Continually evolving viral diseases pose novel and severe threats to the health of human beings. Most recently, the ongoing COVID-19 virus has infected hundreds of millions of people globally, and has caused millions of deaths. Similarly, the influenza virus is characterized as a highly contagious and acute respiratory syndrome, causing hundreds of thousands of deaths annually (Amarelle et al., 2017). Indeed, the rates of hospitalized mortality and requirement for mechanical ventilation systems are similar between both viral diseases, particularly among pediatric patients (Laris-Gonzalez et al., 2021). Therefore, it is of critical importance to identify effective therapies to treat influenza virus infections.

Although there are four types of influenza viruses, including influenza A, B, C and D (Yu et al., 2019), the influenza A virus is the only one known to cause flu pandemics through antigenic shifts and drifts (Samji, 2009). Eight viral ribonucleoproteins (vRNPs) are contained in the influenza virus A (IVA), which encode transcripts for more than 10 essential viral proteins (polymerase acidic protein (PA); polymerase basic protein 1 (PB1); polymerase basic protein 2 (PB2); hemagglutinin (HA); neuraminidase (NA); virus nucleoprotein (NP); matrix protein 1 (M1); matrix protein 2 (M2); viral nonstructural protein 1 (NS1); and viral nonstructural protein 2 (NS2)). Each vRNP is composed of three viral polymerase subunits (PB2; PB1; PA), the NP protein, and viral negative-stranded RNA (Eisfeld et al., 2015). Of note, HA is involved in the entry of virion into the host cell and triggering endocytosis (Samji, 2009). The acidic environment of endosomes can induce fusion of the viral envelope and endosomal membrane via HA conformational transition (Dou et al., 2018). The vRNPs inside the virion dissociate from the M1 protein beneath the viral envelope via the opening of the M2 ion channel (Manzoor et al., 2017). Subsequently, the exposed nuclear localization signal on the vRNP facilitates the transport of vRNP into the nucleus for further transcription and replication (Dou et al., 2018). After the replication of vRNPs in the nucleus, the M1 protein together with NS2 (also known as nuclear export protein (NEP)) mediate the export of progeny vRNPs back into the cytosol (O'Neill et al., 1998). Finally, the co-localized HA and NA associated with the M2 protein as membrane-bending proteins facilitate the budding and spreading of virion (Gottlinger, 2010). Additionally, the M1 protein participates in the budding off of the viral particle at the budding site (Samji, 2009).

After viral infection occurs, the host cell can activate the autophagy process to interrupt viral replication (Zhou et al., 2009); however, various strategies have been developed by the virus to block the autophagy process in host cells (Jordan and Randall, 2012). The influenza virus can escape the antiviral activity of the host cells by inhibiting the fusion of autophagosomes with lysosomes (Gannage et al., 2009), whereby it can direct host cells to replicate and spread the progeny virus. Both Beclin-1and LC3B are important markers of autophagy. More specifically, Beclin-1 can initiate phagophores formation during autophagy (Funderburk et al., 2010). During the elongation stage of autophagy, cytosolic LC3B is converted from LC3B I to LC3B II for the formation of autophagosomes; meanwhile, M2 proteins inhibit the fusion of autophagosomes with lysosomes to form autolysosomes. As a consequence, LC3B II accumulates in the host cell during influenza infection (Rossman et al., 2019). The ratio of LC3B II to LC3B I may be used as an indicator of autophagic flux (Mizushima et al., 2010). These mechanisms help the influenza virus to utilize the autophagic machinery to promote viral replication in the host cells.

Traditional Chinese medicine (TCM) has been used to treat various viral infections for thousands of years, while it has more recently been reported as effective in the treatment of COVID-19 (Qiu et al., 2020). Of particular note, Banlangen is one of the most commonly used herbs in the treatment of upper respiratory tract infections, having been recorded in the Shen Nong Ben Cao Jing, the earliest TCM text which dates back over two thousand years (Yu et al., 2014). Banlangen (Isatidis Radix) is the dried root of the cruciferous plant, Isatis indigotica Fort. (I. indigotica). Various compounds of I. indigotica have been identified and isolated, including alkaloids, nucleosides, amino acids, organic acids, phenylpropanoids, steroids, volatile oils, and polysaccharides (He et al., 2014, Zhou and Zhang, 2013, Chen et al., 2021). Of interest here, studies have reported that I. indigotica possesses several antiviral effects, including against HSV-1, HSV-2, respiratory syncytial virus, and the varicella-zoster virus (Guo et al., 2011, He et al., 2014, Xu et al., 2019). It has also been shown to inhibit influenza virus adsorption via cytoprotective activity (Ke et al., 2012), and suppress influenza A virus-induced inflammation via impeding host TLR3 signaling (Li et al., 2017). Many compounds of I. indigotica, including fractions of water extract with a molecular weight of 3500 to 7000 Da, clemastanin B, epigoitrin, phenylpropanoid portions, alkaloids, and organic acid fractions have been reported to inhibit influenza viral attachment to the host cell surface, and to exhibit direct virucidal activity on MDCK cells (Xiao et al., 2016, Yang et al., 2012), although the exact mechanisms within host cells remain unclear and warrant further investigation. Therefore, we herein aimed to investigate the anti-influenza effects of I. indigotica within host cells, including viral protein production and the effect on the autophagic mechanism. It is important to note that the host cells investigated in this study were human alveolar epithelial cells, which are commonly used for studies of the respiratory system.