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BACKGROUND: Endothelial barrier dysfunction induced by heatstroke contributes to acute lung injury; however, the underlying metabolic mechanisms remain unclear. The glycolytic regulator PFKFB3 is known to fuel inflammation in various pathologies, while the RhoA/ROCK axis controls cytoskeletal dynamics and vascular permeability. Here, we investigated whether PFKFB3-mediated glycolysis activated RhoA/ROCK to disrupt endothelial integrity in heatstroke. METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were exposed to heat stress (43 °C) and lipopolysaccharide(LPS) to model heatstroke in vitro. Glycolytic flux (extracellular acidification rate, ECAR; oxygen consumption rate, OCR; lactate and ATP levels) and transcriptomic profiling (RNA-seq) were used to analyze metabolic and inflammatory pathways. RhoA/ROCK activation was investigated by western blot analysis and pull-down assays, while F-actin immunofluorescence and FITC-dextran permeability were used to investigate cytoskeletal dynamics and barrier integrity. Pharmacological inhibitors (PFK15, Rhosin, Y-27632) and siRNA targeting PFKFB3, RhoA, or ROCK were utilized for mechanistic investigations. RESULTS: Heat-LPS stress activated glycolysis, as demonstrated by increased ECAR, lactate, and glycolytic ATP. Transcriptomic analysis confirmed the association between PFKFB3 upregulation and pro-inflammatory cytokine pathways. This stress triggered the upregulation of PFKFB3 and RhoA/ROCK1/MLC2 signaling, leading to the formation of F-actin stress fibers, VE-cadherin disassembly, and barrier hyperpermeability. PFKFB3 inhibition (PFK15/siRNA) suppressed glycolysis, abrogated RhoA/ROCK activation, and restored barrier function. RhoA (Rhosin/siRNA) or ROCK1 (Y-27632/siRNA) inhibition similarly attenuated cytoskeletal disruption and permeability. CONCLUSION: PFKFB3-driven glycolysis activates RhoA/ROCK1 to disrupt endothelial barriers in heatstroke. Targeting this metabolic-inflammatory axis may mitigate pulmonary vascular leakage, thus providing a therapeutic strategy against heatstroke induced acute lung injury.
Ding et al. (Thu,) studied this question.