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

Figure 2.

Figure 2

Y-27632 pretreatment attenuated rhHMGB1-induced ROCK1/pMLC expression. (A) CCK-8 assay was performed with HPMECs for 24 h with different dosages of Y-27632 as indicated. (B) Effects of Y-27632 on changes in FITC-dextran flux in HPMECs. HPMECs were pretreated with Y-27632 and then treated with 600 ng/ml rhHMGB1 for 60 min. (C) Role of Y-27632 pretreatment in increased barrier permeability induced by rhHMGB1 at 24 h. (D) Effects of Y-27632 on rhHMGB1-mediated morphological change in endothelial F-actin, VE-cadherin and ZO-1. HPMECs were pretreated with Y-27632 for 1 h before rhHMGB1 (600 ng/ml) stimulation for 60 minutes to examine morphology of endothelial F-actin, VE-cadherin and ZO-1 by immunofluorescence. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Y-27632 pretreatment downregulated the ROCK1 expression induced by rhHMGB1 at 60 min. (F) Effects of Y-27632 treatment on rhHMGB1-induced changes in the protein expression levels of VE-cadherin and ZO-1 at 24 h. Y-27632 were added for the last 4 h of the 24 h rhHMGB1 treatment. (G) Pretreatment with Y-27632 attenuated rhHMGB1-induced MLC phosphorylation at 60 min. ECs were pretreated with Y-27632 for 1 h and then stimulated with rhHMGB1 (600 ng/ml) for 1 h. Values were indicated as mean ± SD of 3 separate trials. *p < 0.05 vs. control. # p < 0.05 vs. rhHMGB1 60-min group.