Objective  To investigate the mechanism of action of Shuanghuanglian (SHL) against respiratory syncytial virus (RSV) infection based on GEO datasets, network pharmacology, molecular docking, and molecular dynamics simulations.

Methods  The active ingredients of SHL and their potential targets were screened through the TCMSP and TCMID databases, while the disease targets of RSV infection were obtained from the GEO, GeneCards, OMIM, and TTD databases. The common targets of SHL and RSV infection were then identified. Core target molecules were identified through protein-protein interaction (PPI) network analysis; the key signaling pathway signals were acquired through GO functional and KEGG pathway enrichment analyses; a drug-component-target-pathway network was constructed; and finally, molecular docking and molecular dynamics simulation were performed by AutoDockTools 1.5.6 and AutoDock Vina 4.2 to verify the binding affinity between the key active components of SHL and the core action targets.

Results  A total of 35 active components and 326 gene targets of SHL were obtained in this study. The primary bioactive components of SHL included eriodictyol, kaempferol, luteolin, and quercetin. PPI network analysis identified 10 core targets for SHL in treating RSV, including signal transducer and activator of transcription 1 (STAT1), AKT ser-ine/threonine kinase 1 (AKT1), and prostaglandin-endoperoxide synthase 2 (PTGS2). KEGG enrichment and network analysis indicated that the antiviral effects of SHL are primarily mediated through the TNF, HIF, and PI3K-Akt signaling pathways. Molecular docking and MD simulations confirmed that the active components of SHL exhibit significant binding affinity with these core targets.

Conclusion  This study reveals the potential mechanism of action of SHL in treating RSV infection, which involves the synergistic action of eriodictyol, kaempferol, luteolin, and quercetin on the TNF, HIF, and PI3K-Akt signaling pathways.

1. 李岩异, 吕娜, 马祝青. 人呼吸道合胞病毒感染的预防和治疗 研 究 进 展 [J]. 生 物 技 术 进 展, 2024, 14(1): 26-34. [Li YY, Lyu N, Ma ZQ. Research progress in the prevention and treatment of human respiratory syncytial virus infection[J]. Current Biotechnology, 2024, 14(1): 26-34.] DOI: 10.19586/j.2095- 2341.2023.0140.

2. Rosas-Salazar C, Chirkova T, Gebretsadik T, et al. Respiratory syncytial virus infection during infancy and asthma during childhood in the USA (INSPIRE): a population-based, prospective birth cohort study[J]. Lancet, 2023, 401(10389): 1669-1680. DOI: 10.1016/s0140-6736(23)00811-5.

3. Li Y, Wang X, Blau DM, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis[J]. The Lancet, 2022, 399(10340): 2047-2064. DOI: 10.1016/S0140-6736(22)00478-0.

4. Qiu X, Xu S, Lu Y, et al. Development of mRNA vaccines against respiratory syncytial virus (RSV)[J]. Cytokine Growth Factor Rev, 2022, 68: 37-53. DOI: 10.1016/j.cytogfr.2022.10.001.

5. Borchers AT, Chang C, Gershwin ME, et al. Respiratory syncytial virus-a comprehensive review[J]. Clin Rev Allergy Immunol, 2013, 45(3): 331-379. DOI: 10.1007/s12016-013-8368-9.

6. Gatt D, Martin I, Alfouzan R, et al. Prevention and treatment strategies for respiratory syncytial virus (RSV)[J]. Pathogens, 2023, 12(2). DOI: 10.3390/pathogens12020154.

7. Papi A, Ison MG, Langley JM, et al. Respiratory syncytial virus prefusion f protein vaccine in older adults[J]. N Engl J Med, 2023, 388(7): 595-608. DOI: 10.1056/NEJMoa2209604.

8. Agac A, Kolbe S M, Ludlow M, et al. Host responses to respiratory syncytial virus infection[J]. Viruses, 2023, 15(10): 1999. DOI: 10.3390/v15101999.

9. Jenkins VA, Hoet B, Hochrein H, et al. The quest for a respiratory syncytial virus vaccine for older adults: thinking beyond the F protein[J]. Vaccines (Basel), 2023, 11(2): 382. DOI: 10.3390/ vaccines11020382.

10. Mir WAY, Shrestha DB, Rana W, et al. Successful treatment of respiratory syncytial virus infection in an immunocompromised patient with ribavirin[J]. Cureus, 2021, 13(8): e16930. DOI: 10.7759/cureus.16930.

11. Ohmit SE, Moler FW, Monto AS, et al. Ribavirin utilization and clinical effectiveness in children hospitalized with respiratory syncytial virus infection[J]. J Clin Epidemiol, 1996, 49(9): 963- 967. DOI: 10.1016/0895-4356(96)00137-0.

12. Chen Z, Ye SY. Research progress on antiviral constituents in traditional Chinese medicines and their mechanisms of action[J]. Pharm Biol, 2022, 60(1): 1063-1076. DOI: 10.1080/13880209. 2022.2074053.

13. Gao Y, Fang L, Cai R, et al. Shuang-Huang-Lian exerts anti- inflammatory and anti-oxidative activities in lipo-polysaccharide- stimulated murine alveolar macrophages[J]. Phytomedicine, 2014, 21(4): 461-469. DOI: 10.1016/j.phymed.2013.09.022.

14. Kong XT, Fang HT, Jiang GQ, et al. Treatment of acute bronchiolitis with Chinese herbs[J]. Arch Dis Child, 1993, 68(4): 468-471. DOI: 10.1136/adc.68.4.468.

15. Xu Z, Li K, Pan T, et al. Lonicerin, an anti-algE flavonoid against Pseudomonas aeruginosa virulence screened from Shuanghuanglian formula by molecule docking based strategy[J]. J Ethnopharmacol, 2019, 239: 111909. DOI: 10.1016/j.jep.2019.111909.

16. Ma Q, Liang D, Song S, et al. Comparative study on the antivirus activity of Shuang-Huang-Lian Injectable Powder and its bioactive compound mixture against human adenovirus Ⅲ in vitro[J]. Viruses, 2017, 9(4): 79. DOI: 10.3390/v9040079.

17. Guo B, Zhao C, Zhang C, et al. Elucidation of the anti-inflammatory mechanism of Er Miao San by integrative approach of network pharmacology and experimental verification[J]. Pharmacol Res, 2022, 175: 106000. DOI: 10.1016/j.phrs.2021.106000.

18. Casas AI, Hassan AA, Larsen SJ, et al. From single drug targets to synergistic network pharmacology in ischemic stroke[J]. Proc Natl Acad Sci U S A, 2019, 116(14): 7129-7136. DOI: 10.1073/ pnas.1820799116.

19. Jiao G, Fan X, Wang Y, et al. Dissection of the active ingredients and potential mechanism of Han-Shi-Yu-Fei-decoction in treating COVID-19 based on in vivo substances profiling and clinical symptom-guided network pharmacology[J]. ACS Omega, 2022, 7(41): 36598-36610. DOI: 10.1021/acsomega.2c04589.

20. Jones JM, Fleming-Dutra KE, Prill MM, et al. Use of nirsevimab for the prevention of respiratory syncytial virus disease among infants and young children: recommendations of the advisory committee on immunization practices-United States, 2023[J]. MMWR Morb Mortal Wkly Rep, 2023, 72(34): 920-925. DOI: 10.15585/mmwr. mm7234a4.

21. 杜海涛, 孙铁锋, 王平, 等. 清热药抗呼吸道合胞病毒的研究进展 [J]. 中成药, 2019, 41(10): 2435-2441.[Du HT, Sun TF, Wang P, et al. Research progress of antipyretic drugs against respiratory syncytial virus[J]. Chinese Traditional Patent Medicine, 2019, 41(10): 2435-2441.] DOI: 10.3969/j.issn.1001-1528.2019.10.031.

22. Zhuang Z, Wen J, Zhang L, et al. Can network pharmacology identify the anti-virus and anti-inflammatory activities of Shuanghuanglian oral liquid used in Chinese medicine for respiratory tract infection?[J]. Eur J Integr Med, 2020, 37: 101139. DOI: 10.1016/j.eujim.2020.101139.

23. Shivakumar D, Williams J, Wu Y, et al. Prediction of absolute solvation free energies using molecular dynamics free energy perturbation and the OPLS force field[J]. J Chem Theory Comput, 2010, 6(5): 1509-1519. DOI: 10.1021/ct900587b.

24. Periferakis A, Periferakis AT, Troumpata L, et al. Kaempferol: a review of current evidence of its antiviral potential[J]. Int J Mol Sci, 2023, 24(22): 16299. DOI: 10.3390/ijms242216299.

25. Wang S, Ling Y, Yao Y, et al. Luteolin inhibits respiratory syncytial virus replication by regulating the MiR-155/SOCS1/ STAT1 signaling pathway[J]. Virol J, 2020, 17(1): 187. DOI: 10.1186/s12985-020-01451-6.

26. Sun YL, Zhao PP, Zhu CB, et al. Integrating metabolomics and network pharmacology to assess the effects of quercetin on lung inflammatory injury induced by human respiratory syncytial virus[J]. Sci Rep, 2023, 13(1): 8051. DOI: 10.1038/s41598-023- 35272-8.

27. Franke G, Freihorst J, Steinmüller C, et al. Interaction of alveolar macrophages and respiratory syncytial virus[J]. J Mmunol Methods, 1994, 174(1-2): 173-184. DOI: 10.1016/0022-1759(94)90020-5

28. Neuzil KM, Tang YW, Graham BS. Protective role of TNF-alpha in respiratory syncytial virus infection in vitro and in vivo[J]. Am J Med Sci, 1996, 311(5): 201-204. DOI: 10.1097/00000441- 199605000-00001.

29. Sankuntaw N, Punyadee N, Chantratita W, et al. Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ 1 and CXCL10 expression in human primary bronchial epithelial cells[J]. New Microbiol, 2024, 47(1): 60-67. https://pubmed.ncbi. nlm.nih.gov/38700885/.

30. Morris DR, Qu Y, Agrawal A, et al. HIF-1α modulates core metabolism and virus replication in primary airway epithelial cells infected with respiratory syncytial virus[J]. Viruses, 2020, 12(10): 1088. DOI: 10.3390/v12101088.

31. Kilani MM, Mohammed KA, Nasreen N, et al. RSV causes HIF-1 alpha stabilization via NO release in primary bronchial epithelial cells[J]. Inflammation, 2004, 28(5): 245-251. DOI: 10.1007/ s10753-004-6047-y.

32. Haeberle HA, Dürrstein C, Rosenberger P, et al. Oxygen- independent stabilization of hypoxia inducible factor (HIF)-1 during RSV infection[J]. PLoS One, 2008, 3(10): e3352. DOI: 10.1371/journal.pone.0003352.

33. Chen LF, Cai JX, Zhang JJ, et al. Respiratory syncytial virus co- opts hypoxia-inducible factor-1α-mediated glycolysis to favor the production of infectious virus[J]. mBio, 2023, 14(5): e0211023. DOI: 10.1128/mbio.02110-23.

34. Dunn EF, Fearns R, Connor JH. Akt inhibitor Akt-IV blocks virus replication through an Akt-independent mechanism[J]. J Virol, 2009, 83(22): 11665-11672. DOI: 10.1128/jvi.01092-09.

35. Utpal BK, Mokhfi FZ, Zehravi M, et al. Resveratrol: a natural compound targeting the PI3K/Akt/mTOR pathway in neurological diseases[J]. Mol Neurobiol, 2025, 62(5): 5579-5608. DOI: 10.1007/ s12035-024-04608-4.

36. 梁玉婷, 吕婧, 刘璐, 等. 基于 UPLC-Q-Exactive Orbitrap-MS 联合网络药理学探究双黄连解热抗炎的作用机制[J].中国医院药学杂志, 2022, 42(6): 573-581, 589.[Liang YT, Lyu J, Liu L, et al. Based on UPLC-Q-Exactive Orbitrap-MS combined with network pharmacology to explore the antipyretic and antiinflammatory mechanism of Shuanghuanglian[J]. Chinese Journal of Hospital Pharmacy, 2022, 42(6): 573-581, 589.] DOI: 10.13286/j.1001-5213.2022.06.01.

37. 郑锋, 陆凤娟, 徐颖, 等. 双黄连颗粒联合阿奇霉素干混悬剂治疗小儿支原体肺炎的临床疗效观察 [J]. 中药材, 2015, 38(2): 416-418.[Zheng F, Lu FJ, Xu Y, et al. Clinical observation of Shuanghuanglian granule combined with azithromycin dry suspension in the treatment of Mycoplasma pneumonia inchildren[J]. Journal of Chinese Medicinal Materials, 2015, 38(2): 416-418.] DOI: 10.13863/j.issn1001-4454.2015.02.052.

38. 李徽, 王春彤, 赵占秋, 等. 病毒性心肌炎患者血清 miR、Th 指标变化及双黄连注射液干预效果研究 [J]. 中华医院感染学杂志, 2016, 26(17): 3909-3911. [Li H, Wang CT, Zhao ZQ, et al. Changes of serum miR and Th indexes of patients with viral myocarditis and intervention effect of Shuanghuanglian Injection[J]. Chinese Journal of Nosocomiology, 2016, 26(17): 3909-3911.] DOI: 10.11816/cn.ni.2016-161252.