresis is stopped until bromophenol blue runs to the lower edge of the gel.
(4) Transfer film
(1) A PVDF film is prepared, and the area of the PVDF film is slightly larger than the area of the adhesive surface to be transferred. Transferring a piece of glue requires 8 sheets Bao Lvzhi or 4 sheets of thick filter paper (8X 10 cm). In the case of PVDF membrane, methanol is used for activating for 30-60s before use, and the formulation of the membrane transfer liquid contains methanol.
(2) The clamp, two sponges, filter paper and membrane for transferring membrane are placed in a glass big dish with electrotransfer liquid. The clip is opened to keep the black side horizontal. A piece of sponge paper is placed on the upper surface of the electric rotating liquid machine, and the sponge is pressed by hands to remove bubbles, so that the electric rotating liquid machine can soak the sponge. Two layers of filter paper are arranged on the cushion, one hand is fixed on the filter paper, and the other hand is used for removing bubbles.
(3) And (3) prying the glass plate, peeling, cutting off the glue deformed by extrusion at the lower edge, and stripping the concentrated glue clean. Carefully peel the release gel over the filter paper, gently remove air bubbles by hand, cover the membrane over the gel, and be immovable after the cover. And covering filter paper and sponge, and clamping the clamp.
(4) The clips were placed in an electrotransport cell, glued to the negative electrode and the film to the positive electrode (black-black, red-white). The electric rotating system is in a low-temperature environment by utilizing ice cubes, and 200mA is transferred for 2 hours at constant current.
(5) After the transfer, the membrane was removed with forceps, rinsed 1 time with TBST, and turned over from the side to prevent washing away of proteins.
(5) Antibody incubation
(1) The membrane was first placed in blocking solution (5% skim milk or BSA) and shaken at ambient temperature for 1-4h or overnight at 4 ℃.
(2) Primary antibodies were diluted in proportion with 5% BSA. Adding primary anti-dilution liquid, and shaking at normal temperature for 1-4h or overnight at 4 ℃.
(3) Recovering primary antibody, washing the membrane with TBST solution at normal temperature for 3-4 times, each time for 5min.
(4) Diluting the secondary antibody with 5% milk according to a certain proportion (usually 1:5000), adding secondary antibody diluent, and incubating for 1h at normal temperature by a shaking table.
(5) The secondary antibody was discarded and washed 3-4 times with TBST for 5 min/time.
(6) Development process
(1) The membrane proteins were placed face up on a blackboard, developer a/B solution 1:1, after being evenly mixed (gun head is changed), the mixture is dripped on a film to be fully covered.
(2) The blackboard is placed in an exposure machine, a Marker is shot under the condition of white light, and a target strip is exposed under the condition of self-luminescence.
(7) Data statistics
Data analysis was performed using Photoshop, imageJ and GraphPad Prism.
3. Experimental results
Classical hallucinogens DOM (1 mg/kg) and Psilocin (0.5 mg/kg), non-hallucinogens 5-HT 2A Abdominal administration of the receptor agonist Liquiride (0.1 mg/kg) and TBG (20 mg/kg)The results of the changes in the expression levels of Nogo-A and RhoA proteins in the protein extracts of the cerebral cortex 10min after mice are shown in FIG. 1, and the results show that the changes in the levels of Nogo-A proteins are not obvious and the levels of RhoA proteins are in an upward trend after DOM and Psilocin administration. It has been shown that RhoA proteins may act by altering the amount of protein in the acute effects of classical hallucinogens, whereas Nogo-a proteins may not act by altering the amount of protein, but may act by protein modification or other forms of alteration.
Example 2Phos-tag gel electrophoresis detection of changes in Nogo-A, rhoA after action of hallucinogens 1, experimental Material
The experimental reagents, antibodies and instruments used in this example are shown in tables 6 to 8, respectively.
Table 6 Experimental reagent
TABLE 7 Experimental antibodies
Table 8 laboratory apparatus
The main reagent configuration in this example:
1 x electrophoresis liquid running buffer: electrophoresis liquid: double distilled water is prepared according to the following proportion of 1:9 configuration.
1 x electrotransfer buffer: and (3) converting the electrokinetic liquid: methanol: double distilled water is prepared according to the following proportion of 1:2:7 configuration. 1 XTBST: TBST: double distilled water is prepared according to the following proportion of 1:9 configuration.
5% skim milk powder: 2g of skim milk powder was taken and 40mL of 1 XTBE was added for complete dissolution.
5% BSA: 2g of BSA was taken and 40mL of 1 XTBE was added thereto to dissolve the BSA sufficiently.
Ammonium persulfate: 1.00g of ammonium persulfate was dissolved in 10mL of double distilled water.
Photosbind: 10mg of Phosphind was dissolved in 0.10mL of methanol and 3.2mL of distilled water.
10mmol/L Mncl 2+ :10mg is dissolved in 5mL distilled water.
10mmol/L EDTA:350mg EDTA was dissolved in 120mL of electrotransfer solution.
2. Experimental method
The samples used in this example were classical hallucinogens DOM (1 mg/kg) and Psilocin (0.5 mg/kg) and non-hallucinogens 5-HT 2A Protein extracts of cerebral cortex 10min after intraperitoneal administration of the receptor agonist Liquiride (T61065-Shanghai Tao Shu Biotechnology Co., ltd.) (0.1 mg/kg) and TBG (20 mg/kg) (St. Bei Fu (Beijing) Biotechnology Co., ltd., C57, male, 18-22g,6-8 weeks). The changes in phosphorylation of Nogo-A and RhoA proteins in the above samples were detected by Phos-tag gel electrophoresis. The specific experimental method for detecting the Phos-tag gel electrophoresis is as follows:
(1) Glue making
(1) Selecting a glass plate with a smooth lower edge, cleaning the glass plate and the comb, and then flushing and drying by using distilled water.
(2) The thick glass plate and the thin glass plate are aligned and then put into a clamp to be clamped, and are vertically clamped on a frame. The lower edges of the two glass plates are aligned during operation so as to avoid glue leakage.
(3) Preparing the required separating gel according to the polyacrylamide gel formula, adding TEMED, and immediately shaking to obtain the final product. The 10% split gum formulation is shown in table 9 below and the 5% concentrate gum formulation is shown in table 10 below.
Table 9 10% release gum formulation
Table 10 5% concentrated gum formulation
(2) Filling glue and loading
(1) During glue filling, a gun is used for adding along one side of the glass plate, and the glue surface is lifted to be about 15mm away from the upper edge of the short plate. Then adding a layer of water, and setting the gel surface after liquid sealing more quickly. The glue filling can be started faster, and the glue surface is slowed down when reaching the required height. The glue seal is slow and uniform from left to right, otherwise the glue will be punched out.
(2) When there is a fold line between the water and the gel (about 20min at room temperature), this indicates that the gel has set. And waiting for 3min to solidify the gel sufficiently, pouring out the upper water and sucking the upper water with filter paper.
(3) 5% concentrated gel is prepared according to the polyacrylamide gel formula, and the gel can be filled after being immediately shaken up after being added with TEMED. The remaining space is filled with the concentrated glue from one side and then the comb is inserted into the concentrated glue. The comb is inserted so that one side of the comb is firstly inserted, then the other side of the comb is slowly inserted, and finally whether the comb is horizontal or not is checked.
(4) After gelation, the gel was rinsed with distilled water and placed in an electrophoresis tank. The thin glass plate is on the inside and the thick glass plate is on the outside. If only one glue is run, a plastic plate replacing the glass plate is placed on the other side of the electrophoresis tank.
(5) And (3) filling the inner tank with new electrophoresis liquid, and then preparing for loading, wherein the electrophoresis liquid in the inner tank at least needs to be over the inner side glass plate, and the electrophoresis liquid with the height of about 3cm is added in the outer tank and then over the lower edge of the glass plate. The two sides of the comb are respectively pinched by two hands, the comb is pulled out slightly vertically upwards, the l mL pipetting gun is used for blowing and flushing the sample adding hole, the sample is sucked by the pipetting device, and the gun tip is inserted into the gap between the two plates above the sample adding hole, so that the sample is slowly added. The wells that were not loaded were filled with 1×loading buffer.
(3) Electrophoresis
(1) And (3) electrophoresis is carried out by selecting a constant voltage of 80V, and the voltage can be adjusted to 120V to increase the speed after the sample enters the separation gel. Electrophoresis is stopped until bromophenol blue runs to the lower edge of the gel.
(4) Film washing
After electrophoresis, the manganese ions (Mn) are removed from the gel using a chelating agent (EDTA) prior to transfer 2+ ). This step can increase the transfer efficiency of phosphorylated and non-phosphorylated proteins to PVDF membranes.
(1) After electrophoresis, the gel was immersed in a normal transfer buffer containing 1-10mmol/L EDTA for at least 10 minutes while gently shaking. (10 min. Times.1-3 times). The treatment time and temperature of the EDTA buffer were adjusted according to the gel thickness and the like (for example: 1.5mm thick: 20 minutes. Times.twice).
(2) The gel was immersed in a normal transfer buffer without EDTA for 10 minutes while gently shaking (10 minutes×1).
(5) Transfer film
(1) A PVDF film is prepared, and the area of the PVDF film is slightly larger than the area of the adhesive surface to be transferred. Transferring a piece of glue requires 8 sheets Bao Lvzhi or 4 sheets of thick filter paper (8X 10 cm). In the case of PVDF membrane, methanol is used for activating for 30-60s before use, and the formulation of the membrane transfer liquid contains methanol.
(2) The clamp, two sponges, filter paper and membrane for transferring membrane are placed in a glass big dish with electrotransfer liquid. The clip is opened to keep the black side horizontal. A piece of sponge paper is placed on the upper surface of the electric rotating liquid machine, and the sponge is pressed by hands to remove bubbles, so that the electric rotating liquid machine can soak the sponge. Two layers of filter paper are arranged on the cushion, one hand is fixed on the filter paper, and the other hand is used for removing bubbles in the filter paper
(3) And (3) prying the glass plate, peeling, cutting off the glue deformed by extrusion at the lower edge, and stripping the concentrated glue clean. Carefully peel the release gel over the filter paper, gently remove air bubbles by hand, cover the membrane over the gel, and be immovable after the cover. And covering filter paper and sponge, and clamping the clamp.
(4) The clips were placed in an electrotransport cell, glued to the negative electrode and the film to the positive electrode (black-black, red-white). The electric rotating system is in a low-temperature environment by utilizing ice cubes, and 200mA is transferred for 2 hours at constant current.
(5) After the transfer, the membrane was removed with forceps, rinsed 1 time with TBST, and turned over from the side to prevent washing away of proteins.
(6) Antibody incubation
(1) The membrane was first placed in blocking solution (5% skim milk or BSA) and shaken at ambient temperature for 1-4h or overnight at 4 ℃.
(2) Primary antibodies were diluted in proportion with 5% BSA. Adding primary anti-dilution liquid, and shaking at normal temperature for 1-4h or overnight at 4 ℃.
(3) Recovering primary antibody, washing the membrane with TBST solution at normal temperature for 3-4 times, each time for 5min.
(4) Diluting the secondary antibody with 5% milk according to a certain proportion (usually 1:5000), adding secondary antibody diluent, and incubating for 1h at normal temperature by a shaking table.
(5) The secondary antibody was discarded and washed 3-4 times with TBST for 5 min/time.
(7) Development process
(1) The membrane proteins were placed face up on a blackboard, developer a/B solution 1:1, after being evenly mixed (gun head is changed), the mixture is dripped on a film to be fully covered.
(2) The blackboard is placed in an exposure machine, a Marker is shot under the condition of white light, and a target strip is exposed under the condition of self-luminescence.
(8) Data statistics
Data analysis was performed using Photoshop, imageJ and GraphPad Prism.
3. Experimental results
According to the Phos-Tag experimental principle, as two bands with similar distance appear on the same protein, the upper band is the protein amount with phosphorylation modification, and the lower band is the protein amount without phosphorylation modification. The upper and lower bands represent the total protein amount of the protein.
The results of the phosphorylation changes of Nogo-A and RhoA proteins in protein extract samples of the mouse brain cortex at 10 minutes after intraperitoneal administration of mice with classical hallucinogens DOM (1 mg/kg) and Psilocin (0.5 mg/kg), non-hallucinogenic 5-HT2A receptor agonists Liquiride (0.1 mg/kg) and TBG (20 mg/kg) are shown in FIG. 2, and the results indicate that only one band was detected for Nogo-A protein, and that the phosphorylation changes for Nogo-A protein were not apparent. Two bands are detected by RhoA protein, wherein the upper band is the protein amount which is phosphorylated, the lower band is the protein amount which is not phosphorylated, and the RhoA phosphorylation change of the Psilocin group can be obtained in a descending trend. It was shown that RhoA proteins may play a role in the acute effects of classical hallucinogens by altering the amount of protein and by making phosphorylation modifications. The Nogo-A protein may not function by phosphorylation modification.
EXAMPLE 3 inhibition study of classical hallucinogen-induced hallucination by Fasudil
1. Experimental materials
Experimental animals: SPF class C57 mice, male, body weight 20-22g; the laboratory animal production license, SCXK (Beijing) 2019-0010, was supplied by St Bei Fu (Beijing) Biotechnology Co. The experimental animals are raised in the center of behavior of military medical institute, 8-10 animals/cage. The room temperature (22+/-2 ℃) and the humidity (40+/-20 ℃) are alternated for 12 hours, the water and food are taken freely, and the environment is adapted to three days before the experiment.
Experimental reagent: the experimental reagents used in this example are shown in Table 11 below.
Experimental equipment: the experimental equipment used in this example is shown in table 12 below.
Table 11 Experimental reagent
Table 12 experimental equipment
The main reagent configuration in this example:
DOM solution configuration: weighing a proper amount of sample, dissolving in normal saline, preparing into 0.1mg/mL, and being suitable for intraperitoneal injection of mice.
Psilocin solution configuration: an appropriate amount of sample is weighed and dissolved in 1% DMSO and 99% physiological saline to prepare 0.5mg/mL, and diluted to 0.05mg/mL by physiological saline, and the method is suitable for intraperitoneal injection of mice.
Rho kinase (ROCK) inhibitor Fasudil solution configuration: an appropriate amount of sample was weighed and dissolved in physiological saline to prepare 2mM, and diluted to 1mM and 0.2mM in a gradient for lateral ventricle injection in mice.
Preparing a solution of a P75 NTR inhibitor TAT-Pep 5: an appropriate amount of sample is weighed and dissolved in physiological saline to prepare 1mg/mL, and the sample is diluted into 0.2mg/mL and 0.04mg/mL in a gradient manner and used for lateral ventricle injection of mice.
2. The experiment of the head-shaking reaction proves the inhibition effect of Fasudil on the phantom effect induced by classical inhibitors
(1) Influence of Fasudil on classical inhibitor-induced hallucination
The head-flick response (HTR) is a rapid left-right rotation of the head, in the administration of 5-hydroxytryptamine or other 5-HT hallucinogens to rats and mice 2A The appearance of agonists followed by a head-flick response is widely used as 5-HT 2A Behavior determination of receptor activation. In classical hallucinogen studies, the head-flick response is the most commonly used animal model at present. There is a strong positive correlation between the effect of the mouse's head-flick response and the human-induced effects, i.e. the head-flick response is indicative of the creation of hallucinogenic behaviour.
The experimental mice described in this example were first administered to the lateral ventricle using the above-described tool drug (Fasudil). After waiting 30 minutes, the classical hallucinogens DOM or Psilocin were injected intraperitoneally. Immediately after injection, the mixture is placed in a transparent box for observation of the head-shaking reaction behavior, manual counting is carried out, and behavior test is carried out for 15 or 30 minutes.
Wherein, the drug dosage of DOM is 1mg/kg, and the DOM is injected by an intraperitoneal injection mode; the dosage of Psilocin was 0.5mg/kg and the injection was carried out by intraperitoneal injection. The tool drug (Fasudil) was injected as low (1 nmol/5. Mu.L), medium (5 nmol/5. Mu.L) and high (10 nmol/5. Mu.L) doses, each using lateral ventricular injection, 30min before the administration of the hallucinogen. The lateral ventricle injection administration volume was 5. Mu.L, and the intraperitoneal injection administration volume was 0.2mL/20g.
(2) Effect of the P75 NTR inhibitor TAT-Pep5 on classical inhibitor-induced hallucinations
This example further demonstrates the effect of the P75 NTR inhibitor TAT-Pep5 on classical hallucinogen DOM-induced mouse head flick behavior. In the experiment, the DOM drug dose is 1mg/kg, and the injection is performed by using an intraperitoneal injection mode. TAT-Pep5 was injected as low (0.2. Mu.g/5. Mu.L), medium (1. Mu.g/5. Mu.L) and high (5. Mu.g/5. Mu.L) doses, each by side ventricle injection, 30min before DOM administration.
3. Experimental results
The inhibition effect of Fasudil on the classical inhibitor induced illusion is shown in figures 3 and 4, and the left images in figures 3 and 4 show that Fasudil has a remarkable inhibition effect on the head throwing behavior and shows a change of a dose gradient, which indicates that Fasudil can remarkably inhibit the head throwing behavior of mice induced by classical illusion agents, namely the Fasudil has a remarkable inhibition effect on the illusion effect induced by classical illusion agents; the right panels in figures 3 and 4 show the change in the number of throws in the 15 or 30 minute head-flick response, with cumulative counts every 5 minutes, showing a significant difference in the number of throws that occurs between the Fasudil-dosed group and the control group at about 5 to 10 minutes after the administration of the hallucinogen. The above results further demonstrate that Fasudil can be applied in the preparation of drugs specifically inhibiting the hallucination.
The effect of the TAT-Pep5 inhibitor on the throwing behavior of mice induced by classical hallucinogens is shown in fig. 5, and the left graph in fig. 5 shows that the low, medium and high doses of TAT-Pep have no obvious inhibition or upregulation effect on the throwing behavior of mice, indicating that in Nogo-A/RhoA signaling pathway, the P75 NTR receptor and RhoA activation downstream thereof are not involved in hallucinogenic effects; the right graph in fig. 5 shows the change of the number of times of head shaking in 30 minutes of head shaking experiment, and the cumulative count is carried out every 5 minutes, so that the four groups of head shaking frequency curves have no obvious difference, namely, the proteins in the Nogo-A/RhoA signal channels are not inhibited to inhibit the illusion.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
Claims (10)
- Use of a rho kinase inhibitor in the manufacture of a medicament for specifically inhibiting the hallucination.
- 2. The use according to claim 1, wherein the Rho kinase inhibitor is Fasudil.
- 3. The use according to claim 2, wherein the hallucinations are 5HT 2A Receptor mediated hallucination.
- 4. The use according to claim 3, wherein the medicament further comprises one or more inert, non-toxic excipients that are pharmaceutically acceptable.
- 5. The use according to claim 4, wherein the excipient comprises a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, a binder, a stabilizer, a colorant, a perfume.
- 6. The use according to claim 5, wherein the medicament is in the form of an injection, capsule, granule, drop, lyophilisate, granulate or tablet.
- 7. The use according to claim 6, wherein the medicament is in the form of injection, capsule, granule, drop, freeze-dried product, granule or tablet by adding pharmaceutical auxiliary ingredient to Fasudil of 80% or more by mass fraction.
- 8. A pharmaceutical composition for specifically inhibiting the hallucinations, said pharmaceutical composition comprising an effective amount of a Rho kinase inhibitor.
- 9. The pharmaceutical composition of claim 8, wherein the Rho kinase inhibitor is Fasudil.
- 10. The pharmaceutical composition of claim 9, further comprising one or more inert, non-toxic excipients that are pharmaceutically acceptable.
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