ß-sitosterol ameliorates influenza A virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between RIG-I and IFN/STAT signaling.

ß-Sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol Chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. In this study we investigated the effects of ß-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. We demonstrate that ß-sitosterol (150-450 µg/mL) dose-dependently suppresses inflammatory response through NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling in influenza A virus (IAV)-infected cells, which was accompanied by decreased induction of interferons (IFNs) (including Type I and III IFN). Furthermore, we revealed that the anti-inflammatory effect of ß-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene I (RIG-I) signaling, led to decreased STAT1 signaling, thus affecting the transcriptional activity of ISGF3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the IAV-induced proinflammatory amplification effect in IFN-sensitized cells. Moreover, ß-sitosterol treatment attenuated RIG-I-mediated apoptotic injury of alveolar epithelial cells (AEC) via downregulation of pro-apoptotic factors. In a mouse model of influenza, pre-administration of ß-sitosterol (50, 200 mg·kg-1·d-1, i.g., for 2 days) dose-dependently ameliorated IAV-mediated recruitment of pathogenic cytotoxic T cells and immune dysregulation. In addition, pre-administration of ß-sitosterol protected mice from lethal IAV infection. Our data suggest that ß-sitosterol blocks the immune response mediated by RIG-I signaling and deleterious IFN production, providing a potential benefit for the treatment of influenza.

In vitro anti-influenza virus activities of a new lignan glycoside from the latex of Calotropis gigantea.

A new lignan glycoside, (+)-pinoresinol 4-O-[6″-O-vanilloyl]-ß-D-glucopyranoside (1) and two known phenolic compounds, 6'-O-vanilloyltachioside (2) and 6'-O-vanilloylisotachioside (3) were isolated from the latex of Calotropis gigantea (Asclepiadaceae). The structure of the new compound was elucidated by using spectroscopic and chemical methods. Three isolates (1-3) and one authentic compound, (+)-pinoresinol 4-O-ß-D-glucopyranoside, were screened for A/PR/8/34 (H1N1) inhibitory activity by cytopathic effect (CPE) inhibition assay on MDCK cells. Compound 1 showed inhibitory activity against A/PR/8/34 (H1N1). In sharp contrast, the other three compounds (2, 3 and (+)-pinoresinol 4-O-ß-D-glucopyranoside) did not show such activity. An analysis of structure-activity relationship between 1 and (+)-pinoresinol 4-O-ß-D-glucopyranoside revealed that the presence of a vanilloyl group in the sugar moiety of 1 is crucial for its anti-influenza virus activity. Compound 1 was further evaluated for in vitro inhibitory activities against a panel of human and avian influenza viruses by CPE inhibition assay. It showed inhibitory effect against human influenza viruses in both subtypes A and B (IC50 values around 13.4-39.8 µM with SI values of 3.7-11.4), while had no effect on avian influenza viruses. Its antiviral activity against human influenza viruses subtype A was further confirmed by plaque reduction assay. The time course assay indicated that 1 exerts its antiviral activity at the early stage of viral replication. A mechanistic study showed that 1 efficiently inhibited influenza virus-induced activation of NF-κB pathway in a dose-dependent manner, but had no effect on virus-induced activation of Raf/MEK/ERK pathway. Further studies demonstrated that nuclear translocation of transcription factor NF-κB induced by influenza virus was significantly blocked by 1, meanwhile, nuclear export of viral ribonucleoproteins was also effectively inhibited. These findings suggest that this new lignan glycoside from Calotropis gigantea, may have therapeutic potential in influenza virus infection through inhibition of NF-κB pathway and viral ribonucleoproteins nuclear export.