目的 研究葛根Pueraria lobata水提物的体内外抗病毒作用，基于溶酶体生物发生探究其抗病毒作用的细胞学机制。方法 CCK-8检测葛根水提物对非小细胞肺癌A549细胞活力的影响。利用表达绿色荧光蛋白的水疱性口炎病毒（vesicular stomatitis virus expressing green fluorescent protein，VSV-GFP）感染A549细胞，荧光显微镜和流式细胞术检测葛根水提物对GFP阳性细胞比例的影响；Western blotting检测葛根水提物对VSV病毒G蛋白表达的影响；qRT-PCR检测葛根水提物对甲型流感病毒（influenza A virus，H1N1）、脑心肌炎病毒（encephalomyocarditis virus，EMCV）、单纯疱疹病毒1型（herpes simplex virus type 1，HSV-1）病毒复制的影响。葛根水提物（0.8、1.6 g/kg）治疗H1N1病毒滴鼻感染小鼠，观察记录其对小鼠的生存曲线、体质量变化和肺指数的影响，利用小动物CT、肺组织苏木素-伊红（HE）染色观察药物治疗对肺组织病理变化的影响；qRT-PCR检测肺组织病毒载量、白细胞介素-6（interleukin-6，Il6）和肿瘤坏死因子α（tumor necrosis factor α，Tnfα）的基因表达。葛根水提物处理MEF胚胎成纤维细胞并进行转录组测序分析，qRT-PCR检测溶酶体相关基因表达。流式细胞术检测葛根水提物在转录因子EB（transcription factor EB，TFEB）敲除的鼠永生化骨髓源性巨噬细胞（Tfeb^-/- iBMDM）中对GFP阳性细胞比例的影响。结果 葛根水提物显著抑制细胞内VSV-GFP病毒复制和VSV病毒G蛋白表达（P＜0.001），降低H1N1、EMCV和HSV-1病毒复制（P＜0.001）。葛根水提物能够提升H1N1感染小鼠的存活率，降低肺部病毒载量，改善小鼠肺部病变情况。生物信息学分析表明葛根水提物上调溶酶体相关基因表达，并证明葛根水提物通过TFEB促进溶酶体生物发生抵抗VSV病毒感染（P＜0.001）。结论 葛根水提物通过TFEB促进溶酶体生物发生，从而在体内外抑制病毒感染。

Objective To investigate the anti-viral effects of Pueraria lobata water extract in vitro and in vivo, and explore the cellular mechanisms of its anti-viral effects based on lysosomal biosynthesis. Methods CCK-8 assay was performed to detect cell viability of A549 cells treated with P. lobata water extract. A549 cells were infected by vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), fluorescence microscopy and flow cytometry were used to evaluate the effect of P. lobata water extract on the proportion of GFP positive cells; Western blotting was used to examine the effect of P. lobata water extract on the expression of VSV G protein; qRT-PCR was used to detect the effect of P. lobata water extract on the replication of influenza A virus (H1N1), encephalomyocarditis virus (EMCV), herpes simplex virus type 1 (HSV-1) respectively. P. lobata water extract was used to treat H1N1 drop-nose infected mice at doses of 0.8 and 1.6 g/kg, the survival rate, body weight changes and lung inhibition index of mice were observed; CT images, hematoxylin-eosin (HE) staining of lung tissue were used to observe the effect of drug treatment on pathological changes in lung tissue; qRT-PCR was used to detect the viral load, interleukin-6 (Il6), and tumor necrosis factor α (Tnfα) gene expressions in lung tissue. MEF cells were treated with P. lobata water extract and subjected to transcriptome sequencing analysis, qRT-PCR was used to detect mRNA expressions of lysosomal related genes. Flow cytometry was used to detect effect of P. lobata water extract on the proportion of GFP positive cells in transcription factor EB (Tfeb)^−/− of mouse immortalized bone marrow-derived macrophages (iBMDM). Results P. lobata water extract significantly inhibited intracellular VSV-GFP virus replication and VSV virus G protein expression (P < 0.001), and reduced H1N1, EMCV and HSV-1 virus replication (P < 0.001). P. lobata water extract could improve the survival rate of H1N1 infected mice, reduce the viral load in lung, and improve lung lesions in mice. Bioinformatics analysis showed that P. lobata water extract up-regulated the expressions of lysosomal related genes, and demonstrated that P. lobata water extract promoted lysosomal biogenesis to resist VSV virus infection through TFEB (P < 0.001). Conclusion P. lobata water extract promotes lysosomal biogenesis through TFEB, thereby inhibiting viral infection in vitro and in vivo.

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2023年度北京中医药大学-优莎纳联合研究中心（BURC）基金重点项目（BUCM-2023-JS-KF-032）;中华中医药学会青年人才托举工程项目（CACM-2023-QNRC2-A02）;北京市科技新星计划课题（20230484342）;北京市自然科学基金资助项目（7242239）