Nox2依赖的ROS调节醛固酮合成的关键作用及其机制

Dysregulation of aldosterone biosynthesis has been demonstrated to be involved not only in the pathogenesis of various cardiovascular diseases, but also in the occurrence of primary aldosteronism. Results of our previous studies documented that Nox2 derived reactive oxygen species (ROS) signaling plays an important role in aldosterone biosynthesis; and more importantly, we observed overactivated Nox2 expressions in the adrenal aldosterone-producing adenoma. However, its detailed mechanisms remain to be elucidated. As the ionized calcium (Ca2+ ) is one of the most unequivocal second messengers in cellular signaling pathways regulating aldosterone biosynthesis; and activation of the transcription factor, Nur-Related Factor 1 (Nurr1), is the crucial factor for transcription of CYP11B2, we assume that Nox2 derived ROS might affect cytoplasmic calcium signaling and thereafter Nurr1 nuclear translocation. To verify the hypothesis, we plan to detect levels of ROS, endoplasmic reticulum Ca2+ release, CaM-dependent kinases (CaMK) I, Nurr1, CYP11B2 genes expressions and proteins in the aldosterone-producing adenoma tissue samples and analyze them in regarding with relevant clinical features. Furthermore, in vitro cultured human adrenal cell line H295R, roles of ROS in the direct activation of L-type channel, endoplasmic reticulum Ca2+ release, CaM-dependent kinases will be studied using methods of patch-clamping, laser confocal microscopy, RNA interference, gene transfection and co-immunoprecipitation. We will also test whether Nox2 inhibitor gp91ds-tat can repress the effects of the angiotensin II-induced ROS overproduction and aldosterone biosynthesis in SD rats. Nox2/ROS might be a novel target for therapeutic manipulation of treating end-organ damage of excessive aldosterone.

已经明确醛固酮在心脑血管疾病的发生发展中起着重要的介导作用。醛固酮合成调控过程异常不仅与高血压发生发展密切相关，也与原发性醛固酮增生症的形成有关；而醛固酮分泌调控及其失常机制仍有诸多环节有待阐明。我们前期工作证实ROS是调节醛固酮合成的重要因素，发现NADPH氧化酶亚型Nox2在正常肾上腺球状带细胞高表达，醛固酮瘤组织Nox2的表达较正常肾上腺组织更高，据此推测，Nox2依赖性ROS对调节醛固酮合成起着关键作用。但是尚未明确ROS如何促进醛固酮合成。为此，我们进一步通过激光共聚焦和膜片钳等技术，揭示Nox2依赖的ROS对Ca2+信号通路（包括L-型钙通道开放、内质网钙的释放、钙调蛋白激酶Ⅰ的激活）的直接作用机制；采用免疫共沉淀等技术，了解Nox2依赖的ROS是否调控醛固酮合成酶基因的转录因子Nurr1的核转位。该工作的完成有望为从源头上遏制醛固酮介导的靶器官损害提供新的理论基础和防治依据。

醛固酮是肾上腺球状带细胞合成分泌的盐皮质激素，参与体内水盐代谢和血压的调节，是促进心脑血管疾病的发生发展的重要因素，但醛固酮分泌调节及其失常机制的诸多环节尚不清楚，而我们前期结果显示，醛固酮合成增加的过程伴随着细胞内活性氧（ROS）生成增加，因此本课题为了证实redox-signaling 是调控醛固酮合成的重要通路，并参与自主高分泌的良性肿瘤肾上腺醛固酮瘤的形成，通过体外和体内试验，采用基因芯片技术、siRNA、ChiP、免疫组化、qPCR、酶活性测定方法，证实了醛固酮合成过程伴随ROS水平的增加，在细胞水平还明确了Nox2依赖的ROS在生理性醛固酮合成过程中起重要调控作用，并提出该过程是通过钙通路，即细胞内Ca2+浓度调节CaMKII和CREB磷酸化，从而启动CYB11B2转录来实现的。人体醛固酮瘤中Nox2蛋白等氧化-抗氧化基因表达的升高，提示ROS也可能参与醛固酮瘤形成和醛固酮的自主高分泌行为。在课题经费资助下还积累了大样本醛固酮瘤的临床和生物样本，并因此完成了多项临床研究，包括：术前预测醛固酮瘤的数学模型的建立、分析原发性醛固酮增多症患者靶器官损害的情况（代谢水平、尿酸、PTH水平、腹主动脉钙化等），与性别、年龄、高血压病程、高血压分级匹配的原发性高血压患者相比较，发现醛固酮水平可以直接或通过PTH、氧化应激、花生四烯酸等途径等引起代谢紊乱和靶器官。课题的相关结果对于深入了解醛固酮的生物合成的详细机制、醛固酮瘤的发生机制以及醛固酮对靶器官的作用等方面，均有着重要的作用，对于相关疾病的预防具有重要的意义，还可以为开发新的降压药物提供新的靶点，具有潜在的社会经济效益。