Key Laboratory of Ethnomedicine of Ministry of Education, Center on Translational Neuroscience, School of Pharmacy, Minzu University of China, Beijing, 100081, PR China.
Department of Urology, Affiliated Hospital of Chengde Medical University, Chengde, Hebei, 067000, China.
J Ethnopharmacol. 2021 May 10;271:113907. doi: 10.1016/j.jep.2021.113907. Epub 2021 Feb 6.
Gynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia and Southeast Asia. In clinic, it is mainly used for hyperlipidemia and antitumor. Its antitumor activity was first recorded in "Illustrated Catalogue of Plants". Gypenosides were the main active ingredients of G. pentaphyllum. The anticancer activity of gypenosides in vivo and in vitro had been widely reported. However, the mechanism of gypenosides in renal cell carcinoma (RCC) still unclear.
In this study, we tried to investigate the active constituents from G. pentaphyllum and potential mechanisms in RCC treatment through network pharmacology and in vitro experiments.
MATERIAL/METHODS: Active compounds and their targets were evaluated and screened through TCMSP and Swiss Target Prediction database. Notably, nine preliminary screened components obtained from database were identified by LC-MS and LC-MS/MS. The targets associated with RCC were obtained from OMIM, TTD and GeneCards database. The PPI network and active component/target/pathway networks were constructed to identify the potential drug targets using String database and Cytoscape software. The functions and pathways of targets were analyzed through DAVID database. Finally, AutoDockTools 1.5.6 was used for molecular docking to assess the binding ability between compounds and targets. To support our prediction, we then explore the antitumor effect and mechanism of gypenosides by vitro experiments. CCK8 and flow cytometry were performed to evaluate cell death treated with gypenosides. Quantitative real-time PCR and Western blot were conducted to detect the changes of PI3K/AKT/mTOR signaling pathway.
Nine saponins and 68 targets have been screened. The hub targets covered PIK3CA, VEGFA, STAT3, JAK2, CCND1 and MAPK3. Enrichment analysis showed that the pathways mainly contained PI3K/Akt/mTOR, HIF-1, TNF, JAK-STAT and MAPK signaling pathways. Gypenosides extracted from G. pentaphyllum showed strong activity against 786-O and Caki-1 cells, and cell apoptosis were detected through Annexin V/PI dual staining assay. RT-qPCR showed that gypenosides downregulated the levels of PIK3CA, Akt and mTOR in Caki-1 and 786-O cells. Mechanistically, gypenosides induced apoptosis of RCC cells through regulating PI3K/Akt/mTOR signaling pathway which was implemented though decreasing the phosphorylation level of Akt and mTOR.
Gypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.
绞股蓝（Thunb.）Makino 是一种传统药物，在中国、东亚和东南亚广泛使用。临床上主要用于高血脂和抗肿瘤。其抗肿瘤活性最早记载于《植物图鉴》。绞股蓝皂苷是绞股蓝的主要活性成分。已广泛报道绞股蓝皂苷在体内和体外的抗癌活性。然而，绞股蓝皂苷在肾细胞癌（RCC）中的作用机制尚不清楚。
本研究通过网络药理学和体外实验，试图从绞股蓝中筛选活性成分并探讨其在 RCC 治疗中的潜在机制。
材料/方法：通过 TCMSP 和 Swiss Target Prediction 数据库评估和筛选活性化合物及其靶点。值得注意的是，从数据库中获得的九种初步筛选成分通过 LC-MS 和 LC-MS/MS 进行了鉴定。从 OMIM、TTD 和 GeneCards 数据库中获得与 RCC 相关的靶点。使用 String 数据库和 Cytoscape 软件构建 PPI 网络和活性成分/靶点/通路网络，以鉴定潜在的药物靶点。通过 DAVID 数据库分析靶标的功能和通路。最后，使用 AutoDockTools 1.5.6 进行分子对接，以评估化合物与靶标的结合能力。为了支持我们的预测，我们通过体外实验探索了绞股蓝皂苷的抗肿瘤作用和机制。用 CCK8 和流式细胞术评估用绞股蓝皂苷处理后的细胞死亡情况。定量实时 PCR 和 Western blot 用于检测 PI3K/AKT/mTOR 信号通路的变化。
筛选出 9 种皂苷和 68 个靶点。枢纽靶标包括 PIK3CA、VEGFA、STAT3、JAK2、CCND1 和 MAPK3。富集分析表明，这些通路主要包括 PI3K/Akt/mTOR、HIF-1、TNF、JAK-STAT 和 MAPK 信号通路。从绞股蓝中提取的绞股蓝皂苷对 786-O 和 Caki-1 细胞表现出很强的活性，并通过 Annexin V/PI 双重染色检测到细胞凋亡。RT-qPCR 显示，绞股蓝皂苷下调了 Caki-1 和 786-O 细胞中 PIK3CA、Akt 和 mTOR 的水平。在机制上，绞股蓝皂苷通过调节 PI3K/Akt/mTOR 信号通路诱导 RCC 细胞凋亡，该通路通过降低 Akt 和 mTOR 的磷酸化水平来实现。
绞股蓝皂苷通过调节 PI3K/Akt/mTOR 信号通路诱导 RCC 细胞凋亡。
