Table3_Repeated Ethanol Exposure Alters DNA Methylation Status and Dynorphin/Kappa-Opioid Receptor Expression in Nucleus Accumbens of Alcohol-Preferring AA Rats.docx

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, −3a, and −3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.

越来越多的证据表明，表观遗传机制——如DNA甲基化与去甲基化、组蛋白修饰——参与了酒精与药物成瘾的发生发展。然而，针对酒精使用障碍（Alcohol Use Disorder, AUD）的表观遗传DNA修饰与基因表达变化相关研究，目前结论仍存在分歧。本研究旨在探讨反复乙醇摄入对DNA甲基转移酶、去甲基化酶等表观遗传调控酶的影响，以及此类变化是否会影响伏隔核（Nucleus Accumbens, NAc）内的强啡肽/κ阿片受体系统。本研究使用三组雄性实验大鼠：经选择性繁育获得的高自愿饮酒偏好的Alko Alcohol（AA）大鼠，以及正常雄性Wistar大鼠。其中第一组AA大鼠（AA-ethanol组）在3周周期内，于每周一、周三、周五均可接触10%乙醇溶液，单次饮酒时长为90分钟，以建立稳定的乙醇摄入基线；第二组AA大鼠（AA-water组）与Wistar大鼠（Wistar-water组）仅提供纯水作为饮品。通过qPCR（实时定量聚合酶链式反应）检测发现，与AA-water组及Wistar-water组相比，AA-ethanol组大鼠的Dnmt1、Dnmt3a及Dnmt3b的mRNA水平显著升高，而Tet家族转录本水平无明显变化。DNMT与TET酶活性检测结果与qPCR结果一致：AA-ethanol组的DNMT活性较另外两组显著升高，但三组间TET酶活性未呈现统计学差异。与既往研究结果一致，AA-ethanol组大鼠的全基因组DNA甲基化与羟甲基化水平均较对照组大鼠有所提升。最后，本研究针对强啡肽/κ阿片受体系统中的候选基因（Pdyn、Kor）以及可能与酒精使用障碍相关行为有关的Dnmt3a基因的表达变化进行了分析。基因表达与启动子甲基化分析结果显示，AA-ethanol组大鼠的Pdyn、Kor及Dnmt3a的mRNA水平显著升高，但此类变化仅能部分归因于所选候选基因启动子区域的异常DNA甲基化。综上，本研究结果表明，异常DNA甲基化仅是AA大鼠模型中基因表达调控的多种相关机制之一。