小胶质细胞特有质子通道Hv1调节氧化应激在可卡因成瘾中的作用及机制研究

Drug addiction is a serious social problem. Although the effects of activated microglia on drug addiction have attracted more and more attentions, the relative mechanisms develop slowly because of nonspecific intervention targets. Voltage-gated proton channel (Hv1), which expresses mainly in immune system, only exists on microglia within brain, and mediates microglia continuous activating. Our recent results showed that, microglia were activated after injection of cocaine 7 days, followed by increased Hv1 protein expression, indicating the important role of Hv1 in cocaine addiction. In addition, Hv1 couples to NOX, and is necessary for activated microglia producing excessive reactive oxygen species (ROS) through full activation of NOX2. ROS is another hot and close mechanism relative to drug addiction. So here, we will firstly determine the key effects of Hv1 on cocaine addiction by using cocaine injection and self-administration models, as well as gene knockout and silencing technology. Then we will further investigate the interaction of microglia-Hv1-NOX2 during cocaine addiction with molecular biology, immunofluorescence, brain slice patch-clamp, ESR and optogenetics techniques, and explore the mechanism of Hv1 effects on drug addiction, to find a novel breakthrough for drug addiction treatment and intervention.

药物成瘾是一个严重的社会问题，小胶质细胞因在药物成瘾中被激活并产生毒性而越来越受到关注，但由于缺乏特异性干预靶点，其机制研究进展缓慢。主要表达在免疫系统的电压门控型质子通道（Hv1）脑内为小胶质细胞所特有，并参与小胶质细胞的持续激活。我们近期实验结果发现，可卡因给药7天后随着小胶质细胞的激活，Hv1的表达增加，提示Hv1通道在成瘾过程中的重要作用。此外，Hv1主要与NOX相连，是小胶质细胞NOX2持续激活产生大量ROS所必须的蛋白，而ROS与药物成瘾同样密切相关。由此，本课题将采用被动注射和自身给药两种可卡因成瘾模型，结合基因敲除和沉默的方法首先确定Hv1在可卡因成瘾中的关键作用，再综合应用分子生物学、免疫荧光、脑片膜片钳、ESR和光遗传学等技术研究成瘾过程中小胶质细胞-Hv1通道-NOX2的相互关联和作用，对Hv1影响成瘾行为学的机制进行探讨，以期为药物成瘾的治疗和干预找到新的突破口。

药物成瘾是一个严重的社会问题，小胶质细胞因在药物成瘾中被激活并产生毒性而越来越受到关注，电压门控质子通道（Hv1）特异性表达于小胶质细胞上，参与小胶质细胞的持续激活。其作用是调节细胞内pH值，维持NADPH氧化酶（NOX），主要是NOX2的功能，导致脑内活性氧物质增加。虽然有证据表明可卡因在中枢神经系统中诱导氧化应激，但小胶质细胞特异性的Hv1通道是否参与可卡因成瘾还无人报道。本课题首先采用可卡因被动给药模型，发现NAc脑区Hv1表达特异性增加；随之该脑区的小胶质细胞激活，与NOX2相关的氧化应激反应增强；当给予Hvcn1 shRNA慢病毒载体有效沉默小鼠NAc区Hv1蛋白后，可卡因暴露引起的小鼠条件性位置偏好和行为敏化均减弱，提示Hv1参与可卡因成瘾行为过程；NOX2抑制剂apocynin显著抑制由慢性可卡因暴露引起的条件性位置偏好和行为敏化，并下调Hv1表达和小胶质细胞的激活，提示小胶质细胞上的Hv1蛋白在小鼠成瘾行为中的作用可能与NOX2密切相关。以上研究结果揭示了成瘾过程中小胶质细胞-Hv1通道-NOX2的相互关联和作用，为Hv1作为治疗可卡因成瘾的治疗靶点提供可靠依据。