orexin B alleviates sepsis-associated lung injury through the attenuation of pulmonary endothelial barrier dysfunction by regulating the rho-associated coiled-coil containing protein kinase 2/zonula occludens-1 (ROCK2/ZO-1) axis.

Dysfunction of the alveolar endothelial barrier plays a crucial role in the pathogenesis of septic acute lung injury (ALI). orexin B is a neuropeptide derived from orexin neurons in the lateral hypothalamus and has multiple biological functions. However, the physiological function of orexin B in sepsis is less reported. Here, we aimed to explore the protective effects of orexin B in sepsis-induced ALI and its underlying mechanisms. In this study, we established an ALI in vivo animal model in mice using cecal ligation and puncture (CLP) and an in vitro ALI model using mouse lung microvascular endothelial cells (MLMECs) induced with lipopolysaccharides (LPS). The animal experiments involved four groups: Sham, Sham+orexin B, CLP, CLP+orexin B. First, our results demonstrate that the levels of serum orexin B but not orexin A were reduced in septic mice. Correspondingly, the expression of orexin type 2 receptor (OX2R), but not orexin type 1 receptor (OX1R), was reduced in the lung tissue of septic mice. Administration of orexin B decreased the mortality in sepsis mice and improved M-CASS scores. Hematoxylin-eosin (H&E) staining assay demonstrated that administration of orexin B ameliorated histopathological lung injury. orexin B was also found to inhibit the inflammatory response in the lung tissues of septic mice by reducing the expression of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and recombinant chemokine C-X-C-motif ligand 15 (CXCL15). Additionally, the total cell count and neutrophils in bronchoalveolar lavage fluid (BALF) were reduced by orexin B. Notably, orexin B alleviated vascular endothelial permeability in mice lung tissue by increasing the expression of the tight junction protein zonula occludens-1 (ZO-1) and occludin. In vitro experiments demonstrated that orexin B prevented LPS-induced endothelial permeability in mouse lung microvascular endothelial cells (MLMECs) by upregulating the expression of ZO-1 and occludin. These effects are mediated by rho-associated coiled-coil containing protein kinase 2 (ROCK2). Based on these findings, we conclude that orexin B alleviates sepsis-induced ALI by ameliorating endothelial permeability of lung microvascular endothelial cells.