Objective The expression of Sirt family in amygdala was analyzed by heroin conditioned place preference (CPP) in rat model of chronic heroin administration.The target molecule with significant expression change after heroin administration was screened.The expression of target molecule,Sirt1,in amygdala was then interfered to study its regulationon heroin addiction behavior and the downstream molecules.Methods The rats in chronic heroin group were injected intraperitoneally (i.p.)with 5 mg/kg heroin for 7 days.The expression of Sirt family was detected by RT-PCR and Western blot.Quantitative chromatin immunoprecipitation (ChIP) was used to detect the △FosB-regulated Sirt1 expression.Lentivirus-mediated overexpression and inhibitor were used to upregulate/downregulate the expression of Sirt1 in amygdala.The changes of CPP behavior and BDNF expression in heroin-treated rats were observed.Results After chronic heroin administration,the expression of Sirt1 in amygdala was significantly higher than that in saline control group (t14=8.127,P＜0.01).The expression of Sirt1 was increased 6 h after heroin injection (t14=4.997,P＜0.05),24 h (t14=9.711,P＜0.01),3 d (t14=5.407,P＜0.05).After 24 h of the final heroin injection,Sirt1 (t14=5.016,P＜0.01) and acetylated histone H3K9 (t14=2.416,P＜0.05) in amygdala were significantly higher than those in saline control group.△FosB binding to the promoter region of Sirt1 increased significantly (t14=7.301,P＜0.01).Sina1 overexpression (OE-Sirt1) in the amygdala did not affect the natural preference of rats,but the CPP scores of OE-Sirt1 +heroin group in test 2 were significantly higher than those of OE-Sirt1 +saline group (t20=6.629,P＜0.01);there was no difference of the CPP scores between EX527+heroin group and EX527+saline group from test 1 to test 4.The expression of BDNF in OE-Sirt1 + heroin group and OE-Sirt1+saline group was (1.67+0.32) times and (2.66+0.34) times of that in GFP+saline group,respectively.There was no difference of the BDNF expression between EX527 + heroin group and EX527+saline group.Conclusion Heroin may regulate the expression of Sirt1 by up-regulating △FosB in amygdala,and alter the acetylation level of neuronal chromatin histone,resulting in the expression of downstream genes such as BDNF,and ultimately the change in heroin addiction behavior.%目的 通过慢性海洛因给药分析杏仁核内Sirt家族表达变化,筛选在海洛因给药后具有显著表达改变的目标分子,继而干预杏仁核内目标分子Sirt1的表达,通过海洛因条件位置偏爱大鼠模型研究Sia1对海洛因成瘾行为和下游分子表达调控作用.方法 慢性海洛因给药组大鼠连续7d腹腔注射5 mg/kg海洛因,采用逆转录-聚合酶链反应及蛋白质印迹法检测Sirt家族表达改变.定量染色质免疫沉淀检测△FosB对Sia1的上游调控.过表达慢病毒和抑制剂EX527上调/下调杏仁核内Sirt1表达,观察海洛因条件位置偏爱行为及脑源性神经营养因子(BDNF)表达改变.结果 慢性海洛因给药后,大鼠杏仁核内Sirt1 mRNA表达显著高于盐水对照组(t14=8.127,P＜0.01),Sia1 mRNA在海洛因注射后6h(t14=4.997,P＜0.05)、24h(t14=9.711,P＜0.01)、3d(t14=5.407,P＜0.05)表达升高.海洛因注射24 h后杏仁核内Sia1(t14=5.016,P＜0.01)和乙酰化组蛋白H3K9(t14=2.416,P＜0.05)显著高于盐水对照组,结合于Sia1启动子区的△FosB水平显著升高(t14=7.301,P＜0.01).大鼠杏仁核内注射Sirt1过表达慢病毒(OE-Sirt1)并未影响大鼠的自然偏爱,但在海洛因条件位置偏爱测试2中OE-Sia1+海洛因组条件位置偏爱测试值显著高于OE-Sirt1+盐水组(t20=6.629,P＜0.01);在条件位置偏爱测试1至测试4中EX527+海洛因组条件位置偏爱测试值与EX527+盐水组差异无统计学意义.OE-Sirt1+海洛因组与OE-Sirt1+盐水组BDNF表达分别为绿色荧光蛋白(GFP)+盐水组的(1.67±0.32)倍和(2.66±0.34)倍;EX527+海洛因组BDNF表达与EX527+盐水组差异无统计学意义.结论 海洛因可能通过上调杏仁核内△FosB调控Sia1表达,继而改变神经元染色质组蛋白乙酰化水平,影响BDNF等下游基因表达,从而引起海洛因成瘾行为变化.
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