目的 研究清肺口服液黄酮类成分对呼吸道合胞病毒（respiratory syncytial virus，RSV）感染小鼠坏死性凋亡的影响。方法 将BALB/c小鼠随机分为对照组、模型组及黄酮类成分低、高剂量（30、60 mg/kg）组和利巴韦林（46 mg/kg）组，建立RSV感染的小鼠模型，给药4 d后，苏木素-伊红（HE）染色法观察肺组织病理学变化；qRT-PCR检测肺组织中白细胞介素-6（interleukin-6，IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和RSV-F mRNA表达；流式细胞术检测肺组织细胞坏死性凋亡率；ELISA法检测小鼠肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中乳酸脱氢酶（lactate dehydrogenase，LDH）和高迁移率族蛋白B1（high mobility group protein B1，HMGB1）水平；Western blotting检测肺组织受体相互作用蛋白激酶1（receptor-interacting protein 1，RIP1）、RIP3、混合系列蛋白激酶样结构域（mixed lineage kinase domain-like，MLKL）、磷酸甘油酸变位酶5（phosphoglycerate mutase 5，PGAM5）、动力相关蛋白1（dynamin-related protein 1，DRP1）蛋白表达；免疫荧光检测肺组织RIP1和RIP3的共定位以及p-MLKL与上皮细胞的共定位。结果 与模型组相比，黄酮类成分组小鼠肺组织病理损伤减轻；肺组织中IL-6、TNF-α和RSV-F mRNA表达水平降低（P＜0.05、0.01）；肺组织细胞坏死性凋亡率下降（P＜0.05、0.01）；BALF中HMGB1和LDH水平降低（P＜0.05、0.01）；肺组织RIP1、RIP3、MLKL、PGAM5、DRP1蛋白表达水平均显著降低（P＜0.05、0.01）；肺组织RIP1/RIP3坏死复合物的形成减少，肺上皮细胞中p-MLKL的荧光表达降低。结论 清肺口服液黄酮类成分可能通过调节RIP1/RIP3/MLKL/PGAM5/DRP1信号通路，抑制坏死性凋亡，改善RSV感染的小鼠肺部炎症损伤。

Objective To explore the effect of flavonoids in Qingfei Oral Liquid (清肺口服液) on necroptosis in respiratory syncytial virus (RSV)-infected mice. Methods BALB/c mice were randomly divided into control group, model group, flavonoids low-, high-dose (30, 60 mg/kg) groups and ribavirin (46 mg/kg) group. RSV-infected mice model was established. After continuous administration for 4 d, HE staining was used to observe the pathological changes of lung tissue; qRT-PCR was used to detect interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and RSV-F mRNA expressions in lung tissue; Flow cytometry was used to detect necroptosis rate of lung tissue cell; ELISA assay was used to detect lactate dehydrogenase (LDH) and high mobility group protein B1 (HMGB1) levels in bronchoalveolar lavage fluid (BALF) of mice; Western blotting was used to detect protein expressions of receptor-interacting protein 1 (RIP1), RIP3, mixed lineage kinase domain-like (MLKL), phosphoglycerate mutase 5 (PGAM5), dynamin-related protein 1 (DRP1) in lung tissue; Immunofluorescence was used to detect the co-localization of RIP1 and RIP3, co-localization of p-MLKL with epithelial cells. Results Compared with model group, pathological damage of lung tissue in flavonoids group was alleviated; IL-6, TNF-α and RSV-F mRNA expressions in lung tissue were decreased (P < 0.05, 0.01); Necroptosis rate of lung tissue cells was decreased (P < 0.05, 0.01); HMGB1 and LDH levels in BALF were decreased (P < 0.05, 0.01); RIP1, RIP3, MLKL, PGAM5 and DRP1 protein expressions in lung tissue were decreased (P < 0.05, 0.01); Formation of RIP1/RIP3 necrotic complex in lung tissue was decreased, fluorescence expression of p-MLKL in lung epithelial cells was decreased. Conclusion Flavonoids in Qingfei Oral Liquid may inhibit necroptosis by regulating RIP1/RIP3/MLKL/PGAM5/DRP1 signaling pathway, and improve lung inflammation in RSV-infected mice.

R285.5

国家中医药管理局项目(2019XZZX-ek003);国家自然科学基金资助项目(81873340);国家自然科学基金资助项目(82174436);江苏省研究生实践创新计划项目(KYCX21_1647)