Mechanistic Investigation of Shuanghuanglian Against Infectious Bronchitis in Chickens: A Network Pharmacology and Molecular Dynamics Study

Fuming You ^1,2 ZhangHanZhao ZHz ^1,2 Chuanhong Li ^1,3 Yuxia Yang ^1,3 Yongqiang Wang ⁴ Rigetu Zhao ⁵ Luomeng Chao ^1,2,6,7*

• ¹ Inner Mongolia University for Nationalities, Tongliao, China
• ² College of Animal Science and Technology, Inner Mongolia University for Nationnalities, Tongliao, Inner Mongolia Autonomous Region, China
• ³ School of Computer Science and Technology, Inner Mongolia University for Nationalities, Tongliao, Inner Mongolia Autonomous Region, China
• ⁴ Zhalantun Vocational College, Hulun Buir, China
• ⁵ Chifeng Institute of Agricultural and Animal Husbandry Science, Chifeng, China
• ⁶ Inner Mongolia Rambo Testing Technology Limited Company, Tongliao, China
• ⁷ Inner Mongolia Engineering Technology Research Center for Prevention and Control of Beef Cattle Diseases, Tongliao, China

Introduction: Infectious bronchitis (IB) poses a major challenge to global poultry production, causing substantial economic burdens and underscoring the necessity for novel therapeutic interventions given the limitations of current vaccines and conventional antiviral agents.Shuanghuanglian and their interaction with the key pathological targets of IBV infection. By using advanced computational methods, this study aims not only to identify the therapeutic potential of active ingredients, but also to reveal their mechanism of action against IBV.Methods: Through integrative systems pharmacology approaches, we systematically investigated Shuanghuanglian and its phytochemical constituents against IB, employing multi-omics analysis , ensemble machine learning, and all-atom molecular dynamics (MD) simulations . Network pharmacology revealed 65 target genes associated with Shuanghuanglian's primary bioactive components (quercetin, kaempferol, wogonin, and luteolin), exhibiting high network centrality.Results: Using the TCMSP database, we found 65 target genes associated with key active components, such as quercetin and kaempferol, which exhibited strong connectivity in our network analysis. The GeneCards database also identified 40 common target genes shared by Shuanghuanglian and IB. Importantly, BCL2 and IL6 were recognized as key targets in the proteinprotein interaction (PPI) network analysis, highlighting their roles in apoptosis and inflammation. Furthermore, analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed significant roles in regulating the cell cycle and inflammatory responses. Machine learning techniques identified BCL2 and IL6 as critical genes for therapeutic intervention, supported by molecular docking results that showed strong binding energies. Furthermore, molecular dynamics simulations confirm the stability of the complexes, underscoring the importance of these interactions for treatment efficacy. Conclusion: We used a variety of analytical methods, and finally identified the potential active ingredients of Shuanghuanglian as kaempferol, quercetin, wogonin, and luteolin. The active ingredients target BCL2 and IL6 and play a therapeutic role in avian infectious bronchitis by inhibiting apoptosis and reducing inflammatory response.