黄连生物碱靶向AKR1C3调控胃癌细胞脂代谢的分子机制

Enhanced de novo fatty acid biosynthesis was a hallmark of gastric and other cancer cells. Our previous study showed that Coptidis Rhizoma alkaloids (CRA) had anti-proliferation effect in gastric cancer (GC) cell, and whether this effect was caused by inhibiting cellular lipogenesis need to be illuminated. Docking result indicated that CRA could bind to the active site in AKR1C3 thus inhibit its catalytic activity, and the binding effect was in accordance with the anti-proliferation effect in GC cell. Then we further predicted that AKR1C3 could activate PI3K/Akt/mTORC1 signaling pathway via hindered the conversion of PGD2 into 15dPGJ2, and activate AR signaling pathway via catalytic production of testosterone. The two pathways could work synergistically to promote the expression and activation of SREBP-1 and cellular lipogenesis, finally promote cell proliferation. Correspondently CRA could inhibit the activation of these two pathways via inhibiting the catalytic activity of AKR1C3, and then interfered with lipogenesis in GC cells. This project aims to confirm that the catalytic reaction mediated by AKR1C3 could activate PI3K/Akt/mTORC1 and AR pathway thus promote the expression and activation of SREBP-1 and cellular lipogenesis, thereby to illuminate the mechanism of regulation of fatty acid metabolism in GC cells by CRA via inhibiting AKR1C3. This project could provide a new research idea to reveal the anti-GC mechanism of CR, and supply experimental basis for discovering new target in the GC therapy.

增强的脂肪酸从头合成是胃癌等肿瘤细胞的重要代谢特征，申请者前期研究发现黄连生物碱（CRA）能抑制胃癌细胞增殖，但是否通过抑制脂肪酸生成发挥作用有待明确。申请者通过分子对接发现CRA能与AKR1C3活性抑制区域结合，且结合效果与抑制胃癌细胞增殖效果趋势一致；进一步预测AKR1C3可通过阻碍PGD2向15dPGJ2转化激活PI3K/Akt/mTORC1通路，还可通过催化产生睾酮激活AR通路，两通路协同互补促进SREBP-1表达活化和脂肪酸合成，最终促进胃癌细胞增殖，而CRA可通过抑制AKR1C3发挥双通路抑制作用干预胃癌细胞脂肪酸生成。本项目旨在确认AKR1C3介导的催化反应激活PI3K/Akt/mTORC1和AR通路，促进SREBP-1表达活化和脂肪酸合成的作用，阐明CRA靶向AKR1C3调控胃癌细胞脂代谢的分子机制，为揭示黄连抗胃癌机制、发现胃癌治疗新靶点提供新思路和实验基础。

中药黄连具有清热燥湿的功效，常用于治疗胃肠道疾病，在现代中医临床上还被用于治疗胃癌属湿热证的患者。生物碱是黄连主要有效成分，本项目以黄连碱为代表成分对其抗胃癌作用进行了研究，明确了黄连碱可抑制胃癌细胞的增殖、转移和克隆形成，作用优于小檗碱，但主要不是通过干预细胞DNA复制和诱导细胞凋亡发挥作用。通过反向药效团匹配和分子对接发现AKR1C3为黄连碱重要靶点，并相应构建了AKR1C3基因沉默和过表达胃癌细胞模型进行验证。ELISA检测结果表明黄连碱能抑制AKR1C3催化产生睾酮。CCK-8检测结果表明AKR1C3表达增加能够显著促进胃癌细胞增殖，AKR1C3高表达的胃癌细胞对黄连碱的增殖抑制作用更加敏感。GSEA基因富集分析表明AKR1C3高表达与脂肪酸代谢通路相关。qRT-PCR、Western blot、免疫荧光检测表明AKR1C3表达增加可促进胃癌细胞SCAP的转录和表达，进而促进SREBP-1的剪切活化，进而提高脂肪酸合成关键酶FASN、ACC的表达水平，油红O染色、GC-MS检测表明AKR1C3表达增加可激活脂肪酸合成，而低浓度黄连碱（10μM）即可产生显著干预作用。本项目研究明确了黄连碱通过抑制AKR1C3对睾酮的催化作用，减少SCAP的转录和表达，进而抑制SREBP-1的剪切活化，以及FASN、ACC的表达，最终降低胃癌细胞脂肪酸合成的抗胃癌作用机制，为揭示黄连抗胃癌作用机制提供新研究思路，为发现胃癌治疗的新靶点提供实验依据。