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

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. 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. 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-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. 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-α (TNF-α) 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-β in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 μg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB 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. HCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal 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.

Ling LJ ，Lu Y ，Zhang YY ，Zhu HY ，Tu P ，Li H ，Chen DF ... -  《-》

In vivo effect of quantified flavonoids-enriched extract of Scutellaria baicalensis root on acute lung injury induced by influenza A virus.

Scutellaria baicalensis root is traditionally used for the treatment of common cold, fever and influenza. Flavonoids are the major chemical components of S. baicalensis root. To evaluate the therapeutic effects and action mechanism of flavonoids-enriched extract from S. baicalensis root (FESR) on acute lung injury (ALI) induced by influenza A virus (IAV) in mice. The anti-influenza, anti-inflammatory and anti-complementary properties of FESR and the main flavonoids were evaluated in vitro. Mice were challenged intranasally with influenza virus H1N1 (A/FM/1/47) 2  h before treatment. FESR (50, 100 and 200  mg/kg) was administrated intragastrically. Baicalin (BG), the most abundant compound in FESR was given as reference control. Survival rates, life spans and lung indexes of IAV-infected mice were measured. Histopathological changes, virus levels, inflammatory markers and complement deposition in lungs were analyzed. Compared with the main compound BG, FESR and lower content aglycones (baicalein, oroxylin A, wogonin and chrysin) in FESR significantly inhibited H1N1 activity in virus-infected Madin-Darby canine kidney (MDCK) cells and markedly decreased nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In vitro assays showed that FESR and BG had no anti-complementary activity whereas baicalein, oroxylin A, wogonin and chrysin exhibited obvious anti-complementary activity. Oral administration of FESR effectively protected the IAV-infected mice, increased the survival rate (FESR: 67%; BG: 33%), decreased the lung index (FESR: 0.90; BG: 1.00) and improved the lung morphology in comparing with BG group. FESR efficiently decreased lung virus titers, reduced haemagglutinin (HA) titers and inhibited neuraminidase (NA) activities in lungs of IAV-infected mice. FESR modulated the inflammatory responses by decreasing the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and increasing the levels of interferon-γ (IFN-γ) and interleukin-10 (IL-10) in lung tissues. Although showing no anti-complementary activity in vitro, FESR obviously reduced complement deposition and decreased complement activation product level in the lung . FESR has a great potential for the treatment of ALI induced by IAV and the underlying action mechanism might be closely associated with antiviral, anti-inflammatory and anti-complementary properties. Furthermore, FESR resulted in more potent therapeutic effect than BG in the treatment of IAV-induced ALI.

Zhi HJ ，Zhu HY ，Zhang YY ，Lu Y ，Li H ，Chen DF ... -  《-》

Sangju Cold Granule exerts anti-viral and anti-inflammatory activities against influenza A virus in vitro and in vivo.

Sangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in "Item Differentiation of Warm Febrile Diseases". Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated. Influenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo. Ultrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using plaque reduction and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by Western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count. The main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-β, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P-NF-κB P65 (P-P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-β, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood. SJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment.

Gao T ，Liu J ，Huang N ，Zhou Y ，Li C ，Chen Y ，Hong Z ，Deng X ，Liang X ... -  《-》

The synergistic effect and mechanisms of flavonoids and polysaccharides from Houttuynia cordata on H1N1-induced pneumonia in mice.

Houttuynia cordata Thunb. (HC, Saururaceae family) is a classical Traditional Chinese Medicine used to treat pneumonia clinically. The total flavonoids (HCF) and polysaccharides (HCP) are key medicinal components of H. cordata involved in its beneficial effect on viral pneumonia. The purpose of the study is to investigate the synergistic or complementary effects of combination of HCF and HCP on viral pneumonia as well as the mechanisms underlying. HCF or HCP were administrated separately or combined in different proportions on influenza virus H1N1 - infected mice. The survival and lung weight of mice were recorded. The synergistic effect on HCF and HCP combination was calculated by Chou-Talalay method. H&E staining was performed to detect lung histomorphology. Western blot, immunohistochemistry and enzyme linked immunosorbent assay were done to analyze the representative protein expression in lung and intestine tissues. AB - PAS staining on intestine tissue sections was performed to evaluate the histopathology of intestines. Bacterial genomic DNA was extracted and sequenced for gut microbiota analysis. In H1N1 lethally infected mice, the combined administration of HCF and HCP significantly increased the survival rate and prolonged the life span of mice, compared with mono-drug therapy. The viral pneumonia was remarkably improved by HCF and HCP combination reflected by lower lung index, more intact lung morphology, and less inflammatory cells and mediators. Furthermore, the combination of HCF and HCP regulated intestinal microbiota, significantly reduced the proportion of pathogenic Proteobacteria and the secretion of proinflammatory cytokine in gut. The combined HCF and HCP showed synergistic effect on reducing lung and intestine injury. The complementary interaction was also found in HCF and HCP combined therapy, as HCF provided the significant antiviral activity and HCP markedly improved intestinal physical barrier and increased the protein expression involving removal of edema. Our findings indicated that combination of HCF and HCP from H. cordata synergistically alleviated H1N1-induced viral pneumonia in mice via multimodal regulation of both pulmonary and intestinal homeostasis, which might imply novel therapeutic strategy for treating viral pneumonia.

Ling L ，Ren A ，Lu Y ，Zhang Y ，Zhu H ，Tu P ，Li H ，Chen D ... -  《-》

Houttuynia cordata polysaccharides ameliorate pneumonia severity and intestinal injury in mice with influenza virus infection.

Hottuynia cordata is an important traditional Chinese medicine for the treatment of respiratory diseases including bacterial and viral infections. Polysaccharides isolated from Houttuynia cordata (HCP), as its main ingredients, have been demonstrated to ameliorate the LPS-induced acute lung injury in mice. The study aimed to determine the protective effects of HCP on multiple organ injury in influenza A virus (IAV) H1N1 infected mice and its primary mechanisms in anti-inflammation and immune regulation. Mice were inoculated with IAV H1N1 and then treated with 20 or 40 mg/kg/d of HCP for survival test and acute lung-gut injury test. The treatment with HCP resulted in an increase in the survival rate of H1N1 infected mice and the protection from lung and intestine injury, accompanied with the reduced virus replication. HCP markedly decreased the concentration of pulmonary proinflammatory cytokines/chemokines and the number of intestinal goblet cells, and strengthened the intestinal physical and immune barrier, according to the increase of sIgA and tight junction protein (ZO-1) in intestine. At the same time, the inhibition of inflammation in lung and gut was related to the suppressing of the expression of TLR4 and p-NFκB p65 in lung. These results indicated that HCP ameliorated lung and intestine injury induced by IAV attack. The mechanisms were associated with inhibition of inflammation, protection of intestinal barrier and regulation of mucosal immunity, which may be related to the regulation of gut-lung axis. As an alternative medicine, HCP may have clinical potential to treat IAV infection in human beings.

Zhu H ，Lu X ，Ling L ，Li H ，Ou Y ，Shi X ，Lu Y ，Zhang Y ，Chen D ... -  《-》