Total alkaloids from Alstonia scholaris inhibit influenza a virus replication and lung immunopathology by regulating the innate immune response.

Alstonia scholaris is a folk medicine used to treat cough, asthma and chronic obstructive pulmonary disease in China. Total alkaloids (TA) from A. scholaris exhibit anti-inflammatory properties in acute respiratory disease, which suggests their possible anti-inflammatory effect on influenza virus infection. To assess the clinical use of TA by demonstrating their anti-influenza and anti-inflammatory effects and the possible mechanism underlying the effect of TA on influenza A virus (IAV) infection in vitro and to reveal the inhibitory effect of TA on lung immunopathology caused by IAV infection. Antiviral and anti-inflammatory activities were assessed in Madin-Darby canine kidney (MDCK) and A549 cells and U937-derived macrophages infected with influenza A/PR/8/34 (H1N1) virus. Proinflammatory cytokine levels were measured by real-time quantitative PCR and Bio-Plex assays. The activation of innate immune signaling induced by H1N1 virus in the absence or presence of TA was detected in A549 cells by Western blot. Furthermore, mice were infected intranasally with H1N1 virus and treated with TA (50, 25 and 12.5 mg/kg/d) or oseltamivir (60 mg/kg/d) for 5 days in vivo. The survival rates and body weight were recorded, and the viral titer, proinflammatory cytokine levels, innate immune cell populations and histopathological changes in the lungs were analyzed. TA significantly inhibited viral replication in A549 cells and U937-derived macrophages and markedly reduced cytokine and chemokine production at the mRNA and protein levels. Furthermore, TA blocked the activation of pattern recognition receptor (PRR)- and IFN-activated signal transduction in A549 cells. Critically, TA also increased the survival rate, reduced the viral titer, suppressed proinflammatory cytokine production and innate immune cell infiltration and improved lung histopathology in a lethal PR8 mouse model. TA exhibits anti-viral and anti-inflammatory effects against IAV infection by interfering with PRR- and IFN-activated signal transduction.

Zhou HX ，Li RF ，Wang YF ，Shen LH ，Cai LH ，Weng YC ，Zhang HR ，Chen XX ，Wu X ，Chen RF ，Jiang HM ，Wang C ，Yang M ，Lu J ，Luo XD ，Jiang Z ，Yang ZF ... -  《-》

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 ... -  《-》

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 ... -  《-》

Liu Shen Wan inhibits influenza a virus and excessive virus-induced inflammatory response via suppression of TLR4/NF-κB signaling pathway in vitro and in vivo.

Liu Shen Wan (LSW), first prescribed in "Lei Yunshang Song Fen Tang Fang", traditional Chinese medicine (TCM), is used to cure influenza, tonsillitis, pharyngitis and mumps for more than one hundred years. LSW was proved extensive pharmacological properties, for instance, anti-inflammatory, anticancer, antiviral, analgesic, antibacterial and immunomodulatory activities. Nevertheless, the mechanism of this process and the evaluation of this product is still ambiguous. Hence, the study was designed to investigate the antiviral and anti-inflammatory activities of LSW against the influenza virus in vitro and vivo. The antiviral activities of LSW were assayed in virus-infected cells and mice. To study the antiviral effects of LSW against influenza A/PR/8/34 virus (PR8), we employed CPE inhibition assay with different concentrations of LSW at different times of infection in vitro. The mice were intranasally infected with virus to induce viral pneumonia, then treated with different doses of LSW. The death protection of the mice, the lung index, virus titer and pathological changes in the lung tissue of mice were investigated to estimate the anti-virus effect of LSW. Moreover, RT-qPCR was used to determine the mRNA expression of TNF-α, IL-1β, IL-6, and IFN-γ in the A549 cells and the supernatant of lung tissues, and the concentrations of these four cytokines in serum of mice were determined with ELISA. Western blot was used to determine the expression of TLR4, p-NF-κB p65, NF-κB p65, p-IκBα and IκBα in the A549 cells and lung tissues, which are the key targets of TLR4/NF-κB pathway. Moreover, the immunohistochemical assay was used to determine the expression of the NF-κB p65 in the mice lungs. LSW could significantly inhibit influenza virus at different stages of viral replication (at the process of the pre-, post-, and co-virus infection) in vitro. And LSW (100 mg/kg and 50 mg/kg) could effectively increase the survival time of mice. The virus titres, lung index, pathological changes in the mice lungs also decreased. Moreover, LSW could significantly reduce the contents of IL-1β, TNF-α, IFN-γ and IL-6 in the infected cells and the infected-mice. In addition, LSW could significantly reduce the expression of TLR4, p-NF-κB p65, NF-κB p65 and p-IκBα, while increase the IκBα in the infected cells and in the lung of mice. LSW could significantly not only inhibit virus replication and proliferation in vitro, but also ameliorate pneumonia damage in vivo. The antiviral effect was attributed to down-regulating the expression of inflammatory cytokines induced by influenza virus via regulating the activity of TLR4/NF-кB signaling pathway.

Ma Q ，Huang W ，Zhao J ，Yang Z ... -  《-》

Exploration of the mechanisms of Ge Gen Decoction against influenza A virus infection.

Ge Gen Decoction (GGD), a Traditional Chinese Medicine prescription, is mainly used to treat infectious respiratory diseases and can relieve the symptoms of influenza A virus (IAV) infection. However, the underlying mechanism of GGD against IAV infection remains unclear. In this study, we found that GGD had moderate anti-IAV activity in vitro. GGD was more effective when given before the viral infection and targeted the viral attachment and replication stages rather than the internalization stage. In vivo, GGD treatment reduced thevirus titers of lung tissue significantly and improved the survival rate, lung index, and pulmonary histopathological changes in H1N1-infected mice. We observed the changes in several key immuno-related indexes in GGD administrated H1N1-infected mice with anti-IAV drug oseltamivir phosphate as the control. GGD treatment decreased the expression of TNF-α and improved Th1/Th2 immune balance to reduce the excessive immune response in H1N1-infected mice. Besides, the expression of the toll-like receptor 7 signaling pathway in H1N1-infected mice decreased after GGD treatment. Our results showed that GGD has anti-IAV activity and can modulate the immune system to relieve lung inflammation.

Geng ZK ，Li YQ ，Cui QH ，DU RK ，Tian JZ ... -  《-》