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The Modified Guizhi Fuling Decoction (MGFD) is used in clinical practice to treat multiple tumors. This research focused on the mechanisms of action (MOA) in MGFD that inhibit PCa. Methods: The impact of MGFD on PCa cells (PC3 and DU145) was examined via Cell Counting Kit-8, wound healing assays, and transwell assays. To determine the MOA, high-throughput sequencing based high-throughput screening (HTS2) was utilized along with network pharmacology. Results: The findings indicated that MGFD suppressed the proliferation, migration, and invasion of PCa cells. We then utilized the HTS2 assay to generate 270 gene expression profiles from PCa cells perturbed by MGFD. Large-scale transcriptional analysis highlighted three pathways closely associated with PCa: the TNF signaling pathway, cellular senescence, and FoxO signaling pathway. Through the combination of network pharmacology and bioinformatics, we discovered four primary targets through which MGFD acts on PCa: AKT serine\/threonine kinase 1 (AKT1), Caspase-8 (CASP8), Cyclin-Dependent Kinase 1 (CDK1), and Cyclin D1 (CCND1). Finally, molecular docking demonstrated that the potential bioactive compounds baicalein, quercetin, and 5-[[5-(4-methoxyphenyl)-2-furyl] methylene] barbituric acid strongly bind to CDK1, AKT1, and CASP8, respectively. Conclusions: This research shows that MGFD displays encouraging anticancer effects via various mechanisms. Its multi-target activity profile underscores its promise as a potential therapeutic option for PCa treatment and encourages additional in vivo validation studies.<\/jats:p>","DOI":"10.3390\/ph18091275","type":"journal-article","created":{"date-parts":[[2025,8,27]],"date-time":"2025-08-27T15:49:34Z","timestamp":1756309774000},"page":"1275","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Large-Scale Transcriptome Profiling and Network Pharmacology Analysis Reveal the Multi-Target Inhibitory Mechanism of Modified Guizhi Fuling Decoction in Prostate Cancer Cells"],"prefix":"10.3390","volume":"18","author":[{"given":"Guochen","family":"Zhang","sequence":"first","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Xiang","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingzhou","family":"Li","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingming","family":"Wei","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiankuo","family":"Yu","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8737-3082","authenticated-orcid":false,"given":"Yan","family":"Luo","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6494-5271","authenticated-orcid":false,"given":"Jianping","family":"Chen","sequence":"additional","affiliation":[{"name":"LKS Faculty of Medicine, School of Chinese Medical, The University of Hong Kong, Hong Kong 999077, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xilinqiqige","family":"Bao","sequence":"additional","affiliation":[{"name":"Medical Innovation Center for Nationalities, Inner Mongolia Medical University, Hohhot City 010110, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7877-7440","authenticated-orcid":false,"given":"Dong","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiyi","family":"Zhou","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"17","DOI":"10.3322\/caac.21763","article-title":"Cancer Statistics, 2023. 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