Hydrogen prevents lipopolysaccharide-induced pulmonary microvascular endothelial cell injury by inhibiting store-operated Ca2+ entry regulated by STIM1/Orai1

Abstract:

Background: Sepsis is a type of life-threatening organ dysfunction that is caused by a dysregulated host response to infection. The lung is the most vulnerable target organ under septic conditions. Pulmonary microvascular endothelial cells (PMVECs) play a critical role in acute lung injury (ALI) caused by severe sepsis. The impairment of PMVECs during sepsis is a complex regulatory process involving multiple mechanisms, in which the imbalance of calcium (Ca2+) homeostasis of endothelial cells is a key factor in its functional impairment. Our preliminary results indicated that hydrogen gas (H2) treatment significantly alleviates lung injury in sepsis, protects PMVECs from hyperpermeability, and decreases the expression of plasma membrane stromal interaction molecule 1 (STIM1), but the underlying mechanism by which H2 maintains Ca2+ homeostasis in endothelial cells in septic models remains unclear. Thus, the purpose of the present study was to investigate the molecular mechanism of STIM1 and Ca2+-release-activated- Ca2+ channel protein1 (Orai1) regulation by H2 treatment and explore the effect of H2 treatment on Ca2+ homeostasis in lipopolysaccharide (LPS)-induced PMVECs and LPS-challenged mice.

Methods: We observed the role of H2 on LPS-induced ALI of mice in vivo. The lung wet/dry (W/D) weight ratio, total protein in the bronchoalveolar lavage (BAL) fluid and Evans blue dye (EBD) assay were used to evaluate the pulmonary endothelial barrier damage of LPS-challenged mice. The expression of STIM1 and Orai1 were also detected using epifluorescence microscopy. Moreover, we also investigated the role of H2- rich medium in regulating PMVECs under LPS treatment, which induced injury similar to sepsis in vitro. The expression of STIM1 and Orai1 as well as the Ca2+ concentration in PMVECs were examined.

Results: In vivo, we found that H2 alleviated ALI of mice through decreasing lung W/D weight ratio, total protein in the BAL fluid and permeability of lung. In addition, H2 also decreased the expression of STIM1 and Orai1 in pulmonary microvascular endothelium. In vitro, LPS treatment increased the expression levels of STIM1 and Orai1 in PMVECs, while H2 reversed these changes. Furthermore, H2 ameliorated Ca2+ influx under sepsis-mimicking conditions. Treatment with the sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) inhibitor, thapsigargin (TG), resulted in a significant reduction in cell viability as well as a reduction in the expression of junctional proteins, including VE-cadherin and occludin. Treatment with the store-operated Ca2+ entry (SOCE) inhibitor, YM-58483 (BTP2), increased the cell viability and expression of junctional proteins. Conclusions: The present study suggested that H2 treatment alleviates LPS-induced PMVEC dysfunction by inhibiting SOCE mediated by STIM1 and Orai1 in vitro and in vivo.