CN113893346A - Application of GCS inhibitor in preparation of drug for treating cocaine addiction - Google Patents

Application of GCS inhibitor in preparation of drug for treating cocaine addiction Download PDF

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CN113893346A
CN113893346A CN202010577209.XA CN202010577209A CN113893346A CN 113893346 A CN113893346 A CN 113893346A CN 202010577209 A CN202010577209 A CN 202010577209A CN 113893346 A CN113893346 A CN 113893346A
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cocaine
gcs
mice
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eliglustat
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CN113893346B (en
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岑小波
蒋林宏
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West China Hospital of Sichuan University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/30Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/36Opioid-abuse

Abstract

The invention discloses an application of a GCS inhibitor in preparing a medicine for treating cocaine addiction, belonging to the field of drug rehabilitation medicines. Experiments prove that the GCS inhibitor can specifically reduce the content of glucosylceramide sphingolipids in nucleus accumbens brain areas, reduce the dendritic branch number and dendritic spine density, and effectively inhibit the behavioral effects (behavioral sensitization, reward effect and self-administration behavioral effects) of cocaine on mice. The GCS inhibitor is used for preparing the drug for treating cocaine addiction, and has good application prospect.

Description

Application of GCS inhibitor in preparation of drug for treating cocaine addiction
Technical Field
The present invention belongs to the field of drug-dropping medicine.
Background
Cocaine (Cocaine), also known as Cocaine, chemically known as phenacyl ecgonine (methybizoylecgonine), generally appears as a white crystal, is odorless, bitter and numb in taste, is the strongest natural central stimulant used for local anesthesia and asthma treatment for the earliest time, is abused due to its excitatory effect on the central nervous system, and has become one of the major drugs in the world in 1985.
Cocaine addiction is a chronic recurrent brain disease, belongs to drug dependence (drug dependence) diseases, can cause plasticity changes of brain structures and functions, related brain areas comprise nucleus accumbens, striatum, prefrontal cortex, hippocampus and ventral tegmental area, and health is also harmed in various aspects, including mental abolishness, personality impairment, mental dysfunction, corresponding infection complications and various illegal criminal activities induced by drug addicts who seek and use drugs indiscriminately.
Cocaine addiction is characterized by compulsive solicitation and use to satisfy cravings, uncontrolled quests and solicitations for drugs, loss of interest in things, and profound memory of addiction, even after years of withdrawal treatment, which can induce relapse upon exposure to addiction-related stimuli (e.g., drug friends, circumstances associated with past medications, etc.).
Due to the fact that the cocaine addiction hazard is large, a proper treatment target and a proper medicine are found, and cocaine addiction treatment is imminent.
Glucose Ceramide Synthase (GCS) belongs to the family of glycosyltransferases, widely present in the cell membrane of eukaryotes and essentially consists of 394 amino acids. The current research on GCS and the GCS inhibitor mainly focuses on the occurrence of multidrug resistance mechanism of tumors, and no report on the relationship between GCS and cocaine addiction is found.
Disclosure of Invention
The invention aims to solve the problems that: provides the use of a GCS inhibitor in the preparation of a medicament for the treatment of cocaine addiction.
The term "GCS inhibitor" refers to a substance that specifically inhibits the action of GCS protein, GCS gene or GCS gene and ultimately leads to a reduction in the activity or content of GCS protein, including: small molecule drugs, miRNA, siRNA, shRNA, miRNA mics, miRNA agomir and the like, which can recognize and bind GCS protein.
The technical scheme of the invention is as follows:
use of a GCS inhibitor for the manufacture of a medicament for the treatment of cocaine addiction.
As for the previous use, the GCS inhibitor is Eliglustat, and the structural formula is as follows:
Figure RE-GDA0002613195780000021
the use as aforesaid, the medicament is a pharmaceutical formulation for injection.
The use as described above, wherein the medicament is a medicament for reducing the content of glucosylceramide sphingolipids in the nucleus accumbens brain region.
As previously mentioned, the medicament is a medicament that reduces the number of dendritic branches and dendritic spine density in the nucleus accumbens region.
A medicine for treating cocaine addiction comprises a GCS inhibitor as an active ingredient.
The GCS inhibitor is Eliglustat as the medicine, and the structural formula of the GCS inhibitor is as follows:
Figure RE-GDA0002613195780000022
the medicament as described above, which is a pharmaceutical formulation for injection.
The invention has the beneficial effects that:
the GCS inhibitor can reduce the content of glucose ceramide sphingolipid in nucleus accumbens brain area, reduce the dendritic branch number and dendritic spine density, and effectively inhibit the behavioral effects (behavioral sensitization, reward effect and self-administration behavioral effects) of cocaine on mice. The GCS inhibitor is used for preparing the drug for treating cocaine addiction, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Figure 1 mouse behaviour sensitization experimental box.
FIG. 2 a conditional location preference experimental box.
Figure 3 self-administration experimental box.
FIG. 4 cocaine-induced behavioral sensitization and conditional site preference modeling in mice. (A) Schematic representation of the cocaine-induced behavioral sensitization model operation. (B) Cocaine significantly enhanced spontaneous locomotor activity in mice, t-test,. p < 0.001. Cocaine group n is 14, saline group n is 14. (C) Schematic representation of cocaine-induced conditional location preference model operation. (D) Cocaine significantly reversed the reward effect in mice, t-test,. p < 0.001. Cocaine group n 15, saline group n 15.
FIG. 5 increased expression of GCS in the nucleus accumbens. In the behavior sensitization effect, the expression of GCS in nucleus accumbens brain area of cocaine mice is obviously up-regulated. t test, p <0.05, cocaine group n-4, saline group n-4. In the conditioned place preference behavior, the expression of GCS in the nucleus accumbens brain region of cocaine mice was significantly up-regulated. t test, p <0.05, cocaine group n-6, saline group n-6.
Figure 6 multiple cocaine injections specifically increased nuclear vodka GCS protein expression. Compared with the normal saline group, the level of nucleus accumbens GCS protein is obviously increased, and the level of the GCS protein in the prefrontal cortex, the striatum and the hippocampal brain area does not show obvious change. Prefrontal cortex (PFC), nucleus accumbens (NAc), Striatum (Striatum), hippocampus (Hippoampus), t-test, # p <0.05, cocaine group n-3, saline group n-3.
Figure 7 time-dependent effect of cocaine-induced increased GCS expression from nucleus accumbens. The expression of nucleus accumbens GCS increased statistically and steadily at day 3 with continuous intraperitoneal injection of cocaine compared to normal saline. t-test, p <0.05, p <0.01, p < 0.001. Cocaine group n is 4, saline group n is 4.
Figure 8 food-induced juxtaglomerular GCS expression did not change significantly. (A) Through food-induced mouse CPP training, compared with a control group, a food group mouse forms an obvious reward effect; t-test,. p < 0.01. Cocaine group n-8, saline group n-8. (B) In the food CPP model, compared with a control group, the food group mice have no significant change in the expression of nucleus accumbens GCS; t test, n.s has no significant change, cocaine group n is 4, and normal saline group n is 4.
Figure 9Eliglustat attenuated cocaine-induced behavioral sensitization effects. (A) Behavioral sensitization model operating schematic, Eliglustat was administered site-specifically through the nucleus accumbens brain region 15min prior to cocaine administration. (B) Eliglustat inhibits behavioral sensitization concentration screening, and Eliglustat with high, medium and low doses is set, and the high dose Eliglustat can obviously inhibit the behavioral effect caused by cocaine. Two-way ANOVA analysis closed by bonferroni post-tests, p <0.05and p < 0.01. Cocaine solvent group, n ═ 9; cocaine +5 μ M eligliostatt group, n ═ 6; cocaine +20 μ M eligliostatt group, n ═ 9; cocaine +100 μ M eligliostatt group, n ═ 6. (C) Eliglustat significantly attenuated the cocaine-induced behavioral sensitization effect. Two-way ANOVA analysis closed by bonferroni post-tests, p <0.05and p < 0.01. A physiological saline solvent group, n is 9; saline + Eliglustat group, n is 6; cocaine solvent, n ═ 9; cocaine + eligliistat group, n 6.
Figure 10Eliglustat attenuated cocaine-induced behavioral sensitization effect glucose ceramide content. Eliglustat reduced the content of glucosylceramides with chain lengths of C22: 1and C24: 1. One-way ANOVA, # p <0.05, # p < 0.01. A physiological saline solvent group, n is 6; saline + Eliglustat group, n is 8; cocaine solvent group, n ═ 6; cocaine + eligliistat group, n 6.
FIG. 11Eliglustat attenuated the cocaine-induced conditional locus bias effect. (A) Schematic operating diagram of the conditioned place preference model, Eliglustat was administered site-specifically through the nucleus accumbens brain region 15min prior to cocaine administration. (B) Eliglustat inhibits the conditioned place preference concentration screening, and the Eliglustat is set to high, medium and low doses, and the Eliglustat at high dose can obviously inhibit the conditioned place preference behavioral effect caused by cocaine. One-way ANOVAis fallen by bonferroni post-tests, p < 0.05. Cocaine solvent group, n ═ 9; cocaine +5 μ M eligliostatt group, n ═ 11; cocaine +20 μ M eligliostatt group, n ═ 11; cocaine +100 μ M eligliostatt group, n ═ 8. (C) Eliglustat significantly attenuated the cocaine-induced conditional site preference effect. One-way ANOVA is closed by bonferroni post-tests, p <0.01and p < 0.001. A physiological salt solvent group, n is 12; saline + Eliglustat group, n is 12; cocaine solvent group, n ═ 12; cocaine + eligliistat group, n-12.
Figure 12Eliglustat attenuated cocaine-induced conditional site-bias effect mouse glucose ceramide content at pv septa. One-way ANOVA is closed by bonferroni post-tests, # p <0.05, # p <0.01, # p < 0.001. A physiological salt solvent group, n is 7; saline + Eliglustat group, n is 7; cocaine solvent group, n ═ 7; cocaine + eligliistat group, n ═ 8.
Figure 13Eliglustat attenuated cocaine-induced behavior effects of self-administration. (A) Eliglustat inhibits the effective nasal contact number of self-administration and attenuates the effects of cocaine-induced self-administration. Two-way ANOVA analysis closed by bonferroni post-tests, p <0.05and p < 0.01. A physiological salt solvent group, n is 7; saline + Eliglustat group, n is 6; cocaine solvent group, n ═ 7; cocaine + eligliistat group, n 7. (B) Eliglustat inhibits the number of self-administered doses to attenuate the cocaine-induced self-administered effects. Two-way ANOVA analysis closed by bonferroni post-tests, p <0.05, p <0.01and p < 0.001. A physiological salt solvent group, n is 7; saline + Eliglustat group, n is 6; cocaine solvent group, n ═ 7; cocaine + eligliistat group, n 7.
FIG. 14 Effect of microinjection of nucleus accumbens by Eliglustat at voltage on synaptic plasticity in mice. Panel a. eliglustat significantly attenuated cocaine-induced synaptic branching. One-way ANOVA is closed by bonferroni post-tests,. p < 0.01; panel b. eliglustat significantly attenuated cocaine-induced dendritic spine density. One-way ANOVA is fallen by bonferroni post-tests,. p < 0.05; four mice in each group, 6 neuron statistics are collected from each mouse; image J simulates the delineation of dendritic branches and dendritic spine morphology.
Detailed Description
Example 1 GCS inhibitors of the invention treat cocaine analgesia
1 test reagent
The reagents used in this section were as follows:
cocaine hydrochloride (Chinese food and drug testing institute)
Physiological saline (Sichuan Kelun pharmaceutical Co., Ltd.)
RIPA lysate (Shanghai Biyuntian biotechnology limited, P0013B)
BCA kit (Shanghai Bin Yuntian biotechnology limited, P0010)
5 xSDS-PAGE electrophoresis sample buffer (Shanghai Biyuntian biotechnology, Inc., P0015)
7.5% SDS-PAGE reagent (Guangzhou Baihe Biotechnology Co., Ltd., PG111)
10% SDS-PAGE reagent (Guangzhou Baihe Biotechnology Co., Ltd., PG112)
12.5% SDS-PAGE reagent (Guangzhou Baihe Biotechnology Ltd, PG113)
Methanol (Shanghai chemical reagent Co., Ltd.)
Protein Prestaining marker (Thermo Scientfic, 26616)
Tris-base(Solarbio,G8200)
Glycine (Solarbio, T8060)
anti-GCS antibody (Shanghai biological engineering Co., Ltd.)
anti-Tubulin antibody (Cell Signaling Technology, #15115)
Horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, #8887)
BeyoECL (Shanghai Biyuntian biotechnology limited, P0018)
Tween-20(Biorad,#1706531)
RNA extraction kit (Axygen, AP-MN-P-50)
Isopropanol (Shanghai chemical reagent Co., Ltd.)
100% ethanol (Shanghai chemical reagent Co., Ltd.)
DEPC water (Shanghai Biyuntian biotechnology limited company)
RNAstore sample preservation solution (Tiangen Biochemical technology Co., Ltd., DP408)
BestarTM qPCR RT Kit(
Figure RE-GDA0002613195780000051
Bioscience,DBI-2220)
Stormstar SYBY Green qPCR Mastermix(
Figure RE-GDA0002613195780000052
Bioscience,DBI-2243)
HPLC methanol (Sigma, 34860)
HPLC ethyl acetate (Sigma, 650528)
HPLC isopropanol (Sigma, 34863)
Formic acid (Sigma, 695076)
LC-MS ammonium formate (Sigma, 516961)
LC-MS sodium formate (Sigma, 71539)
LC-MS leucine enkephalin (Waters)
PE(17:0/17:0)(Avanti,830756)
LPC(17:0)(Avanti,855676)
Glucosylceramide(C18:1/16:0)(Avanti,860539)
Glucosylceramide((C18:1/18:0)(Avanti,860548)
Glucosylceramide((C18:1/24:1)(Avanti,860549)
Eliglustat hemitartrate(MedChemExpress,HY-14885A)
DMEM high-sugar medium (Hyclone, 41966052)
Fetal bovine serum (Hyclone, 10100139)
Phosphate buffer (Hyclone, 10010049)
Dimethyl sulfoxide (Sigma, V900090)
Nerve Growth Factor(Thermo Scientfic,13257019)
2 Main Instrument
Electronic analytical balance (Sartorius)
Super clean bench (Shanghai Boxun industry Co., Ltd medical equipment factory)
Refrigerator (Elex)
Deep low temperature refrigerator (Thermo Scientific)
Ice machine (Scotsman)
Flat shaking table (Kaimen kylin medical instrument factory)
Adjustable vortex suspension instrument (SCILOGEX)
Ultrasonic crusher (Ningbo Xinzhi biological science and technology Co., Ltd.)
Constant temperature water bath (Guohua electric appliance Co., Ltd.)
Inverted biological microscope TS100(Nikon)
Multi-tube rack automatic balance centrifuge (Changshan Xiang instrument centrifuge Co., Ltd.)
LifeECO Gene amplification apparatus (Hangzhou Bori science and technology Co., Ltd.)
Electrophoresis apparatus (American Bio-Rad company)
Electrophoresis tank (American Bio-Rad company)
Western blot gel imaging system (Shanghai Qixiang scientific instruments Co., Ltd.)
Constant temperature shaking table (Shanghai Zhicheng analytical instrument manufacturing Co., Ltd.)
Full wavelength plate reader (Thermo Electron Corporation)
CFX96TM Real-Time System (Bio-Rad, USA)
Mouse brain stereotaxic instrument (Shenzhen Riwode Biotech Co., Ltd.)
Mouse spontaneous activity detection box (Shenzhen Riwode Biotech Co., Ltd.)
Mouse conditional position preference detection box (Ningbo' anlai software science and technology limited)
Mouse self-administration detection box (Ningbo' anlai software science and technology Co., Ltd.)
Deep low temperature refrigerator (Thermo Scientific)
5 mul flat head microsyringe (Shenzhen Riwode Biotech Co., Ltd.)
Carbon dioxide incubator IGO150(Jouan)
Super clean bench (Shanghai Boxun industry Co., Ltd medical equipment factory)
Constant temperature water bath (Guohua electric appliance Co., Ltd.)
Inverted biological microscope TS100(Nikon)
UPLC-Qtof-MS/MS (G2-S) separation detector (Waters)
C18 chromatographic separation column (Waters)
Ultrasonic crusher (Ningbo Xinzhi biological science and technology Co., Ltd.)
Mini pipet (Eppendorf)
3 Experimental methods
3.1 test animals
The wild type animals used in this study were male SPF grade healthy mature (8-12 weeks old) C57BL/6J mice, supplied by Beijing Wintolite laboratory animal technology, Inc., weighing 20-22g, and not mated. Feeding conditions are as follows: the temperature of a common animal house of the national (Chengdu) new drug preclinical safety evaluation center is 20-25 ℃, the relative humidity is 55-65%, in the whole experiment process, animals freely eat and drink water, and the feeding environment meets the standard GB14925-2001 in Experimental animal environment and facilities. All animal experimental operations related to the subject meet AAALAC requirements, and experimental animals need to be normally raised for 3-5 days before experiments so as to enable mice to be familiar with and adapt to the environment.
3.2 multiple cocaine injections
Multiple cocaine injections are mainly given according to the literature dosing scheme. The method specifically comprises the following steps: the injection is made by injecting cocaine 20mg/kg once per day for 7 days. And after the cocaine is injected for the last time for half an hour, taking out four brain area samples of prefrontal cortex, striatum, nucleus accumbens and hippocampus according to the stereotaxis of the mouse brain for Western blot detection.
The specific procedures for multiple cocaine injections to detect the time effect were: injecting cocaine 20mg/kg intraperitoneally once a day for 1, 2, 3, 5and 7 days. And taking out nucleus accumbens by referring to the stereotaxic atlas of the mouse brain half an hour after the cocaine is injected for the last time.
3.3 cocaine-induced behavioral sensitization model establishment
The behavior sensitization experiment box is made of organic glass into four square boxes with the same size, a smooth black organic glass plate is arranged around each box, a smooth white organic glass plate is arranged at the bottom of each box, and the behavior sensitization experiment box device is shown in figure 1. The mice were allowed to move freely in the experimental box, acclimatized for three days, 10min a day. The behavioral sensitization experiment is divided into 2 stages, and cocaine and physiological saline are injected according to the table 3-1 in the experimental process. First stage (day 0): without any treatment, the distance traveled by all mice within 15min was recorded as a base value using Ethovision XT from Noldus corporation. Second stage (day1-day 7): cocaine was given to the cocaine group at the same time per day, and the saline group was given an equivalent dose of saline for 7 consecutive days, 1 time per day. The dosing schedule is shown in table 1. Immediately after administration, the mice were placed in a behavior sensitization box and the distance traveled by the mice was recorded for 15 min. The experimental results were analyzed using SPSS statistical software, the differences were expressed as mean. + -. standard error, the difference between cocaine and saline groups was compared using two-tailed t-test analysis, and p <0.05 indicated a statistical difference.
Table 1 behavioral sensitization dosing schedule
Figure RE-GDA0002613195780000071
3.4 cocaine induces the mouse conditional Place Preference model (Conditioned Place Preference, CPP)
The CPP cabinet is shown in FIG. 2. The CPP experiment box consists of a black box and a white box which are made of plastic clapboards and a middle gray box, wherein the black box is provided with black four walls and a round hole rough ground, and the white box is provided with white four walls and a stripe type rough bottom surface. The mice freely move in the experimental box, adapt to the environment for three days, 15min each time. The CPP experiment was divided into 3 stages, the first stage (day 1): mice were tested for natural preference. Second stage (day2-day 7): a training stage, during which, a channel between the boxes is sealed by a plastic partition board, cocaine is injected on 2, 4 and 6 days, the mouse is immediately placed in a non-preference box, physiological saline is injected on 3, 5and 7 days, the mouse is immediately placed in a preference box, and the residence time of the mouse in the box is 15min each time; third stage (day 8): and a testing stage, wherein the plastic partition plate is removed, the mouse is allowed to freely move in the box, and the residence time of the mouse in the black box and the white box is recorded for 15 min. The dosing schedule is shown in table 2. The mice were dissected rapidly within 30min after the test was completed and the nucleus accumbens were removed for subsequent testing.
The results are expressed as a comparison of the time difference between the natural preference state and the preference state, using the time difference between the conditional place preference test period as the preference value after induction, minus the non-preference box dwell time difference. The experimental results were analyzed using SPSS statistical software, the differences were expressed as mean. + -. standard error, the difference between cocaine and saline groups was compared using two-tailed t-test analysis, and p <0.05 indicated a statistical difference.
TABLE 2 conditional site preference dosage regimen
Figure RE-GDA0002613195780000081
3.5 cocaine model establishment
3.5.1 mouse jugular vein cannulation Retention
The jugular vein intubation retention is the basis for establishing an autonomous administration model and is a key step for determining the success of establishing the model. Before operation, the experimental animal adapts to the experimental environment for 3-5 days, and contacts with the experimenters every day, so that the experimental animal is prevented from generating stress reaction. The jugular vein cannula is prepared from an inlet silica gel hard tube (the outer diameter is 0.48mm, the inner diameter is 0.40mm, and the length is about 5mm) and a matched silica gel soft tube (the length is about 3mm), and the soft tube is arranged at the front end of the hard tube so as to avoid stimulation to a mouse.
During operation, according to the mouse weight, the mouse is subjected to intraperitoneal injection of chloral hydrate (10 percent, 10ml/kg) to anaesthetize the mouse, the mouse hair at the clavicle part on the left side of the neck is removed, the head of the mouse is fixed in an supine position towards an experimenter, a longitudinal 1cm incision is formed in the upper end of the clavicle, subcutaneous tissues are separated in a blunt mode, the head of the mouse is pulled away from the external vein of the neck, and a small amount of physiological saline can be dripped in the whole process to prevent the skin and blood vessels from being dry. An oblique opening is cut on the vein by an ophthalmic scissors, the hose end of the previously prepared cannula is inserted into the vein, and the cannula is fixed by knotting a surgical thread. The inserted pre-fabricated catheter pre-fills the entire tubing with heparin sodium to prevent clotting. The catheter cap was then closed and the neck and back wounds were sutured closed. After the operation is finished, the mice are kept warm on an electric blanket until the mice are awakened, and then the mice are placed in clean cages and are bred in five cages. From the next day of operation, small amount of heparin sodium containing antibiotics is injected by intubation every day, so that the catheter is ensured to be unblocked, and wound infection is prevented. The post-operative recovery period for mice is typically 7 days. Mice that have completed this procedure can be used to establish a model for cocaine self-administration.
3.5.2 mice Self-administration (Self-administration)
The mouse self-administration system was manufactured by anlai software technologies ltd (nibo, china) and shown in fig. 3. Every control box all places in the sound insulation cabinet that has the ventilating fan, and equipment mainly includes two nose touch wares in the control box, a cage lamp, infusion connected system, a box lamp and food groove. In addition, the drug delivery system also comprises a syringe pump, an experimental animal behavior recording system and the like.
During the training period of cocaine self-administration in mice, FR1(fixed ratio-1) is the simplest and most basic training mode, i.e. the system automatically pumps out a preset volume of cocaine every time the mouse touches an effective nasal trigger. In the study, FR1 program was used for training, the experimental training time was 120 min/day, the maximum number of injections per round of training was 100, the refractory period of the device was 20s each time, cocaine was injected at 0.75mg/kg, the left nasal palpation was effective nasal palpation, and the right nasal palpation was ineffective nasal palpation. After the nose is effective each time, the cage lamp is turned off, the signal lamp is on, and after the nose is ineffective, no reaction is caused. After the model is successfully established, the mice are dissected within 2h after the last administration, and the nucleus accumbens brain area is taken out for subsequent detection.
The success judgment standard of the self-administration model is as follows: forming a conditional reflection; the frequency stability of cocaine is obtained autonomously; the number of injections for three consecutive days of the animal is within 10% of its mean value.
3.6 food-induced conditional place preference model
Mouse food CPP model establishment is similar to cocaine-induced CPP operation. Prior to modeling, a 7 day starvation acclimation period was first performed, each acclimation for 15 min. During the adaptation period, the mice were allowed to eat only 1h per day, and only water was allowed to drink freely, but no feed was provided for the rest of the time. After the seventh day, the mice were tested for weight change. Mice with a weight not less than 70% of the initial weight were selected at the end of the seventh day for food CPP training. Starting on the eighth day, the original preferences are detected, then randomly grouped, and conditioned training and testing are carried out to alternately establish a food CPP model. During training, the mice subjected to food conditioning training eat food for 1 hour in a feeding cage, and then are immediately placed in a preference box for training for 15 min; the non-food conditioning training is carried out by performing 1h of false feeding (placing in a feeding cage, but not giving feed), immediately placing in a non-preference box, and training for 15 min. The animals in the control group were not fed before training and were fed for 1h after training.
The results are expressed as a comparison of the difference between the conditional place preference test period, the preference box dwell time minus the non-preference box dwell time difference as the post-induction preference, and the natural preference time difference. The experimental results were analyzed using SPSS statistical software, the differences were expressed as mean. + -. standard error, the differences between the food-induced and control groups were compared using two-tailed t-test analysis, with p <0.05 indicating a statistical difference.
3.7 volt nuclear-isolated intracerebral fixed-point injection of GCS inhibitor
3.7.1V nucleus-isolated brain region embedded tube
Brain area positioning and tube embedding are direct means and common technology for brain area fixed-point drug delivery. In the study, 10% chloral hydrate (0.4g/kg body weight) is used for anaesthetizing mice, the tops of the heads of the mice are shaved, the anaesthetized mice are fixed on a brain stereotaxic apparatus, the heads of the mice are adjusted to be in a horizontal state after the fixation, the skin is disinfected by medical alcohol, the skull is exposed, the meninges on the surface of the skull are removed by cotton balls and ophthalmic scissors, the bregma position is found, and a marker pen marks the cephalic bregma position; adjusting a stereotaxic apparatus, arranging and fixing an injection catheter (Shenzhen Riword Biotechnology Limited, #62003, OD 0.48mm X ID 0.34mm), taking a front fontanel part as an origin, moving the catheter to be right above the injection part by referring to a positioning coordinate (nucleus accumbens: AP + 1.5; ML +/-1.2; DV-4.5), adjusting the bottom of the catheter to be just contacted with the skull, adjusting the zero Y coordinate, moving the injection needle downwards to 4.5mm deep, loosening a coordinate arm and moving away, and adjusting the dental powder and the diluent to be pasty fixed cannula. After the dental cement was dried, a catheter cap (Shenzhen Riword Biotech Limited, #62102, OD 0.30mm) was inserted to prevent catheter blockage and suture the top wound of the head. After the operation is finished, the mice are placed on an electric blanket and kept warm until the mice revive, and then the mice are placed into a clean rearing cage, and five mice are reared in the cage. The post-operative recovery period of the mice was 7 days, and whether the catheter was clogged was examined one day before the official experiment. Animals subjected to this surgery were used to study the change in behavioural effects induced by vallecular nucleus injection of the GCS inhibitor Eliglustat to regulate cocaine.
3.7.2 Von nucleus-isolated brain region fixed-point injection inhibitor
Mice, which were cannulated with bilateral septal nuclei, were dosed with the GCS inhibitor, Eliglustat, using a microinjection needle and microinjection catheter, according to the dosing dose in table 3. Eliglustat was formulated as a 100X stock in DMSO and diluted to available concentration with normal saline prior to use.
TABLE 3 GCS inhibitor injection dose
Figure RE-GDA0002613195780000101
3.8 tissue isolation and extraction
After the experimental detection of each group is finished, the neck is rapidly removed within the preset dissection time to kill the mouse, then the brain is rapidly separated, and after the brain is washed for 3 times by physiological saline at 4 ℃, the nucleus accumbens is separated and taken out according to the brain dissection map. Directly placing the nucleus into a prepared 1.5ml EP tube, quickly freezing and storing in liquid nitrogen, and finally completely storing at-80 ℃ until the tissue is collected for subsequent detection.
3.9 real-time fluorescent quantitative PCR
3.9.1RNA extraction
RNA was extracted according to the instructions of the RNA extraction kit (Axygen). The method mainly comprises the following steps: adding the nucleus accumbens brain region obtained by material drawing into 200 μ l Buffer R-I, repeatedly sucking with 1ml syringe for 10-15 times, adding 75 μ l Buffer R-II, vortex shaking for 15-30s, centrifuging at 4 deg.C and 12000g for 5 min. The supernatant was taken, and 125. mu.l of isopropanol was added and mixed well. The preparation tube in the kit is placed in a 2ml centrifuge tube, the mixed solution is added into the preparation tube, and the mixture is centrifuged at 6000g for 1min at 4 ℃. The filtrate was discarded, and the preparation tube was returned to a 2ml centrifuge tube, to which 500. mu.l of Buffer W1A was added, and centrifuged at 12000g for 1min at 4 ℃. Discarding the filtrate, placing the preparation tube back into a 2ml centrifuge tube, adding 700 μ l Buffer W2 into the preparation tube, centrifuging at 4 deg.C and 12000g for 1 min; in the same manner, 700. mu.l of Buffer W2 was washed once more. The filtrate was discarded, and the preparation tube was returned to a 2ml centrifuge tube and centrifuged at 12000g for 1min at 4 ℃. The preparation tube was placed into a clean 1.5ml centrifuge tube and 20. mu.l RNase-free water was added to the center of the preparation tube membrane. Standing at room temperature for 1min, centrifuging at 4 deg.C and 12000g for 1min, and eluting to obtain RNA.
3.9.2RNA reverse transcription and Realtime PCR reaction
Use of
Figure RE-GDA0002613195780000102
Bioscience's reverse transcription kit reverse transcribes the purified extracted RNA into cDNA, and the reaction system is shown in table 4 below:
TABLE 4 reverse transcription reaction liquid System configuration
Figure RE-GDA0002613195780000111
The prepared system is subjected to reverse transcription reaction for 15 minutes at 37 ℃. Then, the reverse transcriptase inactivation reaction was carried out at 98 ℃ for 5 minutes. After the reaction is finished, the reaction product is stored at the temperature of minus 20 ℃ for standby.
Subjecting the prepared cDNA to real-time fluorescent quantitative PCR reaction according to
Figure RE-GDA0002613195780000114
The Bioscience instructions established the reaction system and reaction conditions (table 5). The primers used in this study were purchased from Shanghai Bioengineering Co., Ltd, and the detailed sequences are shown in Table 6.
TABLE 5 reverse transcription reaction System configuration
Figure RE-GDA0002613195780000112
TABLE 6 mRNA detection PCR primer sequence List
Figure RE-GDA0002613195780000113
F, a pre-primer; r, a rear primer.
After a post-reaction system is prepared, carefully covering a PCR eight-tube connection cover, performing vortex centrifugation on the prepared PCR plate, and then placing the PCR plate in a Realtime PCR instrument for PCR reaction according to the following procedures: the reaction was carried out at 95 ℃ for 3min for 40 PCR cycles (single cycle parameters: 95 ℃, 15 sec; 60 ℃, 40 sec). To establish the dissolution curve of the PCR product, after the amplification reaction was completed, the temperature was slowly heated from 65 ℃ to 95 ℃ by gradient heating.
3.9.3 results and calculations
Respectively carrying out qRT-PCR reaction on the target gene and the reference gene of each sample on the same PCR plate, and adopting 2 as data-ΔΔCtThe method performs a differential analysis.
3.10 protein extraction and protein immunoblotting (Western blot)
3.10.1 extraction and quantification of total protein in brain tissue
Taking nucleus accumbens frozen at-80 ℃, adding a proper amount of 100 mu l RIPA lysate, adding protease inhibitors Cocktail and PMSF into the lysate system, and placing on ice for cracking for 15 min. The ultrasonic treatment is carried out in ice bath for 10 times, 5 seconds/time and 3 seconds in each interval, and the ultrasonic treatment aims to break tissue cells and fully release dissolved protein. Then, the mixture was centrifuged at 13000g for 15min at 4 ℃ and the supernatant was aspirated. The protein concentration was quantified using a BCA protein quantification kit (shanghai bi yunnan biotechnology limited) comprising the main steps of: first, BSA was diluted to a concentration gradient of 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025, 0 mg/ml; the extracted protein supernatant was then diluted 30-fold and 20. mu.l of each of the diluted BSA and protein samples were added to the corresponding well of a 96-well plate. Then 200. mu.l of the working solution was added to each well, and incubated at 37 ℃ for 20-25min in the absence of light. Subsequently, the absorbance value at the wavelength of 562nm is measured by a full-wavelength microplate reader, and when the BSA standard curve is linearly related R2>And 0.99 is qualified quantification, and then the corresponding protein concentration of each sample is calculated. Samples were diluted accordingly as required and 5X protein loading buffer was added to each sample to give a final concentration of 1X. Boiling in water bath for 5min, packaging, and storing at-20 deg.C.
3.10.2 Western immunoblotting and Exposure
10% and 7.5% polyacrylamide gel isolates and 5% polyacrylamide gel supernatant concentrates were prepared according to the gel formulation kit. And (3) putting the prepared gel into an electrophoresis tank, adding an electrophoresis buffer solution, and then carrying out spotting. The sample loading volume was adjusted to moderate the sample concentration based on the abundance of each protein in the nucleus accumbens, and approximately 10. mu.l of protein sample was added per sample well. The protein sample was then compressed to the gel separation limit using 60V, and the voltage was increased to 80V after the band ran across the separation limit until the protein of interest was completely separated. Then, the gel loaded with the target protein is transferred from the glass plate to a transfer membrane holder with a filter paper "sandwich" structure, and the PVDF membrane activated by methanol is covered on the gel, the transfer membrane voltage is set as 100V, and the transfer membrane time is set according to the molecular weight of the protein. After the membrane transfer is completed, 5% skimmed milk blocking solution is prepared by TBST buffer solution, and the strips are soaked in the blocking solution and sealed for 1 hour by a shaking table at room temperature. The protein primary antibody was then diluted with the above blocking solution in the required proportions, the hybridizing bands were blocked in the primary antibody, overnight at 4 ℃, shaken on a shaker at 37 ℃ the next day for 1h, and the membrane was washed three times with TBST for 10min each. Selecting corresponding secondary antibody according to the species source of the primary antibody, diluting the secondary antibody according to the ratio of 1:5000, incubating for 1h at 37 ℃, washing the membrane for five times for 10min each time by TBST buffer after finishing, and then washing the membrane for 10min once by TBS buffer. And during exposure, preparing luminescence solution A and luminescence solution B according to the ratio of 1:1, placing the PVDF membrane loaded with the target protein in a luminescence mixed solution for reaction for 1min under the condition of keeping out of the sun, taking out the PVDF membrane, and sucking the redundant luminescence solution by using filter paper. The PVDF film was exposed to a gel imaging system. Data processing: the gray value of each strip of the exposed picture is read by a Clinx Image Analysis system, and the gray value of the Tubulin strip is used as an internal reference for standardized comparison.
3.11 Targeted detection of Glucosylceramide (GlcCer) sphingolipids
GCS is a synthetase of GlcCer sphingolipids, and inhibition of GCS activity results in a reduction in the content of GlcCer sphingolipids as a downstream synthetic product. GCS activity can be reflected by detecting the content of GlcCer sphingolipid.
3.11.1 Standard Curve sample preparation
5mg of glucopyranosamide (C18:1/16:0), 5mg of glucopyranosamide (C18:1/18:0), 5mg of glucopyranosamide (C18:1/24:1) were added to chloroform: methanol was prepared in 5ml of 1:4(v/v) to prepare a stock solution of 1 mg/ml. The three standards were then formulated into the concentration systems of table 8, respectively, to prepare standard curves.
TABLE 8 preparation of Standard samples
Figure RE-GDA0002613195780000131
3.11.2 extraction of sphingolipids
Thawing tissue or cell samples at 4 deg.C, adding 2.5mg tissue extract (isopropanol: water: ethyl acetate ═ 3:1:6(v/v/v), while adding an appropriate concentration of internal standard, sonicating until no particles are visible, vortexing for 10min, centrifuging for 4 deg.C, 6000g, 10min, taking supernatant and transferring to a new EP tube for SolventI, re-extracting the remaining lower layer, adding 2ml isopropanol: water: ethyl acetate ═ 3:1:6(v/v/v), vortexing for 10min, and centrifuging for 10min for 6000g, taking supernatant and transferring to a new EP tube for SolventII, mixing SolventI and SolventII obtained by two extractions uniformly and transferring to the same EP tube, blowing dry with a gentle stream of nitrogen, re-dissolving the blown lipids with 200. mu.l mobile phase B (containing 1mM and 0.2% formic acid methanol solution), if not fully re-dissolved by vortexing, using sonication until complete solubilization, 13000g at 4 ℃ for 10 min. Injecting 1 mu l of sample, and carrying out UPLC-MS/MS separation detection for targeted detection of GlcCer sphingolipids.
3.11.3 chromatographic and mass spectrometric conditions
According to the literature, an ACQUITY UPLC I-class (Waters) high performance liquid chromatograph is used, mobile phase A phase: 0.2% formic acid, 2.0mM aqueous ammonium formate; mobile phase B phase: 0.2% formic acid, 1.0mM methanolic ammonium formate; flow rate: 0.5 ml/min; a chromatographic column: spectra C18 column (Waters); column temperature: 55 ℃; sample introduction volume: 1 μ l. Gradient conditions are as in table 9.
TABLE 9 conditions of mobile phase
Figure RE-GDA0002613195780000141
The mass spectrometry conditions were as follows: the scan pattern is positive ion. 3kV for Capillary; sample Cone: 30, of a nitrogen-containing gas; source Temperature: 140 ℃; desolvation Temperature: 350 ℃; cone Gas Flow: 50L/Hr; desolvation Gas Flow: 800L/Hr; scanning range: 50kDa-1200 kDa. The specific UPLC-MRM scan pattern is shown in Table 10.
TABLE 10 Positive ion MRM Scan conditions
Figure RE-GDA0002613195780000142
3.11.4 data analysis
Concentrations were calculated using MassLynx software plug-in quantlynx analysis. Fitting standard curves of the same standard according to GlcCer sphingolipids with similar chain lengths and abundance, namely C16:0, C18:1, C20:0, C20:1, C16:0, C18:0, C22:0, C26:0 and C26:1 by C18: 0; c22:1, C24:0 and C24:1 were fitted with C24:1 standard curves. The results from the fitting are expressed as means ± standard deviation, the comparisons between the two groups were analyzed by t-test analysis, the comparisons between the four groups were analyzed by one-way ANOVA, and p <0.05 indicated a statistical difference.
4 results of the experiment
4.1 cocaine-induced behavioral sensitization and conditional place preference modeling
The distance of movement of the mice was measured in this experiment by intraperitoneal injection of 20mg/kg cocaine (FIG. 4A, B) for seven consecutive days. The results showed that the cocaine group mice had significantly higher movement distance than the saline group from the first day of administration and the movement distance tended to stabilize on the third day, indicating successful establishment of the behavioral sensitization model (fig. 4C, D).
4.2 expression of GCS in behavior sensitization and conditional location-preference behavior
To further confirm the effect of GCS on cocaine-induced addictive behaviors, Western Blot was used in this assay to detect changes in the post-translational protein level of GCS. The results showed a significant increase in GCS protein levels, about 1.2 fold or so, in the behavioral sensitization and conditional site-preferred reward effects compared to the saline group (figure 5).
4.3 multiple injections of cocaine increased GCS expression in nucleus accumbens brain region
Neurosynaptic plastic brain regions induced by multiple cocaine administrations include the Prefrontal cortex (Prefrontal core), nucleus accumbens (Nuclear accumbens), Striatum (Striatum) and Hippocampus (Hippocampus). In order to study the influence of cocaine administration on GCS expression in each brain region, cocaine with a concentration of 20mg/kg is intraperitoneally injected at the same time point for 7 consecutive days, the neck of the mouse is removed and the mouse is sacrificed half an hour after the last administration, four brain regions of prefrontal cortex, nucleus accumbens, striatum and hippocampus are taken out, and the expression of GCS in the four brain regions is detected by Western blot. Among them, nucleus accumbens (NAcc), also known as the nucleus accumbens, is a group of neurons in the corpus undulatum that is considered the happy center of the brain, responding to stimuli such as food, sex, drugs, etc.
The results showed that upon intraperitoneal cocaine administration to mice, there was a significant increase in GCS expression in the nucleus accumbens brain region (p <0.05), but there was no significant change in the levels of GCS protein in the prefrontal cortex, striatum, and hippocampal brain regions (fig. 6). Thus, the results indicate that multiple cocaine injections can specifically increase the nucleus accumbens GCS protein expression.
4.4 cocaine-induced increase in nucleus accumbens GCS expression over time
In order to explore the change of GCS protein expression along with the prolonging of cocaine administration time, 5 time points are set in the experiment, cocaine is continuously administered for 1 day, 2 days, 3 days, 5 days and 7 days respectively, materials are taken half an hour after the last cocaine injection to obtain nucleus accumbens brain areas, and the nucleus accumbens GCS is detected through Western blot.
The results showed that there was no significant change on days 1and 2 of continuous intraperitoneal administration of cocaine, and that the expression of GCS in mouse nucleus accumbens brain region induced by cocaine was significantly increased from day three and was stably expressed for 5and 7 days after continuous administration, compared with the saline group (fig. 7). The above results indicate that cocaine-induced increase in GCS expression in nucleus accumbens increases with the time of cocaine administration, and that the expression tends to be stable after three days of administration, exhibiting a significant time dependence.
No significant change in GCS in the food-induced conditioned place preference model in the 4.5 volt nucleus septa
To study whether GCS was induced by cocaine drugs, the experiment was modeled using conditional positional preference of food-induced mice, which were fed for 1h before each training, and not fed for the control group, and all mice were fed 1h after training.
As a result, it was found that the preference of the behavior of the mice in the food group was significantly reversed compared to that in the control group. However, the expression of GCS protein was detected by Western Blot, and as a result, it was found that the food group was not significantly changed compared to the control group (fig. 8).
From the results of 4.2 to 4.6, it was found that cocaine specifically stimulates the expression of GCS in nucleus accumbens, but as cocaine stimulates nucleus accumbens to provide happy food, it fails to stimulate the expression of GCS in nucleus accumbens. Indicating that GCS is associated with cocaine stimulation, inhibiting GCS activity is expected to reduce cocaine addiction.
4.6 inhibition of GCS enzymatic Activity significantly attenuates cocaine-induced behavioral sensitization effects
In order to deeply explore the role of GCS in behavior sensitization effect induced by cocaine, a GCS enzyme-specific small molecule inhibitor Eliglustat which can effectively inhibit GCS enzyme activity and reduce GlcCer level is used in the experiment. To examine whether Eliglustat could alter cocaine-induced behavioral effects, the glucose ceramide synthase inhibitor Eliglustat (1 μ l, Intra-NAc) was administered at a site-specific dose to the juxter brain region 15min prior to cocaine administration (20mg/kg, intraperitoneal injection), and the mouse movement distance was measured daily for seven days of continuous administration (fig. 9A). By consulting the literature, we prepared Eliglustat at 5. mu.M, 20. mu.M, and 100. mu.M, respectively, as low, medium, and high dose groups. The results show that the high dose group of 100 μ M Eliglustat was able to attenuate the behavioral sensitization effect of mice, and the low and medium doses did not significantly change the behavioral sensitization effect of mice (fig. 9B). Therefore, the concentration of 100 μ M Eliglustat used in this experiment to inhibit the mouse-induced behavioral sensitization effect.
The results show that the solvent-cocaine group performs a significantly enhanced sensitizing effect compared to the solvent-saline group; whereas the behavioral sensitization effect of mice was significantly reduced after daily Eliglustat administered at the septal site at 15min v prior to cocaine injection (FIG. 9C).
To investigate whether the GlcCer sphingolipid content was altered following Eliglustat administration at the nucleus accumbens site, UPLC-MS/MS targeting was used to measure GlcCer sphingolipid levels in this experiment.
The results show that chain length C22:1, C24:1 significantly increased levels in the solvent-cocaine group compared to the solvent-saline group, and that Eliglustat administered at the septal site 15min earlier per day can reduce GlcCer content (fig. 10).
The above two experimental results show that: GCS and GlcCer sphingolipids are involved in behavior sensitization of cocaine-induced mice, and the content of GlcCer is reduced by reducing the activity of GCS enzyme, so that the behavior sensitization effect induced by cocaine is reduced.
4.7 inhibition of GCS enzymatic Activity significantly attenuated cocaine-induced effects of conditioned place preference behavior
To investigate the role of GCS in the cocaine-induced conditioned place preference behavior effect, Eliglustat, a small molecule inhibitor specific for the GCS enzyme, was used in this experiment to test whether Eliglustat could affect the cocaine-induced reward effect. A glucose ceramide synthetase inhibitor Eliglustat (1 μ l, Intra-NAc) was administered to the nucleus accumbens brain region at a fixed point 15min before CPP training every day for six days, and after six days of CPP training, the mice were examined for changes in behavioral preference on the seventh day (FIG. 11A). According to the literature, Eliglustat was formulated at 1. mu.M, 10. mu.M, 50. mu.M, respectively, into low, medium and high dose groups. The results show that the high dose group of 50 μ M Eliglustat was able to attenuate the reward effect induced by cocaine, and that the low and medium doses had no significant effect on the reward effect induced by cocaine in the mouse conditional place preference model (fig. 11B). Therefore, this experiment used a concentration of 50 μ M Eliglustat to inhibit the mouse-induced conditioned place preference behavior effect.
The results showed that the conditioned place preference effect of the solvent-cocaine group was significantly enhanced compared to the solvent-saline group; whereas the conditioned place preference effect of mice was significantly reduced after daily dosing of Eliglustat at the 15min volt nuclear septation site prior to CPP training (fig. 11C). Meanwhile, UPLC-MS/MS is used for detecting the level of GlcCer sphingolipids in a targeted mode in the experiment.
The results show that the levels of GlcCer sphingolipids of the medium and long chains with the chain lengths 22:0, 22:1, 24:0, 24:1 are significantly increased compared to the solvent-saline group, and that the increase in the cocaine-induced GlcCer sphingolipid content can be reduced by administering Eliglustat 15min earlier in the nucleus septum site per day (fig. 12).
The above two experimental results show that: GCS and GlcCer sphingolipids are involved in cocaine-induced behavioral sensitization of mice, and the content of the GlcCer sphingolipids is reduced by reducing the activity of GCS enzyme, so that the cocaine-induced conditional locus preference effect is reduced.
4.8 inhibition of GCS enzymatic Activity significantly attenuates cocaine-induced behavioral effects of autonomous administration
To investigate the role of GCS in the effects of cocaine-induced self-administration behavior, Eliglustat, a small molecule inhibitor specific for GCS enzyme, was used in this experiment to examine whether Eliglustat could affect cocaine-induced self-administration behavior in mice. Daily mice were dosed with the glucosylceramide synthase inhibitor Eliglustat (1. mu.l, Intra-NAc) at a site-specific dose 15min prior to self-administration, for 7 days, and the number of effective nasal palpations and dosing of the mice was recorded daily. The concentration of 100 μ M Eliglustat was used in this experiment to inhibit the mouse-induced autonomic behavioral effects.
The results show that the effective nasal contact of the solvent cocaine group is significantly enhanced compared to the solvent normal saline group; whereas the effective nasal exposure of mice was significantly reduced after daily Eliglustat given at the 15min volt nuclear septal site prior to cocaine injection (FIG. 13A). Meanwhile, the administration frequency results caused by effective nasal contact show that compared with the solvent physiological saline group, the solvent cocaine group has obviously enhanced administration frequency from the third day and shows gradually weakened trend; whereas the number of doses in mice was significantly reduced after daily Eliglustat dosing at the 15min volt nuclear septal site prior to cocaine injection (FIG. 13B).
The results show that: the GCS inhibitor Eliglustat can inhibit the self-administration behavioral effects induced by mice.
4.9 inhibition of GCS enzymatic Activity significantly reduces dendritic Branch number and dendritic spine Density
Research shows that cocaine can change brain structure within several hours, and the dendritic branch number and dendritic spine density are increased, so that cocaine is the first step of drug addiction.
To investigate whether GCS can modulate cocaine-induced synaptic plasticity changes, this experiment used golgi staining technique to observe mouse synaptic morphology after a fixed point administration of inhibitor Eliglustat in nucleus accumbens brain.
As a result, it was found that the number of dendritic branches and the density of dendritic spines were significantly increased in the cocaine solvent group as compared with the physiological saline solvent group; dendritic branch number and dendritic spine density were significantly reduced following GCS specific inhibitor administration at the nucleus accumbens site-specific site (figure 14).
The results show that: the GCS inhibitor Eliglustat can obviously reduce the dendritic branch number and dendritic spine density and reduce cocaine addiction.
5 conclusion
The invention detects the mouse behavioral effect induced by cocaine by establishing a mouse behavioral model (behavioral sensitization, conditional position preference and autonomous administration), and finds that the cocaine can obviously induce the mouse nucleus accumbens brain region GCS expression to be up-regulated. Cocaine-induced behavioral sensitization, conditioned site preference, and self-administered behavioral effects can be inhibited by administering GCS inhibitors at a site specific for nucleus accumbens.
In conclusion, the GCS inhibitor can effectively weaken the behavioral effect induced by cocaine, and has good clinical application prospect when being used for preparing the medicine for treating cocaine addiction.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
<120> use of GCS inhibitors for the preparation of a medicament for the treatment of cocaine addiction
<130> GY159-2020P0110143CC
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<170> PatentIn version 3.5
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (8)

  1. Use of a GCS inhibitor for the manufacture of a medicament for the treatment of cocaine addiction.
  2. 2. Use according to claim 1, characterized in that: the GCS inhibitor is Eliglustat, and the structural formula is as follows:
    Figure FDA0002551295190000011
  3. 3. use according to claim 1 or 2, characterized in that: the medicament is a pharmaceutical formulation for injection.
  4. 4. Use according to claim 1 or 2, characterized in that: the medicine is used for reducing the content of glucosylceramide sphingolipids in nucleus accumbens brain areas.
  5. 5. Use according to claim 1 or 2, characterized in that: the drug is a drug that reduces the number of dendritic branches and dendritic spine density in the nucleus accumbens brain region.
  6. 6. A medicament for the treatment of cocaine addiction, comprising: the medicament takes a GCS inhibitor as an active ingredient.
  7. 7. The medicament of claim 6, wherein: the GCS inhibitor is Eliglustat, and the structural formula is as follows:
    Figure FDA0002551295190000012
  8. 8. the medicament of claim 6 or 7, wherein: the medicament is a pharmaceutical formulation for injection.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913424A (en) * 2020-07-09 2022-01-11 四川大学华西医院 Adeno-associated virus and application thereof in preparing cocaine addiction treatment drug

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100209542A1 (en) * 2008-11-21 2010-08-19 University Of Massachusetts Medical School Methods For Treating Withdrawal From Addictive Compounds
WO2014150329A1 (en) * 2013-03-14 2014-09-25 University Of Georgia Research Foundation, Inc. Lipid biomarkers of addiction and relapse risk
CN105646442A (en) * 2015-10-27 2016-06-08 北京凯莱天成医药科技有限公司 Preparation method of eliglustat
CN106967042A (en) * 2017-03-21 2017-07-21 浙江奥翔药业股份有限公司 Yi Ligelusita synthetic method and its midbody compound
CN110878079A (en) * 2018-12-31 2020-03-13 北京启慧生物医药有限公司 Preparation method of high-purity eliagliptat

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105153042B (en) * 2015-07-17 2017-11-24 四川大学 A kind of compound and its purposes in the medicine for preparing treatment cocaine habituation
CN105055404B (en) * 2015-08-19 2017-07-18 四川大学 Purposes of the HMGCS2 inhibitor in the medicine for preparing treatment cocaine habituation
CN105055412B (en) * 2015-08-20 2018-06-19 四川大学 Purposes of the NAMPT inhibitor in the drug for preparing treatment cocaine habituation
CN105251006B (en) * 2015-08-21 2018-08-03 四川大学 Purposes of the TLR3 inhibitor in the drug for preparing treatment cocaine habituation
KR20200100697A (en) * 2017-12-15 2020-08-26 젠자임 코포레이션 How to cure Gosche disease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100209542A1 (en) * 2008-11-21 2010-08-19 University Of Massachusetts Medical School Methods For Treating Withdrawal From Addictive Compounds
WO2014150329A1 (en) * 2013-03-14 2014-09-25 University Of Georgia Research Foundation, Inc. Lipid biomarkers of addiction and relapse risk
CN105646442A (en) * 2015-10-27 2016-06-08 北京凯莱天成医药科技有限公司 Preparation method of eliglustat
CN106967042A (en) * 2017-03-21 2017-07-21 浙江奥翔药业股份有限公司 Yi Ligelusita synthetic method and its midbody compound
CN110878079A (en) * 2018-12-31 2020-03-13 北京启慧生物医药有限公司 Preparation method of high-purity eliagliptat

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杜磊 等,: ""Eliglustat",", 《中国药物化学杂志》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113913424A (en) * 2020-07-09 2022-01-11 四川大学华西医院 Adeno-associated virus and application thereof in preparing cocaine addiction treatment drug
CN113913424B (en) * 2020-07-09 2023-08-08 四川大学华西医院 Adeno-associated virus and application thereof in preparation of medicines for treating cocaine addiction

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