CN105055412A - Application of NAMPT inhibitor in preparing cocaine addiction treatment medicine - Google Patents

Abstract

The invention discloses application of an NAMPT inhibitor in preparing cocaine addiction treatment medicine and the cocaine addiction treatment medicine. The cocaine addiction treatment medicine is a preparation prepared from the NAMPT inhibitor serving as the active ingredient and pharmaceutically acceptable auxiliary materials or auxiliary ingredients. The NAMPT inhibitor can effectively weaken the cocaine reward behavior and treat the cocaine addiction and has good clinical application prospects.

Description

The purposes of NAMPT inhibitor in the medicine of preparation treatment cocaine addiction

Technical field

The present invention relates to the purposes of NAMPT inhibitor in the medicine of preparation treatment cocaine addiction.

Background technology

Cocaine (Cocaine) is also known as 2.beta.-carbomethoxy-3.beta.-benzoxytropane, chemistry benzoyl-methyl-ecgonine (methylbenzoylecgonine) by name, general in white crystalline, odorless, bitter in the mouth and numb, it for local anesthesia and treatment asthma, is the strongest natural central stimulant the earliest, because it causes abuse to the excitation of central nervous system, within 1985, rise and become one of worldwide main drugs.

Cocaine addiction, it is a kind of chronic recurrent brain diseases, belong to drug dependence (drugdependence) class disease, cocaine addiction can cause the Changes of Plasticity of brain structure and function, related brain areas comprises nucleus accumbens septi, striatum, prefrontal cortex, Hippocampus and ventral tegmental area, health is also subject to many-sided harm, comprises mental deterioration, personality defect, intelligence dysfunction, concurrent corresponding infection complication and junkie and seeks at all adventures and use drugs and bring out various illegal activity.

Even if the main feature of cocaine addiction shows as patient after knowing the serious consequence of medication, still mandatory ask for and use to meet desire, to medicine seek and ask for out of hand, things is lost interest, Drug addiction is very deep, even if after the withdrawal and treatment several years, contact the stimulation relevant with addiction (as poison friend, the environment etc. relevant with medication in the past) and all may bring out and relapse.

In view of cocaine addiction harm is very large, find suitable therapy target and medicine, treatment cocaine addiction is extremely urgent.

Summary of the invention

In order to solve the problem, the invention provides the medicine of a class treatment cocaine addiction.

NAMPT: Nampt.

NAMPT inhibitor: suppress the material that the enzyme of Nampt is lived or expressed.

The present invention provide firstly the purposes of NAMPT inhibitor in the medicine of preparation treatment cocaine addiction.

Preferably, the medicine that the reduction NAMPT enzyme of described treatment cocaine addiction is alive.Further preferably, the medicine that described reduction NAMPT enzyme is lived is FK866, and its structural formula is as follows:

Present invention also offers a kind of medicine for the treatment of cocaine addiction, it be with NAMPT inhibitor for active component, add the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.

Preferably, described NAMPT inhibitor is reduce NAMPT enzyme medicine alive.Further preferably, the medicine reducing NAMPT enzyme alive is FK866, and its structural formula is as follows:

Preferably, described preparation is ejection preparation.

NAMPT inhibitor effectively can weaken cocaine award behavior, and treatment cocaine addiction, potential applicability in clinical practice is good.

The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.The technology that all contents recorded based on claims of the present invention realize all belongs to scope of the present invention.

Accompanying drawing explanation

Fig. 1 SIRT1 knock out mice genotype identification.1,5,6,7,8,9 homozygote mice is knocked out for SIRT1 midbrain condition; 3 is the SIRT1 hybrid mice containing Wnt1; 2,4 is the wild-type mice containing Wnt1.

Fig. 2 Conditioned place preference case

Fig. 3 Conditioned place preference experimental design schematic diagram

Fig. 4 spontaneous activity in mice case

Fig. 5 pReceiver-Lv201 carrier figure.

Fig. 6 VTA locates collection of illustrative plates

Fig. 7 cocaine Conditioned place preference is tested.Mice is after cocaine CPP trains, and cocaine administration group (CO) is compared with saline control group (SA), and CPP effect increases, * p<0.05.

Fig. 8 cocaine induction spontaneous activity.The distance (cm) that cocaine administration group (CO) records two groups of mices compared with saline control group (SA) has significant difference (* p<0.05); #p<0.05, administration group has significant difference at Day2,3,4,5,6,7 respectively compared with Day1.

The expression of Fig. 9 NAMP Different brain region in cocaine and normal saline group CPP model.(A) WesternBlot detects the expression of NAMPT at prefrontal cortex (PFC), nucleus accumbens septi (NAc), striatum (Striatum), Hippocampus (Hippocampus) and Ventral Midbrain tegmental region [of Forel (VTA).(B) statistic analysis result that in A figure, NAMPT expresses is represented.* p<0.05, represents that cocaine administration group (CO) has significant difference compared with saline control group (SA).

Figure 10 RT-PCR detects the expression of cocaine induction NAMPT.(A), in cocaine CPP model, in NAc and VTA, NAMPT expresses change.The rise that in cocaine induction VTA, NAMPT expresses, * p<0.05, and NAc does not change.(B), in cocaine acute model, in NAc and VTA, NAMPT expresses change.After cocaine single-dose, 24h after 30min, single-dose, successive administration 7d (1 times/day) NAc, VTA brain district NAMPT does not have significant change, cocaine administration group (CO) and saline control group (SA).

FK866 is on ethological impact for the injection of Figure 11 mouse peritoneal.(A) cocaine CPP shot chart.Mouse peritoneal injection FK866 reduces cocaine CPP effect, #p<0.05.(B) spontaneous activity in mice change.Mouse peritoneal injection FK866 can suppress spontaneous activity, p<0.05.Con-SA, solvent-normal saline group; Con-CO, solvent-cocaine group; FK866-SA, FK866-normal saline group; FK866-CO, FK866-cocaine group.

To the impact on mice behavior after FK866 in Figure 12 mice VTA brain.(A) cocaine CPP shot chart.Mice VTA intracerebral injection FK866 cocaine CPP reduces, #p<0.05.(B) spontaneous activity in mice change.Mice VTA intracerebral injection FK866 can suppress spontaneous activity, p<0.05.Con-SA, solvent-normal saline group; Con-CO, solvent-cocaine group; FK866-SA, FK866-normal saline group; FK866-CO, FK866-cocaine group.

NMN is supplemented on the impact of cocaine CPP after Figure 13 mice VTA intracerebral injection FK866.Supplement NMN, cocaine CPP after giving FK866 to recover, #p<0.05.SA represents that abdominal cavity gives normal saline group, and CO abdominal cavity gives cocaine group.Control represents matched group; FK866+SA represents in VTA brain to SA supplementary after FK86630min; FK866+NMN represents in VTA brain to NMN supplementary after FK86630min.

Figure 14 VTA intracerebral injection NAMPT crosses the impact of virus on mice cocaine CPP.(A) WesternBlot detects NAMPT expression.Process LAN (LV-NAMPT) NAMPT compared with contrast (LV-GFP) group expresses and increases, p<0.05.(B) impact on mice cocaine CPP after VTA intracerebral injection NAMPT process LAN virus.NAMPT process LAN (LV-NAMPT) cocaine CPP compared with contrast (LV-GFP) cocaine group increases, #p<0.05.LV-GFPSA, GFP-normal saline group; LV-GFPCO, GFP-cocaine group; LV-NAMPTSA, process LAN NAMPT-normal saline group; LV-NAMPTCO, process LAN NAMPT-cocaine group.

The OSC-PLS-DA Model load figure of NAc and NAc of the cocaine CPP model mice sample of Figure 15 lumbar injection FK866.A, C are respectively the OSC-PLS-DA Model load figure of cocaine group and saline control group VTA and NAc.B, D are respectively the OSC-PLS-DA Model load figure of FK866 cocaine administration group and cocaine group VTA and NAc.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administration group (FK866+CO).

The VTA brain district metabolism spectrum of the cocaine CPP model mice sample of Figure 16 lumbar injection FK866.The cocaine group of A based on 1HNMR and the OSC-PLS-DA shot chart of saline control group.The FK866 administration group of B based on 1HNMR and the OSC-PLS-DA shot chart of cocaine group.The statistical testing of business cycles model that C utilizes PLS-DA arrangement analysis corresponding in A and obtains, R2 represents interpretability variable, and the checking ability variables D that Q2 represents this model utilizes PLS-DA arrangement analysis corresponding in B and the statistical testing of business cycles model obtained.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administration group (FK866+CO).

The NAc brain district metabolism spectrum of the cocaine CPP model mice sample of Figure 17 lumbar injection FK866.The cocaine group of A based on 1HNMR and the OSC-PLS-DA analysis chart of saline control group.The FK866 administration group of B based on 1HNMR and the OSC-PLS-DA analysis chart of cocaine group.The statistical testing of business cycles model that C utilizes PLS-DA arrangement analysis corresponding in A and obtains, R2 represents interpretability variable, and the checking ability variables D that Q2 represents this model utilizes PLS-DA arrangement analysis corresponding in B and the statistical testing of business cycles model obtained.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administration group (FK866+CO).

Figure 18 different dosing group group Different brain region metabolite change box traction substation.(A) VTA brain district; (B) NAc brain district.Line in the middle of square frame represents median; The submeter that rolls off the production line and reach the standard grade of square frame represents the 25th and the 75th quantile; Nethermost line and uppermost line represent minima and maximum respectively.Saline control group (SA), cocaine administration group (CO), FK866 cocaine administration group (FK866CO).

The variation diagram of NAD in Figure 19 different dosing ZuVTANao district.The NAD change compared with matched group of administration group has statistical significance, * p<0.05.Control, intracerebral injection SA; FK866, intracerebral injection FK866; NMN is supplemented after giving FK86630min in FK866+NMN, VTA brain; LV-NAMPT, process LAN NAMPT group.Corresponding SA and CO is respectively saline control group, cocaine administration group.

Detailed description of the invention

Experimental example 1NAMPT inhibitor for treating cocaine addiction

1 foreword

In this research, first we set up cocaine CPP mouse model, detects mice cocaine rewarding effect by behavioristics, detects VTA, NAc Deng Nao district NAMPT express change by the method such as PCR, immunoblotting.Brain inner position injection NAMPT inhibitor, supplementary NMN and NAMPT process LAN viral vector, find that CPP effect suppresses or strengthens.Secondly, adopt the technology based on the metabolism group of nuclear magnetic resonance, NMR and test kit, detect the change of brain district and NAMPT metabolism correlative metabolites, find that NAMPT suppresses or activates, corresponding NAD level reduces or raises.This result illustrates that NAMPT participates in regulation and control cocaine CPP effect by the synthesis affecting NAD.Finally by SIRT1 midbrain conditionality knock-out mice, the effect of checking NAMPT-NAD-SIRT1 approach in cocaine CPP.This research Late Cambrian NAMPT-NAD-SIRT1 path participates in cocaine drug dependence, for deep understanding addiction mechanism and addiction therapy provide new test basis, has also found that NAMPT inhibitor can treat cocaine addiction.

2 experiment materials and method

2.1 experiment materials and equipment

2.1.1 test specimen

Cocaine hydrochloride identifies institute purchased from Chinese pharmaceutical biological product, white powder, and purity is greater than 99%, product batch number: 171210-200803; Normal saline selects Kelun Pharm Ind Co., Ltd., Sichuan, authentication code: the accurate word H51021158 of traditional Chinese medicines, product batch number M12101307.Cocaine hydrochloride with normal saline dilution to desired concn.FK866, β-nicotinamide mononucleotide. (β-NMN) is purchased from Sigma company.

2.1.2 laboratory animal

(1) male SPF level wild type C57BL/6J mice, is provided by laboratory animal Co., Ltd of Beijing Wei Duoli China, non-copulation, body weight 20-22g.

(2) SIRT1 midbrain condition knock-out mice

SIRT1 ^loxp/loxpmice (C57BL/6J mice SIRT1 gene 2,4 exon connects Loxp site) is granted by Biotherapeutics National Key Laboratory of Sichuan University professor Jiang Wei.Wnt1-Cre transgenic mice (Wnt1-Cre localization and expression in midbrain containing the mice of Cre recombinase, with SIRT1 ^loxp/loxpmouse hybrid, Cre recombinase can specific recognition Loxp site, obtains SIRT1 midbrain conditional gene knockout mice) derive from U.S. Jackson test chamber ( http:// www.jax.org/).By SIRT1 ^loxp/loxpmice and the copulation of Wnt1-Cre transgenic mice, obtain F1 generation Wnt1-Cre, SIRT1 ^loxp/-mice.Again by F1 generation Wnt1-Cre, SIRT1 ^loxp/-the copulation of male and female mice, obtains F2 for Wnt1-Cre; SIRT1 ^-/-mice.

Rearing conditions: animal feeding is in new drug safety evaluatio center, national Chengdu SPF level Animal House, and temperature 20-25 DEG C, relative humidity 55-65%, in whole experimentation, animal freely ingests and drinks water.Feeding environment meets GB GB14925-2001, experimental animal licence: SCXK (river) 2003-01.In zoopery, all operations all meets the requirement of international animal welfare (AAALAC).

SIRT1 midbrain condition knock-out mice is identified: by F2 for Mouse Tail-tip clip 0.5-1cm length tail tissue, put into centrifuge tube, preparation LycisBuffer liquid (as table 1), and 0.5mL/ manages, and 55 ° are spent the night, to tail tissue melts.

Table 1LycisBuffer liquid preparation table

Extract DNA: take out tail tissue and put upside down mixing 13000rpm, centrifugal 10min.Pour out supernatant (not drawing).In supernatant, add equal-volume 0.5mL dehydrated alcohol, put upside down mixing 8 times (not vortex).The choicest of 200mL rifle goes out DNA (white filament), is dissolved in 50-100 μ L distilled water, and 55 dissolve, and period per half an hour puts upside down mixing (about 2h)

PCR reaction and agarose gel electrophoresis carry out gene identification: the primer sequence in pcr amplification reaction is as table 2.Pcr amplification reaction system is as table 3.Preparation is containing 1.2% agarose gel of GoldView dyestuff.Molecular weight is DNAMaker and the PCR reacting final product DNA sample loading 2 μ L of 2000, carries out gel electrophoresis.Mousetail agarose gel electrophoresis genotype fragment: SIRT1 homozygote is 750bp, SIRT1 heterozygote is 750bp and 550bp.750bp band (SIRT1 homozygote) is there is and is not SIRT1 midbrain condition knock-out mice containing the mice that the sample of 100bp band (Wnt1) is corresponding in F2 in generation.As in Fig. 1,1,5,6,7,8,9 knock out homozygote mice for SIRT1 midbrain condition; 3 is the SIRT1 hybrid mice containing Wnt1; 2,4 is the wild-type mice containing Wnt1.Choose SIRT1 midbrain condition and knock out homozygote mice for subsequent experimental.

Table 2 gene identification primer sequence

Table 3PCR reaction system

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 type electric heating constant temperature tank, reliablely tests Instrument Ltd. purchased from Shanghai is gloomy

(4) GeneAmpPCRSystem9700, purchased from AppliedBiosystems company

(5) TaKaRaPCRThermalCycler, purchased from precious biological engineering company limited

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

(7) electrophresis apparatus, purchased from American Bio-Rad company

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

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

(10) spectrometer, purchased from German Bruker company

(11) voltage stabilization and current stabilization electrophresis apparatus, vertical slab electrophoresis groove, transferring film groove, equal purchased from American Bio-Rad company

(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 machine Manufacturing Co., Ltd of Wenzhou City

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

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

(16) enzyme-linked immunosorbent assay instrument, purchased from French ThermoFisher company

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

(18) electronic balance, purchased from German sartorius company

(19) Nitrogen evaporator, purchased from Beijing Cheng Meng Science and Technology Ltd.

(20) mice rustless steel metabolic cage tool, purchased from Shanghai with educating medical anatomy model manufacturing company

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

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

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

2.1.4 major experimental reagent

(1) TRIZOL reagent, purchased from purchased from American Invitrogen (LifeTechnologies) company

(2) chloroform, purchased from Solution on Chemical Reagents in Shanghai company limited

(3) methanol, purchased from Solution on Chemical Reagents in Shanghai company limited

(4) deuterated heavy water D2O, purchased from American CIL (CambridgeIsotopeLaboratories) company

(5) TSP, purchased from Sigma-Aldrich company

(6) DNase/RNase-FreeddH2O, purchased from purchased from Beijing Tian Gen biochemical technology company limited.

(7) Reverse Transcriptase kit (ThermoScientificRevertAidFirstStrandcDNASynthesisKit), purchased from DBIBioscience company

(8) StormstarSYBRGreenqPCRMastermix, purchased from DBIBioscience company

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

(10) SDS-PAGE reagent (30% polyacrylamide, Tris-base, SDS, Ammonium persulfate., TEMED, glycine), 0.22 μM of aperture pvdf membrane, all purchased from Bio-Rad company

(11) (PageRulerTMPrestainedProteinLadder, purchased from ThermoScientfic (Fermentas) company for protein pre-dyed marker

(12) chemiluminescence development kit (SuperSignalWestPicoChemiluminescentSubstrate), purchased from American Pierce

(13) Kodak Kodak film, purchased from Kodak

(14) 100% ethanol, purchased from Solution on Chemical Reagents in Shanghai company limited

(15) formaldehyde, purchased from Solution on Chemical Reagents in Shanghai company limited

(16) GoldView dyestuff, Shanghai match Bai Sheng gene technology company limited

(17) agarose, Sheng Gong biological engineering company limited

(18) NAD/NADHAssayKit, purchased from Abcam company

(19) anti-β-actin antibody, purchased from American CST company

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

(21) anti-Sirt1 antibody, purchased from SantaCruze company

All the other are domestic analytical reagent.

2.1.5 the preparation of main agents

(1) 10% (W/V) Ammonium persulfate.: take the ddH that 1g Ammonium persulfate. adds 10mL ₂after O, stirring and dissolving, 4 DEG C of preservations.

(2) 5 × SDS-PAGE electrophoretic buffers: take 15.1gTris-base, 94g glycine and 5gSDS, add the ddH of about 800mL ₂o, uses ddH after stirring and dissolving ₂o is settled to 1000mL, and room temperature preservation is for subsequent use.

(3) film transfering buffering liquid: take 2.9g glycine, 5.8gTris-base and 0.37gSDS, is placed in 1000mL beaker; Add ddH ₂the abundant stirring and dissolving of O; ddH ₂o is settled to 800mL, then adds the methanol of 200mL, room temperature preservation.

(4) 10 × TBS buffer: take Tris-base (tri methylol amino methane) 60.5g, NaCl87.5g, after dissolving completely, drip dense HCl and regulate pH to be 7.4, be finally settled to 1000mL with 800mL distilled water.

(5) dilution proportion 10 × TBS buffer of TBST buffer: 1:10 is to 1 × TBS buffer of 1000mL, adds after 1mLTween-20 fully mixes, 4 DEG C of preservations.

(6) Block buffer: take 5g defatted milk powder TBST buffer and be settled to 100mL, abundant stirring and dissolving, 4 DEG C of preservations (needing matching while using).

2.2 animal behavioral model are set up

2.2.1 the foundation of cocaine Conditioned place preference pattern

Mice Place Preference case is as shown in Figure 2:

(1) start to test front 3-5 days, mice is put into experimental apparatus by same operator and trains 15 minutes by every day, allows animal have certain adaptation to instrument and operator.

(2) mice is put into CPP casing, acclimatization training 3 days, the data measured for the 3rd day are as the data (D0) of pretest.Each adaptive training and testing time are 15min, when the time that animal stops in side (black and white both sides) more than 600s time, should reject.Using the side of animal stay longer as natural preference case.

(3) mice carries out intraperitoneal injection, and dosage following (table 4), administration number of times is 3 times, alternating delivery, continuous 6 days.1st, 3,5 days give with give put into non-natural preference case with cocaine hydrochloride; 2nd, within 4,6 days, nature preference case is put into normal saline.Matched group puts into corresponding case (Fig. 3) to the same with normal saline and with administration group at every turn.

Table 4 cocaine administration approach, dosage, administration volume summary sheet

Within (4) the 7th days, carry out CPP test, mice is put into ash bin successively, allow its 15min that freely shuttles back and forth in large mice position preference case, and record the time that each casing stops.

(5) points for attention: during each animal training, should handle with care, significant impact not caused to the emotion of animal; Each training terminates carry out wiping to casing, to eliminate the abnormal smells from the patient of each animal.Intensity of illumination, the training time of attentional manipulation every day, 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 to test front 3-5 days, mice is put into experimental apparatus by same operator and trains 15min by every day, allows animal have certain adaptation to instrument and operator.

(2) mice is put into spontaneous activity casing, acclimatization training 3 days, the data measured for the 4th day are as the data (D0) of baseline.Each adaptive training and testing time are 15min, the distance (cm) of record mice activity.

(3) the 5th days start, and mice first carries out intraperitoneal injection, once a day, successive administration 7 days, dosage is (table 4) as above, puts into case and detect spontaneous activity, according to data (D1) (Fig. 4) as first day after 3min.Mice is dissected in 30min after spontaneous activity in 7th day detects.

2.3 samples are drawn materials and are prepared

The test of Conditioned place preference terminates quick sacrificed by decapitation C57 mice in rear 30min, then brain is separated rapidly, after 4 DEG C of normal saline flushings 3 times, be separated according to brain anatomical atlas and take out prefrontal cortex, nucleus accumbens septi, striatum, Hippocampus, Ventral Midbrain tegmental region [of Forel.Directly the sample got is put into liquid nitrogen fast, and put into rapidly-80 DEG C of refrigerators and store.

2.4 Western blotting

2.4.1 sample preparation

Take out tissue samples in-80 DEG C of refrigerators, operate on ice, add ice RIPA lysate, be placed in cracking 5min on ice.Ultrasonic 10 times of Ultrasonic Cell Disruptor, 3s/ time (putting with on ice), cell breakage.The centrifugal 15min of 13000g (4 DEG C), abandons precipitation, takes out supernatant.Protein concentration in supernatant is measured with Coomassie Brilliant Blue.Add RIPA lysate and LoadingBuffer adjustment concentration of specimens, loading volume and applied sample amount.Boil sample 5min.Centrifugal 13000 revs/min, 10min, gets supernatant.

2.4.2 glue

Preparative separation glue is as shown in table 5.

Table 5 separation gel preparation preparation figure

The concentrated glue of preparation is as shown in table 6.

Table 6 concentrates glue and prepares preparation figure

2.4.3 electrophoretic separation

Glue is dipped in transfering buffering liquid and balances 10min.Loading 15 ~ 20 μ L to SDS-PAGE glue (10cm × 10cm) electrophoresis.Concentrated glue voltage 70-80V, albumen is to concentrated glue separation gel boundary place, and voltage adds as 90V, until albumen is run through (determining according to albumen size), the time is approximately 120min.

2.4.4 transferring film

The pure methanol of pvdf membrane activated for 3 ~ 5 seconds.According to size clip film and the filter paper 6 of glue, put into transfering buffering liquid and balance 10min.Assembling transfer sandwich: sponge → 3 metafiltration paper → glue → film → 3 metafiltration paper → sponge, every layer put well after, bubble of rushing.Glue is put in negative pole face (black side) and transfer groove is placed in ice bath, put into sandwich (black side is to black side), add transfering buffering liquid, plug electrode, 100V, the transferring film time determines (electric current is about 0.3A) by molecular weight of albumen.After transferring film terminates, cut off the electricity supply, take out hybond membrane and put into the confining liquid got ready.

2.4.5 close

With the Block buffer of the skim milk of TBST preparation 5%, 30mL/ block glue.Pour in plate, by pvdf membrane complete wetting in milk, be placed on shaking table, incubated at room 1.5h.

2.4.6 primary antibodie is hatched

By primary antibodie Block buffer dilution proportion as required, seal with hybrid belt, 4 DEG C are spent the night or 37 DEG C, 1-2h.Film is washed three times, each 5-10min with TBST.

2.4.7 two anti-hatch

Corresponding antibodies Block buffer 1:5000 is diluted, 37 DEG C of shaking table 1-2h, wash film four times with TBST, each 5-10min, then wash film secondary, 5-10min with TBS.

2.4.8 colour developing

Preparation luminescent solution (1mlA liquid: 1mlB liquid), is placed in luminescent solution reaction 1 minute in darkroom by pvdf membrane.Pvdf membrane is taken out, blots unnecessary luminescent solution with filter paper, pvdf membrane is put into magazine, film is faced up and film contact, compress exposure (time length is depended in band brightness), develop a film.

2.4.9 interpretation of result

Immunoblotting film Image-ProPlus6.0 system is read gray value, with β-actin band gray value as internal reference, carries out standardization and compare.

2.5RT-PCR

2.5.1 extract and organize RNA

Reagent prepares: the ddH of Trizol, RNase-free ₂o, chloroform, dehydrated alcohol, 75% ethanol, isopropyl alcohol, the consumptive materials such as EP pipe used, rifle point are the imported product of DNase/RNase-free.

(1) homogenized: tissue sample, to be organized in liquid nitrogen and grind with small size mortar, low temperature (operating) is kept on ice in process of lapping, can not make to organize deliquescing to melt, every 50-100mg tissue adds 1mLTrizol reagent, carry out homogenized with Syrup-homogenizing instrument, be finally transferred in the EP pipe of new 1.5mL.

(2) be separated: hatch 5min for room temperature 15-30 DEG C, treat that in homogenised sample, albumen is separated completely, every 1mLTrizol reagent adds 0.2mL chloroform, carefully builds pipe lid, repeatedly puts upside down mixing 8-10 time, incubated at room 3min, 4 DEG C, the centrifugal 15min of 12,000g.After centrifugal, mixture is divided into three layers, the phenol-chloroform phase of the colourless aqueous phase on upper strata, mesophase spherule and lower floor's redness, and RNA is present in aqueous phase, when aqueous phase volume is about homogeneous added Trizol amount 60%.

(3) RNA precipitation: aqueous phase is transferred in new EP pipe and (notes not being drawn onto intermediate layer, in order to avoid RNA pollutes), during original homogenate, every 1mLTrizol reagent uses 0.5mL isopropyl alcohol, RNA in precipitation aqueous phase, incubated at room sample 10-20min, 12,000g, 4 DEG C of centrifugal 10min.RNA precipitation is general invisible before centrifugation, but centrifugal after at the bottom of pipe, form flocculent deposit.

(4) RNA washing: abandon supernatant, the washing with alcohol RNA of 1mL75% precipitates once, after vortex mixing, 4 DEG C, the centrifugal 5min of 7,500g.

(5) again dissolve RNA: abandon supernatant, wink from, siphon away 75% residual ethanol, dry RNA precipitation (air drying or vacuum drying 5-10min), note not overdrying (its dissolubility can be reduced).Use the ddH of 20-30 μ LRNase-free ₂o dissolves RNA, for subsequent experimental after vortex mixing.When dissolving RNA, the water rifle first added without RNA enzyme is blown and beaten several times repeatedly, then hatches 10min for 55-60 DEG C.RNA solution-80 DEG C of Refrigerator stores obtained.

2.5.2RNA quality testing and quantitatively

(1) electrophoresis detection: the agarose gel using 1 × TBE preparation 1%, get RNA and the rear leakage of electricity swimming of sample-loading buffer mixing of 3 μ L dissolvings, 150V is separated 20min, carries out imaging analysis.The object of electrophoresis is the integrity and their ratio that detect 28S and 18S band, if 28S and 18S band is bright, clear, complete, and the brightness of 28S is more than the twice of 18S band, then think that RNA is up-to-standard, may be used for subsequent experimental.

(2) absorbance detection of RNA: 280,320,230, absorbance under 260nm represents the organic OD values such as nucleic acid, background, salinity and albumen respectively, in general, we only analyze the ratio (Ratio, R) of OD260/OD280.When R is 1.8-2.0, think that albumen or other organic pollutions are acceptables in RNA; As R ﹤ 1.8, to illustrate in RNA albumen or other organic pollutions obvious, generally do not use; As R ﹥ 2.0, illustrate that RNA degrades, unavailable.

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

2.5.3RNA reverse transcription becomes cDNA

As follows according to the description operation of DBIBioscience Reverse Transcriptase kit:

(1), after melting, by also centrifugal a little for each for test kit component vortex mixing, be placed on ice.If RNA template GC content is high or containing secondary structure, mixed gently by above-mentioned mixed liquor, wink from, hatch 5min, insert cooled on ice immediately for 65 DEG C, centrifugal, then be placed on ice.

(2) in the PCR pipe being placed in aseptic nuclease free on ice, reactant liquor (as shown in table 7) is prepared in order:

Table 7 Reverse Transcription preparation table

(3) mix gently, centrifugal.

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

Under (5) 98 DEG C of conditions, carry out the reverse transcriptase inactivation reaction of 5min.Product can be directly used in follow-up PCR and detect, or be stored in-80 DEG C for subsequent use

2.5.4Real-TimePCR

(1) first the gene primer of synthesis is carried out centrifugal 13,300rpm, 5min, guarantee that all powder all concentrate at the bottom of pipe, according to the ddH adding enough nuclease free is described ₂o or TEbuffer is 100 μMs to final concentration, and this is storage liquid concentration, then the liquid diluting 10 times that takes a morsel is for working solution concentration 10 μMs.

(2) according to DBIBioscienceStormstarSYBRGreenqPCRMastermix description preparation reaction system (table 8).

Table 8RT-PCR reaction system preparation table

(3) mix gently, centrifugal.

(4) PCR response parameter is: 95 DEG C of denaturation 2min, 40 circulations (95 DEG C of degeneration 10sec, 60 DEG C of renaturation, extension 30-34sec); Solubility curve analyzes (65-95 DEG C, every 5sec rise 0.5 DEG C), and primer sequence is as table 9.

Table 9RT-PCR primer sequence

2.6 nuclear magnetic resonance, NMR metabolism group

2.6.1 cell lysate is prepared

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

(2) 4ml/g methanol and 0.85mL/g water is by volume added in tissue sample, each 3s of ultrasonication sample, ultrasonic 8-10 time, interval 3s vortex mixing.

(3) add 2mL/g chloroform, vortex mixing 2min, leaves standstill 2min again.

(4) 2mL/g chloroform and 2mL/g distilled water is added, vortex mixing again.

(5) sample to be placed on ice or refrigerator leaves standstill after 15min, 1000g, 15min, 4 DEG C centrifugal (as without obvious layering, then recentrifuge).

(6) by upper water phase transfer in new EP pipe, nitrogen blow after-80 DEG C of preservations.

(7) if do not detect immediately, then water-phase extract is stored in-80 DEG C (are mainly and reduce oxidation reaction), and is stored in-80 DEG C (but preferably not more than 3 days).

(8) aqueous phase metabolite: add 580 μ L heavy water, containing 0.1 ~ 0.5mMTSP, vortex mixes, the centrifugal 5min of 12000g, and 550 μ L samples is placed in nuclear magnetic tube detection [31].

2.6.2 nuclear magnetic resonance spectroscopy

BrukerAvanceII600 type (BrukerBiospin, Germany) nuclear magnetic resonance chemical analyser, experimental temperature 300K is set, NMR instrument calls relaxation edit pulse sequence (Carr-Purcell-Meiboom-Gillsequence, CPMGsequence), presaturation mode is adopted to suppress water peak.Detect spectrum width 12335.5Hz, time delay 5s, sampling time 2.66s, sampled data is counted 64K, and accumulative frequency 64 times, start sampling, obtain free induction decay (FID) signal, the broadening factor of line is-0.3Hz [32].

2.6.3 Data induction and pattern recognition analysis

The initial data of NMR is FID signal, computer software (MestReNova-6.1.1-6384) converts visual NMR spectrogram to through Fourier transform and windowed function, then carries out Treatment Analysis to spectrogram:

(1) calibrate: because the pH value of each sample is different, in order to avoid the error brought of drifting about due to wave spectrum, so will calibrate with a metastable spectrum peak, can be standard with the TSP added (chemical shift is δ 0.0) in this experiment, all spectrum peaks are adjusted.Because the lactic acid peak (1.336ppm ,-CH3 are bimodal) in tissue samples and glucose peaks (5.22ppm ,-CH1 are bimodal) also relatively stable, so also can calibrate with the two.Concrete operations open wave spectrogram with MestReNova software, and object chemical shift amplified, the TMS button in click tools hurdle is calibrated.

(2) phase place adjustment: the spectrogram that usual NMR collects all contains absorption and diffusion component, can obtain pure absworption peak by phase place adjustment, shows and positive quadrant is all adjusted in the peak of reversed image limit by spectrogram exactly.In the instrument of Bruker, first phase place adjustment carries out zero order phase adjustment PH to maximum peak ₀, then with first order phase adjustment PH ₁regulate other peak.

(3) baseline correction: in nuclear magnetic resonance chemical analyser, if the rf frequency composition of transmitter drains in receiver, after detection, these are revealed voltage and just become flip-flop, cause FID signal to have certain Dc bias, occur in a digital signal after digitized, if do not revised, can cause the spike at the odd lot place of spectrum after Fourier transform, this will have influence on the dynamic range of whole spectrum.Namely the main purpose of baseline is adjusted to be exactly improve the outward appearance of spectrum and improve the accuracy of integration.So when pretreatment NMR wave spectrogram, must baseline correction be carried out.

(4) peak overlapping and exclusive PCR peak: after according to above-mentioned three steps all spectrum peaks being carried out pretreatment, all spectrum peaks are carried out overlap, just obtain the spectrum peak figure of a superposition.Because introduce the methanol of external source, chloroform and water when carrying out sample treatment, get rid of 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 is amassed: the limit of integration of employing is 0.5-9.5ppm, and integration size is 0.004ppm

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

(7) data export: through above-mentioned steps, complete the preprocessing process of wave spectrum, just can export data on MestReNova, generally save as TXT form, then transfer 2003 editions Excel to.

(8) pattern recognition analysis: analyze with SIMCA-P10.0 software kit.Variable is concentrated by average and is carried out PCA, PLS-DA, OSC after Pei Ertuotu quantification (pareto-scaled) and analyzes.Quantize to determine whether composition divides meaningful by Pei Ertuotu.Two dimension shot chart is for distinguishing sample; Corresponding load diagram is for distinguishing contributive difference metabolite on wave spectrum.The distribution of nuclear-magnetism chemical shiftsum endogenous metabolites is the information according to document and mankind's metabolism group data base (HumanMetabolomeDatabase) website obtain.Mankind's metabolism group data base is the website of bioinformatics/Chemoinformatics, can obtain the relevant information of metabolite and metabolic enzyme.

2.7 process LAN slow virus carriers build

2.7.1 carrier

(1) carrier information:

Carrier is pReceiver-Lv201, as Fig. 5.

(2) competent cell and conversion is prepared

CaCl ₂it is as follows that legal system carries out transformation experiment step for competent cell:

1. often kind of a competent cell suspension is got 200 μ L and is transferred in sterile eppendorf tubes, and often pipe adds connecting fluid 10 μ L, rotates mixing gently, then puts in ice and places 30min (prepare competent cell, make it have the ability of picked-up foreign DNA).

2. 42 DEG C of heat shock 90s, transfer to cooling cell 1-2min in ice bath fast by centrifuge tube.Often pipe adds 800 μ LLB culture medium, and water-bath is heated to 37 DEG C, then places shaking table incubation 45min with recovery antibacterial.

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

4. clone carries out follow-up PCR qualification.

2.7.2 construction of recombinant plasmid

PCR primer connects into linearisation expression vector reaction system in table 10, reaction condition: 25 DEG C of 30min; 42 DEG C of 15min.

Table 10PCR reaction system

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

2.7.3 virus packaging

Digestion 293T cell, adjusting its density is that every 20mL has 1.2X10 ⁷individual cell, inoculating cell, in culture dish, places 37 DEG C, 5%CO ₂cultivate 24h in incubator, when cell density reaches 70%-80%, can be used for cell transfecting.Cell state is most important for virus packaging, needs to ensure good cell state and less passage number.Before transfection, 2h will be replaced by serum-free medium containing hyclone culture medium.The each DNA solution (pGC-LV carrier 20 μ g, pHelper1.0 carrier 15 μ g, pHelper2.0 carrier 10 μ g) adding preparation in centrifuge tube is mixed homogeneously with respective volume culture medium, and adjustment cumulative volume is 2.5mL, incubation at room temperature 5min.Lipofectamine2000 reagent is softly shaken up, gets 100 μ LLipofectamine2000 reagent in the mixing of another Guan Zhongyu 2.4mLOpti ?MEM culture medium, incubation at room temperature 5min.DNA after dilution is mixed with the Lipofectamine2000 after dilution, mixing of gently running, avoid vibration, and must the interior mixing of 5min.Incubation at room temperature 20min after mixing, is then transferred to mixed liquor in 293T cell culture fluid, mix homogeneously, places 37 DEG C, 5%CO ₂cultivate in incubator.Go culture medium after cultivating 8h, every bottle of cell adds 20mL phosphate buffer (PBS), to wash remaining mixed liquor, removes mixed liquor.Add containing 10% hyclone culture medium 25mL in every bottle of cell, place 37 DEG C, 5%CO ₂cultivation 2 days is continued in incubator.

2.7.4 titer determination

(1) sample preparation

293T passage, 24 Zhong Mei holes, hole add 1 × 10 ⁵individual cell, volume is 500 μ L; Prepare 10 aseptic Ep pipes next day, often in pipe, add 90 μ L culture medium; Getting virus stock solution used 10 μ L to be measured joins in first pipe, mix homogeneously, and the first pipe liquid 10 μ L getting mix homogeneously joins in second pipe and continues an identical operation to the last pipe; Choose required cell hole, suck 90 μ L culture medium.Add the viral solution diluted, place 37 DEG C, 5%CO ₂incubator is cultivated; After 1 day, add fresh culture 500 μ L.Careful operation, extracting RNA after 4 days.

(2) total serum IgE extracting

Remove cell conditioned medium liquid, every hole adds 1mLTrizol, and piping and druming, room temperature leaves standstill 5min, is transferred in another new 1.5mLEp pipe.Often pipe adds 200 μ L chloroforms, and firmly shake 15s, room temperature leaves standstill 15min.The centrifugal 15min of 120000r/min at 4 DEG C.From every pipe, Aspirate supernatant is in another new 1.5mLEp pipe.Add the isopropyl alcohol of equal-volume-20 DEG C of pre-coolings ,-20 DEG C of precipitation 10min after mixing.At 4 DEG C, the centrifugal 10min of 120000r/min, removes supernatant.Add 75% ethanol of 1mL4 DEG C of pre-cooling, washing precipitation and centrifugal tube wall.At 4 DEG C, the centrifugal 5min of 10000r/min, moves and abandons supernatant.At 4 DEG C, 10000r/min recentrifuge 5min, sucks residual liquid, dry under room temperature, does not need bone dry.Add 20 μ L without RNA enzyme (RNase) water to dissolving completely, ultra-violet analysis measures institute's extracting RNA concentration.

(3) RNA reverse transcription obtains cDNA

1 μ LOligodT (0.5 μ g/ μ L) and 2.0 μ g total serum IgE are added PCR tubule, adds pyrocarbonic acid diethyl ester (DEPC) water to 9 μ L.Mix homogeneously, centrifugal, 70 DEG C of temperature bath 10min.And then be inserted in 0 DEG C of ice-water bath.According to the form below ratio, figures out required amount of reagent according to reaction tube.By M-MLV enzyme etc., on ice, mixing obtains reverse transcription reaction liquid.11 μ L reverse transcription reaction liquid are added in each reaction tube, centrifugal after mix homogeneously.Wherein, 11 μ L reverse transcription reaction liquid are containing 5 × RT Buffer 4 μ L, 10mmol/LdNTPs2 μ L, RNA inhibitor 0.5 μ L, M-MLV-RTase1 μ L, DEPC water 3.5 μ L.Carry out 1h at 42 DEG C and complete reverse transcription reaction, rear use 70 DEG C process 10min makes reverse transcriptase inactivation.Reverse transcription reaction product cDNA can be used for PCR, also can-80 DEG C of preservations for a long time.

(4) real-time quantitative PCR detects

Configuration reaction system: often pipe adds SYBRpremixexTaq10 μ L, forward primer (5 μm of ol/L) 1.0 μ L, downstream primer (5 μm of ol/L) 1.0 μ L, cDNA1.0 μ L, ddH ₂o7.5 μ L.Setting program is two-step method real-time quantitative PCR.Denaturation 95 DEG C of 5s; Degeneration 95 DEG C of 5s, anneal 60 DEG C of 30s, extends 60 DEG C of 30s, 40 circulations.Read light absorption value, for making melting curve in the extension stage at every turn.After PCR terminates, at 95 DEG C of degeneration 1min.Then be cooled to 55 DEG C, DNA double chain is fully polymerized.To 95 DEG C from 55 DEG C, each walks increase by 0.5 DEG C, keeps 30s, reads light absorption value simultaneously.After two circulations, set point is raised 0.5 DEG C.

2.7.5 the results of slow virus and concentrated

The 293T cell conditioned medium liquid of 2d after collection transfection, 4 DEG C, the centrifugal 10min of 4000 × g, to remove cell debris.With 0.45 μm of metre filter supernatant in 40mL ultracentrifugation pipe.Viral crude extract sample is added in filter cup.Filter cup is inserted in filtered solution collecting pipe, then 4000 × g is centrifugal to required viral concentration volume, time 15min.Take out centrifugal device after centrifugal end, filter cup and filtered solution collection cups are separated.Tipped upside down on by filter cup 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.Viral concentration liquid is in sample collection cup.

2.8 mice VTA brain district locating injections

Injected in mice 1.5% penta bar, than (40mg/kg, ip) anesthesia under penta sodium, is fixed on Naoliqing capsule.Collection of illustrative plates (Fig. 6) is located according to mice VTA, in left side VTA (3.lmm after bregma, the other 0.7mm of center line, degree of depth 4.5mm), the embedding stainless steel tube (internal diameter 0.34mm, external diameter 0.48mm) being about 0.5mm, skull surface is fixed in again with dental cement, head is fixed in stainless steel tube outer end, sew up wound, covers supporting inner core.Postoperative injection penicillin sodium (0.1 ten thousand units/only, once a day), injection is protected from infection for three days continuously.Post operation carries out the training of corresponding behavioristics on the 7th day, and the drug injection amount of VTA is l μ L, first extracts inner core during injection, and lmin injection is complete, let the acupuncture needle remain at a certain point 2min.During mice VTA both sides injecting virus, be fixed on Naoliqing capsule after mice anesthesia.After the perforation of 10ml syringe needle, inject 0.5 μ L virus (the 0.25 every side of μ L) with spiral micro-sampling pump bilateral, 0.1 μ L/min, let the acupuncture needle remain at a certain point 3min, sew up wound.

2.9NAD/MADH test kit detects

AbcamNAD/NADHAssayKit material composition composition is as shown in table 11.

Table 11NAD/NADHAssayKit material composition composition table

(1) drafting of standard curve

Prepare NADH titer.In 495 μ LNADH/NAD extracting solution, add 5 μ LNADH titers dilute, obtain 10pmol/ μ L (10 μMs) NADH titer.In microcentrifugal tube, dilution standard curve, as shown in table 12.

Table 12NADH titer dilution preparation table

(2) sample prepares

Results organize the amount (20mg tissue) of necessary each mensuration.Tissue is washed with in cold PBS.The NADH/NAD extracting solution adding 400 μ L makes its homogenate.The centrifugal 5min of maximum speed, and the NAD/NADH supernatant that transfer is extracted (operates) on ice in a new pipe.Tissue may comprise the enzyme consuming NADH fast.These enzymes filter by 10kD centrifugal column (ab93349) and are removed, stand-by.

(3) detect

The decomposition step that sample NADH detects:

1. total NAD and NADH: extracting sample does not need reprocessing.

2. NADH:NAD+ needs to be decomposed.Subpackage 200 μ L extracts sample and adds in micro tube.60 DEG C of heating 30min (NADH's under these conditions, all NAD+ still stands intact by decomposition) in a water bath or in heat block.Sample is placed on ice.Quick Centrifuge A sample is to remove the precipitation separated out

3. reacting hole is set:

Gauge orifice=50 μ L standard substance dilution.

Blank control sample hole=1-50 μ L sample (by Extraction buffer adjusted volume to 50 μ L/ holes).

NADt sample well=1-50 μ L sample (by Extraction buffer adjusted volume to 50 μ L/ holes).

NADH sample well=1-50 μ LNAD decomposes sample (by Extraction buffer adjusted volume to 50 μ L/ holes).

4. reactant mixture:

Each response preparation 100 μ L reacts premix, as shown in table 13:

Table 13 reaction mixture preparation table

100 μ L reactant mixtures are added in each standard and sample well.Add 100 μ L background response mixed liquors to blank sample hole.Incubated at room temperature 5min, NAD is transformed into NADH.Adding 10 μ LNADHDeveloper liquid to each hole mixes.At room temperature reaction cycle stirs 1-4h.Under OD450nm wavelength, read 1-4h and get multiple reading.

3 experimental results

3.1 cocaine inductive condition position preferences

By the Conditioned place preference training of 6 days, there is notable difference (Fig. 7) in the time difference that cocaine administration group and normal saline group C57 mice train front and back to stop at preference case and non-preference case.Before testing (test) after the training of cocaine administration group and testing, the time difference of (Pretest) is 295 ± 38s, and saline control group is 44 ± 58s.Above-mentioned result of the test shows, by the training of 20mg/kg cocaine Conditioned place preference, the time that C57 mice stops at cocaine training casing significantly increases, and shows to establish reliable cocaine CPP model.

The spontaneous activity of 3.2 cocaine inductions

After the spontaneous activity training of 10 days, the locomotor activation of contrast cocaine group and normal saline group training front and back 15min, after can finding the training of cocaine administration group, locomotor activation significantly increases (P<0.05) (Fig. 8).The cocaine administration group upon administration locomotor activation of 2,3,4,5,6,7 days, also apparently higher than first day (Day1), and has obvious significant difference.Illustrate that cocaine can increase in inducing mouse spontaneous activity.

The expression of 3.3 cocaine induction Different brain region NAMPT

In order to detect the expression of NAMPT in cocaine administration group and saline control group Different brain region, dissect mice after CPP model training, get prefrontal cortex (PFC), nucleus accumbens septi (NAc), striatum, Hippocampus and Ventral Midbrain tegmental region [of Forel and carry out Westernblot analysis.Result shows, cocaine group is compared with normal saline group, NAMPT does not have significant change at the expression of prefrontal cortex, striatum and hippocampus, but in nucleus accumbens septi and Ventral Midbrain tegmental region [of Forel, NAMPT expression significantly raises (p<0.05) (Fig. 9).

The expression of 3.4 cocaine induction NAMPT

In order to detect the change of NAMPT rna expression in NAc and VTA brain district, we get NAc and the VTA Brain TisX sample of cocaine and normal saline group in CPP model, RNA is organized in extraction, after quality testing, reverse transcription becomes cDNA, carry out Real-TimePCR detection (each primer sequence refers to material component), analyze the relative expression of NAMPT between cocaine group and normal saline group (Figure 10 A), and utilize the two-sample test in T-test to calculate its P value, from analysis result, NAMPT up-regulated in VTA tissue has statistical significance (p<0.05), significant change is not had in NAc.In cocaine acute model: as 24h after 30min, single-dose after single-dose and continuous single-dose 7 days solutions take corresponding Brain TisX, Real-TimePCR detects, and finds the expression no significant difference of NAMPT in NAc and VTA (Figure 10 B-C).

3.5 mouse peritoneal injection FK866 reduce cocaine CPP effect

For exploring the effect of NAMPT in cocaine addiction, we select by the NAMPT of blood brain barrier specific inhibitor---FK866, as probe, studies and affects its neuroethology.We find (as Figure 11), after wild-type mice lumbar injection 4mg/kgFK866, the spontaneous activity of cocaine CPP and cocaine induction obviously reduces (P<0.05), has pointed out NAMPT to play positive acting in cocaine addiction.

3.6VTA brain inner position injection FK866 suppresses cocaine CPP effect

In order to verify the cocaine CPP inhibitory action of FK866 further, in mice VTA brain, pipe laying surgery recovery is after 5-7 days, and it is the FK866 of 10nM that location gives 1 μ L concentration, then carries out cocaine CPP training.Result shows: FK866 is compared with matched group for the injection of VTA brain inner position, and cocaine CPP (as Figure 12 A) and spontaneous activity (as Figure 12 B) all reduce (P<0.05).

The cocaine CPP effect that NMN reverses FK866 is supplemented in 3.7VTA brain

NMN is the direct effect product of NAMPT catalysis nicotiamide metabolism, in order to the effect of NAMPT in cocaine addiction is described.It is after the FK86630min of 10nM that VTA brain inner position injects 1 μ L concentration, then injects the NMN that 1uL concentration is 10mg/mL, then detects cocaine CPP movable.Found that, after injection inhibitor FK866, cocaine CPP reduces; But after supplementing NMN, eliminate inhibitory action (as Figure 13) (P<0.05) of FK866 to cocaine CPP.

In 3.8VTA brain, process LAN NAMPT increases cocaine CPP effect

For the effect of direct-detection NAMPT in cocaine addiction, we construct the process LAN slow virus carrier of NAMPT.VTA locating injection 0.5uL virus (every side 0.25uL) in brain, recovers 5-7 days, then carries out cocaine CPP training.Found that, the process LAN slow virus carrier of mice VTA brain district NAMPT is compared with contrast LV-GFP slow virus carrier, and the expression of NAMPT increases (as Figure 14 A).In VTA, the CPP activity of process LAN NAMPT (LV-NAMPT) cocaine compared with matched group (LV-GFP) increases (P<0.05) (as Figure 14 B).

3.9 abdominal cavities give FK866 inducing mouse metabolite and change

3.9.1 the metabolism spectrum of tissue samples

Giving the change of mice correlative metabolites after FK866 in order to detect abdominal cavity, utilizing and detecting mice NAc and VTA sample based on nuclear magnetic resonance, NMR metabolism group method.We adopt pca model to carry out nuclear magnetic data analysis, to obtain the difference of matched group and drug group mouse brain district metabolite more accurately.Result shows, and the load diagram of OSC-PLS-DA model as shown in the figure (Figure 15), is compared a series of endogenic metabolite relevant to NAMPT metabolism between administration group and contrast and be there occurs obvious change, comprise NMN and NAD.

3.9.2 the pattern recognition analysis between matched group and administration group

In order to more detailed differentiation metabolite change, we apply a kind of means of numerical analysis-OSC-PLS-DA model of multivariable Control, to reduce to change difference interference in potential group, are separated metabolite more perfectly.OSC-PLS-DA model analysis magnetic resonance detection data, there is obvious metabolite profile difference (Figure 16, Figure 17) in result display matched group, cocaine group and FK866 processed group.Further analysis finds, wherein relevant to NAMPT metabolism metabolite shows significant difference (showing 3-1, Fig. 3-9) between matched group and VTA and the NAc brain district of FK866 processed group, comprises NMN and NAD.

According to two-parameter ownership method, in conjunction with the screening conditions of VIP > 1 and P < 0.05, finally determine that cocaine group gives the change of metabolite relevant to NAMPT metabolism after FK866 with normal saline group and abdominal cavity.Found that: cocaine group is compared with normal saline group, NMN and NAD level increases; But after abdominal cavity gives FK866, NMN and NAD level reduces (table 3-1).

Table 3-1 lumbar injection FK866 cocaine CPP model metabolite change sketch plan

Note: compared with control sample, ↑ represent that content increases relatively, ↓ represent that content declines relatively.

3.9.3 the change of key metabolites

NAMPT is the key enzyme of NAD route of synthesis, and NAD is the classical coenzyme in cell Redox reaction.Nicotiamide metabolism is the further metabolism of NMN, NMN by NAMPT is NAD.Cocaine can induce the up-regulated of NAMPT, and FK866 specificity suppresses the activity of NAMPT, therefore affects the synthesis of NAD.We analyze normal saline Normal group, difference metabolite between cocaine processed group and FK866 administration group, have chosen wherein relevant and representative to NAMPT metabolism metabolite, box traction substation is utilized to carry out showing (Figure 18), box traction substation can show scope, median, quartile etc. that metabolite changes, significantly can determine the variation tendency of metabolite.

The change of NAD in 3.10 detection cocaine CPP

In order to determine the change of NAD level further, utilizing NAD/MADH test kit to detect the fresh sample of mice VTA, determining that cocaine administration group and saline control group are in each condition on the impact of NAD.Result display (Figure 19), in CPP model, cocaine can induce NAD level to increase; After giving FK866, NAD reduces; Supplement NMN, NAD after giving FK866 again to recover; Process LAN NAMPT, NAD level increases (P<0.05).

4 conclusions

Metabolism group based on nuclear magnetic resonance, NMR (NMR) detects, and finds that cocaine addiction mice VTA brain district NMN, NAD level increases, but give with FK866 after NMN, NAD level reduce.Detection by quantitative also finds that caine addiction mice VTA brain district NAD level increases; Brain district locating injection FK866 then can reduce NAD level; But NMN, the NAD level of supplementing is restored; After VTA brain district process LAN NAMPT, NAD level also increases.

Set up mice cocaine Conditioned place preference (CPP) model, detect cocaine to the rewarding effect of mice by behavioristics. we send out existing, cocaine can the up-regulated of obviously inducing mouse NAc and VTA brain district NAMPT.By HuoVTANao district, abdominal cavity locating injection after FK866, obviously can suppress mice cocaine CPP effect.Supplement injection NMN in location, VTA brain district, the inhibitory action of FK866 to cocaine CPP can be reversed.At VTA locating injection NAMPT process LAN viral vector, cocaine CPP rewarding effect strengthens.Experimental result illustrates, NAMPT inhibitor FK866 can suppress cocaine CPP rewarding effect.

To sum up, NAMPT inhibitor effectively can weaken cocaine award behavior, and treatment cocaine addiction, potential applicability in clinical practice is good.

Claims (7)

1. The purposes of 1.NAMPT inhibitor in the medicine of preparation treatment cocaine addiction.
2. 2. purposes according to claim 1, is characterized in that: the medicine of the reduction NAMPT enzyme work of described treatment cocaine addiction.
3. 3. purposes according to claim 1, is characterized in that: the medicine that described reduction NAMPT enzyme is lived is FK866, and its structural formula is as follows:
4. 4. treat a medicine for cocaine addiction, it is characterized in that: it be with NAMPT inhibitor for active component, add the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.
5. 5. medicine according to claim 4, is characterized in that: described NAMPT inhibitor is reduce NAMPT enzyme medicine alive.
6. 6. purposes according to claim 1, is characterized in that: the medicine that described reduction NAMPT enzyme is lived is FK866, and its structural formula is as follows:
7. 7. medicine according to claim 4, is characterized in that: described preparation is ejection preparation.