CN109125331A - GABABApplication of the receptor antagonist in terms of blocking the channel TREK-1 - Google Patents
GABABApplication of the receptor antagonist in terms of blocking the channel TREK-1 Download PDFInfo
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Abstract
The invention belongs to biomedicine technical fields, and in particular to GABABApplication of the receptor antagonist in terms of blocking the channel TREK-1.It is in the embodiment of the present invention studies have shown that GABABReceptor antagonist acts on the channel TREK-1 and plays antidepressant effect, this has important theory significance and application value for disclosing the combination drug of the pathogenesis guidance of depression clinically.Therefore, GABABReceptor antagonist is treated in preparation antidepressant, obstruction of biliary tract drug and preparation and will be had a good application prospect in the diseases related drug in the channel TREK-1.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to GABABReceptor antagonist is blocking the channel side TREK-1
The application in face.
Background technique
Depression (depression) is also known as depressive disorder, clinical manifestation be hypothymergasia, retardation of thinking and
Speech movement is reduced.Depression has seriously perplexed the life and work of patient, brings heavy burden to family and society, grinds
The patients with depression for studying carefully display about 15% dies of suicide, has accounted for 2/3rds in total suicide crowd.According to the World Health Organization
There are 3.5 hundred million patients with depression in estimation, the whole world, and China is more up to nearly 100,000,000.Depression has become the fourth-largest illness in the world,
The global disease medical burden of WHO predicts that following 10 years depression will become second-biggest-in-the-world psychosomatic disorder disease.It is so far
Only, the cause of disease of depression is not known, but what is certain is that, biology, heredity, psychology and social environment etc. it is all it is many-sided because
Element takes part in the pathogenic process of depression.Mainly there are monoamine deficiency hypothesis, hpa axis function to lose now the pathogenesis of depression
Hypothesis, growth factor is adjusted to lack hypothesis and nerve regneration obstacle hypothesis.The prefered method for the treatment of depression is controlled at present for drug
It treats, existing first-line drug is largely all based on depression monoamine neurotransmitter hypothesis, main by directly or indirectly increasing
Add the monoamine neurotransmitters such as norepinephrine in synaptic cleft, serotonin and dopamine horizontal and plays antidepression work
With.Contain extremely complex serotonergic system and noradrenergic system in the deep of brain.Many antidepressions
Drug is the reuptake by blocking presynaptic neuron to serotonin and norepinephrine, and then improves synaptic cleft
The content and utilization rate of serotonin and norepinephrine, so that postsynaptic neuron excitability is increased, the anti-suppression played
Yu Zuoyong.Therefore, the effective chemicals of clinical treatment depression are largely all based on depression monoamine nerve and pass at present
Matter hypothesis.However, the pathogenesis of depression again with heredity, neuroendocrine, inhibition and excitement be unbalance, oxidative stress, nerve
Apoptosis, neuron degeneration and environmental factor of first cell etc. are closely related, moreover, anxiety-depression comorbidity considerably increases again
The complexity of depression.Therefore, the complexity of depression mechanism and ambiguity to establish in single mechanism mould
The curative effect of the antidepressant of formula is also far from reaching expection of the modern society to anti depressant therapy.Continue to research and develop novel antidepression
Medicine is following inexorable trend, and finds new target spot and become the hot spot in mental science field by mechanism and multiple treatment depression
Problem.
Tandem-pore-domain potassium channels (Two-pore-domain potassium channels, K2P) are new discoveries in recent years
A kind of potassium-channel family.TREK-1(TWIK-Related K+Channel 1) it is important in tandem-pore-domain potassium channels
A member, and it is wherein distributed the most subclass of most wide expression, there is important physiological function and neuroprotection.GABA is one
Kind of inhibitory neurotransmitter, GABA expression of receptor and dysfunction be also likely to be important link in depression mechanism it
One.
Summary of the invention
This research is carried out by establishing depressed animal model using behaviouristics, immunohistochemistry and Western blot
Intracerebral morphology and biochemical detection, while giving GABAB(γ-gamma aminobutyric acidB) receptor agonism
Agent, GABABReceptor antagonist and TREK-1 channel antagonist are intervened, and find GABABReceptor antagonist is blocking TREK-1 logical
It is had a good application prospect in terms of road.Specifically, in the embodiment of the present invention studies have shown that GABABReceptor antagonist effect
Antidepressant effect is played in the channel hippocampus TREK-1, this is used for disclosing the compatibility of the pathogenesis guidance of depression clinically
Medicine has important theory significance and application value.In addition, GABABReceptor antagonist is in preparation antidepressant, obstruction of biliary tract
Also there is good application prospect in drug and preparation treatment and the diseases related drug in the channel TREK-1.
For application described above, the GABABThe dosage of receptor antagonist is 0.016mg/kg.d.Herein,
Dosage used in zoopery in corresponding embodiment is 0.1mg/kg.d, and converting as the dosage of people is 0.016mg/kg.Other
The dosage of Spadin, converting as the dosage of people is 0.016mg/kg.
For application described above, the GABABReceptor antagonist includes:
CGP55845:3-N[1-(S)-3,4-dichloropheyl)ethyl]amino-2-(S)-hydroxypropyl-
P-benzyl-phosphinic acid；CGP-35348:P-[3-aminopropyl]-P-diethoxymethyl-
phosphinic acid；
CGP52432:[3-[[(3,4-Dichlorophenyl)methyl]amino]propyl]
(diethoxymethyl)-phosphinic acid；
Experimental method is to select SPF grades of male SD rats, and weight 200-250g adapts to environment after a week, according to random point
Group principle, prepares rat depression model under chronic unpredictable stress stimulation.Using spacious field experiment (open field test,
OFT) and syrup preference experiment (sucrose preference test, SPT) carries out Behavior evaluation, success to depression model
It establishes after rat depression model and experimental animal is divided by 9 groups, respectively blank control group (C), suppression according to different pharmaceutical interventions
Strongly fragrant model group (CUS), depression model GABABReceptor stimulating agent (Baclofen) intervention group (CUS+Baclofen), depression model
GABABReceptor antagonist (CGP55845) intervention group (CUS+CGP55845), depression model TREK-1 channel antagonist
(Spadin) intervention group (CUS+Spadin), GABABReceptor stimulating agent intervenes control group (C+Baclofen), GABABReceptor antagonist
Control group (C+CGP55845) is intervened in agent, TREK-1 channel antagonist intervenes control group (C+Spadin), positive controls (CUS+
Citalopram).Changed after pharmaceutical intervention by the difference of spacious field experiment and syrup preference experimental evaluation each group rat Depressive behavior
Become, while using the channel TREK-1 in immunohistochemical experiment and immune protein Blot experiment (Western blot) detection rat brain
And GABABThe change of expression of receptor, further analyzes GABABThe channel receptor modulators diplopore potassium ion TREK-1 is in depression
Effect in mechanism.
Pharmaceutical intervention method
After model foundation success, GABABReceptor antagonist (CGP55845) injection dosage is 0.1mg/kg.d and TREK-1
Channel antagonist (Spadin) injection dosage be 0.1mg/kg.d, pharmaceutical intervention is for 4 weeks, during which chronic stress stimulation normally into
It goes, after pharmaceutical intervention, carries out behaviouristics detection, detection time is after pharmaceutical intervention 30 minutes.
The research of the invention finds that chronic unpredictable stress stimulation rat can simulate the formation of human depression for a long time
Process, the depression model being successfully established；Rat depression sample symptom can be effectively relieved in diplopore potassium ion TREK-1 channel antagonist；
GABABReceptor antagonist can increase the channel hippocampus TREK-1 expression in rat brain in depression model, while alleviate rat suppression
Strongly fragrant sample symptom；GABABReceptor stimulating agent can reduce the channel hippocampus TREK-1 expression in rat brain in depression model, add simultaneously
Great mouse depression sample symptom；GABABThe mechanism of the receptor modulators diplopore channel potassium ion TREK-1 participation depression.
Detailed description of the invention
Fig. 1 is GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene it is chronic it is unpredictable stress
Stimulate the spacious field experimental result of SD rat；
Caption: Total Distance: movement total distance of the experimental rat in spacious field experiment
Time Spent in Central Area: experimental rat tests middle section residence time in spacious field
Crossing Number: horizontal score of the experimental rat in spacious field experiment
Rearing Number: vertical score of the experimental rat in spacious field experiment
C:Control, control group；
CUS:Chronic unpredictable stress, model group；
C+Baclofen:GABABReceptor stimulating agent intervenes control group；
C+CGP55845:GABABReceptor antagonist intervenes control group；
C+Spadin:TREK-1 channel antagonist intervenes control group；
CUS+Baclofen: depression model GABABReceptor stimulating agent intervention group；
CUS+Spadin: depression model TREK-1 antagonist intervention group；
CUS+Citalopram: depression model Citalopram intervenes positive controls
The total distance of A rat motor；Time of the B rat in middle section；The horizontal score of C rat；The vertical score of D rat.
Baclofen and CGP55845 is respectively GABABReceptor stimulating agent and antagonist, Spadin are that the channel TREK-1 is short of money
Anti-agent, Citalopram are positive control medicine serotonin reuptake inhibitor Citalopram；Data are with mean ± standard deviation
It indicates；* p < 0.05, * * p < 0.01；(n=8/group).
Fig. 2 is GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene it is chronic it is unpredictable stress
Stimulate the syrup preference experimental result of SD rat；
Sucrose Preference (%): the sucrose water this consumption ratio of rat in the experiment of syrup preference；
C:Control, control group；
CUS:Chronic unpredictable stress, model group；
C+Baclofen:GABABReceptor stimulating agent intervenes control group；
C+CGP55845:GABABReceptor antagonist intervenes control group；
C+Spadin:TREK-1 channel antagonist intervenes control group；
CUS+Baclofen: depression model GABABReceptor stimulating agent intervention group；
CUS+Spadin: depression model TREK-1 channel antagonist intervention group；
CUS+Citalopram: depression model Citalopram intervenes positive controls
Baclofen and CGP55845 is respectively GABABReceptor stimulating agent and antagonist, Spadin are that the channel TREK-1 is short of money
Anti-agent, Citalopram are positive control medicine serotonin reuptake inhibitor Citalopram；Data are with mean ± standard deviation
It indicates；* p < 0.05, * * p < 0.01；(n=8/group).
Fig. 3 is GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene it is chronic it is unpredictable stress
Stimulate the SD rat hippocampus area channel TREK-1 and GABABThe western blot result of expression of receptor；
GABAB(γ-aminobutyric acid): γ-aminobutyric acidBReceptor
GAPDH (glyceraldehyde-3-phosphate dehydrogenase): glyceraldehyde-3-phosphate dehydrogenase
The channel TREK-1 (TWIK-Related K+Channel 1): diplopore potassium ion TREK-1
C:Control, control group；
CUS:Chronic unpredictable stress, model group；
C+Baclofen:GABABReceptor stimulating agent intervenes control group；
C+CGP55845:GABABReceptor antagonist intervenes control group；
C+Spadin:TREK-1 channel antagonist intervenes control group；
CUS+Baclofen: depression model GABABReceptor stimulating agent intervention group；
CUS+Spadin: depression model TREK-1 channel antagonist intervention group；
CUS+Citalopram: depression model Citalopram intervenes positive controls
The channel hippocampus TREK-1 A and GABABThe western blot result of receptor；
B GABABReceptor gray analysis statistical result；
The channel C TREK-1 gray analysis statistical result.
Statistical result is the gray level ratio result of purpose albumen and internal reference；Baclofen and CGP55845 is respectively GABAB
Receptor stimulating agent and antagonist, Spadin are TREK-1 channel antagonist, and Citalopram is positive control medicine serotonin
Reuptaking inhibitor Citalopram；Data are indicated with mean ± standard deviation；* p < 0.05, * * p < 0.01；(n=5/group).
Fig. 4 is GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene it is chronic it is unpredictable stress
Stimulate the SD rat channel frontal cortex TREK-1 and GABABThe western blot result of expression of receptor；
The channel TREK-1 and GABABThe western blot result of expression of receptor.
GABAB(γ-aminobutyric acid): γ-aminobutyric acidBReceptor
GAPDH (glyceraldehyde-3-phosphate dehydrogenase): glyceraldehyde-3-phosphate dehydrogenase
The channel TREK-1 (TWIK-Related K+Channel 1): diplopore potassium ion TREK-1
C:Control, control group；
CUS:Chronic unpredictable stress, model group；
C+Baclofen:GABABReceptor stimulating agent intervenes control group；
C+CGP55845:GABABReceptor antagonist intervenes control group；
C+Spadin:TREK-1 channel antagonist intervenes control group；
CUS+Baclofen: depression model GABABReceptor stimulating agent intervention group；
CUS+Spadin: depression model TREK-1 antagonist intervention group；
CUS+Citalopram: depression model Citalopram intervenes positive controls
The channel frontal cortex TREK-1 A and GABABThe western blot result of receptor；
B GABABReceptor gray analysis statistical result；
The channel C TREK-1 gray analysis statistical result.
Statistical result is the gray level ratio result of purpose albumen and internal reference；Baclofen and CGP55845 is respectively GABAB
Receptor stimulating agent and antagonist, Spadin are TREK-1 channel antagonist, and Citalopram is positive control medicine serotonin
Reuptaking inhibitor Citalopram；Data are indicated with mean ± standard deviation；(n=5/group)；
Fig. 5 is GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene it is chronic it is unpredictable stress
The ImmunohistochemistryResults Results for stimulating the SD rat hippocampus area channel TREK-1 to express；
Integrated Optical Density: the OD value of immunohistochemical experiment positive expression；
C:Control, control group；
CUS:Chronic unpredictable stress, model group；
C+Baclofen:GABABReceptor stimulating agent intervenes control group；
C+CGP55845:GABABReceptor antagonist intervenes control group；
C+Spadin:TREK-1 channel antagonist intervenes control group；
CUS+Baclofen: depression model GABABReceptor stimulating agent intervention group；
CUS+Spadin: depression model TREK-1 channel antagonist intervention group；
CUS+Citalopram: depression model Citalopram intervenes positive controls
The ImmunohistochemistryResults Results of the Hippocampal CA 1 the A channel TREK-1 expression；
The ImmunohistochemistryResults Results of the area the B hippocampus CA2 channel TREK-1 expression；
The brain area schematic diagram of C hippocampus CA1 and the area CA2；
The OD value statistical result of the positive neuron of the Hippocampal CA 1 the D channel TREK-1 expression；
The OD value statistical result of the positive neuron of the area the E hippocampus CA2 channel TREK-1 expression.
Baclofen and CGP55845 is respectively GABABReceptor stimulating agent and antagonist, Spadin are that the channel TREK-1 is short of money
Anti-agent, Citalopram are positive control medicine serotonin reuptake inhibitor Citalopram；Data are with mean ± standard deviation
It indicates；* p < 0.01；(n=5/group).
Specific embodiment
The present invention will be further explained with reference to the examples below.
The healthy male SD rat of experimental animal selection, SPF grades, weight 200-250g (Dalian Medical Univ's major disease base
Because engineering mode Institute of Botany provides), feeding environment is suitable for temperature: 22 ± 1 DEG C, humidity: 55%-60%, drinking water are real
Test mouse specialized water, ad lib SPF grades of mouse grain of the same race.
Rat spacious field experimental system XR-XZ301 (the glad Soft Inform ation Science and Technology Ltd. in Shanghai)
Concentrated type DAB colour reagent box ZLI-9032 (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；
Immunohistochemical staining kit SP-9000 (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)；
Neutral gum (Beijing Suo Laibao Science and Technology Ltd)；
Holoprotein extracts kit KGP250/KGP2100 (Jiangsu Kai Ji Biotechnology Ltd.)；
BCA protein content detection kit KGPBCA/KGP902-KGP903 (the triumphant limited public affairs of base biotechnology share in Jiangsu
Department)；
30%Acr-Bis (29:1) (the green skies Bioisystech Co., Ltd in Shanghai)；
PageRuler prest Protein Ladder (U.S. Thermo Scientific)；
Special super quick ECL chemical luminescence reagent kit (the green skies Bioisystech Co., Ltd in Shanghai)；
The channel TREK-1 polyclonal antibody (U.S. Abcam)；
GABABReceptor polyclonal antibody (U.S. Abcam)；
GAPDH monoclonal antibody (U.S. Immuno Way)；
Horseradish enzyme marks goat anti-rabbit igg/HRP (U.S. Arigobio)；
Horseradish enzyme marks mountain sheep anti mouse IgG/HRP (Beijing Bo Aosen Bioisystech Co., Ltd)；
Baclofen Baclofen, baclofen (U.S. APExBIO)；
CGP55845 (U.S. APExBIO)；
Citalopram (Citalopram) (U.S. APExBIO)；
Spadin (U.S. APExBIO)；The TREK-1 channel antagonist in addition to include Spadin other than, there are also SID1900,
2- (Alpha-hydroxy amyl) benzoic acid sylvite (Potassium2- (1-Hydroxypentyl)-benzoate, PHPB)；
2,4,6- trinitrophenol (picric acid) (reagent plastics Co., Ltd is resided abroad in Taizhou plain Guangdong)；
Immunohistochemical experiment main solution is prepared:
(1) 4% chloraldurate
Electronic balance weighs the crystallization of 0.6g chloraldurate, and tri-distilled water 10ml dissolution is added, 15ml, room are settled in graduated cylinder
Temperature is kept in dark place spare.
(2)0.01M PBS
It takes one bag of PBS powder to be placed in beaker (1L specification), tri-distilled water is added and is sufficiently dissolved to 1L, it is then fixed in graduated cylinder
Hold to 2L, room temperature preservation is spare.
(3) sucrose
30% sucrose solution: weighing 150g sucrose with electronic balance, is placed in beaker (500ml specification), and 300ml tri- is added
It steams water sufficiently to dissolve, 500ml is then settled in graduated cylinder, 4 DEG C save backup.
20% sucrose solution: measuring 100ml30% sucrose solution with graduated cylinder, and tri-distilled water is added and is settled to 150ml, 4 DEG C of guarantors
It deposits spare.
10% sucrose solution: measuring 50ml30% sucrose solution with graduated cylinder, and tri-distilled water is added and is settled to 150ml, 4 DEG C of preservations
It is spare.
(4) 4% paraformaldehydes: weighing 40g paraformaldehyde powder with electronic balance and be placed in beaker (1L specification), is added three
Water is steamed, places the beaker and is heated to 55 DEG C on magnetic stirring apparatus, pays attention to ventilation, and continue stirring until paraformaldehyde and be completely dissolved,
1L is settled in graduated cylinder, room temperature preservation is spare.
1% paraformaldehyde: taking 200ml4% paraformaldehyde with graduated cylinder, and tri-distilled water is added and is settled to 800ml, room temperature preservation is standby
With.
Western blot tests main solution and prepares
(1) 10% ammonium persulfate solution (Ammonium Persulfate, APS)
0.5g ammonium persulfate is taken, tri-distilled water is added to dissolve and is settled to 5ml, is dispensed into 0.5ml centrifuge tube, -20 DEG C of preservations
It is spare.
(2) preparation (2 plate) of SDS-PAGE running gel
5% concentration glue prepares (4ml): tri-distilled water 2.33ml, 30%Arc-Bis (29:1) 0.67ml, upper layer glue buffering
Liquid (4X) (PH6.8) 1ml, 10%APS0.04ml, TEMED0.004ml.
10% separation gel prepares (15ml): tri-distilled water 6.1ml, 30%Arc-Bis (29:1) 5ml, lower layer's glue buffer
(4X) (PH8.8) 3.75ml, 10%APS0.15ml, TEMED0.006ml.
(3) SDS-PAGE electrophoresis liquid
One bottle of pulvis that can prepare 1LSDS-PAGE electrophoresis liquid is taken, is poured into clean beaker (specification 1L), is added three
Water is steamed to about 900ml, is sufficiently dissolved.Constant volume can be used after being mixed to 1L in graduated cylinder, can room temperature preservation.
(4) Western transferring film liquid
One bottle of pulvis that can prepare 1LWestern transferring film liquid is taken, is poured into clean beaker (specification 1L), three is added and steams
Water is sufficiently dissolved to about 700ml.200ml dehydrated alcohol or 210ml95% ethyl alcohol is then added, mixes.It is steamed in graduated cylinder with three
Water is settled to 1L, and mixing can be used, can room temperature preservation.
(5) Western cleaning solution (TBST)
One bottle of Western cleaning solution (5X) is taken, is poured into clean beaker (specification 1L), tri-distilled water is added and constant volume arrives
1L, mixing is Western cleaning solution (1X), can be used for the washing of film, can room temperature preservation.
(6) 5%BSA (confining liquid)
2.5gBSA powder is weighed, is placed in 50ml centrifuge tube, 30mlTBST is measured with graduated cylinder and is added thereto, sufficiently dissolve,
It is settled to 50ml, -20 DEG C of preservations.
(7) 5% skimmed milk powers (confining liquid)
2.5g skimmed milk power is weighed, is placed in 50ml centrifuge tube, 30mlTBST is measured with graduated cylinder and is added thereto, it is sufficiently molten
Solution, is settled to 50ml, matching while using.
Experimental method
1 chronic unpredictable stress stimulation rat depression model grouping
SPF grades healthy SD rat 16, weight 200-250g, it is randomly divided into two groups every group 8: model group and control group.
Depression model is established to prepare for subsequent experimental.
The chronic unpredictable stress stimulation rat model grouping of 2 pharmaceutical interventions
According to randomly assigne, male and healthy SD rat is randomly divided into blank control group (C), depression model group (CUS), suppression
Strongly fragrant model GABABReceptor stimulating agent intervention group (CUS+Baclofen), depression model GABABReceptor antagonist intervention group (CUS+
CGP55845), depression model TREK-1 channel antagonist intervention group (CUS+Spadin), GABABReceptor stimulating agent intervention control
Group (C+Baclofen), GABABReceptor antagonist intervenes control group (C+CGP55845), TREK-1 channel antagonist intervention control
Group (C+Spadin), positive controls (CUS+ Citalopram), pharmaceutical intervention are intraperitoneal injection, and Baclofen injection dosage is
3mg/kg.d, CGP55845 injection dosage are 0.1mg/kg.d, and Spadin injection dosage is 0.1mg/kg.d, Citalopram
(Citalopram) injection dosage is 5mg/kg.d.Pharmaceutical intervention is for 4 weeks, and during which chronic stress stimulation is normally carried out, drug
Intervene dosage according to bibliography and laboratory preliminary result early period.
The preparation of 3 chronic stresses stimulation depression model
After Katz and Willner method improvement, depression model is prepared with chronic unpredictable stress stimulation
(chronic unpredictable stress, CUS), all rats are raised in the mouse cage of day-night cycle, 8, every cage,
It gives rat free diet before experiment, adapts to environment one week.Feeding environment is suitable for temperature: 22 ± 1 DEG C, humidity 55%-60%.
Environment adapt to after model group a kind of stimulation of rat is given once daily, continue 27 days, every kind stimulation two days in will not continuously occur,
Stimulation type includes: constraint in 30 minutes, fasting in 24 hours, 24 hours taboo water, 15 minutes warm water swimming (31 DEG C), 10 minutes cold water
Swimming (18 DEG C), single cage raising in 24 hours, 24 hours moist paddings add 30 ° of inclination mouse cages, 10 minutes folder rat-tails (away from rat-tail root
1 centimeters) and illuminate all night.Rat after stimulation rest on ensure within experimental site 1-2 hours stress smell disappearance, it is right
Touch processing is only done daily according to group rat.Depression model tests according to spacious field experiment and syrup preference after preparing and detects modeling
Whether succeed.
Embodiment 1
Behaviouristics detection --- spacious field tests (open field test, OFT)
Spacious field experiment is also known as Open field test, is evaluation experimental animal independent behaviour, exploratory behavior and tight in novel environment
A kind of method of tonicity.Rat spacious field experimental box (90cm × 90cm × 40cm) inner wall is black, and bottom surface is equally divided into 9 small sides
Lattice (30cm × 30cm), in Animal Behavior Science analysis system by spacious field experimental box since upper left, by left-to-right, top to bottm suitable
Sequence number consecutively, wherein 5 be middle section, remaining is outer region.One DV of frame at 1 meter of surface, the visual field can
It covers inside entire spacious field.Spacious field illumination is full artificial light (about 40LX), and light is avoided to interfere.Experiment in quiet environment into
Row, the equipment such as experimenter and computer are placed in another room to reduce the interference to animal behavior, laboratory background noise control
System is in 65dB or less.When experiment, rat tail is pinched away from being put down gently in No. 5 grids at root 2/3, experimenter leaves test immediately
Room, while behavior analysis system starts to analyze, and automatically records 5 minutes activity conditions of experimental rat.Analysis indexes include:
Movement total distance, the middle section residence time, level wears lattice number (horizontal score) and fore paw vertically picks up number (vertical score).
Every time after experiment, spacious field experimental box is sterilized with 75% alcohol wipe, a rat smell or excreta is avoided to interfere next
Behavioral experiment data.
Spacious field experimental result
In spacious field experiment (Fig. 2), compared with blank control group, the movement total distance of CUS group rat (C:19885.96 ±
6862.46,CUS:6243.16±2940.12；P < 0.05), middle section residence time (C:6.92 ± 1.43, CUS:2.66
±1.24；P < 0.01), horizontal score (C:53.75 ± 8.53, CUS:11.67 ± 4.72；P < 0.01) and vertical score (C:
18.13±4.88,CUS:3.5±1.05；P < 0.01) significantly reduce, and C+Baclofen group, C+CGP55845 group and C+
Spadin group then no significant difference (p > 0.05)；Compared with CUS group, CUS+CGP55845 group, CUS+Spadin group and CUS+
Citalopram group rat movement total distance (CUS:6243.16 ± 2940.12, CUS+CGP55845:12210.47 ±
3871.13, CUS+Spadin:11368.11 ± 3395.49, p < 0.05；CUS+Citalopram:13887.89±
1971.79, p < 0.01), central area residence time (CUS:2.66 ± 1.24, CUS+CGP55845:5.26 ± 1.34, CUS+
Spadin:6.18±1.29,CUS+Citalopram:5.38±0.67；P < 0.01), horizontal score (CUS:11.67 ±
4.72,CUS+CGP55845:32.83±6.61,CUS+Spadin:31.67±8.64,CUS+Citalopram:26.83±
5.34；P < 0.01) and vertical score (CUS:3.5 ± 1.05, CUS+CGP55845:10.00 ± 2.10, CUS+Spadin:
11.50±2.88,CUS+Citalopram:7.84±1.94；P < 0.01) it increased significantly, CUS+Baclofen group rat
Central area residence time (CUS:2.66 ± 1.24, CUS+Baclofen:0.75 ± 0.56；P < 0.05), horizontal score (CUS:
11.67±4.72,CUS+Baclofen:6.67±1.21；P < 0.05) and vertical score (CUS:3.5 ± 1.05, CUS+
Baclofen:1.00±0.89；P < 0.05) it significantly reduces, move total distance (CUS:6243.16 ± 2940.12, CUS+
Baclofen:3420.87±1071.10；P=0.06) it is reduced trend.As a result chronic unpredictable stress stimulation is passed through in prompt
SD rat afterwards independent behaviour and exploratory behavior in strange environment weaken, TREK-1 channel antagonist, GABABReceptor antagonist
It can be relieved the decrease of above-mentioned behavior, GABA with serotonin reuptake inhibitorBReceptor stimulating agent then promote above-mentioned behavior into
One step weakens.
Fig. 1 .GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene chronic unpredictable stress pierce
Swash the spacious field experimental result of SD rat.The total distance of A rat motor；Time of the B rat in middle section；The horizontal score of C rat；
The vertical score of D rat.Baclofen and CGP55845 is respectively GABABReceptor stimulating agent and antagonist, Spadin TREK-1
Channel antagonist, Citalopram are positive control medicine serotonin reuptake inhibitor Citalopram；Data with mean ±
Standard deviation indicates；* p < 0.05, * * p < 0.01；(n=8/group).
Embodiment 2
Behaviouristics detection --- syrup preference tests (sucrose preference test, SPT)
The experiment of syrup preference is a kind of Behaviors survey method for evaluating rat anhedonia degree.Since pleasant sensation lacks
The core symptom for the depression that can most withdraw deposit is lost, therefore this experiment can largely react the Degree of Depression of rat.Referring to related
Document needs first trained rat to learn and adapts to drink sucrose water before testing formal start.Every cage need to first place two bottle 1% of sugarcane
Syrup allows rat first to attempt to drink sucrose water 24 hours；Subsequent second 24 hours every cage is changed to one bottle of normal rat and drinks
Pure water and one bottle of 1% sucrose water, wherein needing to change within 12 hours two water bottle left-right positions；24 hours every cages of last third place two
Bottle normal rat drinking pure.Between being needed after sucrose water adaptation every two days, then row syrup preference is tested.All rats are single
The normal drinking pure of two bottles of identical weights and 1% sucrose water are solely raised and given, replaces two water bottle positions after 12 hours, 24
Two bottles of water are taken away after hour and measure respective consumption.Syrup preference ratio=syrup consumption (g)/[pure water consumes (g)+syrup
Consume (g)] × 100%.
Fig. 2 .GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene chronic unpredictable stress pierce
Swash the syrup preference experimental result of SD rat.Baclofen and CGP55845 is respectively GABABReceptor stimulating agent or antagonist,
Spadin is TREK-1 channel antagonist, and Citalopram is that the western phthalein of positive control medicine serotonin reuptake inhibitor is general
It is blue；Data are indicated with mean ± standard deviation；* p < 0.05, * * p < 0.01；(n=8/group).
Embodiment 3
Western blot detection
(1) rat cerebral tissue's holoprotein extracts
1) holoprotein extracts kit is used, 10 μ l inhibitors of phosphatases, 1 μ l is added in every cold Lysis Buffer of 1ml
Protease inhibitors and 10 μ l 100mM PMSF are mixed, and several minutes of preservation on ice spare.
2) the subregion brain tissue taken is placed in pre-cooling centrifuge tube, it is cold that 0.5ml is added according to every 100mg solid tissue
Cold Lysis Buffer is added in the standard of Lysis Buffer.With homogenate 30-50 times up and down manually of high-speed homogenization machine, when operation
Keep low temperature environment.
3) centrifuge adjusts the temperature to 4 DEG C of pre-coolings, tissue homogenate is transferred in the centrifuge tube of 1.5 pre-coolings, according to
12000 revs/min of centrifugations, 4 DEG C are centrifuged 5 minutes.
4) supernatant is taken to be transferred in the centrifuge tube of new pre-cooling after being centrifuged, as holoprotein extract.
5) protein sample packing is stored in -80 DEG C of refrigerators, avoids multigelation.
(2) BCA protein quantification
1) it prepares BCA working solution: adding 1 volume BCA reagent B (50:1) to prepare by 50 volume BCA reagent As according to sample size
Suitable BCA reagent is mixed well and is placed in ice chest for use.
2) Protein standards are sequentially added to the standard sample wells of 96 orifice plates by the sequence of 0,1,2,4,8,12,16,20 μ l
In, add deionized water to supply to 20 μ l, then plus 200 μ lBCA working solutions.
3) testing protein sample is taken, suitable concentration is diluted to.
4) protein sample that 20 μ l have diluted is added in each hole, adds prepared 200 μ l of BCA working solution.ELISA Plate
It is placed on concussion instrument to shake 30 seconds, mixes well, be placed in 37 DEG C of incubators 30 minutes.
5) colorimetric estimation under 562nm wavelength.With protein content (ug) for abscissa, light absorption value is ordinate, draws mark
Directrix curve.
6) according to the light absorption value of institute's sample, corresponding protein content (ug) can be calculated to obtain on standard curve, divided by sample
Product dilution total volume (20 μ l) is sample actual concentrations (unit: ug/ μ l) multiplied by sample extension rate.
(3) SDS- polyacrylamide gel electrophoresis (SDS-PAGE)
1) determine that resolving gel concentration is 10% according to detected molecular weight of albumen, concentration gum concentration is 5%
2) glass plate is cleaned, upper cleaning solution is dipped in sponge and gently cleans.Two sides clean after rinsed with tap water, then with steaming
Distilled water is stood after rinsing well dries on offset plate frame.
3) encapsulating: first choice is poured into offset plate with tri-distilled water stands 10 minutes, checks whether leak.First match according to gum concentration
Separation gel processed, separation gel top are added dehydrated alcohol and flatten, be stored at room temperature 40 minutes.After being gelled admittedly, dehydrated alcohol is poured out.Match
System concentration glue, mixes, to glass plate top in implantation glass plate, is rapidly inserted into comb, is stored at room temperature 40 minutes, make separation gel and
Concentration glue sufficiently polymerize.
4) prepare protein sample: by the albumen taking-up of concentration after measured, 5 × SDS sample-loading buffer is added to final concentration
For 1 ×, it is put into dry-type thermostat, 100 DEG C of heating make albuminous degeneration in 5 minutes, pay attention to preventing booster.
5) electrophoresis: prepared offset plate is placed in electrophoresis tank, and the sufficient electrophoresis liquid of injection is loaded to not crossing inside glue
The conductor wire that electrophoresis liquid there was not bottom is also added in hole, periphery, guarantees to be powered.It is vertical to extract comb, remove the bubble in loading hole
And with indenting ball righting glue groove.With the adherent addition protein sample of micropipettor, albumen applied sample amount is 30ug.It is inserted into electrophoresis apparatus electricity
Source, adjusting voltage are constant 90V, and voltage is adjusted to constant 120V after 15 minutes, until bromophenol blue moves to separation gel bottom, are closed
Power supply takes out gel.
(4) transferring film
Gel is cut in the container for being put into and filling transferring film liquid with glue device is cut, and cuts phase according to the size of cut gel
The pvdf membrane for answering size is placed in anhydrous methanol and impregnates activation in 1 minute, places into water and rinse, be finally putting into transferring film liquid
Balance 15 minutes to be used.Transferring film folder, sponge and filter paper are put into the enamel tray added with transferring film liquid.Transferring film is pressed from both sides into black portions
Downward, sponge, 3 layers of filter paper, gel, pvdf membrane, 3 layers of filter paper, sponge are sequentially placed, every step is all needed with cutting glue device above gently
It presses through, drives bubble away.Transferring film is folded up in transferring film slot, black flour of the transferring film folder black flour to slot, red face of the fine flour to slot are made.It adjusts
Constant current 300mA transferring film 90 minutes.Electrophoresis tank need to be placed in ice chest when transferring film, avoiding heat production from destroying transferring film becomes band
Shape.
(5) it closes
Pvdf membrane is taken out from transferring film folder rapidly after to transferring film, is face-up put into the container wash-in equipped with TBST
It washs, is then transferred in the incubation box of prepared 5% skimmed milk power (or 5%BSA), is placed on shaking table, room temperature closing 2 is small
When.Notice that skimmed milk power is ready-to-use, BSA recoverable 2-3 times, -20 DEG C of preservations.
(6) primary antibody is incubated for
Skimmed milk power (recycling BSA) is outwelled, is washed film 3 times, every time 10 minutes with TBST.It is separately added into prepared in proportion
Primary antibody [channel TREK-1 (1:500), GABABReceptor (1:500), GAPDH (1:1000)], 4 DEG C of shaking tables are stayed overnight.
(7) secondary antibody is incubated for
Recycle primary antibody.TBST is washed film 3 times, every time 10 minutes.Be incubated for prepared secondary antibody [goat anti-rabbit igg/HRP (1:
5000), mountain sheep anti mouse IgG/HRP (1:5000)], it is incubated on shaking table after twenty minutes, moves to and be incubated for 30 points in 37 DEG C of insulating boxs
Clock is incubated for 10 minutes on shaking table again later.
(8) chemiluminescence and development
Recycle secondary antibody.TBST is washed film 3 times, every time 10 minutes.According to the ratio preparing developer liquid of 1:1, it is added dropwise with pipettor
Develop on to pvdf membrane, is imaged using gel imager.
(9) Image-Pro Plus6.0 carries out gray analysis
Western Blot result
1.GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist are to hippocampus in depression model rat brain
TREK-1 channel protein and GABABThe influence of receptor protein expression
In the rat hippocampus area channel TREK-1 and GABABIn the Western blot result of receptor protein expression (such as Fig. 4),
Compared with blank control group, CUS group rat hippocampus area's TREK-1 channel protein and GABABReceptor protein expression (TREK-1, C:
1.00±0.00；CUS:0.62 ± 0.07, p < 0.01；GABAB,C:1.00±0.00；CUS:0.55 ± 0.09, p < 0.01)
Significantly reduce；Compared with CUS group, CUS+CGP55845 group, CUS+Spadin group and CUS+Citalopram group rat hippocampus
Expression (CUS:0.62 ± 0.07, CUS+CGP55845:0.88 ± 0.08, CUS+Spadin:0.91 of area's TREK-1 channel protein
±0.09,CUS+Citalopram:0.94±0.09；P < 0.01) obviously increase, and CUS+Baclofen group rat hippocampus
Expression (CUS:0.62 ± 0.07, CUS+Baclofen:0.28 ± 0.06 of area's TREK-1 channel protein；P < 0.01) significantly subtract
It is few；Compared with CUS group, CUS+Baclofen group rat hippocampus area GABABExpression (CUS:0.55 ± 0.09, the CUS of receptor protein
+Baclofen:0.28±0.06；P < 0.05) it significantly reduces, CUS+CGP55845 group rat hippocampus area GABABReceptor protein table
Up to (CUS:0.55 ± 0.09, CUS+CGP55845:0.87 ± 0.05；P < 0.01) significantly increase, CUS+Spadin group rat sea
Horse area GABABExpression (CUS:0.55 ± 0.09, CUS+Spadin:0.62 ± 0.07 of receptor protein；P > 0.05) without conspicuousness
Difference.Gray value statistical result is shown in (Fig. 4, B and C).
Fig. 3 .GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene chronic unpredictable stress pierce
Swash SD rat hippocampus area TREK-1 and GABABThe western blot result of expression.The channel hippocampus TREK-1 A and GABABReceptor
Western blot result；B GABABReceptor gray analysis statistical result；The channel C TREK-1 gray analysis statistical result.
Statistical result is the gray level ratio result of purpose albumen and internal reference；Baclofen and CGP55845 is respectively GABABReceptor agonism
Agent and antagonist, Spadin are TREK-1 channel antagonist, and Citalopram is the suppression of positive control medicine serotonin reuptake transporter
Preparation Citalopram；Data are indicated with mean ± standard deviation；* p < 0.05, * * p < 0.01；(n=5/group).
2.GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist are to frontal lobe skin in depression model rat brain
Layer TREK-1 channel protein and GABABThe influence of receptor protein expression
In the rat channel frontal cortex TREK-1 and GABAB(as schemed in the Western blot result of receptor protein expression
4), each group rat frontal cortex TREK-1 channel protein and GABABReceptor protein is expressed without significant difference (p > 0.05)
Fig. 4 .GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene chronic unpredictable stress pierce
Swash the SD rat channel frontal cortex TREK-1 and GABABThe western blot result of expression of receptor.A frontal cortex TREK-1 is logical
Road and GABABThe western blot result of receptor；B GABABReceptor gray analysis statistical result；C TREK-1 channel gray scale point
Analyse statistical result.Statistical result is the gray level ratio result of purpose albumen and internal reference；Baclofen and CGP55845 are respectively
GABABReceptor stimulating agent and antagonist, Spadin are TREK-1 channel antagonist, and Citalopram is positive control medicine 5- hydroxyl
Reuptake inhibitors Citalopram；Data are indicated with mean ± standard deviation；(n=5/group)
Embodiment 4
Morphologic detection
(1) brain tissue perfusion
1) it anaesthetizes: 4% chloraldurate, 0.01ml/g, intraperitoneal injection.Inserting needle near groin, is walked to ventrimeson direction
Row.If anaesthetic effect is bad, it can suitably increase dosage.
2) fixed: rat four limbs being fixed on cystosepiment with syringe needle, cystosepiment are placed in pallet, wherein one
While riding over tray edge, cystosepiment is tilted, perfusate is facilitated to flow out.
3) perfusion liquid prepares: extracting 150ml1% paraformaldehyde, 100ml4% paraformaldehyde, 1% paraformaldehyde respectively
Before connect sword-shaped needle, connect conventional needle before 4% paraformaldehyde, empty air, it is ensured that syringe needle is unobstructed, spare.
4) perfusion: cutting off abdominal cavity first, then cut off diaphram, cuts off thorax, exposure heart on a small quantity.It is solid with skin pincers or haemostatic clamp
Determine skin and thorax etc., cuts off pericardium.By the apex of the heart into sword-shaped needle, inserting needle is unsuitable too deep, otherwise can puncture heart.There is discovery after inserting needle
Blood backflow usually indicates that position is accurate, cuts off right auricle of heart, slowly injects emitter, at this time if there is kermesinus in heart to sword-shaped needle
Liquid outflow, then inserting needle is accurate.The speed of 50ml/10ml or so is advisable.1% paraformaldehyde is first injected, 4% poly first is reinjected
Rat after the completion of the two equal perfusions of pipe liquid, is placed at room temperature for 30 minutes by aldehyde, keeps its brain tissue sufficiently fixed.
5) it removes brain tissue: carefully removing brain tissue with haemostatic clamp, avoid damaging.
6) fixed: brain tissue being placed in the centrifuge tube containing 4% paraformaldehyde, fixes 3 days.
7) serial dehydration: the paraformaldehyde in the centrifuge tube equipped with brain tissue being discarded, prepared sucrose solution is injected,
Serial dehydration is carried out according to 10%, 20%, 30% (twice) three concentration.Each concentration sucrose is dehydrated 1 day, thorough to brain tissue
It sinks to the bottom to be dehydrated completely, i.e., the sucrose of replaceable high concentration continues to be dehydrated, until dehydration is complete.
(2) brain tissue frozen section
First 3 hours debugging freezing microtome temperature of slice are extremely pre-chilled for -22 DEG C in advance.The complete rat cerebral tissue of dehydration is taken,
Sucrose solution is discarded, brain tissue surface, equating brain tissue bottom are gently wiped with filter paper.Brain tissue is placed on sample carrier and is protected
Its state in a vertical shape is demonstrate,proved, liquid is embedded on surface plus appropriate OTC, is put into the freezing microtome of pre-cooling about 90 minutes, to brain tissue
All after frost, it is sliced.Slice thickness 16um.Every group of slice marks, and -20 DEG C save backup.
(3) rat immunity groupization is tested
1) it from -20 DEG C of taking-up frozen sections, is put into spare in wet box.Slice drying is placed on shaking table, washes 3 with PBS
It is secondary, 5 minutes every time.
2) it takes out slice to be put into wet box, the liquid (keeping tissue in moisture state) on surface is blotted with filter paper.Use liquid relief
Hydrogen peroxide (3%H is added dropwise in device2O2Deionized water) closing endogenous peroxydase, wet box lid is covered, is protected from light, room temperature 5-10 points
Clock, it is unsuitable too long, in order to avoid rotten.
3) it is placed on shaking table, is washed 3 times, every time 5 minutes with PBS.
4) it takes out slice to be put into wet box, blots the liquid (keeping tissue in moisture state) on surface.Reagent A (mountain is added dropwise
Sheep blood serum working solution), wet box lid is covered, 37 DEG C are incubated for 30 minutes, and reclaim reagent A is not washed.
5) diluted primary antibody is added dropwise, guarantees that tissue surface and periphery are completely covered by primary antibody, 4 DEG C overnight.
6) slide is tilted, primary antibody is recycled.It is placed on shaking table, is washed 3 times, every time 5 minutes with PBS.
7) it takes out slice to be put into wet box, blots surface liquid (keeping tissue in moisture state).Reagent B (biology is added dropwise
Elementization secondary antibody working solution), wet box lid is covered, 37 DEG C are incubated for 40 minutes.
8) it is placed on shaking table, is washed 3 times, every time 5 minutes with PBS.
9) it takes out slice to be put into wet box, blots the liquid (keeping tissue in moisture state) on surface.It is (peppery that reagent C is added dropwise
Root enzyme marks streptomysin albumen working solution), wet box lid is covered, 37 DEG C are incubated for 40 minutes.
10) it is placed on shaking table, is washed 3 times, every time 5 minutes with PBS.
11) DAB colour developing is carried out, DAB is ready-to-use, uses, is kept in dark place in 30 minutes.DAB developing solution, Jing Xiaguan is added dropwise
It examines, terminates (PBS buffer solution is dipped in) in due course, terminated in general 8-15 minutes.
12) carry out Gradient elution using ethanol, respectively by slice develop the color be dipped in gradient for 70%, 80%, 90%, 95%,
100%, in 100% ethyl alcohol, 3 minutes every time.It taking out, dimethylbenzene is transparent twice, and 5 minutes every time.
13) mounting, slice be added dropwise rapidly after being taken out in dimethylbenzene neutral tree film (prevent it is dry after organize it is dry and cracked, if envelope
Piece is ineffective, can put back in dimethylbenzene mounting again after dissolving).Bubble is careful not to when mounting, natural gum uses appropriate.From
It so dries, room temperature preservation.
14) using just setting optical electron microscope observation staining conditions and taking pictures, with Image-Pro Plus6.0 to immune
Groupization picture brain area carries out photodensitometry.
Data statistic analysis
Statistical analysis is carried out using SPSS 21.0 (Aramonk, NY, USA).All data use mean ± standard deviation
It indicates, is examined and variance analysis conspicuousness using T.P < 0.05 is to have significant difference.
ImmunohistochemistryResults Results
GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist are to hippocampus CA1 in depression model rat brain
The influence of area's TREK-1 immune response activity
In the immunohistochemical experiment of the CA 1 of Hippocampus channel TREK-1 expression (such as Fig. 5 A), with blank control group phase
Than the Hippocampal CA 1 channel the TREK-1 expression of, CUS group rat positive neuron OD value (C:0.044 ± 0.005, CUS:
0.258±0.003；P < 0.01) it is substantially reduced；Compared with CUS group, CUS+CGP55845 group, CUS+Spadin group and CUS+
Citalopram group CA 1 of Hippocampus TREK-1 channel protein expression positive neuron OD value (CUS:0.258 ±
0.003,CUS+CGP55845:0.040±0.004,CUS+Spadin:0.040±0.005,CUS+Citalopram:0.038
±0.004；P < 0.01) obviously increase, and the positive mind of the CUS+Baclofen group CA 1 of Hippocampus channel TREK-1 expression
Through first OD value (CUS:40.258 ± 0.003, CUS+Baclofen:0.0180 ± 0.005；P < 0.01) it significantly reduces.Light
Density value statistical result such as (Fig. 5, D)
GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist are to hippocampus CA2 in depression model rat brain
The influence of the area channel TREK-1 immune response activity
In the immunohistochemical experiment of the area the rat hippocampus CA2 channel TREK-1 expression (such as Fig. 5 B), with blank control group phase
Than the area the hippocampus CA2 channel the TREK-1 expression of, CUS group rat positive neuron OD value (C:0.030 ± 0.004, CUS:
0.018±0.003；P < 0.01) it is substantially reduced；Compared with CUS group, CUS+CGP55845 group, CUS+Spadin group and CUS+
Citalopram group rat hippocampus CA2 area's TREK-1 channel protein expression positive neuron OD value (CUS:0.018 ±
0.003,CUS+CGP55845:0.028±0.004,CUS+Spadin:0.028±0.003,CUS+Citalopram:0.027
± 0.003:p < 0.01) significantly increase, the positive neurons of the area the group rat hippocampus CA2 channel TREK-1 CUS+Baclofen expression
First OD value (CUS:0.018 ± 0.003, CUS+Baclofen:0.013 ± 0.002；P < 0.01) it significantly reduces.Optical density
Data-Statistics result such as (Fig. 5, E)
Fig. 5 .GABABReceptor stimulating agent or antagonist and TREK-1 channel antagonist intervene chronic unpredictable stress pierce
Swash the ImmunohistochemistryResults Results of the SD rat hippocampus area channel TREK-1 expression.The immunohistochemistry knot of the Hippocampal CA 1 A TREK-1 expression
Fruit；The ImmunohistochemistryResults Results of the area the B hippocampus CA2 channel TREK-1 expression；The brain area schematic diagram of C hippocampus CA1 and the area CA2；D hippocampus
The OD value statistical result of the positive neuron of the area the CA1 channel TREK-1 expression；The expression of the area the E hippocampus CA2 channel TREK-1
The OD value statistical result of positive neuron.Baclofen and CGP55845 is respectively GABABReceptor stimulating agent and antagonist,
Spadin is TREK-1 channel antagonist, and Citalopram is that the western phthalein of positive control medicine serotonin reuptake inhibitor is general
It is blue；Data are indicated with mean ± standard deviation；* p < 0.01；(n=5/group).
For any person skilled in the art, without departing from the scope of the technical proposal of the invention, all
Many possible changes and modifications are made to technical solution of the present invention using the technology contents of the disclosure above, or are revised as equivalent
The equivalent embodiment of variation.Therefore, anything that does not depart from the technical scheme of the invention, according to the technical essence of the invention to
Any simple modifications, equivalents, and modifications that upper embodiment is done should all still fall within the range of technical solution of the present invention protection
It is interior.
Claims (3)
1.GABABApplication of the receptor antagonist in terms of blocking the channel TREK-1.
2. application according to claim 1, it is characterised in that: the GABABThe dosage of receptor antagonist is
0.016mg/kg.d。
3. application according to claim 1, it is characterised in that: the GABABReceptor antagonist includes CGP55845, CGP-
35348 and CGP52432.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114555808A (en) * | 2019-10-22 | 2022-05-27 | 吉尼松公司 | Chimeric polypeptides and uses thereof |
| CN117257815A (en) * | 2023-11-20 | 2023-12-22 | 中国人民解放军军事科学院军事医学研究院 | New application of CGP35348 in preparation of antidepressant drugs |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882598A (en) * | 2003-11-21 | 2006-12-20 | 诺瓦提斯公司 | Phosphinic acid derivatives |
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2018
- 2018-09-13 CN CN201811069657.8A patent/CN109125331A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1882598A (en) * | 2003-11-21 | 2006-12-20 | 诺瓦提斯公司 | Phosphinic acid derivatives |
Non-Patent Citations (1)
| Title |
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| FELIX BRUGGER ET AL.,: "The action of new potent GABA B receptor antagonists in the hemisected spinal cord preparation of the rat", 《EUROPEAN JOURNAL OF PHARMACOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114555808A (en) * | 2019-10-22 | 2022-05-27 | 吉尼松公司 | Chimeric polypeptides and uses thereof |
| CN117257815A (en) * | 2023-11-20 | 2023-12-22 | 中国人民解放军军事科学院军事医学研究院 | New application of CGP35348 in preparation of antidepressant drugs |
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