Hemsleya amabilis Diels，属于葫芦科，是传统中药（TCM）。它被广泛用于治疗各种疾病。然而，这些疾病可能促进RCC的发展。研究了Hemsleya amabilis Diels根提取物（HRE）的抗癌活性，并阐明了其体内和体外的潜在分子机制。用乙酸乙酯提取干燥的Hemsleya amabilis Diels根，用于治疗RCC4，OS-RC-2和ACHN细胞。使用UHPLC-MS分析提取物的化学成分。使用CCK-8和集落形成实验研究细胞增殖。使用PI染色检测细胞周期。使用Annexin-V-FITC，AO/EB和TEM评估细胞凋亡。使用Transwell和伤口愈合实验评估细胞迁移和入侵。使用RNA-seq，网络药理学，虚拟筛选的自动对接和分子动力学模拟分析HRE抑制RCC增殖的潜在分子机制和活性成分。使用LY294002和UC2288分别抑制PI3K和P21的表达。使用IGF-1激活PI3K。建立异种移植瘤模型评估其体内抗肿瘤潜能。使用免疫组织化学和Western blot检测蛋白质表达水平。使用H&E染色探索HRE在体内的副作用。应用生物信息学分析P21对RCC的影响。HRE由739个化合物组成。CCK-8和集落形成实验显示HRE显著抑制RCC细胞增殖。PI染色表明HRE导致G2/M期阻滞。Annexin-V-FITC，AOEB和TEM实验证明HRE显著促进RCC细胞的凋亡。Transwell和伤口愈合实验显示HRE能够抑制RCC细胞的迁移和入侵。RNA-seq显示HRE引起230个基因的改变。网络药理学分析发现HRE-组分-靶点-RCC之间的关系。自动对接发现HRE中的Epitulipinolide二氧化物可以稳定地与PIK3CA结合（-7.22 kJ/mol），分子动力学模拟验证了Epitulipinolide二氧化物与PIK3CA的结合。在RCC4细胞中，IGF-1预处理减弱了HRE诱导的凋亡和G2/M期阻滞。当预处理PIK3抑制剂LY294002时，出现了相反的结果。预处理CDKN1A（P21）抑制剂UC2288减弱了HRE诱导的G2/M期阻滞。异种移植瘤模型显示HRE抑制了肿瘤生长。Western blot分析表明HRE可以调节Bax，Bcl-2，PARP，清除的PARP，Caspase-9，Caspase-8，Caspase-3，Survivin，Cyclin-B1，CDK1，N-钙粘蛋白，蜗牛，slug，E-钙粘蛋白，MMP-9。免疫组织化学染色显示，在处理组中，E-钙粘蛋白，Bax，P21的表达上调，而N-钙粘蛋白，PI3K，AKT和Bcl-2的表达下调。H&E染色显示与对照组相比，HRE治疗组的主要器官没有组织学异常。生物信息学分析表明，在P21高表达组中，RCC患者的总体生存率高于P21低表达组。HRE通过EMT抑制RCC的迁移和侵袭，并在体内和体外抑制增殖。此外，HRE通过促进凋亡和P21诱导的G2/M期阻滞通过PI3K/AKT信号通路抑制增殖。总的来说，这些结果表明HRE可能是一种有前景的RCC化疗药物。版权所有 © 2023. 由Elsevier B.V.出版。

Hemsleya amabilis Diels, belongs to cucurbitaceae, was traditional Chinese medicine (TCM). It is widely used to treat various diseases. However, these diseases may contribute to the development of RCC.investigated the anticancer activities of root extract of Hemsleya amabilis Diels (HRE), and elucidated the underlying molecular mechanism in vivo and in vitro.Dried Hemsleya amabilis Diels roots were extracted by ethyl acetate and used to treat RCC4, OS-RC-2 and ACHN cells. UHPLC-MS was used to analyze the chemical composition of the extract. CCK-8 and colony formation assay were used to investigate proliferation. PI staining was used to detect cell cycle. Annexin-V-FITC, AO/EB and TEM were used to evaluate apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. RNA-seq, Network pharmacology, autodocking for virtual screening and molecular dynamics simulation were used to analyze potential molecular mechanisms and active components of HRE inhibiting proliferation of RCC. LY294002 and UC2288 were used to inhibit PI3K and P21 expression, respectively. IGF-1 was used to activate PI3K. Xenograft tumor model was established to evaluate its anti-tumor potential in vivo. Immunohistochemistry and Western blot were used to test protein expression levels. H&E staining was used to explore the side effects of HRE in vivo. Applying bioinformatics to analyze the effect of P21 on RCC.HRE consists of 739 compounds. CCK-8 and colony formation assay showed that HRE significantly inhibited RCC cells proliferation. PI staining indicated that HRE caused G2/M phase arrest. Annexin-V-FITC, AOEB and TEM experiments revealed that HRE significantly promoted apoptosis of RCC cells. Transwell and wound healing assays showed that HRE can inhibit the migration and invasion of RCC cells. RNA-seq showed that HRE induced 230 gene changes. Network pharmacology analysis found the relationship between HRE-component-target-RCC. Auto-docking found that Epitulipinolide diepoxide in HRE can stably bind to PIK3CA (-7.22 kJ/mol), and molecular dynamics simulation verified the combination between Epitulipinolide diepoxide of PIK3CA. In RCC4 cells, pretreatment with IGF-1, attenuated HRE-induced apoptosis and G2/M arrest. When pretreated with PIK3 inhibitor LY294002, the opposite result appears. Pretreatment with CDKN1A (P21) inhibitor UC2288 attenuated HRE-induced G2/M arrest. Xenograft tumor model showed that HRE inhibited tumor growth. Western blot analysis indicated that HRE can regulating Bax, Bcl-2, PARP, cleared-PARP, Caspase-9, Caspase-8, Caspase-3, Survivin, Cyclin-B1, CDK1, N-cadherin, snail, slug, E-cadherin, MMP-9. Immunohistochemical staining showed that in the treated group, expression of E-cadherin, Bax, P21 was up-regulated, while N-cadherin, PI3K, AKT and Bcl-2 were down-regulated. H&E staining showed that compared to control groups, the main organs in the HRE-treated groups showed no histological abnormalities. The overall survival rate of RCC patients in the high-expression group of P21 was higher than in the low-expression group of P21 on bioinformatics analysis.HRE inhibited RCC migration and invasion through EMT, and inhibited proliferation in vivo and in vitro. In addition, HRE inhibited proliferation through promoting apoptosis and P21-induced G2/M phase arrest via PI3K/AKT signaling pathway. Overall, these results suggest that HRE may be a promising chemotherapy agent for RCC.Copyright © 2023. Published by Elsevier B.V.