Antiviral effect of fufang yinhua jiedu (FFYH) granules against influenza A virus through regulating the inflammatory responses by TLR7/MyD88 signaling pathway.

Fufang-Yinhua-Jiedu Granules (FFYH) optimized from a Yin-Qiao-San, as traditional Chinese medicine (TCM), was used to treat influenza and upper respiratory tract infection and was recommended for the prevention and treatment of SARS in 2003 and current COVID-19 in Anhui Province in 2020. In the clinical studies, FFYH was very effective for the treatment of influenza, but the mechanism of action against influenza A virus remains unclear. In the present study, we investigated the antiviral effect of FFYH against influenza A virus in vitro and vivo. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was investigated for the first time. CPE inhibition assay and HA assay were used to evaluate the in vitro antiviral effects of FFYH against influenza A virus H1N1, H3N2, H5N1, H7N9 and H9N2. Mice were used to evaluate the antiviral effect of FFYH in vivo with ribavirin and lianhuaqingwen as positive controls. RT-PCR was used to quantify the mRNA transcription of TNF-α, IL-6, IFN-γ, IP10, and IL-1β mRNA. ELISA was used to examine the expression of inflammatory factors such as TNF-α, IL-6, IFN-γ, IP10, and IL-1β in sera. The blood parameters were analyzed with auto hematology analyzer. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was also investigated. FFYH showed a broad-spectrum of antiviral activity against H1N1, H3N2, H5N1, H7N9, and H9N2 influenza A viruses. Furthermore, FFYH dose-dependently increased the survival rate, significantly prolonged the median survival time of mice, and markedly reduced lung injury caused by influenza A virus. Also, FFYH significantly improve the sick signs, food taken, weight loss, blood parameters, lung index, and lung pathological changes. Moreover, FFYH could markedly inhibit the inflammatory cytokine expression of TNF-α, IL-6, IFN-γ, IP10, IL-10, and IL-1β mRNA or protein via inhibition of the TLR7/MyD88/NF-κB signaling pathway in vivo. FFYH not only showed a broad-spectrum of anti-influenza virus activity in vitro, but also exhibited a significant protective effect against lethal influenza virus infection in vivo. Furthermore, our results indicated that the in vivo antiviral effect of FFYH against influenza virus may be attributed to suppressing the expression of inflammatory cytokines via regulating the TLR7/MyD88/NF-κB signaling pathway. These findings provide evidence for the clinical treatment of influenza A virus infection with FFYH.

Zhang Y ，Wang R ，Shi W ，Zheng Z ，Wang X ，Li C ，Zhang S ，Zhang P ... -  《-》

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 ... -  《-》

Gegen Qinlian Decoction Downregulates the TLR7 Signalling Pathway to Control Influenza A Virus Infection.

In recent years, Gegen Qinlian decoction (GQD) has been applied to treat influenza virus infection, and its clinical effectiveness has been shown. However, the potential mechanism by which GQD acts on influenza A virus (IAV) has not been fully elucidated. Traditional Chinese medicine (TCM) formulas are well known to have multiple targets and effects. Our previous experiments examined the mechanism by which TCM can be used to treat influenza from the perspective of the influenza immune mechanism. To explore the possible mechanism by which GQD affects mice infected with the FM1 strain of influenza virus. Forty-eight C57BL/6 mice were divided randomly into four groups: a normal control (NG) group, an IAV infection (VG) group, an IAV + oseltamivir (30.44 mg/kg) treatment (VO) group, and an IAV + GQD (9.74 g/kg) treatment (VQ) group. We also grouped forty-eight Toll-like receptor 7 knockout (TLR7-/-) mice in the same manner. The pulmonary mRNA expression of TLR7, myeloid differentiation factor 88 (MyD88), and nuclear factor (NF)-κB p65 was measured by RT-qPCR, and the pulmonary protein expression of TLR7, MyD88, and NF-κB p65 was measured by western blot. The proportions of T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cells were measured by flow cytometry. IAV infection led to low body weights and high viral load. Compared with those in the NG group, the mRNA expression levels of TLR7, MyD88, and NF-κB p65 in the VG group were upregulated (P < 0.05). However, the mRNA and protein expression levels of TLR7, MyD88, and NF-κB p65 were lower in the VO and VQ groups than in the VG group (P < 0.05). IAV infection led to increased proportions of Th1/Th2 and Th17/Treg cells in the VG group. In the VO and VQ groups, both Th2 and Th1 cell numbers were increased, resulting in a lower Th1/Th2 proportion than that in the VG group. GQD downregulated the expression of some key TLR signalling pathway factors. GQD also affected the differentiation of CD4+ T cells, thereby exerting a protective systemic effect on influenza virus infection. In conclusion, GQD activated a balanced inflammatory response in the host to limit immune pathological injury and improve clinical and survival outcomes.

Shi Y ，Xu H ，Xiao Y ，Liu P ，Pang P ，Wu S ，Deng L ，Chen X ... -  《-》

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 ... -  《-》

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 ... -  《-》