基于炎症和NF-κB信号通路的重楼皂苷抗肺癌体内药效物质基础及作用机制研究

Paris polyphylla is a well-known traditional Chinese medicine with heat-clearing and detoxicating (qing re jie du) functions. Many studies showed that the main active ingredients of Paris polyphylla were steroidal saponins, however, the bioavailability of saponins were usually low in vivo，this was inconsistent with the activity. The applicant found that after oral administration of Paris polyphylla steroidal saponins, there might be metabolites transfer into the blood. Furthermore, the steroidal saponins could inhibit lung cancer and cytokines such as NF-κB and MMPs, this effect was consistent with the traditional use of anti-inflammatory and cancer. Thus, the applicant proposed that the effective components of polyphyllins in vivo play anti-lung cancer activity through regulating the inflammatory pathways. To study the effective components in vivo, the metabolism of Paris polyphylla steroidal saponins in rats should be investigated. The components of polyphllins absorbed in blood were determined via analyzing and comparing the compounds both in vivo and in vitro. On the other hand, the metabolites were isolated from the urine and feces by using chromatographic separation and preparation techniques for the first time, therefore to illustrate the effective components in vivo. Furthermore, the anti-lung cancer activity of the metabolites and polyphllins were screened by cell model in vitro and tumor-bearing mice in vivo. And then, the regulation of inflammatory and NF-κB pathways were investigated both in vivo and in vitro by using modern molecular biology techniques to clarify whether the metabolites or the steroidal saponins were responsible for the anti-tumor effect in vivo. This study illustrates the anti-inflammatory and anti-lung cancer activity of Paris polyphylla based on the effective components in vivo of Paris polyphylla steroidal saponins, provides scientific evidences for clinical treatment of lung cancer, and also provides a model for the study of the effective constituent in vivo of saponins from traditional Chinese medicine.

重楼为清热解毒中药，主要有效成分为重楼皂苷。皂苷通常体内生物利用度低，难以解释其体内药效。申请者发现重楼皂苷口服前后，血液中存在差别成分，提示其在体内可能由代谢产物发挥作用，又发现其能抑制肺癌及NF-κB、MMPs等炎症相关因子，这与重楼传统抗炎抗肺癌作用一致，由此我们提出：重楼皂苷的体内药效物质通过影响炎症相关通路，发挥抗肺癌作用。本课题基于中药整体研究思路和重楼传统用药特点，将重楼皂苷与口服后体内成分进行比较，明确其体内存在形式；从尿液、粪便等易富集样品入手，用现代色谱技术分离制备重楼皂苷大鼠体内代谢产物，揭示体内药效物质基础；用细胞及小鼠进行抗肺癌活性验证，结合现代分子生物学技术，从动物和细胞两方面探讨对炎症和NF-κB通路的调控及对肺癌的干预机制。本课题从重楼口服体内活性形式出发揭示其抗炎抗肺癌机制，为其应用于临床治疗肺癌提供科学依据，为研究中药皂苷体内如何发挥药效提供示范参考。

肺癌是临床常见原发恶性肿瘤之一，在我国是致死率最高的恶性肿瘤。随着人口老龄化加速、生存环境变化、雾霾等天气现象频发，肺癌的发病和致死率在未来还有可能呈上升趋势。在中医理论指导下，中药抗肺癌研究近年来进步较快，已显示出独有特色。重楼为清热解毒类中药，民间用于疔疮痈肿、肺痨久咳等，目前临床常用于炎症、肿瘤的治疗，其主要有效成分为重楼皂苷。本课题基于中药整体研究思路和重楼传统用药特点，将重楼皂苷与口服后体内成分进行比较，从尿液、粪便等代谢物样品入手，用现代色谱技术分析重楼皂苷大鼠体内代谢产物，揭示体内药效物质；用细胞及小鼠考查其抗肺癌活性，并探讨对炎症和NF-κB通路的调控及对肺癌的干预机制。在本研究中，我们基于高效液相色谱和质谱联用技术建立了大鼠灌胃重楼总皂苷后血浆中重楼皂苷VII浓度的检测方法，并对其在大鼠体内的药代动力学进行了研究。体内代谢研究发现大鼠灌胃重楼皂苷VII后，大部分以代谢产物的形式存在于大鼠体内，比较了重楼皂苷VII在大鼠粪便、尿液以及胆汁、血浆中的代谢产物，结合文献对这些代谢产物进行了结构推测。使用肺癌荷瘤小鼠对体内抗肿瘤效果进行了研究，重楼总皂苷能显著抑制小鼠体内肿瘤生长，且对小鼠体重影响不显著，脏器指数提示重楼总皂苷对免疫系统抑制作用较低。体外研究发现，重楼皂苷VII能显著抑制肺癌细胞增殖以及集落形成；细胞运动能力研究发现，重楼皂苷VII能抑制肺癌细胞划痕愈合，以及侵袭、迁移能力，具有良好的抗肺癌效果；作用机制研究发现，重楼皂苷VII能够降低肺癌细胞线粒体膜电位，诱导凋亡的发生，其机制与调节PI3K-Akt/NF-κB等炎性通路相关。本课题研究成果表明重楼皂苷类化合物，尤其是重楼皂苷VII有较好的抗肺癌作用效果，通过进一步深入研究，未来有望为临床治疗肺癌提供帮助。