组蛋白去乙酰化酶6抑制剂Tubastatin A对酒精依赖小鼠的条件性位置偏爱行为及海马基质金属蛋白酶-9 mRNA表达的影响

Effects of selective HDAC6 inhibitor tubastatin A on alcohol dependence CPP behavior and expression of MMP-9 in hippocampus of mice

目的研究选择性组蛋白去乙酰化酶6(histone deacetylase 6,HDAC6)抑制剂tubastatin A对酒精依赖小鼠的条件性位置偏爱(conditioned place preference,CPP)行为及海马组织中HDAC6和基质金属蛋白酶-9(matrix metalloproteinases,MMP-9)m RNA表达的影响。方法采用CPP基础值前测试剔除对某侧过度偏爱的小鼠。将36只8周龄SPF级雌性C57BL/6J小鼠随机均分为4组,分别为对照[生理盐水(含1%DMSO)]组、酒精依赖(2.0 g/kg)组、单纯tubastatin A(10mg/kg)组和tubastatin A干预(2.0 g/kg酒精+10 mg/kg tubastatin A)组,每组9只。小鼠于训练阶段第2、4、6、8、10天腹腔注射酒精(2.0 g/kg)后放入伴药箱,第3、5、7和9天腹腔注射等量生理盐水后放入非伴药箱,各停留30 min;采用腹腔注射方式进行染毒,染毒容量为0.01 ml/g;腹腔注射后小鼠放入中间箱,记录15 min在每侧停留时间。采用实时荧光定量PCR法检测小鼠海马组织HDAC6及MMP-9 m RNA的表达水平。结果在测试阶段,酒精依赖组和tubastatin A干预组小鼠在酒精侧停留时间显著高于对照组和单纯tubastatin A组(P<0.01)。与对照组相比,酒精依赖组小鼠出现明显CPP效应(P<0.05),单纯tubastatin A组小鼠CCP效应更加显著(P<0.01)。与对照组比较,单纯tubastatin A组和tubastatin A干预组小鼠海马区HDAC6 m RNA的表达水平显著下调(P<0.01)。与前测阶段比较,酒精依赖组小鼠海马区MMP-9的m RNA表达水平显著上调(P<0.05),tubastatin A干预组小鼠海马区MMP-9的m RNA表达水平的上调更为显著(P<0.01)。结论酒精可明显诱发C57BL/6J小鼠的CPP效应,给予Tubastatin A可增强这种效应。tubastatin A可能通过抑制海马组织HDAC6的表达来增强小鼠酒精依赖性CPP行为,这可能与HDAC6对MMP-9表达的调控有关。

Objective To investigate the effects of selective histone deacetylase 6 （HDAC6） inhibitor tubastatin A on alcohol dependence conditioned place preference （CPP） behavior and the expressions of HDAC6 and matrix metalloproteinases-9 （MMP-9） mRNA in hippocampus of mice. Methods Alcohol-CPP behaviors of mice were tested with conditioned place preference （CPP） video system. The mice excessive preference for one side were removed based on the baseline value. Thirty-six 8-week old C57BL/6J mice were divided equally into four groups： control group （normal saline containing 1% DMSO）, alcohol dependence group（2.0 g/kg body weight）, single tubastatin A group （10 mg/kg body weight） and tubastatin A intervention group （2.0 g/kg alcohol＋10 mg/kg tubastatin A body weight）, nine mice in each group. During the training process,on 2nd day ,4th day, 6th day and 8th day, the mice were placed in the drug-paired box after intra-peritoneal injection （i.p.） of alcohol （2.0 g/kg body weight）;and on 3rd day , 5th day, 7th day and 9th day, mice were placed in the saline-paired box after i.p. with the same amount of normal sodium,stayed for 30 minutes,respectively. The toxicant exposures were intra-peritoneal injection,the volume of toxicant was 0.01 ml/g body weight. The mice were placed in the middle box after the i.p.,records residence time in each box for 15 minutes. Expressions of HDAC6 and MMP-9 mRNA in hippocampus of the mice were analyzed by real time- PCR. Results During the testing phase,the mice of alcohol dependence group and tubastatin A intervention group had significantly longer residence time in the alcohol side than control group and single tubastatin A treatment group （P〈0.01）. Compared with the control group,the mice of alcohol dependence group presented CPP effect （P〈0.05） and single tubastatin A treatment group showed significantly strengthened CPP effect （P〈0.01）. Compared with the control group,the expression of HDA C6 mRNA in single tubastatin A treatment group and tubastatin A intervention group were significantly down-regulated （P〈0.01）. Compared with the pretesting phase,the expression of MMP-9 mRNA in alcohol dependence group was significantlyup-regulated （P〈0.05）,and the expression of MMP-9 mRNA in single tubastatin A treatment group increased significantly （P〈 0.01）. Conclusion Alcohol may induce obvious CPP effect in C57BL/6J mice,tubastatin A may significantly strengthen this effect. Tubastatin A may inhibit expression of HDAC6 in hippocampus of mice to enhance alcohol dependence CPP behavior, this may be related to the regulation of HDAC6 on expression of MMP-9.