中药鸦胆子降三萜类成分抗胰腺癌的构效关系及作用机制研究

Pancreatic cancer has a high degree of malignancy, and the morbidity and mortality are both growing. However, the availability of efficient drugs in clinic is limited, which takes very important position in the surgical prognosis and advanced stage treatment of patients. The applicants found that the nor-triterpene components of Yadanzi, a Chinese medicine from the dry fruits of Bruceae javanica, could significantly inhibit the growth of pancreatic cancer in vitro and in vivo, with satisfied safety to human normal cells and mice. These nor-triterpene components are thus believed to be valuable lead compounds for anti-pancreatic cancers. The present proposal is designed as follows, trying to collect more solid evidences for their potential for anti-pancreatic cancer lead compounds: (1) In vitro or/and in vivo bio-activity evaluation of structurally diverse nor-triterpene components from Bruceae Fructus. (2) Investigation for the structure-activity relationship. And (3) Detailed mechanism investigation to testify the important role of ROS/p38-MAPK/JNK in nor-triterpene-induced apoptosis in pancreatic cancer cells by using N-Acetyl-L-cysteine (NAC), p38-MAPK and JNK inhibitors, as well as gene-knockout by Crispr-Case9 technique.

临床抗胰腺癌药物选择及效用有限，且耐药多发，需要发展新型药物。申请者在前期研究中初步发现中药鸦胆子降三萜类成分体内外抗胰腺癌作用确切，有效剂量下安全，提示基于鸦胆子降三萜类成分发现抗胰腺癌先导物具有潜在价值。然而，鸦胆子降三萜类成分抗胰腺癌的构效关系及具体作用机制尚未阐明。拟开展以下工作：（1）基于结构多样的鸦胆子降三萜，在多细胞系及分子水平上，系统评价其抗胰腺癌活性；经整体动物实验确认、发现高活性抗胰腺癌降三萜。（2）基于体内外抗胰腺癌活性评价，结合计算机辅助模型化方法，探究鸦胆子降三萜抗胰腺癌构效关系。（3）利用Crispr-Case 9基因敲除、N-乙酰-L-半胱氨酸（NAC）、p38及JNK抑制剂等技术手段，体内外实验阐释ROS/p38-MAPK/JNK轴在鸦胆子降三萜诱导胰腺癌细胞凋亡中的作用。为基于中药鸦胆子发现新型抗胰腺癌先导物及开发新型药物提供科学依据。

临床抗胰腺癌药物选择及效用有限，且耐药多发，亟需发展新型药物。本项目完成了中药鸦胆子降三萜成分的制备和结构鉴定，得到25种降三萜成分，其中包括1个新化合物和4个新天然产物。基于降三萜成分的结构及其体外抗胰腺癌活性评价，总结了降三萜类成分对胰腺癌细胞增殖抑制的构效关系。进一步以高活性抗胰腺癌降三萜类成分bruceine A（24 h，IC50 = 28 nM）为分子探针，阐明了bruceine A可以通过PFKFB4/GSK3β信号抑制胰腺癌Warburg效应，进而阻滞细胞周期，诱导细胞凋亡，发挥抗胰腺癌作用；确认了bruceine A可靶向p38 MAPK并激活p38 MAPK的磷酸化，进而抑制胰腺癌细胞增殖。最后，本研究发现鸦胆子中降三萜糖苷bruceoside A基于体内代谢转化成brusatol而发挥抗胰腺癌高活性的机制。综上所述，本研究为胰腺癌治疗先导物的发现及治疗策略提供了新思路，为中药鸦胆子资源利用价值新发现及高值化资源利用提供了理论和技术支撑。