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 inhibits influenza a virus and excessive virus-induced inflammatory response via suppression of TLR4/NF-κB signaling pathway in vitro and in vivo
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DOI：
10.1016/j.jep.2020.112584
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发表时间：
2020-04-24
影响因子：
5.4
通讯作者：
Yang, Zifeng
中科院分区：
文献类型：
--
作者：
Ma, Qinhai;Huang, Wenbo;Yang, Zifeng
民族药理学相关性：六神丸（LSW），最早在中医《雷云上松分汤方》中被规定，用于治疗流感、扁桃体炎、咽炎和腮腺炎，已有一百多年的历史。研究目的：枸杞多糖具有抗炎、抗癌、抗病毒、镇痛、抗菌和免疫调节等广泛的药理作用。然而，这一过程的机制和该产品的评价仍不明确。因此，本研究旨在研究LSW在体外和体内对流感病毒的抗病毒和抗炎活性。材料与方法：在病毒感染的细胞和小鼠中测定LSW的抗病毒活性。为了研究LSW对流感A/PR/8/34病毒（PR8）的抗病毒作用，我们采用了不同浓度LSW在不同感染时间的体外CPE抑制实验。小鼠经鼻内感染病毒诱导病毒性肺炎，然后给予不同剂量的LSW治疗。通过对小鼠的死亡保护作用、肺指数、病毒滴度和肺组织病理变化的研究来评价LSW的抗病毒作用。RT-qPCR检测A549细胞及肺组织上清中tnf - α、IL-1 β、IL-6、ifn - γ mRNA的表达，ELISA检测小鼠血清中这四种细胞因子的浓度。Western blot检测TLR4、p-NF-kappa B p65、NF-kappa B p65、p-I kappa B α和I kappa B α在A549细胞和肺组织中的表达，这是TLR4/NF-kappa B通路的关键靶点。免疫组化法检测NF-kappa B p65在小鼠肺组织中的表达。结果：LSW在体外对流感病毒复制的不同阶段（病毒感染前、感染后和共感染过程）均有明显的抑制作用。LSW （100 mg/kg和50 mg/kg）均能有效延长小鼠的存活时间。小鼠肺部的病毒滴度、肺指数、病理变化均有所下降。LSW还能显著降低感染细胞和感染小鼠体内IL-1 β、tnf - α、ifn - γ和IL-6的含量。此外，LSW还能显著降低TLR4、p- nf - κ B p65、nf - κ B p65和p-I κ B α的表达，增加感染细胞和小鼠肺中I κ B α的表达。结论：LSW不仅能显著抑制体外病毒复制和增殖，还能改善体内肺炎损伤。其抗病毒作用可能是通过调节TLR4/ nf - κ B信号通路活性，下调流感病毒诱导的炎症细胞因子的表达
Ethnopharmacological relevance: 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.Aim of study: 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.Materials and methods: 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 antivirus effect of LSW. Moreover, RT-qPCR was used to determine the mRNA expression of TNF-alpha, IL-1 beta, IL-6, and IFN-gamma 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-kappa B p65, NF-kappa B p65, p-I kappa B alpha and I kappa B alpha in the A549 cells and lung tissues, which are the key targets of TLR4/NF-kappa B pathway. Moreover, the immunohistochemical assay was used to determine the expression of the NF-kappa B p65 in the mice lungs.Results: 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-1 beta, TNF-alpha, IFN-gamma and IL-6 in the infected cells and the infected-mice. In addition, LSW could significantly reduce the expression of TLR4, p-NF-kappa B p65, NF-kappa B p65 and p-I kappa B alpha, while increase the I kappa B alpha in the infected cells and in the lung of mice.Conclusions: 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-kappa B signaling pathway.y