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. 2021 Oct 20;12:697071. doi: 10.3389/fimmu.2021.697071

Figure 4.

Figure 4

FPS-ZM1 treatment alleviated rhHMGB1-mediated RhoA/ROCK1 activation and barrier dysfunction. (A) Cell viability was determined by CCK-8 assay after treated with different dosage of FPS-ZM1 for 24 h. (B) FPS-ZM1 improved lung endothelial permeability at 60 min and 24 h after rhHMGB1 stimulation. (C) FPS-ZM1 significantly downregulated RhoA and ROCK1 expression in HPMECs at 60 min after rhHMGB1 stimulation. (D) Effects of FPS-ZM1 on rhHMGB1-mediated morphological changes in endothelial F-actin, VE-cadherin and ZO-1. ECs were treated with FPS-ZM1 for 1 h prior to stimulation with rhHMGB1 to evaluate morphology of endothelial cytoskeleton F-actin or for the last 4 h of the 24 h rhHMGB1 stimulation to assess morphology of endothelial VE-cadherin and ZO-1 by immunofluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (E) FPS-ZM1 significantly increased VE-cadherin and ZO-1 expression levels in HPMECs at 24 h after rhHMGB1 stimulation. ECs were treated with FPS-ZM1 for the last 4 h of the 24 h rhHMGB1 stimulation. (F) Effects of FPS-ZM1 on rhHMGB1-mediated pMLC expression in cells. ECs were pretreated with FPS-ZM1 for 1 h and then treated with rhHMGB1 for 60 minutes. Mean ± SD of 3 independent trials was shown. *p < 0.05 vs. control. # p < 0.05 vs. rhHMGB1 60-min group or rhHMGB1 24-h group.