莱菔硫烷对肝脏脂肪滴形成的影响及内质网-线粒体结构偶联区域的调控机制

Disorder of lipid metabolism has been considered the precusor of obesity,diabetes and cardiovascular diseases. Both endoplasmic reticulum and mitochondrial are two organs for metabolizing lipids. Our recent findings showed that Sulforaphane could inhibit the formation of lipid droplet induced by oleic acid/ palmitinic acid in human hepatocytes in vitro. Excess accumulation of lipid droplet will damage the liver function and subsequently lead to the disorder of lipid metabolism. To clarify its novel function of SFN in improving the lipid metabolism and its potential in future exploration, this project will be performed from the followings. Firstly, whether SFN could improve the disorders of lipid metabolism induced by high diet through the technology of pathology,serology and proteomics. Secondly, in oleic acid/ palmitinic acid(2:1)-induced hepatocytes sterosis, the mechanisms and the key targets of SFN were studied. Finally, focusing on the mitochondria-endoplasmic reticulum associated membrane, that is MAM area, the biomarkers of MAM and lipid droplet, as well as the calcium gate factors and Nrf2, the classical target of SFN, were analyzed to elucidate their roles in this process using the method of gene silence. Our results will provide the theoretical basis for SFN as functional factor of balancing lipid metabolism in the field of food.

脂代谢紊乱是引起肥胖、糖尿病等营养相关疾病的关键病因。申请者最新发现莱菔硫烷（SFN）能够抑制油酸/软脂酸诱发的肝细胞脂肪滴形成，脂肪滴的大量堆积将直接引起肝细胞功能受损进而导致脂代谢紊乱。为明确SFN是否具有调控脂代谢紊乱的新功能以及其未来的应用开发潜力，本研究拟首先建立高脂膳食诱导的脂代谢紊乱动物模型，通过病理学、血清学、蛋白质组学等分析手段研究SFN的改善作用；随后用油酸/软脂酸诱导人肝细胞脂代谢异常模型探讨SFN调控脂肪滴形成的机制和关键作用靶点；最后以“内质网-线粒体结构偶联部位即MAM区域”为研究中心，采用基因沉默技术，研究SFN如何调控MAM区标志蛋白、脂滴结构标志蛋白以及钙离子门控因子，以及SFN的经典作用靶点Nrf2在上述调控过程中的作用。本研究将阐明SFN对肝脏脂代谢紊乱的改善作用及机制，为SFN作为调节脂代谢平衡、改善脂代谢紊乱的功能因子应用于食品领域提供理论基础。

非酒精性脂肪肝病（non-alcoholic fatty liver disease, NAFLD）是以脂滴过度沉积于肝脏为特征的脂代谢紊乱疾病，可导致脂肪性肝炎，最终发展成肝癌。莱菔硫烷（sulforaphane，SFN）是富含于十字花科蔬菜中的异硫氰酸酯类植物化学物。前期研究发现，SFN能够改善糖尿病、肥胖等糖脂代谢紊乱引起的代谢型疾病，但SFN对高脂摄入引起的NAFLD是否也具有改善作用，及可能的作用机制尚未见报道。本文采用高脂膳食（high fat diet，HFD）建立NAFLD整体动物模型，以脂肪酸（fatty acid，FA）诱导HHL-5肝细胞为体外模型，并以 SFN 为干预因素，内质网与线粒体偶联区（MAM）作为研究核心，探讨SFN对肝脏脂滴形成的影响和改善NAFLD的分子机制。. SFN不仅通过抑制脂肪合成酶DGAT2和ACAT1，降低脂滴核心的脂肪含量，改善肝脏病理损伤，而且通过抑制转录因子PPARγ下调脂滴外周蛋白PLIN2及PLIN5的表达，抑制脂滴形成。ER是合成FA的关键细胞器，SFN明显减轻ER肿胀，缩短ER周长；通过调控内质网应激（ERS）两条途径XBP1-ACC1/SCD1和PERK-SREBP1c-FAS下调脂肪酸合成相关酶ACC1、SCD1和FAS，减少脂质积累，其作用与ERS抑制剂作用类似。SFN减轻线粒体膨胀，拉开线粒体与内质网之间的距离，下调MAM区连接蛋白PACS2的表达；SFN明显降低胞浆及线粒体中Ca2+浓度；下调Ca2+释放通道蛋白IP3R和线粒体内膜上吸收Ca2+的单向转运蛋白MCU的表达；修复MT功能，促进ATP合成，改善能量代谢。. 本研究结果将为SFN作为调节脂代谢平衡、改善脂代谢紊乱的功能因子应用于食品（包括饮料）领域提供理论基础。.