基于SREBP1通路探讨制何首乌对肝癌脂代谢的抑制机制

The development of hepatocellular carcinoma (HCC) depends on the process of energy metabolism including lipogenesis and lipid transformation. Steroid regulatory element binding protein 1 (SREBP1) is a key transcriptional factor in lipid metabolism, which can promote the proliferation of liver cancer cells and transcribe lipid metabolism-related genes (e.g. SCD1) by translocating into the nucleus. Tonics, whose meridians belong to liver, are the most frequently used components in Chinese medicine formulae for anti-HCC, among which Polygoni Multiflori Radix Praeparata (Zhiheshouwu) belong to liver, heart and kidney meridians, can supplement the liver and kidney, transform turbidity and reduce lipid. The pharmacological data showed they exert both anti-tumor and lipid-lowering effects. Our previous data showed that Zhiheshouwu alcoholic extract (HSWE) inhibited the desaturation of fatty acids and induced apoptosis of HCC cells, but the mechanisms are still unknown if the effects of lowering lipid are associated with SREBP1 signaling pathway. Therefore, in this proposal, we aim to investigate if HSWE would induce intrinsic apoptosis and block the synthesis, desaturation and transportation of fatty acids though inhibition of up- and down-stream genes of SREBP1 by knocking-out/knocking-down and over-expressing SREBP1 in vitro and in vivo. The study would provide new insight on studies of the underlying mechanisms of Zhiheshouwu as well as the tonics on both anti-tumor and lipid-lowering.

肝癌的发展有赖于包括脂肪生成与转化在内的能量代谢过程。固醇调节元件结合蛋白1(SREBP1)是脂代谢中关键的转录因子，可通过进入细胞核发挥脂代谢相关因子如脂肪酸去饱和化酶(SCD1)的转录而促进肝癌增殖。归肝经的补虚药在肝癌中医治疗复方中使用最多，而制何首乌归肝心肾经，具补益肝肾，化浊降脂之功效。药理学研究发现其同时具有抗肿瘤和降脂双重作用。我们前期证实制何首乌醇提物抑制肝癌细胞脂肪酸去饱和化并诱导肝癌细胞凋亡，但机制是否与SREBP1信号有关尚不清楚。据此，本课题拟通过敲除、敲低和过表达SREBP1，研究制何首乌提取物是否在体内外抑制SREBP1通路及上下游脂代谢相关基因而抑制脂肪酸合成、去饱和化和转运，并诱导肝癌内源性细胞凋亡，对阐明制何首乌降脂抗癌作用规律和分子机制具有重要意义，为补虚药抗肿瘤机制研究提供新思路。

我们使用制何首醇提液作用于肝癌细胞，发现其可抑制肝癌细胞的增殖，降低肝癌细胞线粒体膜电位，使细胞中不饱和酸亚油酸和棕榈油酸与饱和脂肪酸硬脂酸和棕榈酸比例下调；同时检测了肝癌细胞中SREBP1 和SCD1，发现二者RNA 和蛋白表达水平均下降，表明 SREBP1 可介导下游多种脂代谢信号的转录，其中包括催化饱和脂肪酸如硬脂酸、棕榈酸去饱和化变成不饱和的亚油酸和棕榈油酸的饱和化酶SCD1。而硬脂酸、棕榈酸去饱和化受阻可导致脂肪酸代谢失衡，诱导内源性细胞凋亡。通过文献总结我们发现制何首乌中相关蒽醌类化合物有20 种。我们选取了6种常见的化合物大黄素、大黄酸、芦荟大黄素、大黄素甲醚和大黄素-8-O-β-D-葡萄糖苷、大黄酚，进行抗肿瘤脂代谢的筛选。结果发现其对肝癌细胞的抑制作用顺序大小依次为：大黄素、大黄酸、芦荟大黄素、大黄素甲醚和大黄素-8-O-β-D-葡萄糖苷、大黄酚。其中IC50 值小于 400μM 的有大黄素、大黄酸、芦荟大黄素和大黄素甲醚；对 SREBP1 的 mRNA 和蛋白表达有抑制作用的是大黄素、大黄酸和大黄素甲醚。进一步研究发现单独使用大黄素，其抑制SREBP1作用与制何首乌醇提物类似。但单用大黄素浓度(100μM)远远高于制何首乌中大黄素的浓度（约1.48μM），强烈提示制何首乌中其它成分通过药动学（PK）和/或药效学（PD）促进了大黄素的作用。以PK研究发现制何首乌中其他蒽醌类成分缩短大黄素的Tmax，增加大黄素的Vd、T1/2和CL，提示制何首乌中其它蒽醌类成分不能通过PK促进大黄素的作用。基于上述研究，我们通过建立了BALB/c小鼠皮下种植肿瘤模型，给予制何首乌主要蒽醌类成分大黄素，结果显示脂代谢相关蛋白SREBP1、SCD1、ACACA、FAS、ACLY的表达受到抑制。SCAP和S1P的表达降低，从而诱导肿瘤细胞凋亡。同时下调了Caspase-2表达，表明除了SCAP通路，大黄素也可抑制Caspase-2进而降低S1P的表达，阻滞SREBP1被剪切从而阻断脂代谢，其机制值得下一步深入探讨。