CN105251006A - Application of TLR3 inhibitor in preparation of drug used for treating cocaine dependence - Google Patents

Abstract

The invention discloses application of a TLR3 inhibitor in preparation of a drug used for treating cocaine dependence. The invention also discloses the drug used for treating cocaine dependence. The drug is a preparation prepared from the TLR3 inhibitor which is used as an active component and pharmaceutically acceptable adjuvant materials or adjuvant components. The TLR3 inhibitor provided by the invention can effectively reduce dependence on and craving for cocaine, abates behavioral sensitization induced by cocaine, treats cocaine dependence and has good clinical application prospects.

Description

The purposes of TLR3 inhibitor in the medicine of preparation treatment cocaine addiction

Technical field

The present invention relates to the purposes of TLR3 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.

TLR3:Toll sample receptor 3.

TLR3 inhibitor: suppress the activity of Toll-like receptor 3 or the material of expression.

The present invention provide firstly the purposes of TLR3 inhibitor in the medicine of preparation treatment cocaine addiction.

Preferably, the medicine of described treatment cocaine addiction is the medicine reducing pIKB α and NF-kB expression.

Preferably, described TLR3 inhibitor is Compound I, and its structural formula is as follows:

name is called (R)-2-(3-Chloro-6-fluorobenzo [b] thiophene-2-carboxamido)-3-phenylpropanoicacid.

Present invention also offers a kind of medicine for the treatment of cocaine addiction, it be with TLR3 inhibitor for active component, add the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.

Preferably, the medicine of described treatment cocaine addiction is the medicine reducing pIKB α and NF-kB expression.

Preferably, described TLR3 inhibitor is Compound I, and its structural formula is as follows:

Preferably, described preparation is ejection preparation.

TLR3 inhibitor effectively can weaken the dependence of cocaine and crave for, and weaken the locomotor sensitivity effect of cocaine induction, can treat 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 pReceiver-Lv201 carrier figure

Fig. 2 mice Conditioned place preference case

Fig. 3 self administration system.

Fig. 4 spontaneous activity device.

Fig. 5 nucleus accumbens septi anatomical atlas

The Conditioned place halo effect of Fig. 6 various dose cocaine.* p<0.05, compares with matched group and has significant difference.20mg/kg: cocaine 20mg group; 10mg/kg: cocaine 10mg group; 3mg/kg: cocaine 3mg/kg group; Sa: saline control group

The cocaine CPP behavioristics effect of Fig. 7 TLR3 knock out mice, MyD88 knock out mice.Cocaine (20mg/kg) give genotype mice CPP effect figure.* p<0.05, compares with matched group and has significant difference.WT: wild-type mice group; TLR3KO:TLR3 knock out mice group; MyD88KO:MyD88KO knock out mice group.

The cocaine CPP behavioristics effect of Fig. 8 TLR3 knock out mice, MyD88 knock out mice.Cocaine (10mg/kg) gives genotype mice CPP effect figure figure.* p<0.05, compares with matched group and has significant difference.WT: wild-type mice group; TLR3KO:TLR3 knock out mice group; MyD88KO:MyD88 knock out mice group.

The cocaine CPP behavioristics effect of Fig. 9 TLR3 knock out mice, MyD88 knock out mice.Cocaine (3mg/kg) give genotype mice CPP effect figure.* p<0.05, compares with matched group and has significant difference.WT: wild-type mice group; TLR3KO:TLR3 knock out mice group; MyD88KO:MyD88 knock out mice group.

Figure 10 TLR3 knock out mice and wild-type mice spontaneous activity.Cocaine (20mg/kg) gives wild type (WT) group and TLR3KO group respectively, 9 time point locomotor activations and its baseline ratio after administration.* p<0.05, compares with matched group and has significant difference.TLR3KO:TLR3 knock out mice group; WT: wild-type mice group.

Figure 11 TLR3 knock out mice and wild-type mice spontaneous activity.Cocaine (10mg/kg) gives wild type (WT) group and TLR3KO group respectively, 9 time point locomotor activations and its baseline ratio after administration.* p<0.05, compares with matched group and has significant difference.TLR3KO:TLR3 knock out mice group; WT: wild-type mice group.

Figure 12 TLR3 knock out mice and wild-type mice spontaneous activity.Cocaine (3mg/kg) gives wild type (WT) group and TLR3KO group respectively, 9 time point locomotor activations and its baseline ratio after administration.* p<0.05, compares with matched group and has significant difference.TLR3KO:TLR3 knock out mice group; WT: wild-type mice group.

Figure 13 TLR3 knock out mice and wild-type mice self administration cocaine nose touch number of times comparison diagram.* p<0.05, compares with matched group and has significant difference.The effective nose of TLR3KO-COActive:TLR3 knock out mice cocaine touches number of times; The invalid nose of TLR3KO-COinactive:TLR3 knock out mice cocaine touches number of times; WT-COActive: the effective nose of wild-type mice cocaine touches number of times; WT-COinactive: the invalid nose of wild-type mice cocaine touches number of times.

Figure 14 TLR3 knock out mice and wild-type mice self administration normal saline nose touch number of times.* p<0.05, compares with matched group and has significant difference.The effective nose of TLR3KO-SalineActive:TLR3 knock out mice normal saline touches number of times; The invalid nose of TLR3KO-SalineInactive:TLR3 knock out mice normal saline touches number of times; WT-SalineActive: the effective nose of wild-type mice normal saline touches number of times; WT-SalineInactive: the invalid nose of wild-type mice normal saline touches number of times.

Figure 15 TLR3 knock out mice and wild-type mice self administration cocaine intake comparison diagram.* p<0.05, compares with matched group and has significant difference.TLR3KO:TLR3 knock out mice group; WT: wild-type mice group.

Figure 16 nucleus accumbens septi locating injection slow virus carrier figure

Figure 17 TLR3 knock out mice intracerebral injection TLR3 expresses slow virus carrier and recovers cocaine Conditioned place halo effect.* p<0.05, compares with matched group and has significant difference.LV-TLR3+CO:TLR3 slow virus carrier cocaine group; LV-Mock+CO:TLR3 slow virus contrast cocaine group; LV-TLR3+Sa:TLR3 slow virus carrier normal saline group; LV-Mock+Sa:TLR3 slow virus carrier cocaine group.

Figure 18 TLR3 knock out mice intracerebral injection TLR3 expresses slow virus carrier cocaine spontaneous activity (operating range).* p<0.05, compares with matched group and has significant difference.LV-TLR3+CO:TLR3 slow virus carrier cocaine group; LV-Mock+CO:TLR3 slow virus contrast cocaine group; LV-TLR3+SA:TLR3 slow virus carrier normal saline group; LV-Mock+SA:TLR3 slow virus carrier cocaine group.

Figure 19 TLR3 knock out mice intracerebral injection TLR3 expresses slow virus carrier and strengthens cocaine locomotor sensitivity effect (with baseline ratio).* p<0.05, compares with matched group and has significant difference.LV-TLR3+CO:TLR3 slow virus carrier cocaine group; LV-Mock+CO:TLR3 slow virus contrast cocaine group; LV-TLR3+SA:TLR3 slow virus carrier normal saline group; LV-Mock+SA:TLR3 slow virus carrier cocaine group.

Figure 20 C57 mice nucleus accumbens septi locating injection TLR3 inhibitor suppresses cocaine Conditioned place halo effect.P<0.05, compares with matched group and has significant difference.DMSO+CO: solvent control cocaine group; Inhibitor+CO:TLR3 inhibitor cocaine group; DMSO+CO: solvent control normal saline group; Inhibitor+CO:TLR3 inhibitor normal saline group.

Figure 21 C57 mice nucleus accumbens septi locating injection TLR3 inhibitor weakens cocaine spontaneous activity (displacement).* p<0.05, compares with matched group and has significant difference.(DMSO+CO: solvent control cocaine group; Inhibitor+CO: inhibitor cocaine group; DMSO+CO: solvent control normal saline group; Inhibitor+CO: inhibitor normal saline group)

Figure 22 C57 mice nucleus accumbens septi locating injection TLR3 inhibitor weakens cocaine locomotor sensitivity effect (with baseline ratio).* p<0.05, compares with matched group and has significant difference.DMSO+CO: solvent control cocaine group; Inhibitor+CO: inhibitor cocaine group; DMSO+CO: solvent control normal saline group; Inhibitor+CO: inhibitor normal saline group.

IKK β up-regulated in nucleus accumbens septi after Figure 23 C57 mice cocaine multiple dosing.A:Westernblot result; B:IOD analytic statistics figure.* p<0.05, compares with matched group and has significant difference.Cocaine: cocaine group; Saline: normal saline group.

After Figure 24 C57 mice cocaine multiple dosing, in nucleus accumbens septi, pIkB alpha expression raises.A:Westernblot result; B:IOD analytic statistics figure.* p<0.05, compares with matched group and has significant difference.Cocaine: cocaine group; Saline: normal saline group.

After Figure 25 C57 mice cocaine multiple dosing, nucleus accumbens septi endochylema NF-κ B expresses.A:Westernblot result; B:IOD analytic statistics figure.* p<0.05, compares with matched group and has significant difference.Cocaine: cocaine group; Saline: normal saline group.

After Figure 26 C57 mice cocaine multiple dosing, in nucleus accumbens septi, NF-κ B expresses.A:Westernblot result; B:IOD analytic statistics figure.* p<0.05, compares with matched group and has significant difference.Cocaine: cocaine group; Saline: normal saline group.

Figure 27 C57 mouse brain locating injection TLR3 inhibitor is on the impact of cocaine mice nucleus accumbens septi pIKB alpha expression.* p<0.05, compares with matched group and has significant difference.Inhibitor+Cocaine: inhibitor brain locating injection cocaine group; Inhibitor+Saline: inhibitor brain locating injection normal saline group; DMSO+Cocaine: solvent control brain locating injection cocaine group; DMSO+Saline: solvent control brain locating injection normal saline group.

The impact that Figure 28 C57 mouse brain locating injection TLR3 inhibitor is expressed NF-κ B in cocaine mice nucleus accumbens septi core.* p<0.05, compares with matched group and has significant difference.Inhibitor+Cocaine: inhibitor brain locating injection cocaine group; Inhibitor+Saline: inhibitor brain locating injection normal saline group; DMSO+Cocaine: solvent control brain locating injection cocaine group; DMSO+Saline: solvent control brain locating injection normal saline group.

Figure 29 TLR3 mouse brain locating injection TLR3 expresses slow virus carrier to the impact of pIKB alpha expression in cocaine mice nucleus accumbens septi core.* p<0.05, compares with matched group and has significant difference.LV-TLR3+Cocaine:TLR3 expresses slow virus brain locating injection cocaine group; LV-TLR3+Saline:TLR3 expresses slow virus brain locating injection normal saline group; LV-Mock+Cocaine: slow virus contrast brain locating injection cocaine group; LV-Mock+Saline: slow virus contrast brain locating injection normal saline group.

Figure 30 TLR3 mouse brain locating injection TLR3 expresses the impact that slow virus carrier is expressed NF-κ B in cocaine mice nucleus accumbens septi core.* p<0.05, compares with matched group and has significant difference.LV-TLR3+Cocaine:TLR3 expresses slow virus brain locating injection cocaine group; LV-TLR3+Saline:TLR3 expresses slow virus brain locating injection normal saline group; LV-Mock+Cocaine: slow virus contrast brain locating injection cocaine group; LV-Mock+Saline: slow virus contrast brain locating injection normal saline group.

Detailed description of the invention

Experimental example 1TLR3 inhibitor for treating cocaine addiction

1 foreword

In this research, we are from hereditism and pharmacology's angularly, and utilize gene Knockout, slow virus carrier to build and micromolecular inhibitor etc. carries out multi-angle confirmation, and Late Cambrian TLR3 participates in the process regulating mice cocaine addiction.In experimentation, associating CPP model, self administration model and spontaneous activity model, prove indirectly from another angle clear TLR3 inhibitor more and can treat cocaine addiction.

2 experiment materials and method

2.1 medicine

Cocaine hydrochloride: purchased from Chinese pharmaceutical biological product qualification institute, white powder, purity is greater than 99%, product batch number: 171210-200803.

Normal saline: with 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.

The TLR3 inhibitor that this experiment adopts is compound (R)-2-(3-Chloro-6-fluorobenzo [b] thiophene-2-carboxamido)-3-phenylpropanoicacid, its structural formula:

purchased from Merck Millipore Corp. (614310|TLR3/dsRNAComplexInhibitor – Calbiochem, catalogue numbering 614310-10MGCN).

2.2 laboratory animal

Male and healthy sexual maturity (10 ~ 12 weeks) C57BL/6J mice, body weight 20 ~ 22g, there is provided (production licence number: SCXK (Shanghai) 2007-0005) purchased from Shanghai City Si Laike laboratory animal Co., Ltd, body weight 20 ~ 22g.

TLR3 knock out mice product are B6N.129S1-Tlr3tm1Flv/J, numbering: 009675; MyD88 knock out mice product are B6.129P2 (SJL)-Myd88tm1Defr/J, numbering: 008888.Above two kinds of Gene Knock-Out Animal Model purchased from American Jackson laboratorys, are presented by Biotherapeutics National Key Laboratory of Sichuan University.Two kinds of animals all have fertility.

All animal feeding conditions: new drug safety evaluatio center, national Chengdu SPF level Animal House, temperature 20 ~ 25 DEG C, relative humidity 55 ~ 65%, feeding environment meets GB GB14925-2001, experimental animal licence: SCXK (river) 2003-01.

This problem all zoopery operation all meet International Laboratory Animal feeding and management assessment and accreditation association (AAALAC) requirement.In whole experimentation, animal freely ingests and drinks water, and the certain hour that normally breeds experimental animals before experiment is to be familiar with and to conform.

2.3 key instrument

Electronic analytical balance (Shanghai balance equipment factory)

Clean bench (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.)

DK-8D type electric heating constant temperature tank (Shanghai gloomy reliable test Instrument Ltd.)

High speed low temperature centrifugal machine (German eppendorf company MR1812)

Electric heating constant temperature tank (Shanghai gloomy reliable test Instrument Ltd.)

Mice rustless steel metabolic cage tool (Shanghai is with educating medical anatomy model manufacturing company)

Large mice Place Preference case (Huaibei Zhenghua Biological Instrument Co., Ltd.)

Self administration system (An Lai Instrument Ltd.)

Electrophoresis tank (Bio-Rad company of the U.S.)

Liquid-transfering gun (German eppendorf company)

Liquid nitrogen container (Chengdu liquid nitrogen container factory)

2.4 experiment reagent

100% ethanol (Solution on Chemical Reagents in Shanghai company limited)

75% ethanol (with the preparation of the water of DEPC process)

0.01MEDTA (Huamei Bio-Engrg Co.)

Formaldehyde (Solution on Chemical Reagents in Shanghai company limited)

Formaldehyde (Ambion)

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

Anti-beta-actin antibody (CST company of the U.S.)

Anti-NF-kappa B antibody (CST company of the U.S.)

Anti-pIKB Alpha antibodies (CST company of the U.S.)

Anti-IKK β antibody (CST company of the U.S.)

All the other reagent are domestic analytical pure.

2.5TLR3 expresses the structure of slow virus carrier

2.5.1 implementation sequence

1) carrier information:

Carrier is pReceiver-Lv201, Fig. 1 is carrier figure.Genes of interest fragment TLR3 primer, upstream: 5 '-atgaacggtgttcctcttatctaatgtactcctttgc-3 '; Downstream: 5 '-ggggacttgtccctatggattcttctggtgtctctag-3 '.

2) competent cell and conversion is prepared

CaCl ₂it is as follows that legal system carries out transformation experiment step for competent cell: (1) is respectively got often kind of competent cell suspension 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 place 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 until liquid is absorbed.Then be inverted plate, be placed in 37 DEG C of incubators and cultivate 16h.(4) clone carries out follow-up PCR qualification.

2.5.2 construction of recombinant plasmid

PCR primer connects into linearisation expression vector reaction system in table 1, reaction condition: 25 DEG C of 30min; 42 DEG C of 15min.

Table 1PCR reaction system

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

2.5.3 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.

2.5.4 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, coke booster diethyl carbonate (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.

2.5.5 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.

2.6 nucleus accumbens septi brain district pipe layings

The direct approach of pipe laying Shi Nao district, location, brain district site-specific delivery of drugs and common technology.10% chloral hydrate (0.4g/kg body weight) anesthetized mice is used in this research, head shaves hair, postanesthetic mice is fixed on brain solid positioner, regulate head to level after fixing, medical alcohol sterilization skin, surgical exposure skull, removes the meninges of skull surface with cotton balls and eye scissors, find bregma position, marking pen labelling; Regulate stereotaxic instrument, settle injection catheter (Rui Wode bio tech ltd, Shenzhen, #62003, OD0.48mmXID0.34mm), return to zero in bregma; With bregma position for initial point, reference location coordinate (nucleus accumbens septi: AP+1.6; ML+1.5) entry needle is moved to directly over injection site, entry needle is just contacted with skull, zeroing Y-coordinate, downward slow mobile entry needle is dark to 4.4mm, unclamp coordinate arm and remove, dentistry powder and diluent are adjusted to starchiness fixed sleeving bottom, being connected with skull reaches the object of A/C.Insert conduit cap (Rui Wode bio tech ltd, Shenzhen, #62102, OD0.30mm), prevent catheter blockage, sewing-end top wound.After operation terminates, by mice insulation to reviving, then put into clean rearging cage, single only single cage is raised.From Post operation second day, leg intramuscular injection penicillin sodium solution (160000U/ml, 0.1ml/ mice) after every day, and loosen conduit cap every other day, guarantee clearness of catheter, the convalescence after Miles operation of mice is 7 days.Animal through this operation injects TLR3 inhibitor for studying nucleus accumbens septi.

2.7 nucleus accumbens septi locating injection slow viruss

10% chloral hydrate (0.4g/kg body weight) anesthetized mice, head shaves hair, postanesthetic mice is fixed on brain solid positioner, regulate head to level, medical alcohol sterilization skin, surgical exposure skull, removes the meninges of skull surface with cotton balls and eye scissors, find bregma position, marking pen labelling; Regulate stereotaxic instrument, according to the elements of a fix (AP+1.6; ML ± 1.5; DV-4.4), utilize 33 type entry needles, to inject 0.5 μ l slow virus in 0.1 μ l/min speed bilateral nucleus accumbens septi, after injection, slowly shift out entry needle, sewing-end top skin.After operation terminates, insulation mice, to reviving, then puts into clean rearging cage, and single only single cage is raised.From Post operation second day, leg intramuscular injection penicillin sodium solution (160000U/ml, 0.1ml/ mice) after every day, continuously injection 3 days, the convalescence after Miles operation of mice is 7 days.PRMT1 expression slow virus regulation and control cocaine Conditioned place preference behavioristics's detection and spontaneous activity behavioristics is suppressed to be detected through this animal of performing the operation for studying nucleus accumbens septi injection.

2.8 mouse carotid venous cannulations are detained art

It is the basis that jugular vein self administration model is set up that the outer venous cannulation of cervical region is detained art, is also the committed step determining that whether this model is successfully established.Operation consent, laboratory animal adapts to 3-5 days at ambient conditions, and every day contacts with experimenter, produces stress to avoid laboratory animal in operation and experimentation.Jugular vein intubate adopts the silica gel hose (length is about 3mm) of import silica gel hard tube (length is about 5mm for external diameter 0.48mm, internal diameter 0.40mm) and coupling to be prepared, and flexible pipe is used for hard tube front end, is beneficial to intubate during operation.

During operation, according to Mouse Weight lumbar injection chloral hydrate (10%, 10ml/kg) anesthetized animal, reject shoulder blade portion, back and cervical region left collarbone position Mus hair, mouse head is towards experimenter and carry out supination type and fix, and opens a longitudinal direction about 1cm otch at left clavicle upper limb position, blunt separation subcutaneous tissue, the outer vein of free left neck, whole process can drip a small amount of normal saline, prevents drying.Surgical thread ligation vein distal end, eye scissors cuts diagonal cut joint on vein, with tweezers, the cannula hose end made before is inserted vein, for the fixing intubate of surgical thread knotting.Make a call to a hypodermic tunnel to cervical incision from back channel cervical region is subcutaneous, make the intubate other end pass from back and fix, to be connected with doser line.A small amount of heparin sodium aqua (30U/ml) and antibiotic (160000U/ml, 0.1ml/ mice) be injected in vivo through back of the body cervical region intubate, and prevent from remaining with normal saline flushing, after guaranteeing clearness of catheter, be enclosed within cannula distal end by duct cover, sew up back and cervical region wound.After operation terminates, by mice insulation to reviving, then mice is put into clean cage, single only single cage is raised.From Post operation second day, every day injects a small amount of heparin sodium and antibiotic through intubate, and ensure that intubate is unobstructed, prevent wound infection, the convalescence after Miles operation of mice is 7 days.Cocaine self administration addiction model is used for through this postoperative animal.

2.9 models are set up

2.9.1 the foundation of cocaine condition position preference model

In zoopery, all operations all meets AAALAC requirement, mice Place Preference case as shown in Figure 2:

1) start to test front 3-5 days, every day is stroked mice by same operator, allows animal have certain adaptation to operator.

2) mice is put into CPP casing, acclimatization training 3 days, the data measured for the 3rd day are as the data of pretest.Adaptive training and testing time are 15 minutes, when the time that animal stops in side more than 600 seconds time, knock-out experiment group.

3) using the side longer the animal time of staying as natural preference case, then carry out intraperitoneal injection, dosage following (table 2).

4) administration number of times is three times, alternating delivery.Administration group is given and is put into nature preference case during normal saline; When giving with cocaine hydrochloride, put into non-natural preference case, each training 15 minutes.Matched group is given with normal saline at every turn and is put into corresponding casing.

Table 2 cocaine administration approach, dosage, administration volume summary sheet

5) administration terminates to carry out CPP test in latter second day, and mice is put into intermediate transition case successively, allows it freely shuttle back and forth 15 minutes in Conditioned place preference case, and records the time of each casing stop.

6) points for attention: animal training should action light and slow; Should clean casing after terminating training, eliminate the impact of animal scent.

7) the time statistical result of non-preference case is deducted by the time of preference case, adopt SPSS statistical software analysis design mothod result, difference represents with mean ± standard deviation, compare the difference of cocaine administration group and saline control group by t check analysis method, p<0.05 is for there being significant difference.

2.9.2 the foundation of cocaine self administration model

Select FRI program in this research to train, the Experiment Training time, (SessionTime) was 120 minutes, and in training, maximum injection number of times is 50 times, and per injection cocaine 0.75mg/kg, self administration system as shown in Figure 3.Being set to of experiment: laboratory animal is touched invalid nose and touched not response, touches effective nose and touches and obtain medicine, carry out drug injection, and nose touches lamp and to light and room lamp is closed simultaneously, and the fan in self administration case is opened all the time in experimentation.Modeling is divided into 3 stages, i.e. warming up period, surgery recovery phase, cocaine self administration phase.

1) warming up period

Before starting experiment, natural feeding laboratory animal 3-5 days, experimenter strokes animal every day, reduces because of stress the interference that experimental result causes.

2) operation and convalescent period

Animal accepts jugular vein and puts pipe Post operation, and every day carries out heparin sodium washing pipe, ensures that administration pipeline is unobstructed.Recover after 5-7 days, choose the good animal of recovery and carry out testing into group.

3) the cocaine self administration phase

Laboratory animal is divided into two groups at random, cocaine group (n=16) and normal saline group (n=8).

Whether the medicine before experiment in inspection nose tentaculum, transfusion system and syringe is enough.During experiment, animal is put into self administration case, cervical region conduit is connected with transfusion system, close the door of casing, after allowing animal adapt to 15-20 minute in case, then start training experiment.Laboratory animal Behavior Monitor System automatically records and preserves experimental result, records that effective nose touches number of times, invalid nose touches number of times, cocaine injection number of times.

The standard that addiction model is successfully established:

1) operant conditioned reflex is formed;

2) cocaine injection frequency is stablized;

3) same treated animal continuous three days laxative number of times, within 10% scope of its meansigma methods.

2.9.3 the foundation of cocaine spontaneous activity model

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

1) start to test front 3-5 days, experimenter strokes mice, allows animal adapt to operator.

2) mice is put into spontaneous activity case, acclimatization training 3 days, adaptive training and testing time are 30 minutes.

3) after acclimatization training terminates, animal is divided into cocaine group and matched group, administration before detecting every day, administration number of times is 7 times.

4) points for attention: animal training should action light and slow; Should clean casing after terminating training, eliminate the impact of animal scent.

5) result adopts SPSS statistical software analysis design mothod result, and difference represents with mean ± standard deviation, and p<0.05 is for there being significant difference.

2.10 nucleus accumbens septis are drawn materials and are prepared

Behavior test terminates quick sacrificed by decapitation C57 mice in latter 30 minutes, is then separated brain rapidly, after 4 DEG C of normal saline flushings 3 times, is separated takes out nucleus accumbens septi (Fig. 5 according to brain anatomical atlas.And put into liquid nitrogen flash freezer, after having sampled by sample storage at-80 DEG C of environment.

2.11 micro-injection process

Mice carries out Naoliqing capsule, and head picks hair and sterilizes, and cuts off skin of head with shears, coordinate setting injection site, and syringe needle punctures skull, and entry needle is thrust mouse brain according to coordinate, then inhibitor or viral vector is expelled to nucleus accumbens septi.The injection of slow virus, with the speed injection of 0.1 μ l/ minute, 10 minutes, is total to l μ l.After injection, micro-injection pin is placed in original position 3-5 minute, so that injected material fully spreads at the tip of entry needle.

2.12 prepared by frozen section

Animal exposes heart rapidly with after 10% chloral hydrate intraperitoneal anesthesia, and by left ventricle quick needle insertion, right auricle and right atrium portion cut off breach, successively uses normal saline and pours into rapidly through body circulation first containing the PBS of 4% paraformaldehyde.Take out mouse brain, with containing after fixing (4 DEG C, 2-4h) after the PBS of 4% paraformaldehyde, be successively soaked in the sucrose solution of 20% (4 DEG C, 20-24h) and 30% (4 DEG C, 24-48h) and dewater, until Mus brain sinks to the bottom.Brain after dehydration, with coronalplane, in forebrain plane, cuts 10 μm of thick brain sections with freezing microtome, and-20 DEG C are kept at (PBS containing 30% sucrose and 30% ethylene glycol) in protection liquid.

2.13WesternBlotting

2.13.1 nucleoprotein and plasmosin extract with quantitative

Extract nucleus accumbens septi nucleoprotein according to Nuclear extract and suppressor proteins extraction agent box (green skies bio tech ltd, P0027) description and starch albumen.Key step comprises: dissect the extraction agent A liquid of first few minutes preparation containing 1mMPMSF protease inhibitor of drawing materials, fresh nucleus accumbens septi tissue is put into PBS (1mMPMSF) solution of 500ml, repeatedly tissue is blown and beaten with 200 μ l rifle heads, disrupting tissue is to fine particle, then centrifugal, collection organization is precipitated, then mixed liquor (the 20:1 ratio mixing of Protein Extraction Reagent A and B is added, 1mMPMSF) 100 μ l, the most violent vortex 5 seconds, cell precipitation is exposed the wealth completely and scatter, ice bath 15min, the most violent vortex 5 seconds, ice bath 1min, the most violent vortex 5 seconds, 4 DEG C 13, the centrifugal 5min of 000g, then draw immediately in the plastic tube of supernatant to pre-cooling, be the suppressor proteins that extracting obtains, frozen in-80 DEG C of (attentions: when shifting supernatant, precipitation must not be touched, the supernatant of minimum volume can be retained above precipitation, one side touches precipitation).

For precipitation, exhaust remaining supernatant (if not exhausting the pollution that supernatant can bring suppressor proteins) completely, add the Nuclear extract extraction agent that 30 μ l with the addition of PMSF.The most violent vortex 30 seconds, suspends cell precipitation completely and scatter, then putting back in ice bath, and each 2min violent vortex 30 seconds at a high speed again, is total to 30min; 4 DEG C of 13,000g centrifugal 10min, draws immediately in the plastic tube of supernatant to pre-cooling, is the Nuclear extract that extracting obtains, frozen in-80 DEG C.

2.13.1 total protein extraction is with quantitative

(1) tissue frozen after getting dissection, adds RAPA lysate, is placed in cracking on ice 5 minutes.If tissue extraction albumen, according to organizing size to add RIPA, the RIPA amount that nucleus accumbens septi tissue adds is about 100 μ about l.

(2) ultrasonic 10 times, 2 seconds/time (putting with on ice), object is cell breakage.

(3) 13,000g, 15 minutes, 4 DEG C centrifugal after, take out supernatant, put on ice.

(4) protein quantification

A) BSA is diluted to 1,0.5,0.4,0.3,0.2,0.1,0.05,0mg/ml Concentraton gradient (table 3).

Protein liquid dilution 10-20 times of B) will extract.BSA and protein sample are inhaled 10 μ l respectively and are added other hole.

C) Coomassie brilliant blue G250 of 200 μ l will be added in the hole adding BSA and protein sample.

D) 595nm measures absorbance, and linear dependence is R2>0.99, calculates corresponding protein concentration.

E) be benchmark with Cmin, all the other samples added appropriate RIPA, dilutes for same concentration.

F) each sample adds 5 × loadingbuffer (containing beta-mercaptoethanol), measures 1/5 into albumen volume.

(5) boil sample 5 minutes, subpackage, it is 30 μ about g that volume and concentration calculate final quantity.

Points for attention: whole operating in is carried out on ice.Want accurately during protein quantification, after adding Coomassie brilliant blue G250, lucifuge as far as possible.

BSA dilution ratio table during table 3 protein quantification

2.13.2 preparative separation glue (table 2-2)

Table 4 separation gel preparation preparation table

Attention: finally add 10%APS and TEMED fast successively, fully mixes but does not produce bubble.Encapsulating immediately, every block glue needs about about 5ml; Then add deionized water along glass plate and completely cut off air.Setting time is 15-30 minute.

2.13.3 the concentrated glue (table 5) of preparation

Table 5 concentrates glue and prepares preparation table

Attention: finally add 10%APS and TEMED fast successively, fully mixes but does not produce bubble.Concentrated glue is added in glass plate, plugs comb, solidify 15-30 minute.

2.13.4 electrophoresis prepares

The glass plate solidifying glue is put into electrophoresis tank, adds 30% acrylamide/methene acrylamide.

30% acrylamide/methene acrylamide preparation: acrylamide: 29g; Methene acrylamide: 1g

First dissolve with 50ml distilled water, stir and know that solution is transparent, add distilled water to final volume 100ml, filtration sterilization, investigate this solution PH <7.0, brown bottle 4 DEG C preservation.

2.13.5 point sample

The protein content that each hole adds is about 30 μ g, and 0.75mm point sample is about 20 μ l, and 1mm point sample amount is about 30 μ l.

2.13.6 electrophoresis

Concentrated glue voltage 80V, albumen is to concentrated glue separation gel boundary place, and voltage adds as 120V, until albumen is run through (determining according to albumen size), the time is approximately 60 minutes.

2.13.7 transferring film

By in transferring film liquid everywhere iron pan, glue is put into transferring film liquid separation gel.Transferring film folder is put sponge up and down and respectively puts filter paper one, is put on filter paper by the glue cut after complete wetting.Another preparation culture dish adds methanol, is soaked into wherein, then covers on glue by the pvdf membrane of needs.Transferring film voltage sets is 100V, and the time determines according to albumen size.

2.13.8 close

With the skim milk of TBST preparation 5%, 25ml/ block glue.Put into plate, by pvdf membrane complete wetting in milk, be placed on shaking table and rock 1 hour.

2.13.9 incubate primary antibodie

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

2.13.10 incubate two to resist

With 1:5000 by antibody dilution in skim milk, 37 DEG C of shaking table 1-2 hour, wash film secondary with TBST, each 5-10 minute, then wash film once, 5-10 minute with TBS.

2.13.11 prepare luminescent solution and tabletting

Pvdf membrane is placed in luminescent solution reaction 1 minute in darkroom.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 determines band brightness), develop a film.

2.13.12 gray value collection

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

2.14 data analysis

All data all adopt standard error analysis, and ANOVA is used for the statistical significance of prediction data.All analyses all adopt SPSS11.5 software, and p<0.05 is that data are meaningful statistically.

3 experimental results

3.1 cocaine induction C57 mice Conditioned place preferences

C57 mice carries out the acclimatization training of 3 days and the Conditioned place preference training of 6 days, and 3 cocaine dose groups (n=12) (20mg/kg, 10mg/kg and 3mg/kg) and saline control group (n=12) condition position preference effect are as shown in Figure 6.After the Conditioned place preference training of cocaine, the time that mice stops at cocaine training casing significantly increases (P<0.05), this shows that animal produces drug dependence, define the trained reflex that can obtain cocaine award in a certain environment, wherein 20mg/kg cocaine dose group CPP effect is the strongest.

3.2TLR3 knock out mice cocaine Conditioned place preference weakens

TLR3 knock out mice and MyD88 knock out mice carry out cocaine CPP detection, and cocaine dose is respectively 20mg/kg, 10mg/kg and 3mg/kg, and matched group is wild-type mice (WT) (Fig. 7-Fig. 9).As seen from the figure, compared with matched group, TLR3 knock out mice and the CPP effect of MyD88 knock out mice in the modeling of various dose cocaine condition position preference all variant, wherein TLR3 knock out mice is shorter than wild-type mice in the time of cocaine preference induction case stop, TLR3 knock out mice condition position preference effect significantly reduces, and has statistical significance (p<0.05) compared with wild-type mice.Under 20mg/kg cocaine dose, TLR3 knock out mice condition position preference effect reduces more obvious.Above result shows, the addiction effect of TLR3 knock out mice to cocaine significantly reduces, and has pointed out TLR3 may play certain effect in the formation of cocaine addiction process.

In the experiment of 3 various dose, for MyD88 group, cocaine condition preferences location effect all decreases compared with wild type, but equal no difference of science of statistics between two groups in all experiments.Above experimental result shows, the condition position preference effect that MyD88 disappearance causes for cocaine does not make a significant impact, the drug reward effect that cocaine produces may associate not quite with TLRs/MyD88 Dependent, therefore, experiment backward we study for TLR3/MyD88 non-dependent approach.

The spontaneous activity of 3.3TLR3 knock out mice cocaine weakens

We continue to adopt the effect of spontaneous activity experimental study TLR3 gene in cocaine addiction behavior.TLR3 knock out mice acclimatization training 3 days, 30 minutes every days.Test the activity of front 3 ten minutes animals on the same day as baseline, 4th within ten minutes, start before mouse peritoneal injection give 3,10,20mg/kg cocaine, then after surveying injection continuously, 10min, 20min are until 9 time points such as 90min, each time point interval 10min.

Result of the test (Figure 10-Figure 12) shows, after cocaine injection, wild-type mice behavioral activity amount rises gradually, within 20-30 minute, peaks upon administration, reduces gradually afterwards.TLR3 knock out mice locomotor activation after giving cocaine also increases, but at part-time point locomotor activation lower than wild-type mice group, and have significant difference (p<0.05).Wherein, under 20mg/kg cocaine dose, TLR3 knock out mice locomotor activation reduces more obvious.Above result shows, TLR3 knock out mice reduces cocaine locomotor sensitivity effect, has pointed out TLR3 gene participating in the effect in cocaine addiction benefit.

3.4TLR3 the behavior of knock out mice cocaine self administration weakens

Experiment start first 3 days, animal carries out the adaptation of self administration system environments, every day 30min, to reduce the experimental error because environment causes.Test the 1st day, difference is not had between four indexs, show also not formed in cocaine effect initial stage addiction effect, the nose behavior of touching of animal is the exploratory behavior of itself, along with the propelling of subsequent experimental, find that animal cocaine effective nose touches number of times and constantly increases (Figure 13), and the effective nose of normal saline group touches number of times, in experimentation, significant change (Figure 14 does not occur.Train after 3-4 days, compared with TLR3 knock out mice, the effective nose of wild-type mice cocaine touches number of times significantly to be increased (p<0.05), and invalid nose touches number of times and initial level zero difference, illustrate that wild-type mice has possessed the ability distinguishing effective nose tentaculum and invalid nose tentaculum under the effect of cocaine, set up operant conditioned reflex, on the contrary, this exactly illustrates that TLR3 knock out mice is significantly reduced by the behavior of touching effective nose tentaculum and obtaining drug reward.In the experiment of normal saline treated animal, effective nose of TLR3 knock out mice and wild-type mice touches number of times and invalid nose and touches number of times do not have notable difference in whole experiment process, and both explanations all can not distinguish effective nose tentaculum and invalid nose tentaculum (Figure 15).In addition, from experiment the 5th day, the effective nose of wild-type mice cocaine touches number of times (drug injection number of times) and remains on a more stable level, illustrate that self administration training makes cocaine touch effective nose tentaculum to animal and strengthens to obtain this behavior of drug reward and consolidate, but normal saline does not then play above-mentioned effect.Drawn by Figure 15, TLR3 knock out mice and wild-type mice all present the trend increased gradually to the demand of cocaine, started to tend to be steady by the 5th day, there is a plateau (same treated animal continuous three days laxative number of times are within 10% scope of its meansigma methods), but, compared with wild-type mice, the intake of TLR3 knock out mice cocaine obviously reduces (p<0.05).Above experimental result illustrates that TLR3 gene knockout causes the demand of mice to cocaine to weaken.

3.5TLR3 knock out mice intracerebral injection TLR3 strengthens cocaine CPP effect after expressing slow virus

TLR3 knock out mice brain inner position injection TLR3 expresses slow virus carrier, and injection point is bilateral nucleus accumbens septi.After wounds in animals is recovered, heart perfusion also fixes brain, frozen section, fluorescence microscopy Microscopic observation virus injection position (Figure 16).Animal carries out acclimatization training in 3 days, every day 15-20min.Detected by the Conditioned place preference of cocaine, compared with the animal contrasted with intracerebral injection viral vector, the time that the mice of having injected TLR3 slow virus carrier stops at cocaine induction casing significantly increases (P<0.05), after this illustrates that intracerebral injection TLR3 expresses slow virus, the cocaine addiction effect of TLR3 knock-out mice obviously strengthens, this result (Figure 17) shows: after TLR3 knock-out mice nucleus accumbens septi TLR3 expresses again, can recover cocaine addiction behavior effect.

3.6TLR3 knock out mice intracerebral injection TLR3 expresses slow virus increases cocaine spontaneous activity

In TLR3 knock out mice brain, bilateral nucleus accumbens septi injection TLR3 expresses slow virus carrier, and after wounds in animals is recovered, random packet also carries out environmental adaptation training, each 30min, to reduce the impact of environment on experimental result.Formally start to test and survey spontaneous activity baseline in first 3 days, test the 4th day beginning abdominal cavity and give cocaine.As seen from Figure 18, the four treated animals activity of first 3 days is in same level, basis activity does not have notable difference, but from administration in the 4th day, the locomotor activation that brain locating injection TLR3 expresses slow virus carrier animal has obvious increase, and has significant statistical significance (P<0.05) compared with the animal of injecting lentivirus vehicle Control.For making experimental result more objective, eliminate the impact because each treated animal basis activity difference causes experimental result, own foundation activity before the locomotor activation of every day after every treated animal administration and administration is made ratio, as shown in figure 19, brain locating injection TLR3 expresses locomotor activation remarkable increase compared with the animal of injecting lentivirus vehicle Control of slow virus carrier animal, and has statistical significance (P<0.05) equally.Above experimental result shows: after in TLR3 Gene Knock-Out Animal Model brain, TLR3 is expressed again in nucleus accumbens septi region, and the spontaneous activity of animal increases, and the locomotor sensitivity that cocaine causes is more obvious.

3.7C57 mice TLR3 inhibitor intracerebral injection cocaine CPP effect weakens

C57 mice is carried out TLR3 inhibitor brain locating injection, after wounds in animals is recovered, random packet also carries out cocaine CPP training.For two treated animals of lumbar injection cocaine, brain locating injection TLR3 inhibitor mice condition position preference effect obviously weakens (P<0.05) (Figure 20); But, the unconditional position preference effect of two treated animals of intraperitoneal injection of saline.Above experimental result shows: after C57 mouse brain locating injection TLR3 inhibitor, and animal significantly reduced in the time of staying of cocaine induction case, and animal obviously weakens the demand of cocaine and dependence, and prompting TLR3 gene plays a role in cocaine addiction is formed.

The spontaneous activity of 3.8C57 mice TLR3 inhibitor intracerebral injection cocaine reduces

In C57 mouse brain, nucleus accumbens septi injection TLR3 inhibitor, carries out spontaneous activity detection.As seen from Figure 21, the 4 treated animals activity of first 3 days is in same level, and Activities quantitative baseline does not have notable difference.From the 4th day, the cocaine administration mice activity increase of nucleus accumbens septi locating injection TLR3 inhibitor was starkly lower than solvent control group, and two groups have significant difference (P<0.05).This result illustrates: after suppressing TLR3, can weaken cocaine locomotor sensitivity effect.For making experimental result more objective, eliminate the impact because each treated animal basis activity difference causes experimental result, own foundation activity before the locomotor activation of every day after every treated animal administration and administration is made ratio, as shown in figure 22, the spontaneous activity recruitment of brain locating injection TLR3 inhibitor animal is starkly lower than the animal of matched group, and has statistical significance (P<0.05) equally.

3.8 cocaine multiple dosings activate mice IKB α/NF-κ B signal path

C57 mice is after cocaine CPP trains, and disconnected neck is put to death, and takes out nucleus accumbens septi (NAc) brain district, extracts albumen, carries out Westernblot detection with anti-IKK β, pIKB α and NF-kappa B antibody.Found that, after cocaine CPP trains, in endochylema, (P<0.05) (Figure 23--Figure 25) is obviously raised in the expression of IKK β, pIKB α and NF-κ B.NF-κ B in nucleoprotein is detected, finds that NF-κ B expression raises (Figure 26) (P<0.05) equally in core, to illustrate in endochylema that NF-κ B is separated with phosphorylation I κ B and enters in core and start downstream signal transduction.Above result shows, cocaine CPP training makes the NF-κ B expression inside and outside IKK β in mouse brain, pIkB α and nucleus change, and cocaine CPP training can activate NF-IkB path.Above experimental result shows that TLR3 participates in regulating the process of cocaine addiction, and points out it to realize possibly via TLR3/NF-kB approach.

3.9 suppress or knock out TLR3 to weaken IKB α/NF-κ B signal transduction

For confirmation TLR3 participates in the adjustment of mice cocaine addiction process, cocaine CPP detection is carried out to C57 mice nucleus accumbens septi locating injection TLR3 inhibitor.Mice break neck put to death, take out nucleus accumbens septi, extract albumen, carry out Westernblot detection with anti-pIKB α and NF-kappa B antibody.Found that, after cocaine CPP trains, in endochylema, (Figure 27) (P<0.05) is obviously raised in the expression of pIKB α, and in core, NF-κ B expression raises and has statistical significance (Figure 28) (P<0.05) simultaneously.Above result shows to suppress TLR3 in nucleus accumbens septi can lower the expression of key protein in IKB α/NF-κ B path, namely suppresses TLR3 can weaken the signal transduction of IKB α/NF-κ B.

TLR3 knock out mice nucleus accumbens septi locating injection TLR3 expresses slow virus, and carries out cocaine CPP detection, takes out nucleus accumbens septi sample, carries out Westernblot detection with the antibody of anti-pIKB α and NF-κ B.Found that, after cocaine CPP trains, in the animal endochylema of brain locating injection TLR3 expression slow virus, (Figure 29) (P<0.05) is obviously raised in the expression of pIKB α, simultaneously, in core, NF-κ B expression raises equally and has statistical significance, (Figure 30) (P<0.05), after above result shows that in TLR3 knock out mice nucleus accumbens septi, TLR3 gene recovers expression, the expression of NF-κ B in pIKB α and core has been raised in cocaine CPP training, illustrate that TLR3 gene recovers to express rear cocaine CPP and have activated IKB α/NF-κ B path.

4 conclusions

This problem mainly adopts animal behavioral study and the means such as hereditism and pharmacological intervention to probe into the effect of TLR3 in mice cocaine addiction mechanism.

We find, TLR3 knock out mice cocaine CPP effect significantly reduces, and shows that it weakens the dependence of cocaine.Compared with wild-type mice, TLR3 knock out mice weakens the locomotor sensitivity effect that cocaine is induced.In self administration test, TLR3 knock out mice self administration number of times is obviously less than, and shows to crave for cocaine to weaken.

We construct TLR3 and express slow virus carrier, and express to recover TLR3 at this viral vector of TLR3 knock out mice nucleus accumbens septi locating injection, then carry out related neural Behavior Test.Result of the test shows: significantly raise cocaine addiction after TLR3 knock-out mice nucleus accumbens septi injection TLR3 slow virus carrier.

We continue to adopt TLR3 inhibitor locating injection in C57 mice nucleus accumbens septi, then carry out addictive behavior evaluation.Found that, TLR3 inhibitor can weaken additive to cocaine of C57 mice.

Finally, the key protein of addiction mice TLR3/IKK β/NF-κ B path is detected.Found that, cocaine CPP training makes the equal up-regulated expression of NF-κ B expression inside and outside IKK β in mouse brain, pIkB α and nucleus.But when after C57 mouse brain locating injection TLR3 inhibitor, in endochylema pIKB α and core, NF-kB all lowers expression, in addition, after TLR3 knock out mice recovers to express TLR3 by brain locating injection slow virus carrier, NF-κ B up-regulated expression in endochylema pIKB α and core.These results suggest that TLR3 participates in cocaine addiction process, and may be participate in reconciling by TLR3/IKB α/NF-κ B path.

To sum up, TLR3 inhibitor effectively can weaken the dependence of cocaine and crave for, and weakens the locomotor sensitivity effect of cocaine induction, can treat cocaine addiction.

Claims (7)

1. The purposes of 1.TLR3 inhibitor in the medicine of preparation treatment cocaine addiction.
2. 2. purposes according to claim 1, is characterized in that: the medicine of described treatment cocaine addiction is the medicine reducing pIKB α and NF-kB expression.
3. 3. purposes according to claim 1 and 2, is characterized in that: described TLR3 inhibitor is Compound I, and its structural formula is as follows:
4. 4. treat a medicine for cocaine addiction, it is characterized in that: it be with TLR3 inhibitor for active component, add the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.
5. 5. medicine according to claim 1 has, and it is characterized in that: the medicine of described treatment cocaine addiction is the medicine reducing pIKB α and NF-kB expression.
6. 6. the medicine according to claim 4 or 5, is characterized in that: described TLR3 inhibitor is Compound I, and its structural formula is as follows:
7. 7. medicine according to claim 4, is characterized in that: described preparation is ejection preparation.