Abstract
In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation - fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1β, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.
Highlights
Influenza A virus (IAV) poses a global health and economic threat as the worldwide pandemic
Human Atg5 and Atg12 genes were fused with the N9and C9- fragments of a red fluorescence protein (RFP), respectively
To our knowledge, there were 7 examples that had not been reported to possess anti-IAV activity, i.e. Peucedanum praeruptorum Dunn., Borneolum syntheticum, Ginkgo biloba L., Silybum marianum (L.) Gaertn., Buddleja lindleyana Fort., Eugenia caryophyllata Thunb., and Evodia rutaecarpa Benth, and in these 7 medicinal plants, the crude extract of Evodia rutaecarpa Benth showed the highest activity, so we chose Evodia rutaecarpa Benth as our medicinal plant of interest, and purchased evodiamine (Evo), the major active component of Evodia rutaecarpa Benth, detected the anti-IAV activity of evodiamine and checked the availability of our drug screening method, we explored the mechanisms of evodiamine on anti-IAV activities
Summary
Influenza A virus (IAV) poses a global health and economic threat as the worldwide pandemic. Current antiviral drugs are limited by their negative side effects and by the emergence of drug-resistant strains [1]. Current vaccine production is still problematic due to the difficulty of working with the relatively low immunogenicity of some strains and the need to protect against the large number of strains circulating in the environment [2,3,4,5]. It is still urgent to develop novel drug screening methods. Autophagy inhibition is a good strategy for developing novel anti-IAV drugs
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