基于PI3K-Akt通路探索炒川楝子治疗乳腺良恶性增生的炮制机制

Toosendan Fructus is a Chinese herbal medicine that can promote the circulation of Qi and exert analgesic effect.Because of its ability to disperse the liver depression, it can be successfully used in clinical breast byperplasia treatment, either malignant or non-malignant, however, the processing mechanism remains poorly understood. By exploring the novel pharmacological activity of toosendanin, an active component isolated from Toosendan Fructus,we found that toosendanin was able to inhibit the abnormal proliferation of human breast cells, significantly reverse the triple-negative breast carcinoma drug resistance towards doxorubici.To understand the mechanism, toosendanin was found to selectively suppress the expression of PI3Kinase subunit, p110alpha. Furthermore, the inhibitory effect of fried Toosendan Fructus was significantly higher than that of raw one. We there presume that there are active components from fried Toosendan Fructus that may have more potent inhibitory on p110 expression than that of toosendanin. To prove this hypothesis, we plan to establish drug-screening system based on p110alpha promoter luciferase activity, identify active component from fried Toosendan Fructus that can selectively suppress p110 expression in abormally proliferative breast cells, and meanwhile, investigate its molecular mechanism and chemical transformation. The results of this study will offer preliminary data on the understanding of the processing mechanisms for Toosendan Fructus, provide theoretical basis for the clinical treatment of breast-related disease and candidate drugs.

川楝子长于行气杀虫、解郁止痛，取其疏肝理气之功，它在临床乳腺疾病中的治疗价值逐渐得到肯定。为探索该机制，我们发现川楝子有效成分川楝素能抑制雌二醇作用下乳腺细胞异常增生，显著逆转三阴性乳腺癌细胞对阿霉素的耐药，且川楝素与阿霉素联用抑制肿瘤生长率大于90%。进一步研究发现川楝素能选择性抑制肿瘤靶标PI3K调节亚基p110ɑ的表达，且炒川楝子作用明显强于生品。由此我们推测炒川楝子中含有抑制p110ɑ表达作用更强的活性成分。为验证该推测，本项目拟建立p110ɑ启动子报告基因筛选细胞系，从炒川楝子中寻找对PI3K亚基p110ɑ表达选择性抑制作用强的有效成分，并深入探索其分子作用机制与化学转化过程，以期初步阐明炒川楝子用于乳腺良恶性增生治疗的炮制机制，为临床相关乳腺疾病治疗提供理论参考与候选药物。

川楝子是一味行气中药，取其疏肝理气之功，它在临床乳腺疾病中的治疗价值逐渐得到肯定。川楝子临床上常炮制入药，为探索该机制，我们发现川楝子有效成分川楝素能抑制雌二醇作用下乳腺细胞异常增生，显著逆转三阴性乳腺癌细胞对阿霉素的耐药，进一步研究发现川楝素能选择性抑制肿瘤靶标PI3K调节亚基p110ɑ的表达，且炒川楝子作用明显强于生品，定量研究表明川楝子炒后川楝素的含量降低，由此推测炒川楝子中含有抑制p110ɑ表达作用更强的活性成分。为验证该推测，建立p110ɑ启动子报告基因筛选细胞系，发现炒川楝子正丁醇部位是对PI3K亚基p110ɑ表达选择性抑制作用强的有效部位。分子作用机制研究发现转录因子BNC2在川楝子抑制p110ɑ基因表达中发挥关键作用，BNC2参与p110ɑ基础与激活状态下转录活性。通过抑制BNC2，川楝子能显著抑制p110ɑ基因转录与蛋白表达，进而抑制乳腺癌细胞耐药糖蛋白p-gp的基因与蛋白表达，增强乳腺癌细胞对阿霉素的敏感性。该研究初步阐明炒川楝子用于乳腺良恶性增生治疗的炮制机制，发现川楝素可能是一种新型PI3K选择性抑制剂，为临床相关乳腺疾病治疗提供理论参考与候选药物。