{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T11:05:24Z","timestamp":1779447924098,"version":"3.53.1"},"reference-count":48,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T00:00:00Z","timestamp":1778112000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Yunnan Provincial Science and Technology Talent and Platform Program","award":["202405AF140031"],"award-info":[{"award-number":["202405AF140031"]}]},{"name":"Yunnan Provincial Science and Technology Talent and Platform Program","award":["202405AC350024"],"award-info":[{"award-number":["202405AC350024"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: Bitong Mixture (BTM) is used clinically for rheumatoid arthritis (RA), but its active components and mechanisms remain unclear. Methods: WGCNA, network pharmacology, machine learning, and ROC analysis were integrated to identify candidate targets. We used molecular docking and molecular dynamics simulations for prioritization. In vitro validation was performed in TNF-\u03b1-stimulated MH7A cells. Results: Multiple analytical approaches consistently identified JUN and PPARG. Formononetin and Rhein showed favorable binding and stable interactions. Both compounds significantly reduced IL6 and MMP9 expression and suppressed fibroblast activation. Conclusions: BTM may exert anti-inflammatory effects through a JUN- and PPARG-related regulatory framework, supported by integrated computational and experimental evidence, providing a foundation for further in vivo investigation.<\/jats:p>","DOI":"10.3390\/ph19050735","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T14:05:46Z","timestamp":1778162746000},"page":"735","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Formononetin and Rhein from Bitong Mixture Alleviate Rheumatoid Arthritis-Related Inflammation: An Integrated WGCNA, Machine Learning and In Vitro Study"],"prefix":"10.3390","volume":"19","author":[{"given":"Futing","family":"Tan","sequence":"first","affiliation":[{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiangtao","family":"Wang","sequence":"additional","affiliation":[{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keqing","family":"Fan","sequence":"additional","affiliation":[{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Runzi","family":"Gao","sequence":"additional","affiliation":[{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali 671003, China"},{"name":"Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2925-0428","authenticated-orcid":false,"given":"Zhibin","family":"Yang","sequence":"additional","affiliation":[{"name":"Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical R&D, College of Pharmacy, Dali University, Dali 671003, China"},{"name":"National-Local Joint Engineering Research Center of Entomoceutics, Dali 671003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2023","DOI":"10.1016\/S0140-6736(16)30173-8","article-title":"Rheumatoid arthritis","volume":"388","author":"Smolen","year":"2016","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/j.intimp.2017.11.013","article-title":"Quercetin attenuates collagen-induced arthritis by restoration of Th17\/Treg balance and activation of heme oxygenase-1-mediated anti-inflammatory effect","volume":"54","author":"Yang","year":"2018","journal-title":"Int. Immunopharmacol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1080\/07853890410026025","article-title":"Risk factors for rheumatoid arthritis","volume":"36","author":"Aho","year":"2004","journal-title":"Ann. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1007\/s00296-010-1705-x","article-title":"A PTPN22 promoter polymorphism \u22121123G>C is associated with RA pathogenesis in Chinese","volume":"32","author":"Huang","year":"2012","journal-title":"Rheumatol. Int."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e15122","DOI":"10.1111\/1756-185X.15122","article-title":"The role of microbiota on rheumatoid arthritis onset","volume":"27","year":"2024","journal-title":"Int. J. Rheum. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1016\/j.sjbs.2021.09.037","article-title":"Novel functional polymorphism on PADI-4 gene and its association with arthritis onset","volume":"29","author":"Mukhtar","year":"2022","journal-title":"Saudi J. Biol. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.intimp.2018.07.028","article-title":"Analysis of miRNA expression in patients with rheumatoid arthritis during remission and relapse after a 5-year trial of tofacitinib treatment","volume":"63","author":"Bastian","year":"2018","journal-title":"Int. Immunopharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2205","DOI":"10.1056\/NEJMra1004965","article-title":"The pathogenesis of rheumatoid arthritis","volume":"365","author":"McInnes","year":"2011","journal-title":"N. Engl. J. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"12671","DOI":"10.1038\/s41598-017-12570-6","article-title":"Functional role of PPAR-\u03b3 on the proliferation and migration of fibroblast-like synoviocytes in rheumatoid arthritis","volume":"7","author":"Li","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3605","DOI":"10.4049\/jimmunol.1601330","article-title":"The AP-1 transcription factor c-Jun promotes arthritis by regulating cyclooxygenase-2 and arginase-1 expression in macrophages","volume":"198","author":"Hannemann","year":"2017","journal-title":"J. Immunol."},{"key":"ref_11","unstructured":"Dong, L. (2019). The Bitong Mixture: Research on the Effects in the Adjuvant Arthritis Rat Model. [Master\u2019s Thesis, Nanjing University of Chinese Medicine]."},{"key":"ref_12","first-page":"449","article-title":"Effect of Bitong mixture on synovial tissue in rats with adjuvant arthritis","volume":"8","author":"Dong","year":"2019","journal-title":"J. Pharm. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12953","DOI":"10.1021\/acs.jafc.8b05047","article-title":"Resveratrol alleviates rheumatoid arthritis via reducing ROS and inflammation, inhibiting MAPK signaling pathways, and suppressing angiogenesis","volume":"66","author":"Yang","year":"2018","journal-title":"J. Agric. Food Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1829","DOI":"10.1007\/s00296-012-2657-0","article-title":"Resveratrol inhibits TNF-\u03b1-induced IL-1\u03b2 and MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of the PI3-kinase\/Akt pathway","volume":"33","author":"Tian","year":"2013","journal-title":"Rheumatol. Int."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Sheng, S., Wang, X., Liu, X., Hu, X., Shao, Y., Wang, G., Mao, D., Li, C., Chen, B., and Chen, X. (2022). The role of resveratrol on rheumatoid arthritis: From bench to bedside. Front. Pharmacol., 13.","DOI":"10.3389\/fphar.2022.829677"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1016\/j.chembiol.2007.03.007","article-title":"Glycyrrhizin binds to high-mobility group box 1 protein and inhibits its cytokine activities","volume":"14","author":"Mollica","year":"2007","journal-title":"Chem. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"C917","DOI":"10.1152\/ajpcell.00401.2005","article-title":"High mobility group box 1 protein interacts with multiple Toll-like receptors","volume":"290","author":"Park","year":"2006","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1179\/096805102125001091","article-title":"HMGB1 as a cytokine and therapeutic target","volume":"8","author":"Yang","year":"2002","journal-title":"J. Endotoxin Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2466","DOI":"10.1093\/bioinformatics\/btp465","article-title":"Network analyses in systems pharmacology","volume":"25","author":"Berger","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"105454","DOI":"10.1016\/j.compbiomed.2022.105454","article-title":"Mechanisms underlying the therapeutic effects of Qingfeiyin in treating acute lung injury based on GEO datasets, network pharmacology and molecular docking","volume":"145","author":"Wang","year":"2022","journal-title":"Comput. Biol. Med."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Hu, Z., Li, Y., Zhang, L., Jiang, Y., Long, C., Yang, Q., and Yang, M. (2024). Metabolic changes in fibroblast-like synoviocytes in rheumatoid arthritis: State of the art review. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1250884"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1186\/s13075-023-03208-2","article-title":"Comprehensive multi-omics analysis reveals the core role of glycerophospholipid metabolism in rheumatoid arthritis development","volume":"25","author":"Jian","year":"2023","journal-title":"Arthritis Res. Ther."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1038\/s12276-022-00725-z","article-title":"Lipidome profile predictive of disease evolution and activity in rheumatoid arthritis","volume":"54","author":"Koh","year":"2022","journal-title":"Exp. Mol. Med."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Lei, Q., Yang, J., Li, L., Zhao, N., Lu, C., Lu, A., and He, X. (2023). Lipid metabolism and rheumatoid arthritis. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1190607"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1959","DOI":"10.1002\/art.40569","article-title":"JAK\/STAT blockade alters synovial bioenergetics, mitochondrial function, and proinflammatory mediators in rheumatoid arthritis","volume":"70","author":"McGarry","year":"2018","journal-title":"Arthritis Rheumatol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Langfelder, P., and Horvath, S. (2008). WGCNA: An R package for weighted correlation network analysis. BMC Bioinform., 9.","DOI":"10.1186\/1471-2105-9-559"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1172\/JCI12466","article-title":"c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis","volume":"108","author":"Han","year":"2001","journal-title":"J. Clin. Investig."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Huang, J., Fu, X., Chen, X., Li, Z., Huang, Y., and Liang, C. (2021). Promising therapeutic targets for treatment of rheumatoid arthritis. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.686155"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1111\/cei.13256","article-title":"Dendritic cells, T cells and their interaction in rheumatoid arthritis","volume":"196","author":"Wehr","year":"2019","journal-title":"Clin. Exp. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/1758-2946-6-13","article-title":"TCMSP: A database of systems pharmacology for drug discovery from herbal medicines","volume":"6","author":"Ru","year":"2014","journal-title":"J. Cheminform."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"D1197","DOI":"10.1093\/nar\/gkaa1063","article-title":"HERB: A high-throughput experiment- and reference-guided database of traditional Chinese medicine","volume":"49","author":"Fang","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"W357","DOI":"10.1093\/nar\/gkz382","article-title":"SwissTargetPrediction: Updated data and new features for efficient prediction of protein targets of small molecules","volume":"47","author":"Daina","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1186\/s13321-016-0130-x","article-title":"Improving chemical similarity ensemble approach in target prediction","volume":"8","author":"Wang","year":"2016","journal-title":"J. Cheminform."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"W609","DOI":"10.1093\/nar\/gkq300","article-title":"PharmMapper server: A web server for potential drug target identification using pharmacophore mapping approach","volume":"38","author":"Liu","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/978-1-60761-977-2_1","article-title":"Protein bioinformatics databases and resources","volume":"694","author":"Chen","year":"2011","journal-title":"Methods Mol. Biol."},{"key":"ref_36","first-page":"D845","article-title":"The DisGeNET knowledge platform for disease genomics: 2019 update","volume":"48","author":"Ronzano","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/S0168-9525(97)01103-7","article-title":"GeneCards: Integrating information about genes, proteins and diseases","volume":"13","author":"Rebhan","year":"1997","journal-title":"Trends Genet."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"D1265","DOI":"10.1093\/nar\/gkad976","article-title":"DrugBank 6.0: The DrugBank knowledgebase for 2024","volume":"52","author":"Knox","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"D1465","DOI":"10.1093\/nar\/gkad751","article-title":"TTD: Therapeutic Target Database describing target druggability information","volume":"52","author":"Zhou","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"D991","DOI":"10.1093\/nar\/gks1193","article-title":"NCBI GEO: Archive for functional genomics data sets\u2014Update","volume":"41","author":"Barrett","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e36","DOI":"10.1002\/imt2.36","article-title":"Sangerbox: A comprehensive, interaction-friendly clinical bioinformatics analysis platform","volume":"1","author":"Shen","year":"2022","journal-title":"iMeta"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Tang, D., Chen, M., Huang, X., Zhang, G., Zeng, L., Zhang, G., Wu, S., and Wang, Y. (2023). SRplot: A free online platform for data visualization and graphing. PLoS ONE, 18.","DOI":"10.1371\/journal.pone.0294236"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"17","DOI":"10.2202\/1544-6115.1128","article-title":"A general framework for weighted gene co-expression network analysis","volume":"4","author":"Zhang","year":"2005","journal-title":"Stat. Appl. Genet. Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"D607","DOI":"10.1093\/nar\/gky1131","article-title":"STRING v11: Protein\u2013protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets","volume":"47","author":"Szklarczyk","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"W60","DOI":"10.1093\/nar\/gky311","article-title":"GeneMANIA update 2018","volume":"46","author":"Franz","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"2927","DOI":"10.1093\/bioinformatics\/btq562","article-title":"GeneMANIA Cytoscape plugin: Fast gene function predictions on the desktop","volume":"26","author":"Montojo","year":"2010","journal-title":"Bioinformatics."},{"key":"ref_47","unstructured":"Schr\u00f6dinger, LLC (The PyMOL Molecular Graphics System, 2024). The PyMOL Molecular Graphics System, Version 3.1.6.1."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2785","DOI":"10.1002\/jcc.21256","article-title":"AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility","volume":"30","author":"Morris","year":"2009","journal-title":"J. Comput. Chem."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/5\/735\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T10:14:53Z","timestamp":1779444893000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/19\/5\/735"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,7]]},"references-count":48,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ph19050735"],"URL":"https:\/\/doi.org\/10.3390\/ph19050735","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,7]]}}}