The Role Of SIRT1 Regulating Autophagy In Nucleus Accumbens In Heroin Addiction Behavior

Objective:To investigate whether Silent information regulator 1（SIRT1）is involved in heroin addiction by regulating autophagy,and to preliminarily explore its possible molecular biological mechanisms.Methods:1.Construction of addiction mouse model:according to the daily increasing dose,the addiction model was established by intraperitoneal injection of heroin twice a day for 7 days.Naloxone was given to observe withdrawal symptoms in mice.The mouse CPP model was established by conditioned place reference（CPP）paradigm.Open field experiments detect spontaneous sexual activity in mice.The control group was given an equivalent dose of normal saline.2.To study the expression of autophagy-related proteins in the brain region of nucleus accumbens（NAc）of heroin-addicted mice.（1）Animal grouping:C57BL/6J mice were randomly divided into heroin addiction group and saline control group.（2）Autophagy-related protein expression:the expression of SIRT1 and autophagy-related protein in NAc in each group of mice was detected by western blot（WB）experiment.3.To study the expression of autophagy-related proteins in mouse NAc brain region and and the impact of spontaneous activity by interfering with autophagy by Rapamycin and 3-methyladenine（3-MA）.（1）Animal grouping:C57BL/6J mice were randomly divided into three groups of Rapamycin intervention（24 h,48 h,72 h）,3-MA intervention group（24 h,48 h,72h）,and the corresponding control group according to the drug administration and action time.（2）Stimulant/inhibition autophagy drug（Rapamycin/3-MA）intervention:Microinjection of Rapamycin/3-MA in the lateral ventricle of mice was carried out using brain stereotaxic technology,and heroin was injected intraperitoneally twice a day in each group.WB was used to detect the changes of autophagy protein LC3II/I in NAc in each group of mice,and the spontaneous activity changes of mice were observed by open field experiments.4.To study whether heroin addiction caused by autophagy changes in NAc brain regions is mediated by SIRT1.（1）Animal grouping:Mice with conditional knockout of Sirt1 on Dopamine receptor 1,D1)D1 neurons were constructed by crossbreeding of Sirt1^loxp/loxpand D1-cre double transgenic mice.The experimental animals in this section were divided into C57BL/6J mouse normal saline group,C57BL/6J mouse heroin group,Sirt1^loxp/loxpD1cre（-）mouse heroin group,Sirt1^loxp/loxpD1cre（+）mouse heroin group.（2）The effect of conditional knockout of Sirt1 on autophagy and addictive behavior:the behavior changes of mice in each group were observed in combination with CPP and open field experiments;WB and Real time-PCR detected the expression of SIRT1,autophagy-related protein and m RNA in NAc in each group.Transmission electron microscopy to observe the structure of autophagic bodies in neurons in NAc region.Immunofluorescence staining detected the expression of autophagy-related proteins in NAc in each group of mice.Results:1.Expressions of SIRT1 and related autophagy proteins were increased in NAc of heroin addicted mice.After 7 days of continuous heroin injection,naloxone was injected intraperitoneally,and the mice showed obvious withdrawal symptoms.Mice in the heroin addiction group had a significant preference for heroin-accompanied position,enhanced the reward effect,and successfully established a CPP model.In the open field experiment,heroin mice had a significant increase in spontaneous behavior and increased mental excitability.The WB results showed that compared with the normal saline control group,the protein expression levels of SIRT1 and autophagy-related LC3II/I,ATG-5 and ATG-7 in heroin-addicted mice were significantly increased.2.Intervention in autophagy by Rapamycin/3-MA affects the spontaneous activity of mice.WB results showed that the relative expression of LC3II/I in the NAc brain region of heroin mice was elevated after the intervention of Rapamycin,and the change was most obvious at 72 hours of intervention.Increased spontaneous sexual activity in heroin mice.3-MA can reduce the LC3II/I ratio and reduce the spontaneous activity of heroin mice.3.Mouse tail gene identification verified the successful construction of Sirt1conditional knockout mice in NAc.The CPP results showed that the CPP formation of mice in the strip knocking group was reduced,and the spontaneous activity behavior showed a significant decrease in mental excitability.Transmission electron microscopy observed that the number of autophagies in the NAc brain region of the heroin addiction group was significantly increased compared with that in the control group,and obvious swollen and degenerative mitochondria could be seen in the autophagosomes.The results of WB,q PCR and immunofluorescence showed that compared with the Sirt1^loxp/loxpD1cre（-）group of mice,the expression of LC3II,ATG-5and ATG-7 autophagy-related proteins and m RNA in the NAc of the Sirt1^loxp/loxpD1cre（+）group was significantly reduced.However,although the expression of LC3II/I,ATG-5 and ATG-7 proteins and m RNA in the Sirt1^loxp/loxpD1cre（+）group decreased,it was still higher than that in the saline control group.Conclusions:The results of this study showed that the expression of SIRT1 in the NAc brain region of heroin-addicted mice was increased,and intervention in autophagy could change the addictive behavior of mice.Knocking out the Sirt1 gene in D1 neurons slows heroin-induced autophagy.The above results suggest that inhibition of Sirt1expression may slow down induced addictive behavior by reducing the formation of D1 neuronal autophagy.