The Experimental Study On SIRT1 Regulating Synaptic Plasticity Of Nucleus Accumbens In Heroin Addictive Rats

Addiction is a process of pathological learning and memory,manifested by uncontrolled self-medication and compulsive drug behavior.Synaptic plasticity,neurobiological basis of learning and memory,plays an important role in the process of addiction.NAc is an important part of the brain’s reward system,participating in the body’s cognitive activities related to behavior,learning and memory.Now it is considered to be the main nucleus of reward effect in the body.Silent mating type information regulation 2 homolog 1(SIRT1),the Ⅲ class deacetylation enzyme,is involved in the body’s physiological and pathological activities through the acetylation on the substrates of histone and non-histone.Studies have shown that SIRT1 also engages in active drug abuse such as cocaine and morphine.Yet the role of SIRT1 in heroin addictive rats in NAc is rarely reported.In this study,heroin addictive rat model was established.The expression of silent information regulator two proteins(sirtuins)in heroin addictive rats was detected by WB and q RT-PCR;adeno-associated viruses were transfected into the NAc region.SIRT1 over-expression rats and SIRT1 silence rats were produced.The condition place preference(CPP)and naloxone withdrawal symptoms were observed after heroin injection;PCR chip technology was applied to detect the synaptic plasticity related changes in expression of 84 genes in NAc of rats in each experimental group.Immunohistochemistry and Western blotting were used to get the localization and expression of SIRT1 and synaptic plasticity associated proteins;the synaptic ultrastructure was observed by transmission electron microscopy(TEM).In conclusion,SIRT1 plays an important role in heroin addiction mechanism by adjusting synaptic plasticity in the nucleus accumbens of rats.Part one: The expression of sirtuins in NAc of the heroin addictive ratsObjective: To examine the expression of members of sirtuins(SIRT1-SIRT7)by developing the heroin addictive rat model and heroin conditioned place preference(CPP).Methods:(1)Adult male Sprague-Dawley(SD)rats were randomly divided into three groups as follows: saline control group(SCG),heroin addiction group(HAG)and naloxone withdrawal group(NWG).The HAG and NWG rats were injected with heroin for 9d to reproduce the heroin addictive model,and the SCG rats were injected with equal volume of saline.On 10 d,rats of NWG and SCG were subjected to naloxone to reproduce the naloxone withdrawal.The withdrawal symptoms and body weight change of SCG and NWG rats were recorded.All rats were sacrificed on 10 d and NAc brain regions of rats in each group were extracted.Western blotting and real-time fluorescent quantitative PCR technique were applied to detect SIRT1,SIRT2,SIRT3 and SIRT4,SIRT5,SIRT6 and SIRT7 proteins and m RNA expression.(2)SD rats were divided into the CPP and SCG groups.The CPP rats were placed into the white box after treatment with heroin in the morning,and those treated with saline were placed into the black box in the afternoon.The SCG rats were injected with saline all day.The time of staying in the white box was recorded to detect the heroin CPP.Results:(1)Compared with the SCG,there were significant withdrawal symptoms in HAG rats,such as rearing,jumping,writhing,wet-dog shakes,blepharoptosis and diarrhea.(2)Heroin could induce CPP after the pre-test,training and test periods,time spent in the white box significantly longer SCG(p < 0.05).(3)Western blotting results showed that the expression of SIRT1 protein was higher in NAc in HAG than that in SCG(p < 0.05),while there was no significant difference IN SIRT2-SIRT7 between HAG and SCG.(4)The expression of SIRT1 m RNA was higher in NAc in HAG than that in SCG by q RT-PCR(p < 0.05).Conclusion:SIRT1 protein and SIRT1 m RNA were increased significantly in NAc in HAG,suggesting that SIRT1 is involved in the formation of heroin addiction.Part two: SIRT1 regulats the expression of synaptic plasticity related protein and geneObjective: To explore the relationship between synaptic plasticity and changes in addictive behavior and to work out a novel treatment of drug addiction by observing rat behavior,and detecting the expression of synaptic plasticity associated protein and gene in NAc brain region in different rat models..Methods: r AAV9 r Sirt1 and r AAV9 r Sirt1 Sh RNA adeno-associated viruses were packaged.By using transfection technology,heroin addiction,SIRT1 overexpression,SIRT1 silencing rat models were established to observe the naloxone withdrawal symptoms and CPP changes.PCR gene chip technology was applied to detect the synaptic plasticity related 84 gene expression changes in NAc of HAG and SIRT1 overexpression rats.Immunohistochemistry and Western blotting were used to detect the position and expression of SIRT1 histone substrate and non-histone substrate;the ultrastructure of synapses was observed by transmission electron microscope.Results:(1)The plasmid was successfully constructed and the adeno-associated virus containing the target gene and the silencing target gene was packaged and transfected into NAc;(2)The CPP results showed that there was no significant difference in time spent in the drug-paired compartment of SCG and blank virus group rats before and after training.Behavioral results confirmed Heroin induced a preference for the drug-paired compartment on HAG and SIRT1 overexpression group rats(p < 0.05);the time of SIRT1 silencing group rats in the drug-paired compartment was increased than before training,but significantly decreased compared with HAG.(3)The naloxone withdrawal symptoms appeared apparently in heroin-treated rats,especially in the SIRT1 overexpression group.The symptoms in SIRT1 silencing group were alleviated;(4)Immunohistochemistry and western blotting results showed that Ac-H3 and FOXO1 expression decreased in HAG(p < 0.05),but increased in the SIRT1 silencing group(p < 0.05);the expression of Cdk5,nf-kappa B,PSD95 and Syn was enhancedin the heroin addiction group(p < 0.05),further increased in SIRT1 overexpression group,but reduced in the SIRT1 silencing group(p < 0.05);the expression of Delta Fos B grew after heroin administration(p < 0.05).There was no statistically significant difference among the three groups.(5)The ultrastructural alterations could also be found in synapse: the increased number of synapse in heroin management rats(p < 0.05),mitochondria swelling in the presynaptic membrane,obscure synaptic cleft wider than those in SCG.Conclusions:(1)The PCR gene array results show that SIRT1 and synaptic plasticity related gene in NAc are involved in the regulation of heroin addiction.(2)SIRT1 overexpression could increase the behavioral sensitization in NAc of rats,and SIRT1 silencing would ease withdrawal symptoms and CPP.