去势诱导的雄激素缺乏小型猪肝脏胆固醇代谢的转录组学研究

In recent years, the relationship between serum testosterone levels and atherosclerosis in males attracts people's attention. However, the mechanisms of accelerated atherosclerosis in androgen deficiency males is not very clear. Serum lipids, especially elevated serum cholesterol levels is the main pathogenesis factor of atherosclerosis. In our previous research, we have confirmed that castration-induced testosterone deficiency resulted in a disorder of lipid metabolism, and serum cholesterol levels increased significantly in castrated male pigs. It is known that the liver is the most important organ for cholesterol metabolism, its function contained cholesterol synthesis, absorption and transformation. The abnormalities of liver enzyme or the receptor can lead to a higher serum cholesterol levels. We hypothesized that, the testosterone levels may affect blood cholesterol levels by regulating the expression of genes related to cholesterol metabolism in the liver. In the research, a hypercholesterolemia minature swine model were induced by castration and high-fat diet. A new generation of high-throughput transcriptome sequencing ( RNA-Seq ) technology were employed to analyze the liver transcriptome from castrated, intact and castrated plus testosterone treatment miniature pigs. This is the first time that we explore the molecular mechanisms of elevated serum cholesterol levels in androgen deficiency pigs by using a whole transcriptome sequencing technique. This may provide a new thinking for the prevention and treatment of atherosclerosis caused by low androgen levels in males.

近年来雄激素与男性动脉粥样硬化之间的关系倍受人们关注，但迄今为止，低雄激素水平加速男性动脉粥样硬化形成的机理尚不十分清楚。血脂，尤其是血胆固醇水平升高是动脉粥样硬化的主要发病因素。前期我们已证实，去势诱导的雄激素缺乏导致猪脂代谢紊乱，血胆固醇水平显著升高。资料表明，肝脏是体内胆固醇代谢的最重要器官，其功能涉及胆固醇的合成、吸收和转化，与上述过程相关的肝脏酶类或受体异常均可导致血胆固醇水平升高。我们推测，低雄激素水平可能通过调控肝脏胆固醇代谢基因转录来影响血胆固醇水平。本项目建立高脂饮食诱导下，雄激素调控小型猪肝脏胆固醇代谢的动物模型，采用新一代高通量转录组测序（RNA-Seq）技术，分析去势、非去势和去势加睾酮处理小型猪肝脏转录本表达、结构及功能变化。本课题首次从转录组学角度探讨雄激素缺乏小型猪血胆固醇水平升高的分子机制，为防治低雄激素水平引起的男性动脉粥样硬化等心血管疾病提供新思路。

血脂代谢紊乱，尤其是胆固醇水平升高是AS的主要发病因素。近年来研究发现，雄激素缺乏能够引起男性血清胆固醇水平升高并诱发AS形成，然而具体机制却不清楚。本项目建立了高脂饮食环境下雄激素缺乏调控胆固醇代谢的小型猪模型，并采用RNA-Seq技术探讨了雄激素缺乏调控高脂饮食小型猪肝脏胆固醇代谢的机制。主要结果如下：.（1）小型猪去势后，血清睾酮水平显著降低，但TC、LDL-C和TG水平显著升高，并且采用睾酮处理能够恢复去势引起的性激素和血脂水平变化。.（3）去势（CM）小型猪肝脏TC和TG含量、血清AST和MDA含量显著高于，而SOD活性显著低于其他两组小型猪。.（4）CM小型猪腹主动脉较其他两组小型猪具有较多的脂质斑块沉积。.（5）功能富集分析结果表明，CM小型猪肝脏上调的基因主要和TG沉积、免疫和炎症反应、氧化应激反应以及凋亡等生物学过程有关，下调的基因主要和脂肪酸氧化（FAO）、胆固醇代谢、胆汁酸合成、糖代谢等过程有关。.（6）CM小型猪肝脏LDLR 基因和蛋白表达显著降低，而肝脏PCSK9基因表达和血清蛋白含量却显著升高。另外，CM小型猪血清和肝脏总胆汁酸含量显著升高，而肝脏FXR及其靶基因SHP的表达水平却显著降低。.（7）CM小型猪CPT1A和PPARD等FAO基因表达水平显著降低，而MCP1、CXCL9和IRF7等免疫和炎症基因表达水平显著升高。.综合以上结果表明，雄激素缺乏不仅诱发高脂饮食小型猪血脂代谢紊乱，还促进小型猪肝脏脂质沉积和早期AS形成。FXR激活可以抑制肝细胞PCSK9表达，而PCSK9参与肝脏LDLR表达的转录后调控。我们猜测，CM小型猪肝脏FXR表达降低可能导致PCSK9上调，从而使得肝脏LDLR表达减少并降低LDL-C的清除能力，最终导致其血清胆固醇水平升高。另外，CM小型猪肝脏代谢功能受损、炎症和免疫反应以及氧化应激诱导的细胞凋亡增加可能是其肝脏脂质沉积的重要原因。本研究结果有助于揭示雄激素缺乏引起血脂代谢紊乱和肝脏脂质沉积的分子机制，为今后防治男性AS和非酒精性脂肪肝等代谢性疾病提供了实验依据。