Cyanidin-3-rutinoside(C3R) Attenuates Free Fatty Acid-Induced Hepatic Lipid Accumulation in HepG2 Cells

Non-alcoholic fatty liver disease (NAFLD) is characteristic by pathological lipid accumulation in hepatocytes. This study investigates how Cyanidin-3-rutinoside(C3R), a natural anthocyanin with antioxidant and metabolic regulatory properties, counteracts this process. In a free fatty acid (FFA)-induced HepG2 steatosis model, C3R significantly reduced intracellular triglycerides, total cholesterol, and LDL-C levels, suppressed lipid droplet formation, and alleviated oxidative stress by enhancing SOD/CAT activities and GSH content while lowering MDA and ROS. RNA-seq analysis revealed that C3R reversed FFA-induced transcriptomic alterations. Mechanistically, C3R inhibited SREBP2-mediated cholesterol synthesis, activated the PCSK9/LDLR pathway to promote cholesterol clearance, and upregulated PPAR?-dependent CYP7A1 expression to stimulate bile acid synthesis from cholesterol. C3R also enhanced autophagic flux, facilitating lipid droplet degradation. Molecular docking confirmed high-affinity binding of C3R to INSIG1, PCSK9, and PPAR?, with hydroxyl groups forming specific hydrogen bonds at active sites, supporting direct target engagement. These findings establish a multi-targeted mechanism for C3R in ameliorating hepatic steatosis and provide a molecular basis for anthocyanin-mediated regulation of lipid metabolism. Overall design: To elucidate the lipid-lowering mechanism of C3R in the high-fat HepG2 cell model, transcriptome sequencing was performed to analyze differentially expressed genes. The experimental design comprised five groups: a blank control (NC), a high-fat model induced with 500 µM FFA (FFA), and three C3R intervention groups at 25 µM (FCL), 50 µM (FCM), and 100 µM (FCH), each with three biological replicates. Following the treatment protocol in section 2.5, cells were trypsinized, washed with PBS, and total RNA was extracted using TRIzol reagent (1 mL per 5×106 cells). RNA concentration and purity were measured with a Nanodrop2000, integrity was verified by agarose gel electrophoresis, and RQN values were determined using an Agilent 5300 system. Qualified RNA samples were submitted to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for library preparation (Illumina NovaSeq Reagent Kit) and sequencing. Sequencing reads were aligned to the human reference genome (Ensembl: Homo_sapiens) using HiSat2, followed by gene expression quantification with RSEM.

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）以肝细胞内病理性脂质蓄积为核心病理特征。本研究聚焦于矢车菊素-3-芸香糖苷（Cyanidin-3-rutinoside, C3R）——一种兼具抗氧化与代谢调节活性的天然花青素，探讨其拮抗该病理进程的作用机制。在游离脂肪酸（FFA）诱导的HepG2细胞脂肪变性模型中，C3R可显著降低细胞内甘油三酯、总胆固醇及低密度脂蛋白胆固醇（low-density lipoprotein cholesterol, LDL-C）水平，抑制脂滴形成，并通过提升超氧化物歧化酶（Superoxide Dismutase, SOD）、过氧化氢酶（Catalase, CAT）活性与谷胱甘肽（Glutathione, GSH）含量，同时降低丙二醛（Malondialdehyde, MDA）与活性氧（Reactive Oxygen Species, ROS）水平，有效缓解氧化应激。RNA测序（RNA-sequencing, RNA-seq）分析结果显示，C3R可逆转FFA诱导的转录组表达紊乱。机制层面研究表明，C3R可通过抑制固醇调节元件结合蛋白2（Sterol Regulatory Element Binding Protein 2, SREBP2）介导的胆固醇合成通路、激活前蛋白转化酶枯草溶菌素9/低密度脂蛋白受体（Proprotein Convertase Subtilisin/Kexin Type 9/Low-Density Lipoprotein Receptor, PCSK9/LDLR）通路以促进胆固醇清除，以及上调过氧化物酶体增殖物激活受体γ（Peroxisome Proliferator-Activated Receptor γ, PPARγ）依赖的胆固醇7α-羟化酶1（Cytochrome P450 Family 7 Subfamily A Member 1, CYP7A1）表达，从而推动胆固醇向胆汁酸的转化合成。此外，C3R可增强自噬流，加速脂滴降解过程。分子对接实验证实，C3R可与胰岛素诱导基因1（Insulin Induced Gene 1, INSIG1）、PCSK9及PPARγ发生高亲和力结合，其分子结构中的羟基可在靶标活性位点形成特异性氢键，直接验证了C3R与靶标蛋白的结合作用。上述研究结果明确了C3R改善肝细胞脂肪变性的多靶点作用机制，为花青素介导的脂质代谢调控提供了坚实的分子生物学依据。
实验整体设计：为阐明C3R在高脂诱导HepG2细胞模型中的降脂作用机制，本研究通过转录组测序分析差异表达基因。本次实验共设置5组处理：空白对照组（Negative Control, NC）、500 μM FFA诱导的高脂模型组（FFA），以及25 μM（FCL）、50 μM（FCM）、100 μM（FCH）三个C3R干预组，每组均设置3次生物学重复。按照2.5章节的标准化处理流程，对各组细胞进行胰酶消化，经磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤后，采用TRIzol试剂（每5×10^6个细胞加入1 mL）提取总RNA。使用Nanodrop2000超微量核酸蛋白测定仪检测RNA的浓度与纯度，通过琼脂糖凝胶电泳验证RNA完整性，并采用安捷伦5300系统测定RNA的RQN值。将质检合格的RNA样本送至上海美吉生物医药科技有限公司，采用Illumina NovaSeq测序试剂盒完成文库构建与测序实验。使用HiSat2软件将测序得到的读段比对至人类参考基因组（Ensembl数据库：Homo_sapiens），随后通过RSEM工具完成基因表达水平的定量分析。