Abstract:

Objective

To investigate the mechanism of dihydroartemisinin （DHA） against influenza A virus （IAV） in human non-small cell lung cancer cells （A549） and normal human lung epithelial cells （BEAS-2B）.

Methods

The CC₅₀ values of DHA on A549 and BEAS- 2B cells was calculated after treatment with different concentrations of DHA for 24 hours. A549 cells were infected with H1N1 for 3 hours， then treated with different concentrations of DHA （0.1， 1， and 10 μmol/ L）for 24 hours， and cell viability was measured using the CCK-8 assay. The inhibitory effect of DHA on H1N1 replication was detected by plaque assay. Metabolomic was employed to analyze the differential metabolites induced by DHA following H1N1 infection， and the KEGG pathway enrichment analysis was conducted to map the pathways associated with these differential metabolites.The contents of reduced glutathione （GSH） and oxidized glutathione （GSSG）， intracellular reactive oxygen species （ROS）， and mitochondrial function were measured using commercial kits. The expression of RIG1， MAVS， p-TBK1， and p-IRF3 proteins was detected by Western blot. The mRNA expression and secretion levels of IFN-α and IFN-β were measured by real-time quantitative PCR（RT-qPCR） and enzyme-linked immunosorbent assay （ELISA）， respectively. The comparison of means between two groups was performed using an independent samples t-test， the comparison of means among multiple groups was conducted using one-way ANOVA， and pairwise comparisons of means among multiple groups were tested using the Tukey's test method.

Results

The CC₅₀ values of DHA in A549 and BEAS-2B cells was 18.26 μmol/ L and 28.61 μmol/L， respectively. At a concentration of 1 μmol/L， DHA enhanced cell viability and reduced plaque formation. DHA inhibited the metabolism of glutamate， glycine， and ornithine induced by influenza virus infection by regulating glutathione metabolism， and preventing GSH depletion as well as the formation of GSSG and ROS，thereby alleviating mitochondrial damage. Furthermore， DHA treatment increased the expression of RIG1 and MAVS proteins， promoting the aggregation of MAVS to mitochondria， and activating the phosphorylation of TBK1 and IRF3 proteins， while enhanced both mRNA expression and protein secretion of IFN-α and IFN-β.

Conclusion

DHA exerts its antiviral effects against influenza virus by regulating intracellular glutathione homeostasis， reducing ROS formation， alleviating mitochondrial damage， and enhancing the innate immune response.

Key words: Influenza A virus, Dihydroartemisinin, Metabolomics, Innate immune