补肾化痰祛瘀方对Wilson病TX小鼠卵泡发育障碍的作用及ERK1/2信号通路调控机制研究

Wilson's disease is an autosomal recessive genetic disorder, in which copper progressively accumulates in tissues due to defects in copper metabolism. It mostly causes damage in the brain and the liver. In addition, it may also cause considerable damage in the reproductive system of women . However, such damage in the reproductive system of women has long been ignored. Our previous clinical studies showed that the traditional Chinese medicine (TCM) formulation for reinforcing kidney， resolving phlegm and removing blood stasis, could effectively manage the menoxenia symptoms in Wilson’s disease female patients. Based on our preliminary clinic studies and the researches, which are sponsored by National Natural Science Fundation of China, we propose that the TCM formulation for reinforcing kidney ， resolving phlegm and removing blood stasis may activate the ERK1/2 signaling pathways and consequently ameliorate follicular maldevelopment induced by Wilson’ disease. To test this hypothesis, we will use a classic TX mouse model for Wilson's disease to determine the effect of the TCM formulation for reinforcing kidney， resolving phlegm and removing blood stasis on ovulation, ovarian endocrine, oocytes developmental potential, development of different stage follicular, ultrastructure, follicular cell proliferation and apoptosis and define the effect of the formulation on the status of the ERK1/2 pathway. Furthermore, we will use small molecule inhibitors to block the ERK1/2 signaling pathway in order to validate that activation of the ERK signaling pathway is indeed a key molecular mechanism by which the TCM formation for reinforcing kidney， resolving phlegm and removing blood stasis improves the follicular development. The study would help to elucidate the molecular mechanism of the TCM formulation for reinforcing kidney， resolving phlegm and removing blood stasis in treating Wilson’s disease. The study is intended to provide a theory basis of modern molecular biology and a new idea in the traditional Chinese medicine therapy of the reproductive system damage in Wilson’s disease.

Wilson病是一种常染色体隐性遗传的慢性进行性铜代谢障碍性疾病，可造成以脑和肝为主的损害。女性患者生殖系统损害亦十分显著且长期被忽视。前期临床研究显示补肾化痰祛瘀方可改善Wilson病所致月经不调。综合文献、结合前期课题研究发现和预实验研究基础，我们提出补肾化痰祛瘀方可通过激活ERK信号通路改善Wilson病卵泡发育障碍。本课题将运用经典的Wilson病TX小鼠模型，观察补肾化痰祛瘀方对Wilson病模型小鼠排卵、卵泡内分泌功能、卵母细胞发育潜能、各级卵泡发育、卵泡超微结构、卵泡细胞增殖和凋亡以及ERK1/2信号通路的影响，并运用工具药阻断ERK1/2信号通路激活的方法反向验证ER1K/2信号通路在其中的作用。研究旨在阐明补肾化痰祛瘀方对Wilson病卵泡发育障碍的作用及ER1K1/2信号通路调控机制，可为Wilson病生殖系统损害的中医药治疗提供现代分子生物学理论基础。

目的：探讨Wilson病卵泡发育障碍的机制，研究ERK1/2信号通路和BDNF在补肾化痰祛瘀中药改善卵泡细胞发育中的作用，为中药治疗女性Wilson病伴有生殖系统异常提供依据。.方法：小鼠随分为空白组、Wilson病模型组、DMSA组、补肾化痰祛瘀方组、DMSA+补肾化痰祛瘀方组、补肾化痰祛瘀方+U0126组。运用HE染色法观察各级卵泡发育情况；电镜观察卵巢中颗粒细胞及卵泡细胞超微结构的改变；免疫组化结合荧光及TUNEL法，分析BDNF与卵泡细胞增殖及凋亡的相关性；免疫印迹法检测 p-ERK 及 ERK蛋白水平；孤雌激活法观察小鼠卵母细胞发育潜能； .结果：与空白组比较，各组卵巢组织中的正常次级卵泡及窦卵泡数目出现明显的减少,闭锁卵泡的数目增加显著；卵巢组织可见显著增多的异常变性空泡化的线粒体，BDNF阳性表达量及卵泡内阳性增殖细胞平均光密度降低，凋亡细胞数量增加，BDNF蛋白表达量降低，平均光密度降低。卵母细胞的囊胚形成率显著降低，ERK1 / 2磷酸化蛋白相对表达量显著降低。与Wilson病模型组比较，各治疗组卵巢内正常次级卵泡及窦卵泡数目增加，而闭锁卵泡数目减少，正常线粒体数目增加，异常变性及空泡化线粒体数目减少，而DMSA+补肾化痰祛瘀方组卵巢内颗粒细胞凋亡现象减轻，正常线粒体数目增加，异常变性及空泡化线粒体数目减少；各治疗组卵巢组织BDNF阳性表达量及卵泡内阳性增殖细胞平均光密度增加，平均光密度显著增高，凋亡颗粒细胞数量降低，BDNF蛋白表达量增加，DMSA+补肾化痰祛瘀方组凋亡细胞数量显著降低，BDNF蛋白表达量增加。与DMSA组比较，补肾化痰祛瘀方组及补肾化痰祛瘀方+DMSA组效果更优。补肾化痰祛瘀方可以明显提高卵母细胞的囊胚形成率，增加ERK1 / 2磷酸化蛋白相对表达量明显增加。与补肾化痰祛瘀方组比较，补肾化痰祛瘀方+U0126组卵巢内正常次级卵泡及窦卵泡数目减少，闭锁卵泡数目增加，卵巢内颗粒细胞凋亡现象加重，正常线粒体数目减少，异常变性及空泡化线粒体数目增加。.结论：Wilson病通过抑制卵巢ERK信号通路和BDNF表达水平导致卵泡发育障碍，补肾化痰祛瘀方可能通过激活ERK信号通路、提高BDNF表达水平改善Wilson病模型小鼠卵泡发育。