Effect of Haoqin Qingdan Tang on influenza A virus through the Janus kinase/signal transducer and activator of transcription (JAK/ STAT) pathway

Objective: Influenza, a viral respiratory illness, leads to seasonal epidemics and occasional pandemics. Given the rising resistance and adverse reactions associated with anti-influenza drugs, Traditional Chinese Medicine (TCM) emerges as a promising approach to counteract the influenza virus. Specifically, Haoqin Qingdan Tang (HQQDT), a TCM formula, has been employed as an adjuvant treatment for influenza in China. However, the active compounds and underlying mechanisms of HQQDT remain unknown. Aim: The aim of this study was to investigate HQQDT ' s antiviral and anti-inflammatory activities in both in vivo and in vitro , and further reveal its active ingredients and mechanism. Methods: In vivo and in vitro experiments were conducted to verify the antiviral and anti-inflammatory activities of HQQDT. Subsequently, the active ingredients and mechanism of HQQDT were explored through combining high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF MS) analysis and network pharmacology. Finally, the examinations of cell cytokines and signaling pathways aimed to elucidate the predicted mechanisms. Results: The results indicated that HQQDT exhibited inhibitory effects on influenza viruses A/PR/8/34 (H1N1), A/HK/1/68 (H3N2), and A/California/4/2009 (H1N1) in vitro . Furthermore, HQQDT enhanced the survival rate of influenza-infected mice, reduced the lung index and lung virus titer, and mitigated lung tissue damage in vivo . The proinflammatory cytokine expression levels upon influenza virus infection in PR8-induced A549 cells or mice were suppressed by HQQDT, including IL-6, IL-1 beta , CCL 2 , CCL 4 , IP-10, interferon beta 1 (IFN- beta 1), the interferon regulatory factor 3 (IRF3), and hemagglutinin (HA). Twenty-two active components of HQQDT against influenza were identified using HPLC-Q-TOF-MS analysis. Based on network pharmacological predictions, the JAK/STAT signaling pathway is considered the most relevant for HQQDT ' s action against influenza. Finally, western blot assays revealed that HQQDT regulated the protein level of the JAK/STAT signaling pathway in PR8-infected A549 cells and lung tissue. Conclusion: These findings verified the antiviral and anti-inflammatory effects of HQQDT through JAK-STAT signaling pathway in influenza infections, laying the foundation for its further development.