Skip to main content
. 2021 Oct 20;12:697071. doi: 10.3389/fimmu.2021.697071

Figure 5.

Figure 5

rhHMGB1-induced RhoA/ROCK1 pathway activation via RAGE in HPMECs. (A) ECs were transfected with RAGE siRNA. Western blots were used to determine the expression of RAGE in endothelial cells. (B) Cytotoxicity of RAGE siRNA was assessed by CCK-8 assay after transfected with different concentration of RAGE siRNA for 24 h or transfected with 100 nM RAGE siRNA for the different times. No evidence of cytotoxicity was found in RAGE siRNA transfected cells. (C) Treatment with RAGE siRNA ameliorated endothelial barrier dysfunction induced by rhHMGB1 at 60 min and 24 h. (D) ECs were transfected with RAGE siRNA and then stimulated with rhHMGB1 for 60 min and 24 h. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was determined by fluorescence microscopy. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Knockdown of RAGE by siRNA reduced the rhHMGB1-induced MLC phosphorylation at 60 min as detected by western blot. (F) Effects of inhibition of RAGE with siRNA on increased expression of RhoA and ROCK1 induced by rhHMGB1 at 60 min in HPMECs. (G) Role of RAGE siRNA in the VE-cadherin and ZO-1 protein expression levels in ECs at 24 h after rhHMGB1 treatment. Mean ± SD of 3 independent trials was shown. *p < 0.05 vs. corresponding control group. # p < 0.05 vs. rhHMGB1 60-min group or rhHMGB1 24-h group. NC, negative control.