沙棘叶结合态多酚通过调控PKM选择性剪接抑制Warburg效应的抗肿瘤活性成分及作用机制

Sea buckthorn (Hippophae rhamnoides L.) Elaeagnaceae, a sheepberry with small berries, has recently gained worldwide attention mainly for its medicinal and nutritional potential. Previous studies have shown that polyphenols in Hippophae rhamnoides leaves exhibited significant antitumor effects, however, the antitumor active components and underlying molecular mechanism remain unclear. PKM1 and PKM2 are two common subtypes of pyruvate kinase (PK), which are generated through PKM alternative splicing. Compared with differentiated tissues, tumor cells prefer to adopt aerobic glycolysis called Warburg effect, which provides an advantage for tumor growth, and the high expression of PKM2 plays an important role in it. Currently, we found that bound polyphenols from sea buckthorn leaf (SBLBP) inhibit PKM2 expression and promote PKM1 expression by regulating PKM alternative splicing to reverse Warburg effect, thereby inhibiting the growth of colon cancer cells. This project aims to explore the active components and molecular mechanisms of SBLBP to inhibit the proliferation of colon cancer cells. The contents are as follows: separating and purifying the chemical components of SBLBP using macroporous resin, sephadex column and UPLC technology, identifying the compound structures by ESI-MS and spectral method (13C-NMR, 1H-NMR and IR); Obtaining a large amount of SBLBP components with preparative HPLC method, confirming the active ingredient in SBLBP that possess antitumor effect in vitro and in vivo, named SBLBP-A; determining the key splicing factor that regulating PKM alternative splicing, elucidating the molecular mechanism of SBLBP-A inhibiting tumor proliferation by reversing Warburg effect. Based on the resource advantage of our province, the project provides a theoretical basis for the exploitation and utilization of wild sea buckthorn resources in the field of biological medicine.

沙棘属多年生落叶灌木，是非常珍贵的药食同源植物。已有研究表明沙棘叶多酚具抗肿瘤活性，但其抗肿瘤活性成分及作用机制尚不明确。丙酮酸激酶PKM1和PKM2由PKM基因选择性剪接产生，其中PKM2在肿瘤细胞Warburg效应中发挥重要作用。前期研究发现，沙棘叶结合态多酚（SBLBP）通过调控PKM选择性剪接逆转Warburg效应，进而抑制肿瘤细胞生长。本项目将确定SBLBP中抑制肿瘤细胞增殖的活性成分及分子机制。研究内容包括：采用多种色谱技术分析SBLBP成分并鉴定单体结构；分离获得SBLBP各成分，通过细胞水平和裸鼠模型实验确定SBLBP中抗肿瘤活性成分SBLBP-A；确定SBLBP-A调控PKM选择性剪接，抑制PKM2表达的关键剪接因子，阐明SBLBP-A通过逆转Warburg效应抑制肿瘤增殖的分子机制。本项目立足我省资源优势，为当地野生沙棘资源在生物医药领域的开发利用提供理论依据。

沙棘属多年生落叶灌木，是非常珍贵的药食同源植物。沙棘叶多酚种类多、含量高，具有抗氧化、消炎、增强免疫力及抑癌等作用。与正常细胞不同，肿瘤细胞即使在氧气存在的情况下也主要进行糖酵解代谢，这种现象称为 Warburg效应。丙酮酸激酶PKM1和PKM2由PKM基因选择性剪接产生，其中PKM2在肿瘤细胞Warburg效应中发挥重要作用。本研究从沙棘叶中提取结合态多酚，分离鉴定了其中的多酚成分，并探讨其通过逆转 PKM 选择性剪接介导的肿瘤糖酵解发挥抗结肠癌效应的具体分子机制。研究内容和结果包括：（1）确定沙棘叶中的多酚成分。研究发现甲醇提取自由态多酚、碱水解法提取沙棘叶结合态多酚为最优提取方法；HPD600为最优纯化树脂，60%乙醇为最佳洗脱液。采用LC-MS分离鉴定了 8 种多酚成分，主要为山奈酚及其衍生物。（2）明确沙棘多酚的抗结肠癌效应。通过MTT 法、WB 实验及流式分析，发现沙棘多酚可诱导结肠癌细胞凋亡、G1期阻滞来抑制结肠癌的生长。（3）探讨沙棘多酚发挥抗结肠癌效应的分子机制。结果表明沙棘多酚处理后葡萄糖消耗、乳酸和 ATP 生成均降低；PKM1表达升高，PKM2及剪接因子hnRNPA1、PTBP1表达下降；miR-339-5p 的水平升高，且直接与剪接因子的3’UTR区结合，进而抑制 PKM2 的表达。综上所述，此研究分离鉴定了沙棘结合态多酚中的抗肿瘤活性成分，并阐明其通过miR-339-5p/PTBP1/PKM2信号轴重塑肿瘤糖代谢，进而发挥抗肿瘤活性的分子机制。此外，还从沙棘果中分离获得抗肿瘤活性多酚HPs60，在细胞水平及裸鼠成瘤模型发现其可通过调控miR-195/497介导的细胞周期阻滞，及miR-195-5p/ miR-497-5p/ miR-1247-3p介导的细胞凋亡发挥抗结肠癌效应。以上研究为结肠癌的治疗提供新思路，为充分利用沙棘资源奠定理论基础。