基于性激素和胰岛素信号转导途径的对话环节探讨胡芦巴丸改善糖代谢紊乱的分子机制

The goal of the proposed research is to define the pathophysiological relevance of connections between sex hormone dysfunction and insulin resistance in type 2 diabetes. In this project, the proposed research will investigate the mechanisms by which Yang-intensifying formula Fenugreek pills improve glucose and lipid metabolic dysregulation and reverse hypogonadism. Preliminary results of this project demonstrate the beneficial effects of Fenugreek pills on reversing metabolic abnormalities of type 2 diabetes. Given that Fenugreek pills also improve hypogonadism and that hypogonadism contributes to insulin resistance, it is hypothesized that Fenugreek pills act through improving the crosstalk between sex hormone signaling and insulin signaling pathways to produce anti-diabetic effects. To test this central hypothesis, the proposed research will characterize the functional interaction between sex hormone signaling and insulin signaling pathways. Accordingly, the effect of sex hormone on insulin signaling pathway will be examined using molecular and cellular approaches including immunoprecipitation, Western blots, enzyme-linked immunoassay, and real-time quantitative PCR. The pertinent downstream functions will then be analyzed in animals and cells using pathological analyses and metabolic quantifications. Similar approaches will be used to examine the effect of insulin on sex hormone signaling pathways. As the translational extension of the point(s) where sex hormone signaling and insulin signaling converge, the proposed research will address the mechanisms of action of Fenugreek pills. For this purpose, the proposed research will also include both animal and cell experiments to determine the effect of Fenugreek pills on the interaction between sex hormone signaling and insulin signaling using the above approaches. The successful completion of the proposed research will illustrate the mechanisms of actions of Fenugreek pills. Additionally, the proposed research will provide theoretical basis for using Yang-intensifying approaches to treating type 2 diabetes.

为揭示2型糖尿病性激素紊乱与胰岛素抵抗密切关联的病理生理基础，探讨温阳方胡芦巴丸改善糖脂代谢紊乱和纠正性功能低下的分子机制，本课题采用动物实验与细胞实验相结合、病理结构观察与机能代谢检测相结合的方法，以性激素与胰岛素信号转导途径的对话环节为切入点，采用实时定量RT-PCR、ELISA、免疫沉淀与蛋白印迹等细胞与分子生物学新技术，系统探讨性激素与胰岛素在效应与信号转导途径方面相互影响的交叉共有环节及其分子机制；同时以温阳方胡芦巴丸干预实验动物和细胞模型，观察其对性激素和胰岛素信号转导途径的影响；从而验证"性激素与胰岛素信号转导途径的对话可能是2型糖尿病性激素紊乱和胰岛素抵抗密切相关的机制之一；中医温阳方葫芦巴丸治疗2型糖尿病的分子机制与其改善性激素和胰岛素信号转导途径的对话环节密切相关"的假说，为温阳法在2型糖尿病中的应用提供理论依据。

本研究以链脲佐菌素（STZ）尾静脉注射和高糖高脂饮食建立大鼠糖尿病模型，以高胰岛素诱导HepG2细胞建立胰岛素抵抗模型，棕榈酸诱导NIT-1胰岛细胞建立胰岛细胞凋亡模型，采用ELISA、免疫荧光、RT-PCR和Western blot等实验技术，系统地探讨了胡芦巴丸基于性激素和胰岛素信号转导途径的对话环节改善胰岛素抵抗的机制。动物实验表明，胡芦巴丸能显著改善糖尿病大鼠胰岛素抵抗和血脂异常，改善肾功能，增加血清睾酮浓度，减轻肾小球硬化和睾丸组织中生精细胞的凋亡，提高抗氧化应激物质活性，下调肾脏和睾丸组织中氧化应激相关蛋白PKCα和P47phox的磷酸化水平；离体实验表明胡芦巴丸中的单体成分（薯蓣皂苷元、补骨脂素）能显著改善高胰岛素诱导的HepG2细胞胰岛素抵抗，增加葡萄糖摄取而减少糖异生；上调GLUT-4和PI3K的表达，上调ER、src、Akt、GSK-3β等相关通路蛋白的磷酸化水平；胡芦巴丸中单体成分胡芦巴内酯可以减少棕榈酸诱导的胰岛细胞NIT-1的凋亡，改善胰岛素分泌和脂质紊乱，提高抗氧化应激物质的活性，下调氧化应激相关蛋白PKCα和P47phox的磷酸化水平。因此，本研究提示胡芦巴丸治疗2型糖尿病的机制与其调节性激素和胰岛素信号转导途径的对话环节有关。