蒺藜果是植物学中的Tribulus terrestris L.（TT）的拉丁名，在《神农本草经》中首次有记载，被中国人用作镇肝、祛痰、促进血液循环、改善视力和缓解瘙痒等草药已有数千年历史。此外，古代中国还用它来治疗乳腺癌。然而， TT提取物在乳腺癌上的药理活性受到的关注较少。本研究对这种草药的抗乳腺癌效应和可能的作用机制进行了调查。网络药理学分析研究了TT的抗乳腺癌效应的可能性。随后，使用Autodock软件进行TT7/TT8与血管内皮生长因子受体2（VEGFR2）的分子对接，并通过Western blot分析相关蛋白质表达以验证此效应。体内实验：通过注射4T1细胞建立乳腺癌小鼠模型。然后，药物每天一次经口给药给小鼠连续十四天。实验结束时记录体重、肿瘤大小和肿瘤重量。此外，计算肿瘤抑制率。最后，分别通过HE染色和荧光染色法评估乳腺癌组织的病理变化和细胞凋亡。蛋白质组学和代谢组学分析：选择肿瘤组织进行联合分析。首先，发现差异蛋白质和代谢物。随后，通过软件分析它们的功能。最后，在肿瘤组织中进行了SGPP1、SPHK1和p-SPHK1的免疫荧光染色。网络药理学研究获得TT的12个活性成分、127个活性成分的靶向蛋白、15,253个乳腺癌的靶向蛋白、1,225个乳腺肿瘤的靶向蛋白以及123个重叠基因。TT7/TT8与VEGFR2之间存在紧密的连接。此外，与模型组相比，TT组的肿瘤大小和重量显著减少。TTM组的肿瘤抑制率超过26%。药物治疗后，发现许多脂肪细胞和肿瘤之间的裂缝以及细胞凋亡。Western blot结果显示，TT水提物降低了VEGFR2、ERK1/2、p-ERK1/2（Thr202, Tyr204）和Bcl2的水平，同时提高了Bax的水平和Bax/Bcl2比值。此外，在TTM组和模型组之间发现了495个差异蛋白质和76个差异代谢物，其中鞘脂代谢通路富集。最后，TT治疗显著降低了肿瘤组织中SGPP1、SPHK1和p-SPHK1的水平。总之，TT在乳腺癌小鼠模型中展现了治疗效果，并且其作用机制涉及鞘脂代谢信号通路的调节。这项研究支持TT提取物作为乳腺癌治疗的药理潜力。版权所有 © 2023 Elsevier GmbH。保留所有权利。

Tribulus terrestris L. (TT) was initially documented in Shen-Nong-Ben-Cao-Jing and has been used for thousands of years in China as a herb to calm liver, dispel melancholy and wind, promote blood circulation, improve eyesight, and relieve itching. Moreover, it was also used to treat breast cancer in ancient China. However, the pharmacological activities of TT extract on breast cancer have received little attention.In this study, we investigated the anti-breast cancer effects and possible mechanisms of action of this herbal drug.Network pharmacology analysis the study of network pharmacology was done to analyze the possibility of TT's anti-breast cancer effect. And then, molecular docking between TT7/TT8 and vascular endothelial growth factor receptor 2 (VEGFR2) were performed by Autodock software as well as the related protein expressions were analyzed by western blot to verify this effect. In vivo experiment: The mouse model of breast cancer was established by injection of 4T1 cells. Then drugs were intragastrically administered to the mice once daily for fourteen days. Body weight, tumor size, and tumor weight were recorded at the end of the experiment. Moreover, tumor inhibitory rate was calculated. Finally, pathological changes and apoptosis of breast cancer tissues were respectively evaluated by HE and Hoechst staining. Proteomics and metabonomics analyses: The tumor tissues were chosen to perform conjoint analysis. Firstly, differential proteins and metabolites were found. Furthermore, the functional analyses of them were analyzed by software. At the last, immunofluorescent staining of SGPP1, SPHK1 and p-SPHK1 in tumor tissue were done.12 active ingredients of TT, 127 targets of active ingredients, 15,253 targets of breast cancer, 1,225 targets of Ru yan, and 123 overlapping genes were obtained in the network pharmacology study. There was firm conjunction between TT7/TT8 and VEGFR2. Besides, tumor size and weight were markedly reduced in TT groups compared to the model group. The tumor inhibitory rate was more than 26% in TTM group. After drug treatment, many adipocytes and cracks between tumor and apoptosis were discovered. The western blot results showed that TT aqueous extract lowered the levels of VEGFR2, ERK1/2, p-ERK1/2 (Thr202, Tyr204) and Bcl2, while increasing the levels of Bax and the ratio of Bax/Bcl2. Furthermore, 495 differential proteins and 76 differential metabolites were found between TTM and model groups with the sphingolipid metabolism pathway being enriched. At last, TT treatment significantly reduced the levels of SGPP1, SPHK1 and p-SPHK1 in tumor tissue.In conclusion, TT demonstrates therapeutic effects in a mouse model of breast cancer, and its mechanism of action involves the regulations of sphingolipid metabolism signaling pathways. This study lends credence to the pharmacological potential of TT extract as a breast cancer therapy.Copyright © 2023 Elsevier GmbH. All rights reserved.