Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

Background: Influenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata. Purpose: This study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice. Methods: Flavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-kappa B p65 phosphorylation (NF-kappa B p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. Results: HCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-beta in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-kappa B p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 mu g/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-kappa B p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells. Conclusion: HCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenza) NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.