CN105055412B - Purposes of the NAMPT inhibitor in the drug for preparing treatment cocaine habituation - Google Patents

Abstract

The invention discloses purposes of the NAMPT inhibitor in the drug for preparing treatment dependence producing drug habituation.The invention also discloses a kind of drugs for treating cocaine habituation, it is using NAMPT inhibitor as active constituent, in addition the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared.NAMPT inhibitor of the present invention can effectively weaken cocaine award behavior, treat cocaine habituation, potential applicability in clinical practice is good.

Description

Purposes of the NAMPT inhibitor in the drug for preparing treatment cocaine habituation

Technical field

The present invention relates to purposes of the NAMPT inhibitor in the drug for preparing treatment cocaine habituation.

Background technology

Cocaine (Cocaine) is also known as cocaine, the entitled benzoyl-methyl-ecgonine (methyl of chemistry Benzoylecgonine), general white lenticular, it is odorless, bitter and it is numb, for local anaesthesia and treat asthma earliest, It is most strong natural central stimulant, causes to abuse due to the excitation of its Central nervous system, as generation from 1985 One of main drugs of criticality.

Cocaine habituation is a kind of chronic recurrent brain diseases, belongs to pharmacological dependence (drug dependence) class disease Disease, cocaine habituation can cause the Changes of Plasticity of brain structure and function, and related brain areas includes nucleus accumbens septi, corpus straitum, forehead Cortex, hippocampus and ventral tegmental area, health are disorderly including mental deterioration, personality defect, intelligence function also by various harm Disorderly, concurrent corresponding infection complication and drug addict seek and use drugs and induce various delinquent work at all adventures It is dynamic.

Even if the main feature of cocaine habituation shows as patient after the serious consequence of medication is known, sex cords is still forced Take and use with meet desire, to drug seek and ask for it is out of hand, lose interest to things, Drug addiction it is very deep It carves, after the withdrawal and treatment several years, the contact stimulation related with habituation is (such as poison friend, the environment related with medication in the past Deng) all may induce relapse.

In view of cocaine habituation harm is very big, suitable therapy target and drug are found, treatment cocaine habituation is compeled in eyebrow Eyelash.

Invention content

To solve the above-mentioned problems, the present invention provides the drugs of a kind for the treatment of cocaine habituation.

NAMPT：Nampt.

NAMPT inhibitor：Inhibit the enzyme activity of Nampt or the substance of expression.

Present invention firstly provides purposes of the NAMPT inhibitor in the drug for preparing treatment cocaine habituation.

Preferably, the drug of the reduction NAMPT enzyme activity of the treatment cocaine habituation.It is further preferred that the reduction The drug of NAMPT enzyme activity is FK866, and structural formula is as follows：

The present invention also provides a kind of drugs for treating cocaine habituation, it is using NAMPT inhibitor as active constituent, is added The preparation that upper pharmaceutically acceptable auxiliary material or complementary ingredient are prepared.

Preferably, the NAMPT inhibitor is the drug for reducing NAMPT enzyme activity.It is further preferred that reduce NAMPT enzymes Drug living is FK866, and structural formula is as follows：

Preferably, the preparation is ejection preparation.

NAMPT inhibitor can effectively weaken cocaine award behavior, treat cocaine habituation, potential applicability in clinical practice is good It is good.

The specific embodiment of form by the following examples makees further specifically the above of the present invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following embodiment.It is all to be wanted based on right of the present invention The technology that the content that secretary carries is realized is asked to all belong to the scope of the present invention.

Description of the drawings

Fig. 1 SIRT1 knock out mice genotype identifications.1st, 5,6,7,8,9 is small for SIRT1 midbrain condition knockout homozygote Mouse；3 be the SIRT1 hybrid mices containing Wnt1；2nd, 4 be the wild-type mice containing Wnt1.

Fig. 2 Conditioned place preference casees

Fig. 3 Conditioned place preference experimental design schematic diagrames

Fig. 4 spontaneous activity in mice casees

Fig. 5 pReceiver-Lv201 carrier figures.

Fig. 6 VTA position collection of illustrative plates

Fig. 7 cocaine Conditioned places preference is tested.Mouse through cocaine CPP training after, cocaine administration group (CO) with Saline control group (SA) is compared, and CPP effects increase, * p<0.05.

Fig. 8 cocaines induce spontaneous activity.Cocaine administration group (CO) measures two compared with saline control group (SA) The distance (cm) of group mouse has significant difference (* p<0.05)；#p<0.05, administration group respectively Day2,3,4,5,6,7 with Day1, which is compared, significant difference.

The expression of Fig. 9 NAMP Different brain region in cocaine and physiological saline group CPP models.(A)Western Blot detects NAMPT in prefrontal cortex (PFC), nucleus accumbens septi (NAc), corpus straitum (Striatum), hippocampus (Hippocampus) With the expression of Ventral Midbrain tegmental region (VTA).(B) statistic analysis result that NAMPT is expressed in A figures is represented.*p<0.05, it represents Cocaine administration group (CO) has significant difference compared with saline control group (SA).

The expression of Figure 10 RT-PCR detection cocaine inductions NAMPT.(A) in cocaine CPP models, in NAc and VTA NAMPT expression variations.The up-regulation that NAMPT is expressed in cocaine induction VTA, * p<0.05, and NAc does not change.(B) cocaine In acute model, NAMPT expression variation in NAc and VTA.After cocaine single-dose after 30min, single-dose for 24 hours, it continuously gives Medicine 7d (1 times/day) NAc, VTA brain areas NAMPT does not have significant change, cocaine administration group (CO) and saline control group (SA)。

Figure 11 mouse peritoneals inject FK866 to ethological influence.(A) cocaine CPP shot charts.Mouse peritoneal is injected FK866 reduces cocaine CPP effects, #p<0.05.(B) spontaneous activity in mice changes.Mouse peritoneal injection FK866 can inhibit certainly Hair activity, p<0.05.Con-SA, solvent-physiological saline group；Con-CO, solvent-cocaine group；FK866-SA, FK866- physiology Brine group；FK866-CO, FK866- cocaine group.

Figure 12 mouse VTA intracerebrals to after FK866 to the influence of mice behavior.(A) cocaine CPP shot charts.Mouse VTA Intracerebral injection FK866 cocaines CPP is reduced, #p<0.05.(B) spontaneous activity in mice changes.Mouse VTA intracerebral injections FK866 can Inhibit spontaneous activity, p<0.05.Con-SA, solvent-physiological saline group；Con-CO, solvent-cocaine group；FK866-SA, FK866- physiological saline groups；FK866-CO, FK866- cocaine group.

Influences of the NMN to cocaine CPP is supplemented after Figure 13 mouse VTA intracerebral injections FK866.It is supplemented after giving FK866 NMN, cocaine CPP restore, #p<0.05.SA represents that physiological saline group is given in abdominal cavity, and CO gives in abdominal cavity cocaine group.Control Represent control group；FK866+SA represents VTA intracerebrals to supplementing SA after FK86630min；FK866+NMN represents that VTA intracerebrals are given NMN is supplemented after FK86630min.

Figure 14 VTA intracerebral injections NAMPT influences of the virus to mouse cocaine CPP excessively.(A) Western Blot are detected NAMPT expressions.Being overexpressed (LV-NAMPT) NAMPT expression compared with control (LV-GFP) group increases, p<0.05.(B)VTA Intracerebral injection NAMPT is overexpressed the influence to mouse cocaine CPP after virus.NAMPT is overexpressed (LV-NAMPT) with compareing (LV-GFP) cocaine group increases compared to cocaine CPP, #p<0.05.LV-GFP SA, GFP- physiological saline groups；LV-GFP CO, GFP- cocaine groups；LV-NAMPT SA are overexpressed NAMPT- physiological saline groups；LV-NAMPT CO are overexpressed NAMPT- cocker Because of group.

The OSC-PLS-DA models of the NAc and NAc of the cocaine CPP model mice samples of Figure 15 intraperitoneal injections FK866 carry Lotus is schemed.A, C is respectively cocaine group and the OSC-PLS-DA Model load figures of saline control group VTA and NAc.B, D distinguishes For FK866 cocaine administrations group and the OSC-PLS-DA Model load figures of cocaine group VTA and NAc.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administrations group (FK866+CO).

The VTA brain area metabolism spectrums of the cocaine CPP model mice samples of Figure 16 intraperitoneal injections FK866.A is based on 1H NMR's The OSC-PLS-DA shot charts of cocaine group and saline control group.FK866 administration group and cocaine group of the B based on 1H NMR OSC-PLS-DA shot charts.C statistical testing of business cycles models obtained from PLS-DA arrangement analysis corresponding in A, R2 are represented Interpretability variable, Q2 represent the verification ability variables D of the model obtained from PLS-DA arrangement analysis corresponding in B Statistical testing of business cycles model.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administrations group (FK866+CO).

The NAc brain area metabolism spectrums of the cocaine CPP model mice samples of Figure 17 intraperitoneal injections FK866.A is based on 1H NMR's The OSC-PLS-DA analysis charts of cocaine group and saline control group.FK866 administration group and cocaine group of the B based on 1H NMR OSC-PLS-DA analysis charts.C statistical testing of business cycles models obtained from PLS-DA arrangement analysis corresponding in A, R2 are represented Interpretability variable, Q2 represent the verification ability variables D of the model obtained from PLS-DA arrangement analysis corresponding in B Statistical testing of business cycles model.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administrations group (FK866+CO).

Figure 18 different dosing group groups Different brain region metabolin variation box traction substation.(A) VTA brain areas；(B) NAc brain areas.In box Between line represent median；An offline and point table of reaching the standard grade for box represents the 25th and the 75th quantile；Nethermost line and most Line above represents minimum value and maximum value respectively.Saline control group (SA), cocaine administration group (CO), FK866 cocker Because of administration group (FK866CO).

The variation diagram of NAD in Figure 19 different dosing group VTA brain areas.NAD variations have statistics to administration group compared with the control group Meaning, * p<0.05.Control, intracerebral injection SA；FK866, intracerebral injection FK866；FK866+NMN, VTA intracerebral are given NMN is supplemented after FK86630min；LV-NAMPT is overexpressed NAMPT groups.Corresponding SA and CO be respectively saline control group, Cocaine administration group.

Specific embodiment

1 NAMPT inhibitor for treating cocaine habituations of experimental example

1 foreword

In this research, we initially set up cocaine CPP mouse models, and mouse cocaine award effect is detected by behaviouristics Should, by detecting the brain areas NAMPT such as VTA, NAc expression variations the methods of PCR, immunoblotting.Intracerebral locating injection NAMPT inhibits Agent, supplement NMN and NAMPT are overexpressed viral vectors, it is found that CPP effects inhibit or enhance.Secondly, using based on nuclear magnetic resonance The technology and kit of metabolism group, detection brain area and the variation of NAMPT metabolism correlative metabolites, it is found that NAMPT inhibits or swashs Living, corresponding NAD levels reduce or raising.The result illustrates that NAMPT participates in regulation and control cocaine CPP by influencing the synthesis of NAD Effect.Finally by SIRT1 midbrain conditionity knock-out mices, work of the verification NAMPT-NAD-SIRT1 approach in cocaine CPP With.This research find for the first time NAMPT-NAD-SIRT1 accesses participate in cocaine drug habit, for it is deep understanding habituation mechanism and Addiction therapy provides new test basis, it was found that NAMPT inhibitor can treat cocaine habituation.

2 experiment materials and method

2.1 experiment materials and equipment

2.1.1 test specimen

Cocainehydrochloride is purchased from Chinese pharmaceutical biological product and identifies institute, white powder, and purity is more than 99%, product batch number： 171210-200803；Physiological saline selects Kelun Pharm Ind Co., Ltd., Sichuan, authentication code：Chinese medicines quasi-word H51021158, product batch number M12101307.Cocainehydrochloride normal saline dilution to required concentration.FK866, β-niacinamide Mononucleotide (β-NMN) is purchased from Sigma companies.

2.1.2 experimental animal

(1) SPF grades of wild type C57BL/6J mouse of male, by Beijing, experimental animal Co., Ltd of Wei Duoli China carries For, it did not mated, weight 20-22g.

(2) SIRT1 midbrain condition knock-out mice

SIRT1^loxp/loxpMouse (C57BL/6J mouse SIRT1 genes 2,4 exons connection Loxp sites) is big by Sichuan Biological therapy National Key Laboratory professor Jiang Wei is learned to grant.(Wnt1-Cre localization and expressions are in for Wnt1-Cre transgenic mices The mouse of the recombinase containing Cre of brain, with SIRT1^loxp/loxpMouse hybrid, Cre recombinases can with specific recognition Loxp sites, Obtain SIRT1 midbrain conditional gene knockouts mouse) from Jackson laboratories of the U.S. (http://www.jax.org/)。 By SIRT1^loxp/loxpMouse mates with Wnt1-Cre transgenic mices, obtains F1 generation Wnt1-Cre, SIRT1^loxp/-Mouse.Again will F1 generation Wnt1-Cre, SIRT1^loxp/-Male and female mouse mates, and obtains F2 for Wnt1-Cre；SIRT1^-/-Mouse.

Rearing conditions：Animal feeding is in national center SPF grades of animal house of Chengdu new drug safety evaluatio, 20-25 DEG C of temperature, Relative humidity 55-65%, in whole experiment process, animal freely ingests and drinks water.Feeding environment meets national standard GB14925- 2001, experimental animal licensing：SCXK (river) 2003-01.All operations meet international animal welfare in zoopery (AAALAC) requirement.

SIRT1 midbrain conditions knock-out mice is identified：By F2 for Mouse Tail-tip clip 0.5-1cm length tail tissues, it is put into In centrifuge tube, Lycis Buffer liquid (such as table 1) is prepared, 0.5mL/ pipes, 55 ° overnight, until tail tissue melts.

Table 1Lycis Buffer liquid is with tabulation

Extract DNA：It takes out tail tissue and overturns mixing 13000rpm, centrifuge 10min.Pour out supernatant (not drawing).Upper Isometric 0.5mL absolute ethyl alcohols are added in clear, overturn mixing 8 times (not being vortexed).200mL pipette tips choose DNA (white filament), molten In 50-100 μ L distilled waters, during which 55 dissolvings overturn mixing (2h or so) per half an hour

PCR reacts and agarose gel electrophoresis carries out identified for genes：Primer sequence such as table 2 in pcr amplification reaction.PCR Amplification reaction system such as table 3.Prepare 1.2% Ago-Gel of the dyestuffs of View containing Gold.Molecular weight is 2000 DNA 2 μ L of Maker and PCR reacting final product DNA samples loading carry out gel electrophoresis.Mousetail agarose gel electrophoresis genotype Segment：SIRT1 homozygotes are 750bp, and SIRT1 heterozygotes are 750bp and 550bp.F2 750bp bands occurs in generation, and (SIRT1 is pure Zygote) and be SIRT1 midbrain condition knock-out mices without the corresponding mouse of sample of 100bp bands (Wnt1).In Fig. 1, 1st, 5,6,7,8,9 homozygote mouse is knocked out for SIRT1 midbrain condition；3 be the SIRT1 hybrid mices containing Wnt1；2nd, 4 be containing The wild-type mice of Wnt1.It chooses SIRT1 midbrain condition and knocks out homozygote mouse for subsequent experimental.

2 identified for genes primer sequence of table

3 PCR reaction systems of table

2.1.3 key instrument

(1) electronic analytical balance, purchased from Shanghai balance equipment factory

(2) superclean bench, purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.

(3) DK-8D types electric heating constant temperature sink reliable tests Instrument Ltd. purchased from Shanghai is gloomy

(4) Gene Amp PCR System 9700, purchased from Applied Biosystems companies

(5) TaKaRa PCR Thermal Cycler, purchased from precious bioengineering Co., Ltd

(6) high speed low temperature centrifugal machine, purchased from German eppendorf companies

(7) electrophoresis apparatus, purchased from Bio-Rad companies of the U.S.

(8) vortex vortex mixer, purchased from its woods Bell Instrument Ltd. of Haimen City

(9) micro oscillator, purchased from new health medicine Instrument Ltd. of Jiangyan City

(10) spectrometer, purchased from German Bruker companies

(11) voltage stabilization and current stabilization electrophoresis apparatus, vertical slab electrophoresis groove, transferring film slot are purchased from Bio-Rad companies of the U.S.

(12) constant temperature culture oscillator, purchased from Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.

(13) plastic film sealing machine, purchased from Ya Tong package packing machinies Manufacturing Co., Ltd of Wenzhou City

(14) constant water bath box of digital display three, purchased from Jin Cheng Guo Sheng laboratory apparatus factory of Jintan City of Jiangsu Province

(15) ultraviolet spectro-photometric analysis instrument, purchased from Japanese SHIMADZU companies

(16) enzyme-linked immunosorbent assay instrument, purchased from French Thermo Fisher companies

(17) fluorescent electronic inverted microscope, purchased from Zeiss, Germany optical instrument ZEISS companies

(18) electronic balance, purchased from German sartorius companies

(19) nitrogen evaporator, purchased from Beijing into sprouting Science and Technology Ltd.

(20) mouse stainless steel metabolism cage tool educates medical anatomy model manufacturing company purchased from Shanghai is same

(21) big mouse Place Preference case, purchased from Huaibei Zhenghua Biological Instrument Co., Ltd.

(22) liquid-transfering gun, purchased from German eppendorf companies

(23) liquid nitrogen container, purchased from Chengdu liquid nitrogen container factory

2.1.4 major experimental reagent

(1) TRIZOL reagents, purchased from purchased from U.S. Invitrogen (Life Technologies) company

(2) chloroform, purchased from Solution on Chemical Reagents in Shanghai Co., Ltd

(3) methanol, purchased from Solution on Chemical Reagents in Shanghai Co., Ltd

(4) deuterated heavy water D2O, purchased from U.S. CIL (Cambridge Isotope Laboratories) company

(5) TSP, purchased from Sigma-Aldrich companies

(6) DNase/RNase-Free ddH2O, purchased from purchased from Beijing Tiangeng biochemical technology Co., Ltd.

(7) Reverse Transcriptase kit (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit), purchased from DBI Bioscience companies

(8) Stormstar SYBR Green qPCR Mastermix, purchased from DBI Bioscience companies

(9) RIPA lysates, purchased from the green skies Bioisystech Co., Ltd in Shanghai

(10) SDS-PAGE reagents (30% polyacrylamide, Tris-base, SDS, ammonium persulfate, TEMED, glycine), 0.22 μM of aperture pvdf membrane, is purchased from Bio-Rad companies

(11) protein pre-dyed marker (PageRulerTM Prestained Protein Ladder, purchased from Thermo Scientfic (Fermentas) company

(12) chemiluminescence development kit (SuperSignal West Pico Chemiluminescent Substrate), purchased from U.S. Pierce

(13) Kodak Kodak films, purchased from Kodak

(14) 100% ethyl alcohol, purchased from Solution on Chemical Reagents in Shanghai Co., Ltd

(15) formaldehyde, purchased from Solution on Chemical Reagents in Shanghai Co., Ltd

(16) Gold View dyestuffs, Shanghai match Bai Sheng gene technology Co., Ltd

(17) agarose, Sheng Gong bioengineering Co., Ltd

(18) NAD/NADH Assay Kit, purchased from Abcam companies

(19) anti-β-actin antibody, purchased from CST companies of the U.S.

(20) anti-NAMPT antibody, purchased from Abcam companies

(21) anti-Sirt1 antibody, purchased from Santa Cruze companies

Remaining is domestic analytical reagents.

2.1.5 the preparation of main agents

(1) 10% (W/V) ammonium persulfate：Weigh the ddH that 1g ammonium persulfates add in 10mL₂After O, stirring and dissolving, 4 DEG C of guarantors It deposits.

(2) 5 × SDS-PAGE electrophoretic buffers：15.1g Tris-base, 94g glycine and 5g SDS are weighed, is added in about The ddH of 800mL₂O uses ddH after stirring and dissolving₂O is settled to 1000mL, and room temperature preservation is spare.

(3) film transfer buffer solution：2.9g glycine, 5.8g Tris-base and 0.37g SDS are weighed, is placed in 1000mL burnings In cup；Add in ddH₂O is sufficiently stirred dissolving；ddH₂O is settled to 800mL, adds the methanol of 200mL, room temperature preservation.

(4) 10 × TBS buffer solutions：Tris-base (tri methylol amino methane) 60.5g, NaCl 87.5g is weighed, is used After 800mL distilled water is completely dissolved, it is 7.4 that dense HCl, which is added dropwise, and adjusts pH, is finally settled to 1000mL.

(5) TBST buffer solutions：1：10 10 × TBS of dilution proportion buffer solutions are added in 1 × TBS buffer solutions of 1000mL After the abundant mixings of 1mL Tween-20,4 DEG C of preservations.

(6) Block buffer：It weighs 5g skimmed milk powers and is settled to 100mL with TBST buffer solutions, be sufficiently stirred dissolving, 4 DEG C It preserves (needing matching while using).

2.2 animal behavioral models are established

2.2.1 the foundation of cocaine Conditioned place preference pattern

Mouse Place Preference case is as shown in Figure 2：

(1) start before testing 3-5 days, mouse is put into laboratory apparatus by same operating personnel daily and is trained 15 minutes, is allowed Animal has instrument and operator certain adaptation.

(2) mouse is put into CPP babinets, acclimatization training 3 days, number of the data that third day measures as pretest According to (D0).Each adaptation training and testing time are 15min, when animal is more than in side (black and white both sides) residence time During 600s, it should reject.Using the side of animal stay longer as nature preference case.

(3) mouse carries out intraperitoneal injection, and dosage is following (table 4), and administration number of times is 3 times, alternating delivery, continuously 6 days.It gives cocainehydrochloride within 1st, 3,5 day and is put into non-natural preference case；It gives physiological saline and is put into nature within 2nd, 4,6 day Preference case.Control group gives physiological saline and is put into corresponding case (Fig. 3) as administration group every time.

4 cocaine administration approach of table, dosage, administered volume summary sheet

It carries out CPP tests within (4) the 7th days, mouse is sequentially placed into cinder box, allows its freedom in big mouse position preference case Shuttle 15min, and record each babinet residence time.

(5) points for attention：During each animal training, it should handle with care, not cause significant impact to the mood of animal； Training terminates wipe babinet every time, to eliminate the smell of each animal.Intensity of illumination pays attention to controlling daily instruction Practice the time, training duration, noise etc..

2.2.2 the foundation of spontaneous activity model

Spontaneous activity in mice instrument is as shown in Figure 4：

(1) start before testing 3-5 days, mouse is put into laboratory apparatus training 15min by same operating personnel daily, is allowed Object has instrument and operator certain adaptation.

(2) mouse is put into spontaneous activity box body, acclimatization training 3 days, the data measured are as baseline within the 4th day Data (D0).Each adaptation training and testing time are 15min, record the distance (cm) of mouse activity.

Start within (3) the 5th days, mouse first carries out intraperitoneal injection, and once a day, successive administration 7 days, dosage is such as Above (table 4), detection spontaneous activity in case is put into after 3min, according to the data (D1) (Fig. 4) as first day.Spontaneous activity in 7th day Dissection mouse in 30min after detection.

2.3 samples are drawn materials and are prepared

Quick sacrificed by decapitation C57 mouse in 30min, are then rapidly separated brain after Conditioned place preference is tested, After 4 DEG C of normal saline flushings 3 times, detached according to brain anatomical atlas and take out prefrontal cortex, nucleus accumbens septi, corpus straitum, hippocampus, in Brain ventral tegmental area.Directly the sample taken is quickly put into liquid nitrogen, and is put into rapidly in -80 DEG C of refrigerators and stores.

2.4 Western blotting

2.4.1 sample preparation

Tissue samples are taken out in -80 DEG C of refrigerators, are operated on ice, ice RIPA lysates is added in, is placed in and cracks 5min on ice.It is super Sound crushes instrument ultrasound 10 times, 3s/ time (put and on ice), clasmatosis.13000g centrifugation 15min (4 DEG C), abandon precipitation, take out Supernatant.Protein concentration in supernatant is measured with Coomassie Brilliant Blue.Add in RIPA lysates and Loading Buffer adjustment samples This concentration, loading volume and applied sample amount.Boil sample 5min.13000 revs/min of centrifugation, 10min takes supernatant.

2.4.2 glue

Preparative separation glue is as shown in table 5.

5 separation gel of table is prepared with drawing

It is as shown in table 6 to prepare concentration glue.

Table 6 concentrates glue and prepares with drawing

2.4.3 electrophoretic separation

Glue is dipped in transfering buffering liquid and balances 10min.15~20 μ L of loading are electric to SDS-PAGE glue (10cm × 10cm) Swimming.Concentrate glue voltage 70-80V, for albumen at concentration glue separation gel boundary, voltage adds as 90V, until albumen is run through (according to egg Bai great little is determined), the time is about 120min.

2.4.4 transferring film

Pvdf membrane is activated 3~5 seconds with pure methanol.According to the size clip film of glue and filter paper 6, it is put into transfering buffering liquid Middle balance 10min.Assembling transfer sandwich：Sponge → 3 layer filter paper → glue → film → 3 layer filter paper → sponge, after every layer is put well, catches up with Remove bubble.Glue is put in cathode face (black side) and transfer groove is placed in ice bath, is put into sandwich (black side is to black side), adds and turns Buffer solution is moved, plugs electrode, 100V, the transferring film time is determined (electric current is about 0.3A) by molecular weight of albumen.After transferring film, cut-out Power supply takes out hybond membrane and is put into the confining liquid got ready.

2.4.5 closing

The Block buffer of 5% skim milk, 30mL/ block glue are prepared with TBST.It pours into plate, pvdf membrane is complete Infiltration is placed in milk on shaking table, is incubated at room temperature 1.5h.

2.4.6 primary antibody is incubated

By dilution proportion of the primary antibody with Block buffer as required, sealed with hybrid belt, 4 DEG C overnight or 37 DEG C, 1- 2h.Film is washed with TBST three times, each 5-10min.

2.4.7 secondary antibody is incubated

By corresponding antibodies Block buffer 1：5000 dilutions, 37 DEG C of shaking table 1-2h wash film four times, each 5- with TBST 10min, secondary, the 5-10min that then washes film with TBS.

2.4.8 colour developing

Prepare luminescent solution (1ml A liquid：1ml B liquid), pvdf membrane is placed in luminescent solution in darkroom and is reacted 1 minute.It will Pvdf membrane takes out, and blots extra luminescent solution with filter paper, pvdf membrane is put into magazine, by film face-up and film contact, pressure Tight exposure (band brightness depends on time length), develops a film.

2.4.9 interpretation of result

Immunoblotting film 6.0 systems of Image-Pro Plus are read into gray value, with β-actin band gray values As internal reference, it is standardized and compares.

2.5 RT-PCR

2.5.1 extract tissue RNA

Reagent prepares：The ddH of Trizol, RNase-free₂O, chloroform, absolute ethyl alcohol, 75% ethyl alcohol, isopropanol are used EP pipes, the consumptive materials such as rifle point be DNase/RNase-free imported product.

(1) homogenized：Tissue in liquid nitrogen with trumpet mortar is ground, low temperature is kept in process of lapping by tissue sample (operating on ice), it is impossible to tissue be made to soften and melted, tissue adds in 1mL Trizol reagents per 50-100mg, is carried out with Syrup-homogenizing instrument even Slurry processing, is finally transferred in the EP pipes of new 1.5mL.

(2) it is separated：15-30 DEG C of incubation 5min of room temperature, treats that albumen is kept completely separate in homogenised sample, per 1mL Trizol examinations 0.2mL chloroforms are added in agent, carefully cover pipe lid, overturn mixing repeatedly 8-10 times, are incubated at room temperature 3min, 4 DEG C, 12,000g centrifuge 15min.After centrifugation, mixture is divided into three layers, and the phenol-chloroform phase of the colourless aqueous phase on upper strata, interphase and lower floor's red, RNA is deposited Be in water phase, water phase volume when being about homogeneous added Trizol amounts 60%.

(3) RNA precipitate：Water phase is transferred in new EP pipes (it is careful not to be drawn onto middle layer, in order to avoid RNA pollutes), just RNA in water phase is precipitated using 0.5mL isopropanols per 1mL Trizol reagents during beginning homogenization, is incubated at room temperature sample 10-20min, 12,000g, 4 DEG C of centrifugation 10min.RNA precipitate is generally invisible before centrifugation, but forms flocculent deposit in tube bottom after centrifugation.

(4) RNA is washed：Supernatant is abandoned, the ethyl alcohol washing RNA precipitate of 1mL 75% is primary, after vortex mixing, 4 DEG C, 7,500g Centrifuge 5min.

(5) RNA is re-dissolved：Abandon supernatant, wink from, siphon away remaining 75% ethyl alcohol, dry RNA precipitate (be air-dried or It is dried in vacuo 5-10min), it is careful not to overdrying (its solubility can be reduced).Use 20-30 μ L RNase-free's ddH₂O dissolves RNA, and subsequent experimental is used for after vortex mixing.Dissolve RNA when, first add in no RNA enzyme water blown and beaten repeatedly with rifle it is several It is secondary, then it is incubated 10min for 55-60 DEG C.- 80 DEG C of refrigerators of RNA solution of acquisition preserve.

2.5.2 RNA quality testings and quantitative

(1) electrophoresis detection：1% Ago-Gel is prepared using 1 × TBE, the RNA and sample-loading buffer that 3 μ L is taken to dissolve Electrophoresis is run after mixing, 150V separation 20min carry out imaging analysis.The purpose of electrophoresis is to detect the complete of 28S and 18S bands Property and their ratio, if 28S and 18S bands become clear, clearly, completely, and the brightness of 28S is more than twice of 18S bands, Then think that RNA is up-to-standard, can be used for subsequent experimental.

(2) absorbance detection of RNA：280th, 320,230, the absorbance under 260nm represents that nucleic acid, background, salt are dense respectively The OD values of the organic matters such as degree and albumen, in general, we only analyze the ratio (Ratio, R) of OD260/OD280.When R is During 1.8-2.0, it is believed that the pollution of albumen or other organic matters is acceptable in RNA；As R ﹤ 1.8, illustrate egg in RNA White or other organic matters pollutions are obvious, do not use generally；As R ﹥ 2.0, illustrate that RNA has degraded, it is unavailable.

(3) concentration (μ g/ μ L) of RNA can be calculated simultaneously on spectrophotometer：OD260 × extension rate × 40/ 1000。

2.5.3 RNA reverse transcriptions are into cDNA

It is operated according to the specification of DBI Bioscience Reverse Transcriptase kits as follows：

(1) it after melting, centrifuges kit each component vortex mixing and slightly, is placed on ice.If RNA template G/C contents It is high or contain secondary structure, by above-mentioned mixed liquor gently mixing, wink from, 65 DEG C of incubation 5min are immediately inserted into cooled on ice, from The heart, then be placed on ice.

(2) in the PCR pipe for being placed in sterile nuclease free on ice, reaction solution (as shown in table 7) is prepared in sequence：

7 Reverse Transcription of table is with tabulation

(3) gently mixing, centrifugation.

Under the conditions of (4) 37 DEG C, the reverse transcription reaction of 15min is carried out.

Under the conditions of (5) 98 DEG C, the reverse transcriptase inactivation reaction of 5min is carried out.Reaction product can be directly used for subsequent PCR Detection or be stored in -80 DEG C it is spare

2.5.4 Real-Time PCR

(1) gene primer of synthesis is subjected to centrifugation 13,300rpm, 5min first, it is ensured that all powder all concentrate on Tube bottom, according to the ddH for illustrating to add in enough nuclease frees₂For O TE buffer to final concentration of 100 μM, this is storing liquid Concentration, then it is 10 μM of working solution concentration to take a small amount of 10 times of liquid diluting.

(2) it is prepared according to DBI Bioscience Stormstar SYBR Green qPCR Mastermix specifications anti- Answer system (table 8).

8 RT-PCR reaction systems of table are with tabulation

(3) gently mixing, centrifugation.

(4) PCR response parameters are：95 DEG C of pre-degeneration 2min, 40 cycles (95 DEG C of denaturation 10sec, 60 DEG C of renaturation, extensions 30-34sec)；Solubility curve analysis (65-95 DEG C, rise 0.5 DEG C per 5sec), primer sequence such as table 9.

9 RT-PCR primer sequence of table

2.6 nuclear magnetic resonance metabolism group

2.6.1 prepare cell lysate

Take out sample to be tested in (1) -80 DEG C of refrigerator, weigh 20-200mg frozen tissues, and prepare pre- cold methanol, chloroform, MiniQ water.

(2) it adds in 4ml/g methanol and 0.85mL/g water to tissue sample, each 3s of ultrasonication sample, surpasses by volume Sound 8-10 times is spaced 3s and the mixing that is vortexed.

(3) 2m L/g chloroforms are added in, are vortexed again for mixing 2min, stand 2min.

(4) 2mL/g chloroforms and 2mL/g distilled waters are added in, is vortexed again for mixing.

(5) sample is placed in after standing 15min on ice or in refrigerator, 1000g, 15min, 4 DEG C of centrifugations are (as without apparent point Layer then centrifuges again).

(6) upper strata aqueous phase is transferred in new EP pipes, -80 DEG C of preservations after nitrogen is blown.

(7) if not being detected immediately, water-phase extract is stored in -80 DEG C (predominantly reducing oxidation reaction), and It is stored in -80 DEG C (but preferably not more than 3 days).

(8) water phase metabolin：580 μ L heavy water are added in, containing 0.1~0.5mM TSP, vortex mixing, 12000g centrifuges 5min, And 550 μ L samples are placed in detection [31] in nuclear magnetic tube.

2.6.2 nuclear magnetic resonance spectroscopy

600 types of Bruker Avance II (Bruker Biospin, Germany) nuclear magnetic resonance chemical analyser, setting experiment temperature 300K is spent, relaxation edit pulse sequence (Carr-Purcell-Meiboom-Gill sequence, CPMG are called on NMR instrument Sequence), water peak is inhibited using presaturation mode.Spectrum width 12335.5Hz, delay time 5s, sampling time 2.66s are detected, Sampled data is counted 64K, and accumulative frequency 64 times starts to sample, and obtains free induction decay (FID) signal, the broadening factor of line for- 0.3Hz[32]。

2.6.3 Data induction and pattern recognition analysis

The initial data of NMR is FID signal, and Fourier is passed through on computer software (MestReNova-6.1.1-6384) Transformation and windowed function are converted into visual NMR spectra, then carry out processing analysis to spectrogram：

(1) it calibrates：Since the pH value of each sample is different, in order to avoid error caused by being drifted about due to wave spectrum, so will It is calibrated with a metastable spectral peak, it be standard that the TSP (chemical shift is δ 0.0) added in can be used in this experiment, to institute Some spectral peaks is adjusted.Because lactic acid peak (1.336ppm ,-CH3 are bimodal) and glucose peaks in tissue samples (5.22ppm ,-CH1 are bimodal) is also stablized relatively, so can also be calibrated with the two.Concrete operations are to use MestReNova softwares open wave spectrogram, purpose chemical shift are amplified, the TMS buttons in click tools column are calibrated.

(2) phase adjustment：The usual collected spectrograms of NMR all containing absorb with diffusion component, can be with by phase adjustment Pure absorption peak is obtained, shows on spectrogram to be exactly that the peak that reversed image limits all is adjusted to positive quadrant.In the instrument of Bruker, Phase adjustment first carries out maximum peak zero order phase adjustment PH₀, PH is then adjusted with first order phase₁To adjust other peaks.

(3) baseline correction：In nuclear magnetic resonance chemical analyser, if the rf frequency ingredient of transmitter is drained in receiver, warp After detection, these leakage voltages reform into flip-flop, and FID signal is caused to have certain Dc bias, is appeared in after digitlization In digital signal, if do not corrected, the spike at the odd lot of spectrum can be caused after Fourier transform, this will influence entire spectrum Dynamic range.The main purpose for adjusting baseline is exactly the appearance for improving spectrum and the accuracy for improving integration.So pre-processing During NMR spectra figure, have to carry out baseline correction.

(4) overlap of peaks and exclusive PCR peak：It, will be all after according to above-mentioned three step, all spectral peaks are pre-processed Spectral peak is overlapped, and just obtains the spectral peak figure of a superposition.Because introduced when sample treatment is carried out the methanol of external source, Chloroform and water exclude Interference Peaks (residual water peak, the about 4.7ppm of three when carrying out subsequent analysis；Methanol peak, about 3.37- 3.34ppm；Chloroform peak, about 7.8-7.6ppm).

(5) segmentation product：The limit of integration of use is 0.5-9.5ppm, and integration size is 0.004ppm

(6) it normalizes：It is normalized relative to full spectral integral area, the concentration difference between each tissue samples can be made up It is different.

(7) data export：By above-mentioned steps, the preprocessing process of wave spectrum is completed, it is possible to defeated on MestReNova Go out data, generally save as TXT forms, then switch to 2003 editions Excel.

(8) pattern recognition analysis：It is analyzed with SIMCA-P10.0 software packages.Variable neutralizes Pei Ertuotu amounts by mean value collection Change (pareto-scaled) and carry out PCA, PLS-DA, OSC analysis afterwards.It is whether intentional that decision group ingredient is quantified by Pei Ertuotu Justice.Two-dimentional shot chart is used to distinguish sample；Corresponding load diagram is used to distinguish contributive difference metabolin on wave spectrum.Nuclear magnetisation The distribution of displacement study and endogenous metabolites is according to document and mankind's metabolism group database (Human Metabolome Database) the information obtained on website.Mankind's metabolism group database is the website of bioinformatics/Chemoinformatics, can To obtain the relevant information of metabolin and metabolic enzyme.

2.7 are overexpressed slow virus carrier structure

2.7.1 carrier

(1) carrier information：

Carrier is pReceiver-Lv201, such as Fig. 5.

(2) competent cell and conversion are prepared

CaCl₂It is as follows that method prepares competent cell progress transformation experiment step：

1. each competent cell suspension respectively takes 200 μ L to be transferred in sterile eppendorf tubes, often pipe adds in 10 μ of connection liquid L, gently rotates mixing, then puts and 30min (preparing competent cell, make it have the ability of intake exogenous DNA) is placed in ice.

2. 42 DEG C of heat shock 90s, are quickly transferred to cooling cell 1-2min in ice bath by centrifuge tube.Often pipe adds in 800 μ L LB culture mediums, water-bath are heated up to 37 DEG C, then place shaking table and incubate 45min with recovery bacterium.

3. 150 μ L transformed competence colibacillus cells are transferred on the LB agar mediums of AMP (100 μ g/ml) resistance.Tablet Room temperature to liquid is placed in be completely absorbed.Then plate is inverted, is placed in 37 DEG C of incubators and cultivates 16h.

4. clone carries out follow-up PCR identifications.

2.7.2 construction of recombinant plasmid

PCR product is connected is shown in Table 10 into linearisation expression vector reaction system, reaction condition：25℃30min；42℃ 15min。

10 PCR reaction systems of table

X：The volume number of linearized vector DNA；Y：PCR product fragmental volumes number after purification.

2.7.3 virus packaging

293T cells are digested, it is to have 1.2X 10 per 20mL to adjust its density⁷A cell, inoculating cell are put in culture dish 37 DEG C are put, 5%CO₂It is cultivated in incubator for 24 hours, can be used for cell transfecting when cell density reaches 70%-80%.Cell state pair It is most important in virus packaging, it needs to ensure good cell state and less passage number.2h will contain tire ox blood before transfection Clear culture medium is changed to serum free medium.Added in into centrifuge tube preparation each DNA solution (20 μ g of pGC-LV carriers, 15 μ g of pHelper1.0 carriers, 10 μ g of pHelper 2.0 carrier) it is uniformly mixed with respective volume culture medium, adjustment total volume is 2.5mL incubates 5min at room temperature.Lipofectamine2000 reagents are softly shaken up, take 100 μ L Lipofectamine2000 reagents are mixed in another Guan Zhongyu 2.4mL Opti-MEM culture mediums, incubate 5min at room temperature.Dilute DNA after releasing is mixed with the Lipofectamine2000 after dilution, and mixing of gently running avoids vibrating, and must be mixed in 5min It closes.20min is incubated after mixing at room temperature, then mixed liquor is transferred in 293T cell culture fluids, is uniformly mixed, places 37 DEG C, 5%CO₂It is cultivated in incubator.Culture medium is removed after culture 8h, every bottle of cell adds in 20mL phosphate buffers (PBS), with The remaining mixed liquor of washing, removes mixed liquor.It is added in every bottle of cell and contains 10% fetal calf serum culture medium 25mL, 37 DEG C of placement, 5% CO₂Continue culture 2 days in incubator.

2.7.4 titer determination

(1) sample preparation

293T cells pass on, and add in 1 × 10 in 24 holes per hole⁵A cell, volume are 500 μ L；Next day preparation 10 is sterile Ep is managed, per 90 μ L culture mediums of Guan Zhongjia；10 μ L of virus stock solution used to be measured is taken to be added in first pipe, are uniformly mixed, take mixing equal Even 10 μ L of the first pipe liquid, which are added in second pipe, continues an identical operation to the last pipe；Cell hole needed for selection is inhaled Remove 90 μ L culture mediums.Add the viral solution diluted, place 37 DEG C, 5%CO₂Incubator culture；After 1 day, fresh cultured is added in 500 μ L of base.Careful operation, extracting RNA after 4 days.

(2) total serum IgE extracts

Cell supernatant is gone, 1mL Trizol are added in per hole, piping and druming is stored at room temperature 5min, is transferred to another new 1.5mL In Ep pipes.Often pipe plus 200 μ L chloroforms, firmly shake 15s, are stored at room temperature 15min.120000r/min centrifuges 15min at 4 DEG C.From Often in pipe in Aspirate supernatant to another new 1.5mL Ep pipes.The isopropanol of isometric -20 DEG C of precoolings is added in, -20 DEG C after mixing Precipitate 10min.120000r/min centrifuges 10min at 4 DEG C, removes supernatant.Add in 75% ethyl alcohol of 1mL4 DEG C of precooling, washing Precipitation and centrifugation tube wall.10000r/min centrifuges 5min at 4 DEG C, and supernatant is abandoned in shifting.10000r/min is centrifuged again at 4 DEG C 5min sucks raffinate, dries at room temperature, is not required to be completely dried.Add 20 μ L without RNA enzyme (RNase) water to being completely dissolved, ultraviolet point Analysis measures institute's extracting RNA concentration.

(3) RNA reverse transcriptions obtain cDNA

1 μ L Oligo dT (0.5 μ g/ μ L) and 2.0 μ g total serum IgEs are added in into PCR tubules, add pyrocarbonic acid diethyl ester (DEPC) Water is to 9 μ L.It is uniformly mixed, centrifugation, 70 DEG C of warm bath 10min.And then it is inserted into 0 DEG C of ice-water bath.According to the form below ratio, according to anti- Should pipe figure out required amount of reagent.M-MLV enzymes etc. are uniformly mixed so as to obtain reverse transcription reaction liquid on ice.Add 11 in each reaction tube μ L reverse transcription reaction liquid, centrifuges after mixing.Wherein, 11 μ L reverse transcription reactions liquid containing 5 × RT Buffer, 4 μ L, 10mmol/LdNTPs2 μ L, 0.5 μ L of RNA inhibitor, M-MLV-RTase1 μ L, 3.5 μ L of DEPC water.It is inverse that 1h completions are carried out at 42 DEG C Responsive transcription, it is rear to inactivate reverse transcriptase with 70 DEG C of processing 10min.Reverse transcription reaction product cDNA can be used for PCR, also can -80 It is DEG C long-term to preserve.

(4) real-time quantitative PCR detects

Reaction system is configured：Often pipe add in SYBRpremix ex Taq10 μ L, sense primer (5 μm of ol/L) 1.0 μ L, under Swim primer (5 μm of ol/L) 1.0 μ L, cDNA1.0 μ L, ddH₂O 7.5μL.Setting program is two-step method real-time quantitative PCR.Pre-degeneration 95℃5s；95 DEG C of 5s are denaturalized, anneal 60 DEG C of 30s, extends 60 DEG C of 30s, 40 cycles.Light absorption value is read in the extension stage every time, For making melting curve.After PCR, 1min is denaturalized at 95 DEG C.55 DEG C are subsequently cooled to, DNA double chain is made fully to polymerize.From 55 DEG C start to 95 DEG C, and each step increases by 0.5 DEG C, keep 30s, while read light absorption value.Set point is increased after two cycles 0.5℃。

2.7.5 the harvest of slow virus and concentration

Collect the 293T cell supernatants of 2d after transfecting, 4 DEG C, 4000 × g centrifugation 10min, to remove cell fragment.With In 0.45 μm of filter filtering supernatant to 40mL ultracentrifugation pipes.Viral crude extract sample is added in filter cup.It will filtering Cup is inserted into filtered solution collecting pipe, then 4000 × g is centrifuged to required viral concentration volume, time 15min.It is taken out after centrifugation Centrifugal device separates filter cup and filtered solution collection cups.Filter cup is tipped upside down on sample collection cup, centrifugal force is no more than 1000 × g, time 2min (excessive speeds can make sample loss).Filter cup is removed from sample collection cup.Sample collection cup In be viral concentration liquid.

2.8 mouse VTA brain area locating injections

Mouse injects under 1.5% penta bars of penta sodium of ratio (40mg/kg, ip) and anaesthetizes, and is fixed on Naoliqing capsule.According to mouse VTA positioning collection of illustrative plates (Fig. 6), it is embedding to be about 0.5mm in left side VTA (3.l mm, center line side 0.7mm, depth 4.5mm after bregma) Stainless steel tube (internal diameter 0.34mm, outer diameter 0.48mm), then be fixed in skull surface with dental cement, will be outside stainless steel tube Head is fixed at end, is sewed up a wound, is covered mating inner core.Postoperative injection Benzylpenicillin sodium salt (0.1 ten thousand units/only, once a day), even Continuous injection prevents from infecting for three days.Corresponding behaviouristics training is carried out within the 7th day after operation, the drug injection amount of VTA is l μ L, is injected When first extract inner core, l min injections finish, let the acupuncture needle remain at a certain point 2min.During mouse VTA both sides injecting virus, it is fixed on after mouse anesthesia On Naoliqing capsule.After the perforation of 10ml syringe needles, injecting 0.5 μ L viruses with spiral micro-sampling pump bilateral, (0.25 μ L are every Side), 0.1 μ L/min, let the acupuncture needle remain at a certain point 3min sew up a wound.

2.9 NAD/MADH kits detect

Abcam NAD/NADH Assay Kit material compositions composition is as shown in table 11.

Table 11NAD/NADH Assay Kit material compositions form table

(1) drafting of standard curve

Prepare NADH titers.5 μ LNADH titers are added in 495 μ LNADH/NAD extracting solutions to be diluted, and are obtained 10pmol/ μ L (10 μM) NADH titers.Dilution standard curve in microcentrifugal tube, as shown in table 12.

The dilution of 12 NADH titers of table is with tabulation

(2) sample prepares

The amount (20mg tissues) each measured necessary to harvest tissue.Tissue is washed in cold PBS.Add in 400 μ L's NADH/NAD extracting solutions make its homogenate.Maximum speed centrifuges 5min, and the NAD/NADH supernatants for shifting extraction are newly managed to one In (on ice operate).Tissue may include the enzyme of quick consumption NADH.These enzymes can be filtered by 10kD centrifugal columns (ab93349) And be removed, for use.

(3) it detects

The decomposition step of sample NADH detections：

1. total NAD and NADH：Extraction sample does not need to reprocess.

②NADH：NAD+ needs are decomposed.200 μ L extraction samples are dispensed to add in micro tube.In a water bath or heat block In 60 DEG C heating 30min (under these conditions, all NAD+ will decompose and NADH still stand intact).Sample is placed on ice On.Quick Centrifuge A sample is to remove the precipitation of precipitation

3. reacting hole is set：

The dilution of the μ L standard items of gauge orifice=50.

Blank control sample hole=1-50 μ L samples (adjusts volume to 50 μ L/ holes) with Extraction buffer.

NADt sample wells=1-50 μ L samples (adjust volume to 50 μ L/ holes) with Extraction buffer.

NADH sample wells=1-50 μ L NAD decompose sample (adjusting volume to 50 μ L/ holes with Extraction buffer).

4. reaction mixture：

Each reaction prepares 100 μ L reaction premixs, as shown in table 13：

13 reaction mixture of table is with tabulation

100 μ L reaction mixtures are added in each standard and sample well.100 μ L background responses mixed liquors are added to blank sample Sample wells.It is incubated 5min at room temperature, NAD is transformed into NADH.10 μ L NADH Developer liquid are added in each hole and mixing. Reaction cycle stirring 1-4h at room temperature.Under OD 450nm wavelength, read 1-4h and take multiple readings.

3 experimental results

3.1 cocaine inductive condition position preferences

Trained by the Conditioned place preferences of 6 days, cocaine administration group and the training of physiological saline group C57 mouse it is front and rear There is notable difference (Fig. 7) in preference case and non-preference case residence time difference.(test) is tested after the training of cocaine administration group Time difference with (Pretest) before test is 295 ± 38s, and saline control group is 44 ± 58s.Above-mentioned result of the test Show to train by 20mg/kg cocaine Conditioned places preference, C57 mouse show in cocaine training babinet residence time Writing increases, and shows that reliable cocaine CPP models have had been established.

The spontaneous activity of 3.2 cocaines induction

After the spontaneous activity training of 10 days, 15min's is spontaneous before and after comparison cocaine group and physiological saline group training Activity, it can be found that locomotor activation dramatically increases (P after the training of cocaine administration group<0.05) (Fig. 8).Cocaine administration group The locomotor activation of 2,3,4,5,6,7 days is also apparently higher than first day (Day1), and have apparent significant difference upon administration.It says Bright cocaine can be increased with inducing mouse spontaneous activity.

3.3 cocaines induce the expression of Different brain region NAMPT

In order to detect expressions of the NAMPT in cocaine administration group and saline control group Different brain region, CPP models Mouse is dissected after training, prefrontal cortex (PFC), nucleus accumbens septi (NAc), corpus straitum, hippocampus and Ventral Midbrain tegmental region is taken to carry out Westernblot is analyzed.The result shows that cocaine group is compared with physiological saline group, NAMPT prefrontal cortex, corpus straitum and The expression of hippocampus does not have significant change, but NAMPT expressions significantly increase in nucleus accumbens septi and Ventral Midbrain tegmental region (p<0.05) (Fig. 9).

3.4 cocaines induce the expression of NAMPT

In order to detect the variation of NAMPT rna expressions in NAc and VTA brain areas, we take cocaine and life in CPP models NAc the and VTA Brain TisX samples of brine group are managed, extract tissue RNA, after quality testing, reverse transcription is carried out into cDNA Real-TimePCR detects (each primer sequence refers to material part), and NAMPT is between cocaine group and physiological saline group for analysis Relative expression (Figure 10 A), and calculate its P value, from the point of view of analysis result, NAMPT using the two-sample test in T-test Up-regulated expression has statistical significance (p in VTA tissues<0.05), there is no significant change in NAc.Cocaine acute model In：As 7 days solutions of single-dose take corresponding Brain TisX, Real- for 24 hours and continuously after 30min, single-dose after single-dose Time PCR are detected, and find expression no significant differences (Figure 10 B-C) of the NAMPT in NAc and VTA.

3.5 mouse peritoneals injection FK866 reduces cocaine CPP effects

To explore effects of the NAMPT in cocaine habituation, we select that the specificity of the NAMPT of blood-brain barrier can be passed through For inhibitor --- FK866 as probe, studying influences its neuroethology.We have found that (such as Figure 11), wild-type mice abdomen After chamber injection 4mg/kg FK866, the spontaneous activity of cocaine CPP and cocaine induction is substantially reduced (P<0.05) it, prompts NAMPT plays positive acting in cocaine habituation.

3.6VTA intracerebral locating injections FK866 inhibits cocaine CPP effects

In order to further verify the cocaine CPP inhibiting effect of FK866, mouse VTA intracerebral pipe layings surgery recovery 5-7 days Afterwards, the FK866 of 1 a concentration of 10nM of μ L is given in positioning, then carries out cocaine CPP training.As a result it shows：VTA intracerebral locating injections Compared with the control group, cocaine CPP (such as Figure 12 A) and spontaneous activity (such as Figure 12 B) reduce (P to FK866<0.05).

3.7VTA intracerebrals supplement NMN reverses the cocaine CPP effects of FK866

NMN is the direct effect product of NAMPT catalysis niacinamide metabolism, in order to illustrate NAMPT in cocaine habituation Effect.After the FK86630min of 1 a concentration of 10nM of μ L of VTA intracerebrals locating injection, then the NMN of a concentration of 10mg/mL of 1uL is injected, Then detection cocaine CPP activities.As a result, it has been found that after injection inhibitor FK866, cocaine CPP is reduced；But after supplementing NMN, disappear In addition to FK866 is to inhibiting effect (such as Figure 13) (P of cocaine CPP<0.05).

3.8VTA intracerebrals, which are overexpressed NAMPT, increases cocaine CPP effects

Directly to detect effects of the NAMPT in cocaine habituation, the overexpression slow virus that we construct NAMPT carries Body.Intracerebral VTA locating injections 0.5uL viruses (per side 0.25uL), restore 5-7 days, then carry out cocaine CPP training.As a result it sends out Existing, compared with compareing LV-GFP slow virus carriers, the expression of NAMPT increases the overexpression slow virus carrier of mouse VTA brain areas NAMPT Add (such as Figure 14 A).The CPP activities of NAMPT (LV-NAMPT) cocaines compared with control group (LV-GFP) are overexpressed in VTA to be increased (P<0.05) (such as Figure 14 B).

Give the change of FK866 inducing mouses metabolin in 3.9 abdominal cavities

3.9.1 the metabolism spectrum of tissue samples

In order to detect the change that mouse correlative metabolites after FK866 are given in abdominal cavity, using based on nuclear magnetic resonance metabolism group Method detects mouse NAc and VTA sample.We carry out nuclear magnetic data analysis using pca model, to obtain more accurately control group With the difference to medicine group mouse brain area metabolin.The results show that the load diagram of OSC-PLS-DA models is as shown in the figure (Figure 15), Apparent change has occurred with the relevant endogenic metabolin of NAMPT metabolism compared to a series of between administration group and control, wraps Include NMN and NAD.

3.9.2 the pattern recognition analysis between control group and administration group

In order to it is more detailed differentiation metabolin variation, we using a kind of means of numerical analysis of multivariable Control- OSC-PLS-DA models are interfered with reducing variation in potential group, detach metabolin more perfectly.OSC-PLS-DA moulds As a result type analysis magnetic resonance detection data show that there are apparent metabolite profiles for control group, cocaine group and FK866 processing group Difference (Figure 16, Figure 17).Further analysis is found, wherein being metabolized relevant metabolin in control group and FK866 processing with NAMPT Significant difference (table 3-1, Fig. 3-9) is shown between VTA the and NAc brain areas of group, including NMN and NAD.

According to two-parameter ownership method, with reference to the screening conditions of VIP ＞ 1 and P ＜ 0.05, cocaine group and physiology are finally determined The variation for being metabolized relevant metabolin after FK866 with NAMPT is given in brine group and abdominal cavity.As a result, it has been found that：Cocaine group and life Reason brine group is compared, NMN and NAD levels increase；But after FK866 is given in abdominal cavity, NMN and NAD levels reduce (table 3-1).

Table 3-1 intraperitoneal injection FK866 cocaine CPP models metabolin variation sketch plans

Note：Compared with control sample, ↑ represent content relative increase, ↓ expression content relative drop.

3.9.3 the variation of key metabolites

NAMPT is the key enzyme of NAD route of synthesis, and NAD is the classical coenzyme in cell Redox reaction.NAMPT Niacinamide is metabolized as NMN, NMN is further metabolized as NAD.Cocaine can induce the up-regulated expression of NAMPT, and FK866 is special Property inhibit NAMPT activity, therefore influence NAD synthesis.We analyze physiological saline Normal group, cocaine processing group and Difference metabolin between FK866 administration groups has chosen metabolin wherein related and representative to NAMPT metabolism, profit It is shown (Figure 18) with box traction substation, box traction substation can show range, median, quartile etc. of metabolin variation, can be with The variation tendency of apparent determining metabolin.

The variation of NAD in 3.10 detection cocaine CPP

In order to further determine the variation of NAD levels, the detection fresh samples of mouse VTA are detected using NAD/MADH kits This, determines cocaine administration group and saline control group in each condition to the influence of NAD.As a result (Figure 19) is shown, CPP moulds In type, cocaine can induce NAD levels to increase；NAD is reduced after giving FK866；NMN is supplemented after giving FK866, NAD is extensive It is multiple；NAMPT is overexpressed, NAD levels increase (P<0.05).

4 conclusions

Metabolism group detection based on nuclear magnetic resonance (NMR), it is found that cocaine habituation mouse VTA brain areas NMN, NAD are horizontal Increase, but give NMN, NAD level reduction after FK866.Quantitative detection is it has also been found that cacaine habituation mouse VTA brain area NAD levels increase Add；Brain area locating injection FK866 can then reduce NAD levels；But NMN is supplemented, NAD levels are restored；VTA brain areas are overexpressed After NAMPT, NAD levels also increase.

Mouse cocaine Conditioned place preference (CPP) model is established, prize of the cocaine to mouse is detected by behaviouristics Appreciate effect.It was found that cocaine can apparent inducing mouse NAc and VTA brain area NAMPT up-regulated expression.By abdominal cavity or After VTA brain area locating injections FK866, it can significantly inhibit mouse cocaine CPP effects.Supplement injection NMN is positioned in VTA brain areas, Inhibiting effect of the FK866 to cocaine CPP can be reversed.Viral vectors, cocaine CPP are overexpressed in VTA locating injections NAMPT Rewarding effect enhances.The experiment results show that NAMPT inhibitor FK866 can inhibit cocaine CPP rewarding effects.

To sum up, NAMPT inhibitor can effectively weaken cocaine award behavior, cocaine habituation be treated, before clinical practice Scape is good.

Claims (5)

1. Purposes of the 1.NAMPT inhibitor in the drug for preparing treatment cocaine habituation,

   The NAMPT inhibitor is FK866, and structural formula is as follows：
2. 2. purposes according to claim 1, it is characterised in that：The drug of the treatment cocaine habituation is to reduce NAMPT The drug of enzyme activity.
3. 3. one kind is using NAMPT inhibitor as active constituent, and pharmaceutically acceptable auxiliary material or complementary ingredient prepare and Into preparation prepare treatment cocaine habituation drug in purposes；

   The NAMPT inhibitor is FK866, and structural formula is as follows：
4. 4. purposes according to claim 3, it is characterised in that：The NAMPT inhibitor is the medicine for reducing NAMPT enzyme activity Object.
5. 5. purposes according to claim 3, it is characterised in that：The preparation is ejection preparation.