犀角地黄汤合银翘散影响PKC-SSeCKS介导的F-actin变构抑制流感病毒诱导的PMVEC通透性增加

Xijiao Dihuang decoction combined with Yinqiao powder inhibits influenza virus-induced hyperpermeability by affecting PKC-SSeCKS-mediated F-actin reorganization

目的:基于蛋白激酶C(PKC)-Src抑制的蛋白激酶C底物(SSeCKS)介导的纤维形肌动蛋白(F-actin)变构观察犀角地黄汤合银翘散(XDY)对流感病毒诱导的肺微血管内皮细胞(PMVEC)通透性变化的影响,探讨XDY治疗病毒性肺炎的机制。方法:原代培养大鼠PMVEC,测流感病毒FM1半数组织培养感染剂量(TCID50),分组为对照组、模型组(100TCID50 FM1)和XDY组(100TCID50 FM1+XDY含药血清),不同因素干预24 h后检测跨PMVEC电阻(TER)、 PKC活性、SSeCKS mRNA和蛋白表达水平,激光共聚焦显微镜观察SSeCKS定位和F-actin结构变化。结果:流感病毒感染导致PMVEC的TER降低(通透性增强)、PKC活性增高,XDY可增高TER、降低PKC活性;XDY可下调流感病毒诱导升高的SSeCKS mRNA和蛋白表达,并使集中分布在核周的SSeCKS趋于均匀分布在细胞中;模型组细胞周边F-actin致密束基本消失,XDY可明显改善这种变构。结论:犀角地黄汤合银翘散可以抑制PKC-SSeCKS通路激活并影响F-actin的变构从而干预流感病毒诱导的PMVEC通透性增高。

Objective:To investigate the mechanisms of the Xijiao Dihuang decoction combined with Yinqiao powder(XDY) in treating viral pneumonia by observing effects of XDY on influenza-induced hyperpermeability of pulmonary microvascular endothelial cells(PMVEC) based on the protein kinase C(PKC)-Src-suppressed C kinase substrate(SSeCKS)-mediated F-actin reorganization.Methods:After median tissue culture infectious dose(TCID50) had been detected,primary cultured PMVECs were devided into control group,model group(100 TCID50 FM1) and XDY group(100 TCID50 FM1+XDY-contained serum).After the 24-hour pretreatment,the transendothelial electrical resistance(TER),the activity of PKC and the expression of SSeCKS at mRNA and protein of primarily cultured PMVEC were detected.In addition,the laser scanning confocal microscopy was used to observe the location of SSeCKS and the structure of F-actin.Results:Influenza virus decreased the TER(increased the permeability) and enhanced the PKC activity,while XDY could increase the TER and reduce the PKC activity.Compared with the model group,XDY could down-regulate the mRNA and protein expression of SSeCKS and made the SSeCKS translocate from the perinuclear to the cytoplasm.Besides,F-actin reorganization occurred in moldel group as dense peripheral bundle surrounding the cells almost disappeared,which could be significantly improved by XDY.Conclusion:XDY inhibits the activation of PKC-SSeCKS pathway and affect the reorganization of F-actin to make interventions in the influenza virus-induced PMVEC hyperpermeability.