FSH通过调控HMGCR对肝脏胆固醇合成的影响及分子机制

It is well known that postmenopausal women prone to have dyslipidemia. The underlying mechanism is widely thought to be estrogen loss. However, elevated levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) have been also observed in premenopausal status, in which the estrogen level had no change, only the gonadotropin, follicular stimulating hormone (FSH) levels increased. Thus, the development of dyslipidemia in premenopausal status can not be explained only by the role of estrogen.. In the present sthudy, we have demonstrated that functional FSHR was localized to the hepatocyte surface. In ovariectomized (OVX) mice which were maintained the serum estrogen level by estrogen supplementation, we found the activity of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis, and the rate of cholesterol synthesis increased in a dose-dependent manner with FSH treatment. By far, there has been no clear molecular mechanism to c explain how FSH affects cholesterol metabolism in the liver.. The project intends to develop a series of in vivo (like ovariectomized mouse model, conditional knockout hepatic or ovarian Fshr mouse model) and in vitro (like silencing or overexpressing some key molecules in hepatocytes) experiments to confirm that functional FSHR expresses in liver, and that FSH increases cholesterol synthesis in the liver, and to explore how FSH regulates cholesterol synthesis via HMGCR. In this way, we could clearly explain the molecular mechanism that FSH increases the hepatic cholesterol synthesis via HMGCR. Our work will provid a noval perspective to explain the pathophysiological mechanism of hypercholesterolemia in postmenopausal status, which will be benificial to develop effective intervention therapies.

传统观点认为绝经期妇女血脂水平紊乱是雌激素（E2）减少的结果。但围绝经期早期的女性雌激素水平与生育期女性相近，仅卵泡刺激素(FSH)水平升高，其胆固醇和LDL-C水平也升高,无法单纯用E2降低作用解释。我们前期研究发现肝细胞存在功能性FSH受体（FSHR）；卵巢去势小鼠补充雌激素维持生理水平后，FSH增加肝脏胆固醇合成限速酶（HMGCR）表达和新生胆固醇，呈量效关系，但其具体机制不清，国内外未见报道。本项目拟采用体内（摘除卵巢去势小鼠、条件性敲除肝脏或卵巢Fshr基因小鼠模型）和体外（干扰、激活肝细胞关键分子表达）结合策略，从整体、细胞、分子三个水平探讨：①肝脏FSHR表达及其生物学活性、②FSH对肝脏胆固醇合成的影响和③建立FSH调控HMGCR信号通路,阐明FSH通过FSHR调控肝脏HMGCR增加胆固醇合成的机制。为揭示绝经期胆固醇代谢异常病生机制和有效的干预治疗提供新的依据和策略。

FSH 通过与卵巢FSHR 结合，发挥其作用。 证明肝细胞表面有FSH受体存在，是阐明FSH 调节肝脏胆固醇的基础。因此，以卵巢的FSHR 为阳性对照，进行了如下实验：肝脏FSHR mRNA 序列验证。设计FSHR细胞膜外、内引物，分别检测到来源于人和小鼠肝脏存在FSHR 产物，序列与卵巢的FSHR序列相同。原位杂交验证，人和小鼠肝脏检测到FSHR表达。验证蛋白结构域位点。针对N-,C-端，选用不同抗体进行western blotting . 显示蛋白KD 与卵巢FSHR 的KD一致。蛋白定位，免疫荧光实验证实FSHR 存在于肝细胞表面。体外实验获得相似结果，选用HepG2 和小鼠 NCTC 1469细胞系（卵巢 CHO为阳性对照）进行体外实验， 获得与体内相似的结果。放射性竞争结合实验显示FSH与FSHR 结合具有特异性。FSH主要通过与细胞表面FSHR结合完成细胞内信号传递过程，而FSHR属于G蛋白偶联受体家族（GPCR）。通过对GPCR活化后的一些激酶进行筛查，发现FSH可以显著促进丝氨酸/苏氨酸激酶（Akt）的磷酸化（p-Akt）。同时，肝细胞内加入Akt不同阻断剂后，FSH对SREBP-2及HMGCR的上调作用都显著降低；发现当β-arrestin2敲减后FSH上调p-Akt和HMGCR作用显著减弱。