Re Du Ning exerts anti-influenza activity by counteracting viral NS1-mediated suppression of host type I interferon signaling

Re Du Ning (RDN) injection is a modern Chinese herbal formulation with heat-clearing, wind-dispersing, and detoxifying properties. It is clinically used for the treatment of influenza-related high fever and upper respiratory tract infections, with validated clinical efficacy and a favorable safety profile. Although its anti-inflammatory activity has been extensively documented, the antiviral effects of RDN against influenza A virus (IAV) remain controversial, and the underlying mechanisms are not fully elucidated.

To investigate the mechanisms and active constituents through which RDN exerts anti-influenza activity by counteracting viral nonstructural protein 1 (NS1)-mediated suppression of host type I interferon (IFN-I) signaling.

The anti-IAV efficacy of RDN was evaluated by monitoring mortality, body weight, lung index, pulmonary viral load, and histopathological changes. qRT-PCR and Western blotting were used to quantify IFN-α/β and interferon-stimulated genes (ISG15, MX1, OAS1, and PKR) at defined time points in vivo, as well as to assess the activation of the IFNAR1/IFNAR2-JAK1/TYK2-STAT1/STAT2 signaling pathway. The contribution of STAT1 and STAT2 to the antiviral effects of RDN was examined using siRNA-mediated knockdown. The effects of NS1 on IFN-I signaling and the inhibitory role of RDN were assessed by evaluating interferon-stimulated response element (ISRE)-luciferase reporter activation, JAK1/TYK2-STAT1/STAT2 phosphorylation, STAT1/STAT2 nuclear translocation, and NS1-associated protein complexes. Chromatographic profiles of RDN and its polarity fractions were obtained by HPLC, and peak constituents were identified by HPLC-Q-TOF-MS/MS. Anti-NS1 constituents were identified using spectrum-effect relationship analysis and subsequently validated.

RDN provided significant protection against IAV infection in mice, reducing mortality, attenuating body weight loss, lowering lung index and viral load, and alleviating lung injury. After infection, IFN-α/β expression remained consistently lower in the RDN (H) group than in the model group. In contrast, ISG15, MX1, OAS1, and PKR levels in the RDN (H) group were comparable to those in the model group on days 1 and 2 post-infection, whereas ISG15 and MX1 were markedly elevated on day 4. Consistently, STAT1 and STAT2 phosphorylation was transiently increased on day 4 relative to the model group. siRNA-mediated knockdown of STAT1 or STAT2 reduced, but did not abolish, the anti-IAV activity of RDN. RDN dose-dependently inhibited NS1 (A/Puerto Rico/8/34, PR8)-mediated suppression of ISRE-luciferase activity. NS1 suppressed phosphorylation of JAK1, TYK2, STAT1, and STAT2 and impaired STAT1/STAT2 nuclear translocation. RDN mitigated NS1-mediated suppression of STAT1 and STAT2 phosphorylation and nuclear translocation both in vitro and in vivo. No detectable association was observed between NS1 and either exogenously expressed STAT1 or STAT2; however, NS1 was found to associate with exogenously expressed JAK1 and TYK2, and these associations were attenuated by RDN. Chlorogenic acid was identified as one of the major contributing constituents that weakened the NS1-JAK1 association and mitigated NS1-mediated suppression of STAT1/STAT2 phosphorylation. By contrast, neochlorogenic acid alleviated NS1-mediated inhibition of STAT1 phosphorylation but did not significantly affect NS1-JAK1 or NS1-TYK2 associations.

This study demonstrates that RDN exerts significant anti-IAV activity. JAK1 and TYK2 may be previously underappreciated host kinases associated with PR8 NS1. RDN partially restored host IFN-I-JAK/STAT-ISG antiviral signaling, potentially by attenuating NS1-JAK1/TYK2 associations. Chlorogenic acid was identified as one of the major constituents contributing to the anti-NS1 activity of RDN. These findings provide mechanistic insight into the host-directed antiviral activity of RDN.