目的 研究盐酸黄连碱对甲型流感病毒（influenza A virus，IAV）的抑制作用及其潜在分子机制。方法 采用CCK-8法检测盐酸黄连碱对犬肾细胞（Madin-Darby canine kidney，MDCK）、人胚肾293T细胞（human embryonic kidney 293T cells，293T）活力的影响；通过体外病毒感染模型，检测盐酸黄连碱对IAV复制及病毒核蛋白（nucleoprotein，NP）表达的调控作用，并计算其半数有效浓度（half effective concentration，EC₅₀）与半数细胞毒性浓度（half cytotoxic concentration，CC₅₀）。构建H1N1-UI182感染小鼠模型，给予盐酸黄连碱干预后，检测小鼠生存率、体质量、肺指数、肺组织病毒滴度及病毒载量变化；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察肺组织病理损伤情况；免疫组化法检测肺组织NP蛋白表达；采用Western blotting和qRT-PCR检测盐酸黄连碱对瞬时受体电位香草酸亚型4（transient receptor potential vanilloid 4，TRPV4）/核因子-κB（nuclear factor-κB，NF-κB）信号通路的调控作用，以及对肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、IL-1β、γ干扰素（interferon-γ，IFN-γ）等细胞因子表达的影响。结果 在细胞水平，与模型组比较，盐酸黄连碱显著抑制IAV复制，降低NP蛋白表达水平（P＜0.05、0.001）；盐酸黄连碱在MDCK细胞中EC₅₀为15.93 μg/mL、CC₅₀为83.42μg/mL，在293T细胞中EC₅₀为18.44 μg/mL、CC₅₀为69.90μg/mL。在H1N1-UI182感染小鼠模型中，与模型组比较，盐酸黄连碱显著改善小鼠体质量下降情况，提高小鼠生存率，并显著降低小鼠肺指数、肺组织病毒滴度及病毒载量（P＜0.05、0.01、0.001）。组织病理学结果显示，盐酸黄连碱显著减轻病毒感染引发的肺部炎性浸润与肺泡结构破坏。免疫组化结果表明，盐酸黄连碱可显著减少肺组织中NP蛋白表达。Western blotting和qRT-PCR结果显示，与模型组比较，盐酸黄连碱可调控TRPV4/NF-κB信号通路，显著抑制TRPV4过度活化（P＜0.001），阻断NF-κB核转位（P＜0.001），进而下调TNF-α、IL-6、IL-1β等促炎因子表达（P＜0.05、0.01、0.001）。结论 盐酸黄连碱可通过直接抑制IAV复制，并调控TRPV4/NF-κB信号通路改善病毒诱导的炎症反应，发挥抗甲型流感病毒作用。

Objective To study the inhibitory effect and its potential molecular mechanism of coptisine hydrochloride on influenza A virus (IAV). Methods CCK-8 method was used to detect the effect of coptisine hydrochloride on viability of Madin-Darby canine kidney (MDCK) and human embryonic kidney 293T cells (293T). By using an in vitro viral infection model, the regulatory effect of coptisine hydrochloride on IAV replication and viral nucleoprotein (NP) expression was detected, and its half effective concentration (EC₅₀) and half cytotoxic concentration (CC₅₀) were calculated. A mouse model infected with H1N1-UI182 was constructed, coptisine hydrochloride was given for intervention, changes in survival rate, body weight, lung index, lung tissue virus titer and viral load were detected. Hematoxylin-eosin (HE) staining was used to observe the pathological damage of lung tissue. Immunohistochemistry was used to detect the expression of NP protein in lung tissue. Western blotting and qRT-PCR were used to detect the regulatory effect of coptisine hydrochloride on transient receptor potential vanilloid 4 (TRPV4)/nuclear factor-κB (NF-κB) signaling pathway, as well as its effect on the expressions of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and interferon-γ (IFN-γ). Results At the cellular level, compared with model group, coptisine hydrochloride significantly inhibited IAV replication and reduced NP protein expression level (P < 0.05, 0.001). Coptisine hydrochloride had an EC₅₀ of 15.93 μg/mL and a CC₅₀ of 83.42 μg/mL in MDCK cells, and an EC₅₀ of 18.44 μg/mL and a CC₅₀ of 69.90 μg/mL in 293T cells. In the H1N1-UI182 infected mouse model, compared with model group, coptisine hydrochloride significantly improved the decline in body weight of mice, increased survival rate, and significantly reduced lung index, lung tissue virus titer and viral load (P < 0.05, 0.01, 0.001). The histopathological results showed that coptisine hydrochloride significantly reduced the inflammatory infiltration and alveolar structural damage caused by viral infection in lungs. The immunohistochemical results showed that coptisine hydrochloride significantly reduced the expression of NP protein in lung tissue. Western blotting and qRT-PCR results showed that compared with model group, coptisine hydrochloride could regulate TRPV4/NF-κB signaling pathway, significantly inhibit TRPV4 overactivation (P < 0.001), block NF-κB nuclear translocation (P < 0.001), and subsequently downregulate the expressions of pro-inflammatory factors such as TNF-α, IL-6, IL-1β (P < 0.05, 0.01, 0.001). Conclusion Coptisine hydrochloride could directly inhibit IAV replication and regulate TRPV4/NF-κB signaling pathway to improve virus induced inflammatory response, exerting anti-IAV effects.

R285.5

国家自然科学基金资助项目（32170539）