RESUMO
Endothelial barrier disruption plays a key role in the pathophysiology of heat stroke (HS). Knockout of DNAJA1 (DNAJA1KO) is thought to be protective against HS based on a genomewide CRISPRCas9 screen experiment. The present study aimed to illustrate the function of DNAJA1KO against HS in human umbilical vein endothelial cells. DNAJA1KO cells were infected using a lentivirus to investigate the role of DNAJA1KO in HSinduced endothelial barrier disruption. It was shown that DNAJA1KO could ameliorate decreased cell viability and increased cell injury, according to the results of Cell Counting Kit8 and lactate dehydrogenase assays. Moreover, HSinduced endothelial cell apoptosis was inhibited by DNAJA1KO, as indicated by Annexin VFITC/PI staining and cleavedcaspase3 expression using flow cytometry and western blotting, respectively. Furthermore, the endothelial barrier function, as measured by transepithelial electrical resistance and FITCDextran, was sustained during HS. DNAJA1KO was not found to have a significant effect on the expression and distribution of cell junction proteins under normal conditions without HS. However, DNAJA1KO could effectively protect the HSinduced decrease in the expression and distribution of cell junction proteins, including zonula occludens1, claudin5, junctional adhesion molecule A and occludin. A total of 4,394 proteins were identified using proteomic analysis, of which 102 differentially expressed proteins (DEPs) were activated in HSinduced wildtype cells and inhibited by DNAJA1KO. DEPs were investigated by enrichment analysis, which demonstrated significant enrichment in the 'calcium signaling pathway' and associations with vascularbarrier regulation. Furthermore, the 'myosin lightchain kinase (MLCK)MLC signaling pathway' was proven to be activated by HS and inhibited by DNAJA1KO, as expected. Moreover, DNAJA1KO mice and a HS mouse model were established to demonstrate the protective effects on endothelial barrier in vivo. In conclusion, the results of the present study suggested that DNAJA1KO alleviates HSinduced endothelial barrier disruption by improving thermal tolerance and suppressing the MLCKMLC signaling pathway.