| Objective: It has the same key characters that opioids are abused andultimately developed to addiction. At present, a large number of studies haveshown that repeated use of addictive drugs can cause the structure of brainneuron dendritic spines, which associated with motivation of emotion, rewardand study, changing continuously. It suggests that addictive drugs inducedsynaptic connections have plasticity changes In the process of dendritic spines’plastic changes and neurons’ morphological changes, skeleton actin plays animportant role. In the process of skeleton actin refactoring, it hsd not beenelucidated what the role morphine plays and what its mechanism is. Thisexperiment is beginning from researching the skeleton actin, combiningmethods such as ethology and molecular cell biology, to study the effect ofmorphine addiction and withdrawal to the rat skeleton actin remodeling. Thiswork deepens the knowledge of understanding the molecular mechanism ofopioid addition.Methods:1The establishment of rat models with morphine acute treatment: Rats wererandomly divided into3groups with3in each. Including control group, whichinjected physiological saline in intraperitoneal, morphine45minutes group,which injected morphine10mg.kg-1in intraperitoneal at45min, morphine2hour group, which injected morphine10mg.kg-1in intraperitoneal at2hour.Three groups were respectively decapitated at the time of45min and2h aftermorphine injected and separated related brain areas.2The establishment of rat models with y-27632injected in intraventricular:According the acute treatment results,which showed that intraperitonealinjection of morphine in45min changed significantly, blocking time was chosen for45min. After y-276320.5mg.kg-1was injected in intraventricular,injected morphine10mg.kg-1in intraperitoneal. Decapitated at the time of45min after morphine injected and separated related brain areas.3. The establishment of morphine dependence rat models: Rats were randomlydivided into4groups with6in each. Including: A: Physiological salinecontrol group, B: Morphine dependence group, C: Morphine withdrawalgroup, D: Morphine withdrawal control group. B and C groups wereestablished with morphine hydrochloride for7days. By the way of increasingdoses of morphine hydrochloride two times per day through intraperitonealinjection. The daily dose of morphine was increased as5,10,15,20,25,30,35mg.kg-1.C and D groups were injected with5mg/kg-1naloxone at d8tourged with drawal. Changes recorded within30minutes. All the rats weredecapitated and separated related brain areas. Intraventricular injectionblocking drugs rat models: Respectively injected MK-8010.5mg.kg-1, FK-5060.05mg.kg-1and MASPGVAVSDGVIKVFN0.5mg.kg-1in intraventricular ofrats. Injected morphine in Intraperitoneal and processing method as above.4. Determination of actin and actin binding protein levels in rat brain regionsby Western-blot: brain tissues were homogenized, then cofilin, P-cofilin,P-LIMK, LIMK, SSH, drebrinA/E, cortactin and β-actin were detected fromtotal protein. F-actin in cystoskeleton fraction was separated from G-actin inendochylema with different buffer, then drebrin, cortactin, F-actin and G-actinwere detected by western-blot.5. Immunohistochemistry: The brain tissues of morphine dependence rats werefixed and sliced. After that, incubated with rabbit mAb P-cofilin（1:100）,drebrinA/E（1:100） and cortactin （1:100） and observed under microscope afterstained.Results:1Acute treatment rat results1.1The ratio of F-actin and G-actin: Compared with the control group45mingroup was down-regulated by20%in Hip; there is no significant changes in2h group; y-27632group was up-regulated by15%in Pfc. 1.2The change of drebrin: In Pfc, y-27632group was up-regulated by25%intotal protein, the binding protein was up-regulated by30%, the freeendochylema protein was down-regulated by15%, the ratio was up-regulatedby15%. In Hip, y-27632group was up-regulated by20%in total protein, thefree endochylema protein was down-regulated by20%, the ratio wasup-regulated by35%. In Str,45min group was down-regulated20%in totalprotein, the free endochylema protein was down-regulated by15%, the ratiowas down-regulated by20%. Y-27632group was up-regulated by45%in totalprotein, the ratio was up-regulated by50%.In Amy,45min group was up-regulated by25%in total protein, the bindingprotein was up-regulated by15%, the ratio was up-regulated by30%. Y-27632group was up-regulated by35%in total protein, the binding protein wasup-regulated by25%, the ratio was up-regulated by15%.1.3The change of cortactin: In Pfc,45min group was down-regulated by20%in free endochylema protein. Y-27632group was down-regulated by40%intotal protein, the binding protein was down-regulated by25%, the ratio wasdown-regulated by50%. In Hip,45min group was up-regulated by20%intotal protein, the binding protein was up-regulated by25%, the freeendochylema protein was up-regulated by15%, the ratio was down-regulatedby20%.2h group was up-regulated by30%in total protein,the binding protein was up-regulated by45%. Y-27632group wasdown-regulated by30%in total protein, the binding protein wasdown-regulated by30%, the free endochylema protein was up-regulated by25%, the ratio was down-regulated by40%. In Str, there is no significantchanges. In Amy,45min group was up-regulated by25%in total protein, theratio was up-regulated by15%. Y-27632group was down-regulated by15%intotal protein, the free endochylema protein was down-regulated by20%, theratio was up-regulated by15%.1.4The change of cofilin:45min group was up-regulated by15%in Hip.2hgroup was down-regulated by15%in Str and down-regulated by25%in Amy.Y-27632group was down-regulated by30%in Hip and down-regulated by 40%in Hip.1.5The ratio of P-cofilin and cofilin:45min group was down-regulated by15%in Pfc and Str.2h group was up-regulated by25%in Pfc andup-regulated by15%in Str. Y-27632group was up-regulated by30%,25%,45%in Pfc, Str and Amy.1.6The change of SSH: Y-27632group was down-regulated by30%,25%and15%in Pfc, Str and Amy.1.7The change of LIMK:45min group was up-regulated by15%in Hip,down-regulated by20%in Str. Y-27632group was down-regulated by35%inPfc, up-regulated by50%in Hip.1.8The ratio of P-LIMK and LIMK: Y-27632group was up-regulated by20%in Pfc, down-regulated by15%in Amy.2morphine dependence rat results2.1Naloxone withdrawal behavior results: The grade of morphine dependencegroup withdrawal reaction was significantly higher than withdrawal contrastgroup.2.2The ratio of F-actin and G-actin: In Pfc, morphine dependence group,morphine withdrawal group and MK801group was respectively up-regulatedby25%,75%and20%. In Hip, morphine dependence group and Smallmolecule peptide group was respectively up-regulated by30%and20%. In Str,morphine dependence group, Morphine withdrawal group, MK801group,FK506group and Small molecule peptide group was respectively up-regulatedby170%,130%,90%,40%, and170%. In Amy, morphine dependence group,MK801group and Small molecule peptide group was respectivelyup-regulated by35%,20%and30%.2.3The change of drebrin: In Pfc, dependence group and withdrawal groupwas respectively down-regulated by30%and35%, MK801group, FK506group and Small molecule peptide group was respectively up-regulated by30%,45%and15%in total protein. Dependence group, MK801group, FK506group was respectively up-regulated by50%,30%,15%in binding protein.Dependence group, withdrawal group, withdrawal contrast group, MK801 group was respectively down-regulated by35%,25%,20%,40%, FK506group and Small molecule peptide was respectively up-regulated by130%and25%in free endochylema protein. Dependence group, withdrawal group,withdrawal contrast group, MK801group and FK506group was respectivelyup-regulated by140%,100%,130%,120%,45%in the ratio. In Hip,dependence group, withdrawal group was respectively up-regulated by40%,30%, MK801group, FK506group, Small molecule peptide was respectivelydown-regulated by20%,25%,25%in total protein. Dependence group,withdrawal group was respectively up-regulated by50%,40%, withdrawalcontrast, MK801group, FK506group was respectively down-regulated by40%,15%,40%in binding protein. MK801group, FK506group, smallmolecule peptide was respectively down-regulated by55%,65%,40%in freeendochylema protein. Dependence group, withdrawal group, MK801group,FK506group was respectively up-regulated by90%,65%,55%,25%,withdrawal contrast was down-regulated by20%in the ratio. In Str,dependence group, withdrawal group, MK801group was respectivelydown-regulated by50%,65%,15%, FK506group was up-regulated by80%in total protein. Dependence group, withdrawal group, MK801group, FK506group was respectively down-regulated by30%,35%,25%,35%in bindingprotein. FK506group was up-regulated by120%in free endochylema protein.Dependence group, withdrawal group, MK801group, FK506group wasrespectively down-regulated by50%,20%,20%,25%in the ratio. In Amy,withdrawal group, withdrawal contrast group, FK506group was respectivelydown-regulated by20%,30%,25%in total protein. Withdrawal group,withdrawal contrast group, FK506group was respectively down-regulated by50%,65%,40%in binding protein. Withdrawal group, withdrawal contrastgroup, FK506group was respectively down-regulated by50%,65%,55%inthe ratio.2.4The change of cortactin: In Pfc, dependence group, withdrawal group,FK506group was respectively up-regulated by25%,30%,20%, MK801group was down-regulated by30%in total protein. Dependence group, withdrawal group, FK506group was respectively up-regulated by60%,75%,55%, MK801group was down-regulated by25%in the binding protein.Dependence group was down-regulated by20%in free endochylema protein.Dependence group, withdrawal group, FK506group was respectivelyup-regulated by100%,130%,45%, MK801group was down-regulated by55%in the ratio. In Hip, dependence group, withdrawal group, withdrawalcontrast group, MK801group, Small molecule peptide was respectivelyup-regulated by30%,30%,20%,25%,15%, FK506group wasdown-regulated by30%in total protein. Dependence group, withdrawal group,withdrawal contrast group, MK801group, small molecule peptide wasrespectively up-regulated by80%,75%,30%,35%,30%in binding protein.Dependence group, withdrawal group, MK801group, FK506group wasrespectively down-regulated by35%,30%,30%,40%in free endochylemaprotein. Dependence group, withdrawal group, withdrawal contrast group,MK801group, FK506group, Small molecule peptide was respectivelyup-regulated by60%,120%,20%,70%,60%,80%in the ratio. In Str,Dependence group, withdrawal group, MK801group was respectivelydown-regulated by50%,40%,40%, FK506group was up-regulated by30%in total protein. Withdrawal contrast group, MK801group, FK506group wasrespectively down-regulated by15%,30%,25%in binding protein.Dependence group, withdrawal group was respectively down-regulated by40%,50%, MK801group, FK506group was respectively up-regulated by20%,40%in free endochylema protein. Dependence group, withdrawal groupwas respectively up-regulated by60%,60%, MK801group, FK506group wasdown-regulated by25%,15%in the ratio. In Amy, withdrawal group，FK506group was respectively down-regulated by15%,30%in total protein.Dependence group, withdrawal group， FK506group was respectivelydown-regulated by20%,20%,30%in binding protein. Withdrawal group wasdown-regulated by20%in free endochylema protein. MK801group, FK506group was respectively down-regulated by15%,15%in the ratio.2.5The change of cofilin: In Pfc, dependence group, MK801group respectively was down-regulated by20%,30%. In Hip, dependence group,withdrawal group was respectively up-regulated by20%,95%. Smallmolecule peptide was down-regulated by15%. In Str, dependence group,withdrawal group was respectively up-regulated by50%,85%. MK801groupwas down-regulated by15%. In Amy, dependence group, withdrawal group,withdrawal contrast group, small molecule peptide was respectivelyup-regulated by40%,45%,35%,75%.2.6The ratio of P-cofilin and cofilin: In Pfc, dependence group, withdrawalgroup, MK801group, small molecule peptide was respectively up-regulatedby20%,35%,60%,30%. In Hip, dependence group, withdrawal group,MK801group, FK506group, small molecule peptide was respectivelyup-regulated by60%,15%,70%,20%,65%. In Str, dependence group,withdrawal group, withdrawal contrast group, MK801group, FK506group,small molecule peptide was respectively up-regulated by55%,15%,15%,80%,15%,50%. In Amy, dependence group was up-regulated by40%.2.7The change of SSH: In Pfc, MK801group was up-regulated by55%. InHip, dependence group, MK801group was respectively up-regulated by30%,15%. In Str, dependence group, MK801group, FK506group was respectivelyup-regulated by30%,35%,20%. In Amy, small molecule peptide wasdown-regulated by15%.2.8The change of LIMK: In Pfc, FK506group was up-regulated by15%,small molecule peptide was down-regulated by30%. In Hip, dependencegroup, small molecule peptide was respectively down-regulated by25%,25%.In Str, dependence group was up-regulated by40%, MK801group, FK506group, small molecule peptide was respectively down-regulated by30%,25%,30%.In Amy, dependence group, MK801group was respectively up-regulatedby60%,35%.2.9The ratio of P-LIMK and LIMK: In Pfc, dependence group wasdown-regulated by30%. In Hip, dependence group was down-regulated by30%, MK801group, FK506group was respectively up-regulated by15%,45%. In Str, MK801group, FK506group was respectively up-regulated by 20%,20%. In Amy, dependence group, MK801group was respectivelydown-regulated by40%,35%, FK506group was up-regulated by15%.Conclusion: In the process of rats morphine dependence and withdrawalthe actin rearrangement in different brain regions, It’s the P-cofilin that playsthe key role in this process; the actin rearrangement Induced by morphine wasrelated to the Content change of drebrin, cortactin and binding with F-actin;the actin rearrangement Induced by acute treatment May be related to relatedto ROCK2/LIMK pathway; and the dependence rats May be related to relatedto NMDA/Ca2+/Calcineurin/SSH pathway. |
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