基于ER-SHP通路探讨补肾降浊法治疗绝经后糖脂代谢紊乱的机制研究

The incidence of glycolipid metabolism disorder(GMD) increase significantly after menopause，which is closely related to the deficiency of estrogen. The pathway of estrogen receptor (ER)-SHP play a important role on the liver glycolipid metabolism for women. The previous studies showed that the intervention of “Tonifying Kidney and Descending Turbidity therapy” can improve the glycolipid metabolism to postmenopausal women, and can up-regulate the express of ER in the liver on OVX rats. Therefore, we speculate that “Tonifying Kidney and Descending Turbidity therapy” cure the postmenopausal GMD through the pathway of ER-SHP. To confirm this hypothesis, GMD model would be construct on the OVX Wild and ER-/- mice, the effect of “Tonifying Kidney and Descending Turbidity therapy” on the the glycolipid metabolism and its signal pathway of ER-SHP would be evaluated. The isolation Primary mouse hepatocytes were cultured by high Glucose and high Fatty Acid solution to construct the GMD cell model. The effect of “Tonifying Kidney and Descending Turbidity therapy” on the model cell and the pathway of ER-SHP would be observed by RNA interference and gene silence technique .This research will clarify the mechanism and the signal pathway of “Tonifying Kidney and Descending Turbidity therapy” on postmenopausal GMD, which is of great significance for the prevention and treatment of postmenopausal cardiovascular diseases.

女性绝经后糖脂代谢紊乱(glycolipid metabolism disorder, GMD)发生率显著增加，与雌激素下降密切相关。近期研究发现肝脏雌激素受体(ER)-SHP通路在女性糖脂代谢中发挥重要作用。前期研究显示补肾降浊法对绝经后女性具有良好的调脂降糖作用，且可上调去势大鼠肝脏ER表达，我们推测补肾降浊法经ER-SHP通路发挥防治绝经后GMD的作用。为证实假说，项目拟通过野生小鼠和ER基因敲除小鼠去势后建立GMD模型，观察补肾降浊法对模型小鼠糖脂代谢表型及通路蛋白表达的影响；分离肝脏原代细胞，建立高脂高糖损伤模型，通过RNA干扰技术和基因沉默技术等方法，基于ER-SHP通路观察中药对模型细胞的保护作用及其信号通路。本项目将从新视角阐明补肾降浊法防治绝经后GMD的作用机制及信号通路，对绝经后心血管疾病的防治具有重大意义。

绝经后糖脂代谢紊乱（disorder of glycolipid metabolism，DLM）的发生率显著增加。前期研究显示补肾降浊法对于绝经后DLM具有良好的临床疗效，但作用机制不明。为探讨其作用疗效和机制，课题组分三部分开展研究。.1.补肾降浊法对模型小鼠糖脂代谢表型及通路蛋白表达的影响：去势联合高脂高热量饮食构建绝经后DLM小鼠模型，给予高中低剂量中药及阳性药物干预。结果显示，中药可显著降低绝经后DLM小鼠体重、腹围、血糖、TC、TG、胰岛素抵抗，并减少肝脏脂肪病变。同时可降低小鼠肝脏糖脂代谢基因G6PC、PEPCK、FBP1、Srebp1、SCD1表达，提高SHP蛋白表达，降低GSK3β磷酸化，抑制PI3K/AKT信号通路。但肝脏雌激素受体（ER）未出现显著增加。.2.中药对ER缺陷小鼠糖脂代谢紊乱的影响：分别采用ER-/-小鼠和ER拮抗剂结合高热量饮食构建ER缺乏绝经后DLM小鼠模型，与非ER阻断进行对照。结果显示，在阻断ER后，中药仍能改善模型小鼠糖脂代谢表型，但相比无ER阻断下的中药作用减弱。阻断ER不影响中药增加SHP蛋白表达及抑制PI3K/AKT通路。提示中药通过ER调节绝经后DLM，但ER并非唯一作用靶点，且中药对肝脏SHP的调节并非通过ER实现。.3.中药对肝脏细胞糖脂代谢紊乱的调节作用及其对ER-SHP信号通路蛋白表达的影响：中药能够改善AML12细胞脂毒性损伤和HepG2细胞的脂肪沉积，降低细胞炎症因子水平，上调SHP蛋白表达，改善SHP上游信号阻断（FXR抑制剂）引起的脂代谢紊乱。AML12细胞实验显示，在ER抑制的情况下，中药对PI3K、AKT通路蛋白的表达水平的影响与无ER抑制的情况下类似。.综上，补肾降浊方可显著改善去势小鼠DLM。ER是其作用通路和靶点之一。通过PI3K/AKT信号通路调节FXR-SHP蛋白表达，可能是补肾降浊方调节绝经后DLM的主要通路。