Integrating bioinformatic analysis, network pharmacology, molecular docking and experimental validation to explore the mechanism of Tian Long Cha against influenza virus

Ethnopharmacological relevance

Tong Long Cha (TLC) has been employed in clinical treatment for respiratory diseases, such as influenza, for over three decades. However, the precise mechanisms underlying its defense against influenza remain poorly understood.

Aim of study

This study aimed to investigate the bioactive compounds, pharmacological effects, and underlying mechanisms of TLC in combating influenza viruses.

Methods

Ultrahigh-performance liquid chromatography (UPLC)-Q-Exactive analysis was employed to identify the bioactive compounds of TLC. Key therapeutic targets and pathways involved in TLC's treatment for influenza were predicted using bioinformatics analysis, network pharmacology, and molecular docking. The antiviral effects of TLC and its principal active compound against various strains of influenza A viruses (A/Aichi/2/1968 (H3N2), A/PR/8/34 (PR8), A/California/04/2009 (CA04), A/HK/Y280/97(H9N2)) and influenza B virus (B/Lee/1940 (Lee)) were assessed using the cytopathic effect (CPE) inhibition assay and plaque reduction assay in MDCK cells. The therapeutic effects of TLC were evaluated using an influenza H3N2 virus-infected Balb/c mouse model. Quantitative PCR and Western blot analyses were employed to quantify the expression levels of key targets involved in TLC's potential mechanisms within A549 cells and the lungs of mice, as well as to investigate BCL's (baicalin) mechanism in A549 cells post-H3N2 infection. A co-culture model using Jurkat T cells and H3N2-infected A549 cells was also established to verify the modulation of key targets by TLC and BCL by using Western blot analyses.

Results

A total of 25 bioactive compounds were identified in TLC, with BCL being the predominant compound. TLC and BCL significantly inhibited the replication of the aforementioned influenza virus strains in MDCK cells. Additionally, TLC reduced weight loss, lung index, viral titers, lung tissue lesions, and levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IP-10, TNF-α), interferon (IFN-γ), and chemokine (MCP-1) in H3N2-infected mice. Mechanistic studies revealed that TLC and BCL upregulated Interleukin (IL)-2-inducible T cell kinase (ITK) and Tyrosine-protein kinase (FYN) expression while downregulating mitogen-activated protein kinase (MAPK14) expression, thereby modulating the T cell receptor signaling pathway, as predicted by bioinformatics analysis, network pharmacology, and molecular docking.

Conclusion

TLC could inhibit influenza virus replication and mitigate excessive inflammatory responses by modulating the T cell receptor signaling pathway, suggesting that it may serve as a promising therapeutic agent in traditional Chinese medicine for the treatment of influenza.