DNAJA1‑knockout alleviates heat stroke‑induced endothelial barrier disruption via improving thermal tolerance and suppressing the MLCK‑MLC signaling pathway

Li,Lei; Wang,Ya-Wei; Chang,Xin; Chen,Jue-Lin; Wang,Man; Zhu,Jia-Qi; Li,Jin-Feng; Ren,Li-Jun; Dai,Xiao-Yu; Yan,Lang; Fan,Xin-Chen; Song,Qing; Zhu,Jiang-Bo; Chen,Ji-Kuai; Xu,Shuo-Gui

doi:10.3892/mmr.2024.13211

DNAJA1‑knockout alleviates heat stroke‑induced endothelial barrier disruption via improving thermal tolerance and suppressing the MLCK‑MLC signaling pathway

Authors:
- Lei Li
- Ya-Wei Wang
- Xin Chang
- Jue-Lin Chen
- Man Wang
- Jia-Qi Zhu
- Jin-Feng Li
- Li-Jun Ren
- Xiao-Yu Dai
- Lang Yan
- Xin-Chen Fan
- Qing Song
- Jiang-Bo Zhu
- Ji-Kuai Chen
- Shuo-Gui Xu
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Affiliations: Department of Emergency, Changhai Hospital, Naval Medical University, Shanghai 200433, P.R. China, Department of Gastroenterology, Changhai Hospital, Shanghai 200433, P.R. China, Department of Rehabilitation, Changhai Hospital, Shanghai 200433, P.R. China, Department of Cardiology, Changhai Hospital, Shanghai 200433, P.R. China, Department of Health Toxicology, Faculty of Naval Medicine, Naval Medical University, Shanghai 200433, P.R. China, College of Basic Medical Sciences, Naval Medical University, Shanghai 200433, P.R. China, Heatstroke Treatment and Research Center, Hainan Hospital, Chinese People's Liberation Army General Hospital, Sanya, Hainan 572022, P.R. China
Published online on: March 26, 2024 https://doi.org/10.3892/mmr.2024.13211
Article Number: 87
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Endothelial barrier disruption plays a key role in the pathophysiology of heat stroke (HS). Knockout of DNAJA1 (DNAJA1‑KO) is thought to be protective against HS based on a genome‑wide CRISPR‑Cas9 screen experiment. The present study aimed to illustrate the function of DNAJA1‑KO against HS in human umbilical vein endothelial cells. DNAJA1‑KO cells were infected using a lentivirus to investigate the role of DNAJA1‑KO in HS‑induced endothelial barrier disruption. It was shown that DNAJA1‑KO could ameliorate decreased cell viability and increased cell injury, according to the results of Cell Counting Kit‑8 and lactate dehydrogenase assays. Moreover, HS‑induced endothelial cell apoptosis was inhibited by DNAJA1‑KO, as indicated by Annexin V‑FITC/PI staining and cleaved‑caspase‑3 expression using flow cytometry and western blotting, respectively. Furthermore, the endothelial barrier function, as measured by transepithelial electrical resistance and FITC‑Dextran, was sustained during HS. DNAJA1‑KO was not found to have a significant effect on the expression and distribution of cell junction proteins under normal conditions without HS. However, DNAJA1‑KO could effectively protect the HS‑induced decrease in the expression and distribution of cell junction proteins, including zonula occludens‑1, claudin‑5, 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 HS‑induced wild‑type cells and inhibited by DNAJA1‑KO. DEPs were investigated by enrichment analysis, which demonstrated significant enrichment in the ‘calcium signaling pathway’ and associations with vascular‑barrier regulation. Furthermore, the ‘myosin light‑chain kinase (MLCK)‑MLC signaling pathway’ was proven to be activated by HS and inhibited by DNAJA1‑KO, as expected. Moreover, DNAJA1‑KO 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 DNAJA1‑KO alleviates HS‑induced endothelial barrier disruption by improving thermal tolerance and suppressing the MLCK‑MLC signaling pathway.

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