基于“心-肾-子宫轴”理论经验方“清心滋肾汤”调节下丘脑KNDy神经元通路治疗绝经综合征潮热作用机制

Menopausal syndrome,a common disease which affects women’s life quality,has limited methods in treating hot flushes which appears early and has big impact in a long duration."Qingxin Zishen Decoction",based on the theory of "Heart-Kidney-Uterine axis"from Xia Guicheng,who is the master of national physician,has shown the unique efficacy in alleviating symptoms such as hot flushes in clinical applications.Preliminary experiments have showed that this decoction can regulate the content of neurotransmitter in the hypothalamus which may initially be a hint of the relationship between hot flushes of menopausal syndrome and the neuroendocrine mechanism. Furthermore,it has been confirmed recently by the articles in National Academy of Sciences that KNDy neurons,which are the "power switch" of human reproductive endocrine,participate in the thermoregulation and are associated with postmenopausal hot flushes.Therefore,it is hypothesized that "Qingxin Zishen Decoction" can act on the pathway of KNDy neurons of the hypothalamus to play a role of treatment.The functional target for thermoregulation,the mechanism of relieving hot flushes and the effect to HPO axis will be illuminated through this study by observing the changes of reproductive endocrine peptides and receptors,the telemetry temperature of rats,related factor expression in peripheral blood as well as the activity of neurons in vivo in this KNDy neuronal pathway after the intervention.Thereby,the core meaning of diagnosis and treatment of "heart" in menopausal syndrome (hot flushes) and the clinical guiding effect of "Heart-Kidney-Uterine axis" will be proved.

绝经综合征是影响女性生活质量的常见病，潮热是出现早、影响大、持续长的绝经相关症状，目前治疗方法有限。基于国医大师夏桂成教授“心－肾－子宫轴”理论验方“清心滋肾汤”临床缓解潮热疗效显著，但作用机制未明。前期实验表明，该方能调控下丘脑神经递质含量，初步提示其治疗绝经期潮热与调控神经内分泌机制相关。结合美国国家科学院刊新近发文：生殖内分泌“启动源”--下丘脑KNDy神经元参与体温调节并与绝经期潮热密切相关，本项目提出假说：“清心滋肾汤”通过调节下丘脑KNDy神经元通路治疗绝经期潮热。研究拟通过实验明确本方干预后大鼠KNDy神经元通路生殖内分泌肽及受体、大鼠遥测体温、外周血清血管舒缩因子的变化情况及体外神经元活性变化，阐明本验方对体温调节产生影响的作用靶点、缓解潮热的机制及对HPO轴的影响，印证“心”在绝经综合征（潮热）诊治过程中的核心意义及“心-肾-子宫轴”理论对临床的指导作用。

绝经综合征潮热是影响绝经期女性生活质量的典型症状。“清心滋肾汤”临床缓解潮热疗效显著，但作用机制未明。项目对“清心滋肾汤”进行指纹图谱研究及15种成分定量分析，为质量标准提供了依据。1）采用大鼠双侧卵巢摘除动物模型，使用DEM实验动物植入式遥测系统监测大鼠核心体温（模拟潮热）变化，结果显示：“清心滋肾汤”显著降低双侧卵巢摘除大鼠模型夜间平均核心体温、降低昼夜核心体温升高次数。2）WB检测“清心滋肾汤”对双侧卵巢摘除大鼠下丘脑弓状核KNDy神经元通路神经肽及受体蛋白表达的影响，结果显示：与模型组比较，戊酸雌二醇显著下调NKB表达，“清心滋肾汤”显著下调NKB表达，显著上调Dynorphin蛋白，提示“清心滋肾汤”对于KNDy神经元通路的调节是通过调节神经肽实现的，其作用的发挥并非通过直接改变Kisspeptin，而是通过下调与其共表达的NKB、上调与其共表达Dynorphin蛋白实现的，这一实现途径与雌二醇通过下调NKB作用存在部分不同。3）进一步检测“清心滋肾汤”对模型大鼠KNDy神经元通路神经肽mRNA表达的影响，结果显示：与假手术组比较模型大鼠Kiss1和NKB mRNA显著上调，结果与PNAS上研究结果一致；与雌二醇作用相似，“清心滋肾汤”显著下调模型大鼠Kiss1 mRNA表达。qRT-PCR检测“清心滋肾汤”对模型大鼠KNDy神经元通路神经肽受体表达的影响，与假手术组比较，模型大鼠GPR54和NKB3R mRNA显著下调；“清心滋肾汤”显著上调模型大鼠GPR54 mRNA表达，而雌二醇未见此调控作用。故明确了“清心滋肾汤”调节KNDy神经元网络通路治疗潮热的机制是通过双向调节与Kisspeptin共表达的神经肽Dynorphin和NKB的蛋白表达，进一步下调kiss-1 mRNA，上调GPR54 mRNA实现的。4）研究同时检测了外周血血管舒缩因子，明确了“清心滋肾汤”在双侧卵巢摘除大鼠外周血中通过促进NO合成，调整NO/ET影响外周血管舒缩功能，达到降低核心体温的作用。综上研究，项目阐明了“清心滋肾汤”对体温调节产生影响的作用靶点、缓解潮热的机制，印证了“心（脑）”在“心（脑）-肾-子宫轴”理论运用于绝经前后诸证辨治过程中的核心理论地位。