RESUMO
AIM:To investigate the molecular mechanism of Xijiao Dihuang decoction combined with Yinqiao powder (XDY) in treating viral pneumonia, and the effects of XDY on TNF-α-induced permeability in pulmonary microvascular endothelial cells (PMVEC) and the role of PKC-SSeCKS pathway involved.METHODS:The electric conductivity method was used to detect transendothelial electrical resistance (TER) of primarily cultured PMVEC on Transwell chamber at different time points to determine the permeability of PMVEC.After pretreatment for 24 h, the activity of PKC, TER, and the expression of SSeCKS at mRNA and protein levels were detected.Laser scanning confocal microscopy was used to observe the location of SSeCKS and construction of F-actin in PMVEC.RESULTS:The permeability of PMVECs induced by TNF-α reached the peak at 24 h.Compared with control group, the TER in TNF-α group was decreased, and the activity of PKC was increased.Compared with TNF-α group, the activity of PKC in TNF-α with PKC inhibitor group and TNF-α with XDY group was decreased, while the TER was increased, without difference from control group.Compared with control group, the mRNA expression of SSeCKS and phospho-SSeCKS was increased in PMVEC of TNF-α group, but decreased in TNF-α with XDY group compared with TNF-α group.In control group, F-actin was mainly located around the nucleus and at cytoplasmic borders of PMVEC, forming the dense peripheral bundle, and SSeCKS was evenly scattered in the cell.In TNF-α group, the dense peripheral bundle of F-actin surrounding the cells almost disappeared, and SSeCKS was concentrated around the nucleus.Compared with TNF-α group, the distribution and the structure of F-actin and SSeCKS nearly returned to normal in TNF-α with XDY group.CONCLUSION:XDY inhibits the activation of PKC signaling pathway in PMVEC caused by TNF-α to reduce the mRNA expression of SSeCKS and the phosphorylation of SSeCKS, thus preventing the deformation of endothelial cells and reducing the permeability of PMVEC.