糖皮质激素肾阳虚模型稳态形成的表观遗传机制及干预研究

One limitaion of the traditional animal model for Shen-Yang deficiency syndrome is that inhibition induced by glucocorticoids in hypothalamic-pituitary-adrenal(HPA) axis often recovered rapidly after glucocorticoids withdrawal. According to the principle that 'prolonged disease should damage Shen', our previous studies showed that greatly prolonged application of low does of glucocorticoids, mimicking chronic stress, resulted in increased urine volume, decreased reproduction and decreased ability of coping with cold stress in animal which formed a phenotype combination termed Shen Yang deficiency syndrome in traditional Chinese medicine. Especially, the hypofunction of HPA axis retained after glucocorticoids withdrawal. We also obtained genes which we called "alternative genes for stable state of hypofunction of HPA axis". They were up- or down-regulated by glucocorticoids, but did not restore to the normal level. This type of formation of novel stable state in HPA axis was termed HPA programming in foreign literature, which is mainly related to epigenetic modifications by rearranging the set-point of HPA axis in hypothalamus. In this project, we will use ChIP-QPCR and ChIP-Seq techniques to investigate which genes among above are subjected to DNA methylation and/or histone modifications. Next, we will confirmed whether these epigenetic modifications are related to HPA programming in this modified model. At the same time, the mechanisms underlies these epigenetic modifications will be investigated. At last, the effects and mechanisms of Shen yang-nourishing Chinese herbs on HPA programming will be probed. This project is significant for preparation of novel animal model, core mechanisms research of Shen Yang deficiency syndrome of traditional Chinese medicine.

传统的糖皮质激素肾阳虚模型，激素停用后，动物下丘脑-垂体-肾上腺皮质（HPA）轴功能抑制迅速恢复；借鉴"久病及肾"的思路，我们改用长期低剂量糖皮质激素造模（模拟慢性应激），结果动物出现生殖功能降低、尿量增多、抗冷应激能力降低等典型肾阳虚表型，并且其HPA轴功能在撤激素后稳定低下。在此模型我们也筛选到"HPA轴功能低下稳态"备选基因：表达被糖皮质激素上调或下调，撤激素后不能恢复。HPA轴功能的稳定改变，国外称为"HPA轴编程"，机制是下丘脑有关基因受到表观遗传修饰使HPA轴功能设定点发生重置。据此本项目首先采用ChIP-QPCR和ChIP-Seq技术研究上述备选基因发生DNA甲基化和/或组蛋白修饰的情况；进而向下游探查被表观遗传修饰基因中，哪些真正参与HPA轴编程；并向上游追溯形成这些表观修饰的信号机制；最后研究补肾阳药的针对性干预作用。本项目对肾阳虚证新型模型制作及机制研究均具有重要意义。

糖皮质激素给与是制备中医肾阳虚证模型的常见方法，但糖皮质激素撤除后，动物外观及各项生理生化指标迅速恢复，这与中医肾阳虚证难以自动恢复不一致。课题组曾通过低剂量长期给与糖皮质激素，发现给与SD大鼠10周左右时间，再撤除激素，动物HPA轴功能虽有恢复，但在应激情况下，不能完全恢复正常，我们推测HPA轴，尤其是动物的下丘脑可能发生了表观遗传修饰，使调节CRH的基因或其本身表达下降。本项目首先观察了10周100 μg/ml皮质酮给与SD大鼠，大鼠表现出肾阳虚证样表型组合：听力敏感性减退、尿量增多、性能力下降、怕冷等。并发现动物休息2个月后，给与寒冷应激，模型组皮质酮水平升高仍低于对照组。在给动物糖皮质激素过程中，我们发现动物的静息能量代谢率先升高后降低，认为与使用糖皮质激素早期出现“阴虚火旺”，晚期“阴阳两虚”的中医描述吻合，静息能量代谢率可作为糖皮质激素造模后，处于肾阴虚阶段或肾阳虚阶段的指标。采用全基因组mRNA表达谱分析激素应用2个月和撤退后两个月下丘脑基因表达，筛选到13个基因表达在糖皮质激素撤退后仍然维持不变。随后采用CHIP-sequence技术，采用甲基化胞嘧啶的抗体和组蛋白3赖氨酸4三甲基化（H3K4me3）抗体，沉淀染色质，随后测序，发现Phox2a基因启动子区有去甲基化现象，但该基因转染下丘脑神经元，对CRH合成和分泌仅有轻微影响。糖皮质激素长期应用对下丘脑CRH和GR甲基化和组蛋白乙酰化都无明显影响。本项目还观察到补肾阳药物金匮肾气丸能提高大鼠的能量代谢率，而知柏地黄丸能降低其代谢率。补肾药对HPA轴功能低下有改善作用。本实验显示糖皮质激素撤退后，HPA轴和外周损伤会一定程度恢复，HPA轴功能在应激状态下仍低下可能源于调节CRH的某些旁路信号发生了变化。补肾阳药物和滋阴泻火中药对动物静息能量代谢率有显著的调节作用。本项目对理解肾阳虚证稳定性和动态性及补肾药物改善肾阳虚证的机制有一定意义。