CN118370834A

CN118370834A - Application of pregnane X receptor as target in preparation of antidepressant and prepared antidepressant

Info

Publication number: CN118370834A
Application number: CN202410287810.3A
Authority: CN
Inventors: 毕惠嫦; 王鹏; 欧阳辉; 杨潇; 方坚鸿
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2024-03-13
Filing date: 2024-03-13
Publication date: 2024-07-23

Abstract

The invention discloses application of a pregnane X receptor serving as a target spot in preparation of a medicament for preventing and treating depression and the prepared antidepressant medicament. The invention finds a new anti-depression target, namely a pregnane X receptor, and experimental results show that pregnane X receptor agonist pregnenolone-16 alpha-carbonitrile (PCN) can obviously increase sugar water preference rate and open field activity of depressed mice, obviously shorten tail suspension immobility time and forced swimming immobility time, obviously regulate neurotransmitter level in brain of depressed mice, obviously increase neuron dendrite length and dendrite branch number of depressed mice, and has obvious anti-depression effect superior to that of a positive control drug fluoxetine, and has the advantages of obvious anti-depression effect and quick effect. In contrast, PCN loses its antidepressant effect after knockout of the pregnane X receptor, i.e., PCN ameliorates depression by acting on the pregnane X receptor. According to the invention, the pregnane X receptor can be used as a target spot for preventing and treating depression, and pregnane X receptor agonists such as PCN can be developed into high-efficiency novel clinical antidepressants.

Description

Application of pregnane X receptor as target in preparation of antidepressant and prepared antidepressant

Technical Field

The invention belongs to the technical field of medicines. More particularly, the application of the pregnane X receptor as a target spot in preparing the medicament for preventing and treating the depression and the prepared antidepressant medicament.

Background

Depression is a common mental disorder associated with suicidal tendency, typical symptoms include low mood, insomnia or hypersomnia, appetite change, fatigue, feeling of spell or no value, excessive self-responsibility, difficulty in concentrating, etc., serious people may have symptoms such as hallucinations, delusions, etc., even suicidal ideation or behavior, seriously affect physical and mental health and quality of life of patients, and according to the research of world health organization, about 2.8 billions of people worldwide are saturated with the trouble of depression, and become one of global important disease burden. Moreover, the age-reduced depression is on an ascending trend and is increasingly lower in age, and children of 14 to 30 years old and even eight to nine years old account for about one fourth of thousands of people undergoing mental disease treatment each year; over 70 ten thousand people suicide each year for depression, and suicide is the fourth leading cause of death in teenagers aged 14-30 years.

The cause of depression is complex, the pathogenesis of the depression is not clear, and the present tendency considers that factors such as biology, psychology, social environment and the like are involved in the pathogenesis of depression, in particular the interaction between genetics and environmental or stress factors. Biological studies suggest that depression may be associated with a variety of factors such as reduced neurotransmitter secretion, apoptosis of nerve cells, inflammatory responses, and dysregulation of intestinal flora. Monoamine neurotransmitters such as norepinephrine, 5-hydroxytryptamine and dopamine have been implicated in depressed mood, anxiety, motor inhibition, inability to cope with symptoms such as irritation, anorexia, sleep disorders, circadian rhythm disorders and endocrine dysfunction in depressed patients. With the acceleration of the development pace of modern society, the life and work rhythm is also continuously accelerated, people can encounter a plurality of stress events in life, and chronic and long-term stress events can lead to long-term psychological stress, which is an important social environment factor for inducing depression.

The main means of clinical treatment of depression is still traditional antidepressants. Commonly used antidepressants such as selective 5-hydroxytryptamine reuptake inhibitors SSRIs (e.g., fluoxetine, fluoxetine), 5-hydroxytryptamine and norepinephrine reuptake inhibitors SNRIs, norepinephrine and specific 5-hydroxytryptamine antidepressants NaSSA, 5-hydroxytryptamine receptor antagonists and 5-hydroxytryptamine reuptake inhibitors SARIs act primarily by modulating the concentration of synaptic cleavamine neurotransmitters (e.g., 5-hydroxytryptamine, norepinephrine, dopamine, etc.). However, the medicines have the problems of low clinical cure rate, slow onset of action and the like. The initial antidepressant treatment is only effective for about 55% of patients with depression, and the vast majority of antidepressant single drug treatments only reach clinical cure for 30% of patients.

Therefore, the search for a novel antidepressant drug with remarkable antidepressant effect and quick response has great clinical demands and is also a hotspot of the current international research.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the existing antidepressant medicaments and provide a novel antidepressant medicament with remarkable antidepressant effect and quick response. The research of the invention shows that the pregnane X receptor (Pxr) can be used as a target spot for treating depression, and the pregnane X receptor agonist pregnenolone-16 alpha-carbonitrile (PCN) has obvious antidepressant effect and quick response.

The first object of the invention is to provide the application of pregnane X receptor as target in preparing medicine for preventing and treating depression.

A second object of the present invention is to provide the use of a pregnane X receptor agonist in the manufacture of a medicament for the prevention and treatment of depression.

A third object of the present invention is to provide the use of a pregnane X receptor agonist in combination with other antidepressant ingredients for the preparation of a medicament for the prevention and treatment of depression.

The above object of the present invention is achieved by the following technical scheme:

The research data of the invention show that pregnenolone-16 alpha-carbonitrile (PCN) serving as an agonist of a pregnenX receptor (Pxr) has the effect of preventing and treating depression, and is specifically shown as follows: in the CRS and CUMS two classical depression animal model experiments, pregnenolone-16 alpha-carbonitrile has the effect of rapidly resisting depression, the depression symptoms of mice begin to be relieved within one day after 50mg/kg of pregnenolone-16 alpha-carbonitrile is injected, the sugar water preference rate and open field activity of the CRS and CUMS mice are obviously increased after 7 days of pregnenolone-16 alpha-carbonitrile injection, and the tail suspension immobility time and forced swimming immobility time are obviously shortened. Meanwhile, pregnenolone-16 alpha-carbonitrile can obviously regulate the level of neurotransmitters in brain of a depressed mouse, obviously increase the length of dendrites and the number of dendrite branches of neurons of the depressed mouse, and has antidepressant effect superior to that of a positive control drug fluoxetine. On the other hand, after the pregnane X receptor (Pxr) is knocked out, the PCN loses the antidepressant effect, and the fact that the pregnane X receptor is taken as a target is very critical to the antidepressant effect of the PCN, namely, the PCN can prevent and treat the depression by acting on the pregnane X receptor.

The present invention therefore protects the following scheme:

The application of pregnane X receptor as target in preparing medicine for preventing and treating depression.

The pregnane X receptor is used as a target spot in screening medicaments for preventing and treating depression.

Use of pregnane X receptor in the manufacture of a medicament for the prevention and treatment of depression.

Use of a pregnane X receptor in combination with other antidepressant ingredients for the manufacture of a medicament for the prevention and treatment of depression.

Use of a pregnane X receptor agonist in the manufacture of a medicament for the prevention and treatment of depression.

Use of a pregnane X receptor agonist in combination with other antidepressant ingredients for the preparation of a medicament for the prevention and treatment of depression.

Use of a pregnane X receptor or an agonist thereof in combination with a pharmaceutically acceptable carrier for the manufacture of a medicament for the prevention and treatment of depression.

As an alternative, the pregnenolone X receptor agonist is pregnenolone-16 a-carbonitrile or a salt thereof.

More specifically, the salt is a sodium salt or a potassium salt.

More specifically, the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.

In addition, in the clinic, as an alternative embodiment, it is contemplated that a synergistic effect may be expected to be achieved by combining a pregnane X receptor agonist with other antidepressant ingredients such as any one or more of monoamine oxidase inhibitors, tricyclic antidepressants, selective 5-hydroxytryptamine reuptake inhibitors, 5-hydroxytryptamine and norepinephrine reuptake inhibitors, noradrenergic and specific 5-hydroxytryptamine receptor antagonists, 5-hydroxytryptamine antagonism and reuptake inhibitors, melatonin and 5-HT2C receptor antagonists, 5-hydroxytryptamine modulators and agonists, norepinephrine and dopamine reuptake inhibitors, ketamines.

The tricyclic antidepressant is selected from any one or more of imipramine, amitriptyline, clomipramine, doxepin and tenecteplatine; the selective 5-hydroxytryptamine reuptake inhibitor is selected from any one or more of fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram and escitalopram; the 5-hydroxytryptamine and the norepinephrine reuptake inhibitor are selected from any one or more of venlafaxine and duloxetine; the noradrenergic and specific 5-hydroxytryptamine receptor antagonists include mirtazapine; the 5-hydroxytryptamine antagonism and reuptake inhibitor is selected from any one or more of nefazodone and trazodone; the melatonin and 5-HT2C receptor antagonist comprises agomelatine; 5-hydroxytryptamine modulators and agonists include voathixetine; norepinephrine and dopamine reuptake inhibitors include bupropion; the ketamine comprises S-ketamine.

The medicine can be prepared into different dosage forms according to actual needs by a person skilled in the art, and the dosage forms of the medicine can be any one of powder, granules, tablets, capsules, pills, solutions and suspensions.

Meanwhile, the medicine of the invention can be prepared into common preparations, and can also be sustained release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.

For the purpose of preparing the administration unit into a tablet, various carriers well known in the art can be widely used. The carrier may be selected from: diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, and the like; humectants and binders, such as water, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate, -alpha-hydroxy acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may further be formulated as coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or as bi-or multi-layered tablets.

For the preparation of the dosage unit into a pill, various carriers well known in the art can be widely used. The carrier may be selected from: diluents and absorbents such as dextrose, lactose, starches, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, and the like; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, and the like; disintegrants, for example, agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, and the like.

For preparing the dosage unit into suppositories, various carriers well known in the art can be widely used. The carrier may be selected from: polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semisynthetic glycerases, etc.

For encapsulating the administration unit, various carriers known in the art can be widely used, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The carrier may be selected from: polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semisynthetic glycerases, etc.

For the preparation of the dosage unit into an injectable preparation, such as a solution, suspension, emulsion, lyophilized powder for injection, which may be aqueous or nonaqueous, one or more pharmaceutically acceptable carriers may be used, such as diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxy isostearyl alcohol, polyoxyethylene sorbitol lipase, and the like. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added.

Also, colorants, preservatives, flavors, flavoring agents, sweeteners or other materials may be added to the pharmaceutical formulation.

In addition, the present invention does not limit the dosage of antidepressant drugs. The appropriate dosage may be determined clinically based on clinical factors such as the severity of depression, sex, age, weight, character and individual response of the patient or animal suffering from depression, route or frequency of administration, etc. The therapeutic doses of the medicaments of the present invention can thus vary widely. In general, the therapeutic doses of the medicaments of the present invention are well known to those skilled in the art and can be suitably adjusted to achieve the desired therapeutically effective amount of the medicament according to the actual effective amount contained in the pharmaceutical formulation of the present invention. The antidepressant of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.

The administration route of the antidepressant can be intestinal tract or parenteral tract, such as oral administration, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum, etc., and the administration mode can be instillation or injection administration, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc.; the subject is a mammal, including a human.

The research results of the invention adopt various behavioral detection means to evaluate the drug effect of PCN, including sugar water preference experiments, tail suspension experiments, forced swimming experiments and open field experiments. Depression is accompanied by a variety of behavioral manifestations, one of which is a hedonic deficit (Anhedonia), and a sugar water preference test (Sucross PREFERENCE TEST, SPT) can be used to test for hedonic deficit symptoms, which are not prone to drinking Sucrose solutions when rodents are in a model of depression caused by chronic stress. Forced SWIMMING TEST (FST) and mouse tail suspension Tail Suspension Test (TST) are two commonly used animal behavior destinationally depressive disorder model experiments, and can better ensure the reliability of experimental results. The Open field test (Open FIELD TEST, OFT) of mice is a method for evaluating the autonomous behavior, exploring behavior and tension of experimental animals in a new heterogeneous environment.

The invention has the following beneficial effects:

The invention provides application of a pregnane X receptor (Pxr) serving as a target in preparation of a medicament for preventing and treating depression and the prepared antidepressant medicament. The research results of the invention show that in the experiments of two classical depressive animal models of CRS and CUMS, the pregnenolone-16 alpha-carbonitrile (PCN) serving as a pregnenolone X receptor agonist has the effects of remarkable antidepressant effect and quick effect, the depressive symptoms of mice begin to be relieved within one day after 50mg/kg PCN is dosed, the sugar water preference rate and open field activity of CRS and CUMS mice are remarkably increased after 7 days of PCN is dosed, and tail suspension immobility time and forced swimming immobility time are remarkably shortened; meanwhile, PCN can obviously regulate the level of neurotransmitter in brain of a depressed mouse, obviously increase the length of dendrite of neurons and the number of dendrite branches of the depressed mouse, and has antidepressant effect close to that of a positive control drug fluoxetine. On the other hand, after the pregnane X receptor (Pxr) is knocked out, the PCN loses the antidepressant effect, and the fact that the pregnane X receptor is taken as a target is very critical to the antidepressant effect of the PCN, namely, the PCN can prevent and treat the depression by acting on the pregnane X receptor. Thus, pregnane X receptors as targets are useful for the prevention and treatment of depression.

The invention not only provides a new reverse direction for the development of antidepressant drugs, namely a new target pregnane X receptor; also provided is a novel class of antidepressant drugs, namely pregnenolone X receptor agonists, including pregnenolone-16 alpha-carbonitrile (PCN).

In addition, the pregnane X receptor pregnenolone-16 alpha-carbonitrile (PCN) has the advantages of obvious antidepressant effect and quick response, is obviously better than a positive control drug fluoxetine in antidepressant effect, can be developed into a novel efficient clinical antidepressant drug, and has good clinical application value and prospect.

Drawings

Fig. 1: pharmacodynamic effects of PCN on CRS-induced depressed mice. A: CRS model animal experiment flow; b: CRS mice quality change; c: CRS mice sugar water preference rate change; d: the time change condition of tail suspension of the CRS mice; e: CRS mice forced swimming immobility time-varying conditions.

Fig. 2: pharmacodynamic effects of PCN on CUMS-induced depressed mice. A: animal experiment flow of CUMS model; b: the condition of change of the constitution of CUMS mice; c: CUMS mice sugar water preference rate change condition; d: the tail suspension time change condition of the CUMS mice; e: the time change condition of the forced swimming of the CUMS mice; f: the movement distance change condition of the CUMS mice in open field experiment; g: CUMS mice test the motion trail change condition in open field.

Fig. 3: effect of PCN on CRS mouse hippocampal neurotransmitter levels (hippocampal metabolite concentration).

Fig. 4: effect of PCN on hippocampal dendrite complexity in CUMS mice. Graph A is a dendritic shape; the upper graph in panel B shows the dendrite length and the lower graph shows the dendrite branching number.

Fig. 5: in Pxr knockout homozygote C57BL/6 mice, PCN was shown to have pharmacodynamic effects on CRS-induced depressed mice. A: CRS model animal experimental flow based on Pxr knockout homozygote C57BL/6 mice; b: CRS mice quality (BW) change; c: CRS mice sugar water preference rate (SPT) variation; d: CRS mice forced swimming immobility time (FST) change; e: CRS mice tail suspension immobility time (TST) variation; f: CRS mice open field experimental movement track change condition; g: CRS mice were subjected to open field experimental range of motion (OFT) change.

Fig. 6: in Pxr homozygous C57BL/6 mice, the effect of PCN on the dendrite complexity (A panel for dendrite shape; B upper panel for dendrite length, lower panel for dendrite branching number) and hippocampal neurotransmitter level (hippocampal metabolite concentration) of CRS-induced depressed mice (C).

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Unless otherwise indicated, reagents and materials used in the following examples were those available commercially.

The animal experiment materials, animal model construction methods and administration schemes referred to in the following examples are referred to as "experiment materials and methods".

Experimental materials and methods

Experimental medicine (a):

Pregnenolone-16α -carbonitrile (Pregnenolone 16α -carbonitrile, PCN): CAS number 1434-54-4, chemical formula C ₂₂H₃₁NO₂, relative molecular weight 416.5g/mol, purity 99.87%, purchased from Chengomant Biol.Co., ltd, and its structural formula and physicochemical properties are shown in formula I below:

fluoxetine (Fluoxetine): CAS number 54910-89-3, available from Shanghai Milin Biochemical technologies Co., ltd., product number F830634.

(II) experimental equipment: a cross bar; an adhesive tape; a camera; JUNSO multifunction timer; the mouse is put in a field behavior test box; centrifuge TDL-5 Centrifuge.

(III) experimental animals:

C57BL/6 mice: male, 20-25 grams in weight, supplied by Guangdong Kangdong Biotechnology Co., ltd., license number: SCXK (Yue) 2020-0054. Animals are fed in separate cages, the light and dark period is 12h/12h, the temperature is 20-22 ℃, water is free, feed is provided by the animal center of the university of south medical science, and experiments are carried out after 1 week.

Pxr knockout homozygote C57BL/6 mice: male, 20-25g, supplied by the Proprietary national institutes of health FRANK GONZALEZ, cultivated by the university of medical science, south and fed to the university of medical science laboratory animal center SPF-grade environment. Animals are fed in separate cages, the light and dark period is 12h/12h, the temperature is 20-22 ℃, water is free, feed is provided by the animal center of the university of south medical science, and experiments are carried out after 1 week.

(IV) experimental animal model construction method, experimental grouping and dosing scheme:

One) chronic constraint stress (chiral RESTRAINT STRESS, CRS) animal model

1. CRS animal model modeling method, experimental grouping and dosing regimen based on C57BL/6 mice

(1) The molding method comprises the following steps: after adaptively feeding male C57BL/6 mice for 1 week, carrying out chronic restraint stress for 4 hours in the same time period every day for 21 days; if during the dosing treatment, chronic restraint stress is performed 30min after the dosing. Chronic constraint stress refers to: mice were placed in a 50mL home-made restraint device and fasted for water deprivation during restraint.

(2) Experimental grouping: the model mice were randomly divided into model group (CRS), PCN group (crs+pcn) and fluoxetine group (crs+ Fluoxetine), while the non-model mice were randomly divided into blank control group (Con) and control PCN group (con+pcn), 8 each.

(3) Dosing regimen: the drug administration treatment is carried out on the 15 th day after the beginning of the molding, and the specific drug administration mode is as follows:

PCN group (crs+pcn): mice were injected intraperitoneally (ip) 1 time per day with 50mg/kg PCN and 10mL/kg corn oil (corn oil as solvent, mixed and injected) for 7 days;

Fluoxetine group (crs+ Fluoxetine): mice were injected intraperitoneally (ip) 1 time 20mg/kg fluoxetine (Flu) and 10mL/kg corn oil (corn oil as solvent, mixed and injected) for 7 days;

Model group (CRS): mice were intraperitoneally injected (ip) 1 time per day with 10mL/kg of corn oil for 7 days;

Blank control (Con): mice (not molded) were given 10mL/kg of corn oil 1 time per day by intraperitoneal injection (ip) for 7 days;

Control PCN group (con+pcn): mice (not molded) were injected intraperitoneally (ip) 1 time per day with 50mg/kg PCN and 10mL/kg corn oil (corn oil as solvent, mixed and injected) for 7 days.

2. CRS animal model modeling method, experimental grouping and dosing regimen based on Pxr knockout homozygote C57BL/6 mice

(1) The molding method comprises the following steps: male Pxr knockout homozygous C57BL/6 mice were subjected to chronic restraint stress for 4 hours for 21 days per day for the same period of time after being subjected to adaptive breeding for 1 week. Chronic constraint stress refers to: mice were placed in a 50mL home-made restraint device and fasted for water deprivation during restraint.

(2) Experimental grouping: the model mice were randomly divided into Pxr knockout model groups (Pxr KO +crs), pxr knockout model+pcn groups (Pxr KO +crs+pcn), 7 each.

Pxr knockout model+pcn group (Pxr KO +crs+pcn): mice were injected intraperitoneally (ip) 1 time per day with 50mg/kg PCN and 10mL/kg corn oil (corn oil as solvent, mixed and injected) for 7 days;

Pxr knockout model group (Pxr KO +crs): mice were given 10mL/kg of corn oil 1 time per day by intraperitoneal injection (ip) for 7 consecutive days.

(II) Chronic unpredictable Wen Heying laser (chronic unpredictable MILD STRESS, CUMS) animal model construction, experimental grouping and dosing regimen

(1) The molding method comprises the following steps: after adaptively feeding male C57BL/6 mice for 1 week, selecting 1 stimulation mode daily from seven stimulation modes of forbidden water 24h, ice water swimming at 4 ℃ for 5min, wet padding 24h, tail clamping 2min, day-night inversion 24h, noise stimulation 3h and constraint 4h, and randomly implementing 1 time for 28 days; if during the dosing treatment, chronic unpredictable Wen Heying challenges are randomized 1 of them 30min after dosing.

(2) Experimental grouping: the model mice were randomly divided into model groups (CUMS) and PCN groups (cums+pcn), while the non-model mice were randomly divided into blank control groups (Con) and control PCN groups (con+pcn), 8 each.

(3) Dosing regimen: the drug administration treatment was performed on day 22 after the beginning of the molding, and the specific drug administration method was as follows:

PCN group (cums+pcn): mice were injected intraperitoneally (ip) 1 time with 50mg/kg PCN and 10mL/kg corn oil (corn oil as solvent, mixed and injected) for 7 days;

Model Set (CUMS): mice were injected intraperitoneally (ip) 1 time with 10mL/kg of corn oil for 7 days;

blank control (Con): mice (not molded) were injected intraperitoneally (ip) 1 time with 10mL/kg of corn oil and administered for 7 consecutive days;

Control PCN group (con+pcn): mice (not molded) were injected intraperitoneally (ip) 1 time with 50mg/kg PCN and 10mL/kg corn oil (corn oil as solvent, mixed and injected) and administered for 7 days.

EXAMPLE 1 influence of PCN on body Mass of depressed mice

CRS mice: at the same time points of modeling on days 0, 7, 14 and 21, the quality of each group of CRS mice was measured, and the quality change condition from the initial modeling to the modeling on days 7, 14 and 21 was observed.

CUMS mice: at the same time points of modeling on days 0, 7, 14, 21 and 28, the body mass of each group of CUMS mice was measured, and the body mass change condition from the initial modeling to the modeling on days 7, 14, 21 and 28 was observed.

The results are shown in the graph B in FIG. 1 and the graph B in FIG. 2, and the detailed description is shown in the experimental results and analysis section.

Example 2 effect of PCN on sugar preference in depressed mice

CRS mice: the sugar water preference test was conducted 1 st time before the start of molding, and the sugar water preference test was conducted 7 th, 14 th, 15 th, 17 th, 19 th and 21 th days after the start of molding. If the test is performed 1h after the administration period of 15 to 21 days.

CUMS mice: the sugar water preference test was conducted 1 st day before the start of molding, and the sugar water preference test was conducted 7 th, 14 th, 21 st, 22 nd, 24 th, 26 th and 28 th days after the start of molding. If the test is performed 1h after the administration period of days 22 to 28.

The sugar water preference test method comprises the following steps: after water is forbidden for 24 hours, each mouse cage is placed with 2 quantitative water bottles, 1 bottle of pure water and 1 bottle of 1% syrup, and after 1 hour, the mice are removed, and the syrup preference rate (syrup consumption/total liquid consumption) of the mice is used as an evaluation index.

The results are shown in the graph C in FIG. 1 and the graph C in FIG. 2, and the detailed description is shown in the experimental results and analysis section.

Example 3 effect of PCN on tail-holding time in depressed mice

CRS mice: the tail suspension experiment is carried out on the 21 st day of molding, and the specific method is as follows: the 3/4 part of the mouse tail is fixed on the hook, the camera is placed in the horizontal direction with the mouse hanging device, then the time is counted down for 6min, and the immobility time of the mouse in the last 4min is recorded.

CUMS mice: the tail suspension experiment is carried out on the 28 th day of molding, and the specific method is as follows: the 3/4 part of the mouse tail is fixed on the hook, the camera is placed in the horizontal direction with the mouse hanging device, then the time is counted down for 6min, and the immobility time of the mouse in the last 4min is recorded.

The results are shown in the graph D in FIG. 1 and the graph D in FIG. 2, and the detailed description is shown in the experimental results and analysis section.

EXAMPLE 4 effect of PCN on forced swimming time in depressed mice

CRS mice: the forced swimming experiment is carried out on the 21 st day of molding, and the specific method is as follows: the mice were placed in a forced swimming bucket filled with water, allowed to swim in the bucket for 6min, and the immobility time of the mice was recorded for the last 4 min.

CUMS mice: the forced swimming experiment is carried out on the 28 th day of molding, and the specific method is as follows: the mice were placed in a forced swimming bucket filled with water, allowed to swim in the bucket for 6min, and the immobility time of the mice was recorded for the last 4 min.

The results are shown in the graph E in FIG. 1 and the graph E in FIG. 2, and the detailed description is shown in the experimental results and analysis section.

Example 5 effect of PCN on open field Activity in depressed mice

The open field experiment is carried out on the 28 th day of CUMS modeling, and the specific method comprises the following steps: and (3) placing the CUMS mouse in the central position of an open field experiment box, and recording the movement track of the mouse within 5 minutes by a camera.

The results are shown in the F graph and the G graph in FIG. 2, and the specific description is shown in the experimental results and the analysis part.

Example 6 influence of PCN on neurotransmitters in depressed mice

On day 22 of CRS modeling, the CRS mice were dissected and then separated, placed in an EP tube, 10mg of the mouse hippocampus was weighed, 100 μ L H ₂ O was added, 400 μL of MeOH/ACN (v: v=1:1) was added after homogenization, vortexing for 30 seconds, sonicated for 10 minutes (4 ℃ water bath), allowed to stand at-20 ℃ for 1 hour, centrifuged at 13000r/min and 4 ℃ for 15 minutes, the supernatant was taken, evaporated to dryness at 4 ℃ using a vacuum concentrator, re-melted with 80 μL of methanol was added, vortexing for 5 minutes, vortexing for 10 minutes at 4 ℃ and 13000r/min, and the supernatant was placed in a sample injection vial for LC-MS detection under the following specific detection conditions:

(1) Conditions of liquid chromatography

Mobile phase: 0.1% formic acid (A) and acetonitrile (B).

Gradient elution procedure: 0-2.5 min,5% B; 2.5-4 min,5% -20% of B; 4-7 min, 20-60% B; 7-7.5 min, 60-5% B; 7.5-10 min,5% B.

Flow rate: 0.2mL/min.

Column temperature: 30 ℃.

Sample injection amount: 3. Mu.L.

(2) Mass spectrometry conditions

Electrospray ion source (ESI): a positive ion mode;

multiple reaction monitoring mode (MRM) gas curtain gas: 40.0psi;

Collision gas: n ₂;

spray voltage: 5500V;

Ion source temperature: 500 ℃;

GS1：50psi，GS2：50psi；

gas flow rate: 12L/min.

The results are shown in FIG. 3, and the detailed description is shown in the experimental results and analysis section.

Example 7 effect of PCN on neuronal dendrites in depressed mice

On the 29 th day of CUMS molding, equal amounts of solution A and solution B were mixed according to the instructions of the Golgi staining kit to prepare an impregnating solution, the brains of all experimental mice were taken out, the brains of the mice were placed in the impregnating solution, after 14 days of standing, the brains were transferred to the C solution and stored for 3 days, and after the brain tissues in the C solution were cut into 150 μm coronal whole brain sections using a frozen microtome, staining was performed strictly according to the steps of the instructions of the Golgi staining kit. The mouse hippocampal CA1 cone neuron dendrites were observed using an optical microscope, and dendrite branch counts and dendrite length measurements were performed using Image J1.54 software.

The results are shown in FIG. 4, and the detailed description is shown in the experimental results and analysis section.

Example 8 pharmacodynamic effects of PCN on CRS-induced depressed mice in Pxr knockout mice

At the same time point of the molding day 21, the quality of the CRS mice was measured, and the quality change from the initial molding to the molding day 21 was observed.

The sugar water preference test is carried out for 1 time on the 21 st day of molding, and the specific method is as follows: after water is forbidden for 24 hours, each mouse cage is placed with 2 quantitative water bottles, 1 bottle of pure water and 1 bottle of 1% syrup, and after 4 hours, the mice are removed, and the syrup preference rate (syrup consumption/total liquid consumption) of the mice is used as an evaluation index.

1 Forced swimming experiment is carried out on the 21 st day of molding, and the specific method is as follows: the mice were placed in a forced swimming bucket filled with water, allowed to swim in the bucket for 6min, and the immobility time of the mice was recorded for the last 4 min.

1 Tail suspension experiment is carried out on the 21 st day of molding, and the specific method is as follows: the 3/4 part of the mouse tail is fixed on the hook, the camera is placed in the horizontal direction with the mouse hanging device, then the time is counted down for 6min, and the immobility time of the mouse in the last 4min is recorded.

1 Time of open field experiment is carried out on the 21 st day of molding, and the specific method is as follows: and (3) placing the CRS mice in the central position of an open field experiment box, and recording the movement track of the mice within 5 minutes by a camera.

The results are shown in FIG. 5, and the detailed description is shown in the experimental results and analysis section.

Example 9 effects of PCN on neuronal dendrites and neurotransmitters in Pxr knockout mice in CRS-induced depressed mice

On day 22 of CRS modeling, the mouse brain was removed and golgi stained and examined as in example 7.

On day 22 of CRS modeling, CRS mice were dissected and hippocampus isolated, placed in EP tubes, and sampled and LCMS on-board analyzed as in example 6.

The results are shown in FIG. 6, and the detailed description is given in the experimental results and analysis section.

Experimental results and analysis:

As shown in fig. 1 and 2, the weights (B panels in fig. 1 and 2) and sugar water preference rates (C panels in fig. 1 and 2) of CRS-depressed mice and CUMS-depressed mice were significantly reduced, tail suspension immobility times (D panels in fig. 1 and 2) and forced swimming immobility times (E panels in fig. 1 and 2) were significantly prolonged (P < 0.05), and the open field locomotion distances (F and G panels in fig. 2) of CUMS mice were significantly shortened (P < 0.05) compared to the blank control group (Con). After PCN administration, the weight and sugar preference rate of CRS and CUMS mice can be obviously up-regulated, the tail suspension and forced swimming immobility time (P < 0.05) can be obviously down-regulated, the open field exercise level of the CUMS mice can be improved, and the open field exercise distance of the CUMS mice can be obviously increased. Meanwhile, the drug effect of PCN on the body mass and sugar water preference rate is stronger than that of fluoxetine, which shows that PCN can obviously improve the mouse depression-like behavior caused by CRS and CUMS.

As shown in fig. 3, 5-HT, DA, GABA and 5-HIAA were significantly reduced in the hippocampus of CRS mice compared to the placebo group (Con), glu, KA, kyn was significantly increased (P < 0.05), and post PCN administration, levels of 5-HT, DA, GABA, 5-HIAA, glu, KA and Kyn in the hippocampus of CRS mice were significantly reversed (P < 0.05), revealing that PCN was able to significantly modulate neurotransmitter levels in the brain of depressed mice.

As shown in fig. 4, compared with the blank control group (Con), the CUMS mouse neuron dendrite length was significantly reduced, the dendrite branching number was significantly reduced, and the CUMS mouse dendrite length and dendrite branching number after PCN administration could be significantly increased.

As shown in fig. 5, compared with Pxr KO +crs, PCN administration did not affect the sugar water preference rate, tail suspension immobility time, forced swimming immobility time, and open field exercise level of Pxr KO +crs+pcn mice, indicating that Pxr is very critical for the antidepressant effect of PCN.

As shown in fig. 6, no effect on Pxr KO +crs+pcn mouse neuron dendrite length, dendrite branch number and neurotransmitter level was observed after PCN administration compared to Pxr KO +crs, indicating that Pxr is critical for the antidepressant effect of PCN.

In conclusion, PCN can remarkably improve the depression-like state caused by CRS and CUMS, can regulate neurotransmitter and dendritic complexity of depressed mice, can start to relieve the depression symptoms of the mice within one day after PCN administration, can remarkably improve the depression symptoms of the mice after PCN administration for 7 days, has stronger drug effect than fluoxetine, and has the advantages of quick and good effect. Meanwhile, after the pregnane X receptor (Pxr) is knocked out, the PCN loses the anti-depression effect, and the pregnane X receptor serving as a target is very critical to the anti-depression effect of the PCN. That is, the invention proves that the PCN can prevent and treat the depression by acting on the pregnane X receptor, so that the pregnane X receptor can be used as a target spot for preventing and treating the depression, and the pregnane X receptor agonist PCN can be developed into a novel efficient clinical antidepressant.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The application of pregnane X receptor as target in preparing medicine for preventing and treating depression.

2. The pregnane X receptor is used as a target spot in screening medicaments for preventing and treating depression.

3. Use of pregnane X receptor in the manufacture of a medicament for the prevention and treatment of depression.

4. Use of a pregnane X receptor in combination with other antidepressant ingredients for the manufacture of a medicament for the prevention and treatment of depression.

5. Use of a pregnane X receptor agonist in the manufacture of a medicament for the prevention and treatment of depression.

6. Use of a pregnane X receptor agonist in combination with other antidepressant ingredients for the manufacture of a medicament for the prevention and treatment of depression.

7. Use of a pregnane X receptor agonist in combination with a pharmaceutically acceptable carrier for the manufacture of a medicament for the prevention and treatment of depression.

8. The use according to any one of claims 5 to 7, wherein the pregnenolone X receptor agonist is pregnenolone-16 α -carbonitrile or a salt thereof.

9. The use according to claim 8, wherein the salt is a sodium or potassium salt.

10. The use according to any one of claims 1 to 7, wherein the pharmaceutical dosage form is any one of a powder, a granule, a tablet, a capsule, a pill, a solution, a suspension.