清肺口服液黄酮类成分对呼吸道合胞病毒感染小鼠坏死性凋亡的影响

Effect of flavonoids in Qingfei Oral Liquid on necroptosis in RSV-infected mice

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 protein1,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 m RNA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-αand 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.