State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, Guangdong, China.
State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, Guangdong, China; State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau.
J Ethnopharmacol. 2021 Sep 15;277:114066. doi: 10.1016/j.jep.2021.114066. Epub 2021 Mar 23.
Liu Shen Wan (LSW) is a traditional Chinese medicine (TCM) with detoxification and antiphlogistic activity; it is composed of bezoar, toad venom, musk, pearl powder, borneol and realgar. In recent years, LSW has been widely used in traditional medicine for the treatment of influenza, tonsillitis, pharyngitis, mumps, cancer and leukaemia.
The anti-influenza virus properties of LSW and its inhibition of the inflammatory response was demonstrated in our previous research; however, the effect and potential mechanism of LSW against influenza induced secondary bacteria have remained obscure. Therefore, in the present study, a model of influenza virus PR8 with secondary infection by Staphylococcus aureus (S. aureus) in vitro and in mice was established to examine the effect and potential mechanism by which LSW inhibits bacterial adhesion and subsequent severe pneumonia after viral infection.
We investigated the effect of LSW on the PR8-induced adhesion of live S. aureus in A549 cells. RT-qPCR was used to detect the expression of adhesion molecules. Western blotting was used to determine the expression of CEACAM1, RIG-1, MDA5, p-NF-κB, and NF-κB in A549 cells. Inflammatory cytokines were detected using a Bio-Plex Pro Human Cytokine Screening Panel (R&D) in A549 cells and Mouse Magnetic Luminex Assays (R&D) in mice infected with PR8 virus and secondarily with S. aureus, respectively. Moreover, the survival rate, lung index, viral titre, bacterial loads and pathological changes in the lung tissue of mice infected with PR8 and S. aureus were investigated to estimate the effect of LSW in inhibiting severe pneumonia.
LSW significantly decreased S. aureus adhesion following influenza virus infection in A549 cells, which may have occurred by suppressing expression of the adhesion molecule CEACAM1. In addition, treatment with LSW dramatically suppressed the induction of proinflammatory cytokines (CCL2/MCP-1 and CXCL-9/MIG) and chemokines (IL-6 and TNF-α) by PR8 infection following secondary LPS stimulation in A549 cells. Upregulation of related signalling proteins (RIG-I, MDA5 and NF-κB) induced by viruses and bacteria was suppressed by LSW in A549 cells. LSW significantly decreased the viral titres and bacterial load, prolonged survival time, and ameliorated lung inflammation and injury in mice with S. aureus infection secondary to PR8 infection.
We demonstrated that LSW prevents S. aureus adherence to influenza virus-infected A549 cells, perhaps by inhibiting the expression of the adhesion molecule CEACAM1. The upregulation of proinflammatory cytokines and related signalling proteins induced by viruses and bacteria was suppressed by LSW in A549 cells. LSW significantly ameliorated lung injury caused by viral and secondary bacterial infection. These findings provide a further evaluation of LSW and suggest a beneficial effect of LSW for the prevention of secondary bacterial infection and related complications.
Liu Shen Wan（LSW）是一种具有解毒和消炎作用的中药，由牛黄、蟾酥、麝香、珍珠粉、冰片和雄黄组成。近年来，LSW 在传统医学中被广泛用于治疗流感、扁桃体炎、咽炎、腮腺炎、癌症和白血病。
我们之前的研究表明 LSW 具有抗流感病毒的特性，并能抑制炎症反应，但 LSW 对流感诱导的继发性细菌的作用及其潜在机制仍不清楚。因此，本研究建立了流感病毒 PR8 与金黄色葡萄球菌（S. aureus）继发感染的体外和小鼠模型，以研究 LSW 抑制病毒感染后细菌黏附和随后发生严重肺炎的作用和潜在机制。
我们研究了 LSW 对 A549 细胞中活的 S. aureus 诱导黏附的影响。采用 RT-qPCR 检测黏附分子的表达。采用 Western blot 检测 A549 细胞中 CEACAM1、RIG-1、MDA5、p-NF-κB 和 NF-κB 的表达。采用 Bio-Plex Pro 人细胞因子筛选试剂盒（R&D）检测 A549 细胞中的炎性细胞因子，采用 Mouse Magnetic Luminex Assays（R&D）检测感染 PR8 病毒和继发感染 S. aureus 的小鼠中的炎性细胞因子。此外，还通过检测感染 PR8 和 S. aureus 的小鼠的存活率、肺指数、病毒滴度、细菌负荷和肺组织病理变化来评估 LSW 抑制严重肺炎的效果。
LSW 显著降低了流感病毒感染 A549 细胞后 S. aureus 的黏附，这可能是通过抑制黏附分子 CEACAM1 的表达来实现的。此外，LSW 处理还显著抑制了 A549 细胞中 PR8 感染后继发 LPS 刺激诱导的促炎细胞因子（CCL2/MCP-1 和 CXCL-9/MIG）和趋化因子（IL-6 和 TNF-α）的诱导。LSW 还抑制了 A549 细胞中病毒和细菌诱导的相关信号蛋白（RIG-I、MDA5 和 NF-κB）的上调。LSW 还显著降低了继发于 PR8 感染的 S. aureus 感染小鼠的病毒滴度和细菌负荷，延长了小鼠的存活时间，并改善了小鼠的肺部炎症和损伤。
我们证明了 LSW 可防止 S. aureus 黏附于流感病毒感染的 A549 细胞，这可能是通过抑制黏附分子 CEACAM1 的表达来实现的。LSW 还抑制了 A549 细胞中病毒和细菌诱导的促炎细胞因子和相关信号蛋白的上调。LSW 显著改善了病毒和继发细菌感染引起的肺部损伤。这些发现进一步评价了 LSW，并提示 LSW 对预防继发性细菌感染和相关并发症具有有益作用。
