Ghrelin pretreatment enhanced the protective effect of bone marrow-derived mesenchymal stem cell-conditioned medium on lipopolysaccharide-induced endothelial cell injury

Background: Lung endothelial barrier injury plays a crucial role in the pathophysiology of acute respiratory distress syndrome. It has been demonstrated that bone marrow-derived mesenchymal stem cells-conditioned medium (BMSCs-CM) and ghrelin have a protective effect. This study investigated if ghrelin pretreatment enhanced the protective effect of BMSCs-CM on lipopolysaccharide (LPS)-induced endothelial cell injury. Methods: BMSCs were isolated from rat bone marrow, expanded, then phenotypically tested for mesenchymal stem cell-identifying criteria by flow cytometry. The effects of the conditioned medium derived from ghrelin-pretreated BMSCs (BMSCs-(ghrelin-pretreated)-CM) on LPS-injured endothelial cells were evaluated by migration, apoptosis, permeability, and pro-inflammatory factor (e.g., tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-6) assays in endothelial cells. Further, AKT/GSK3 beta pathway activation in endothelial cells was examined by Western blot, and the gene expression profiles of ghrelin-pretreated BMSCs were examined by RNA sequencing. Results: BMSCs-(ghrelin-pretreated)-CM had a greater protective effect on LPS-induced endothelial cell injury than BMSCs-CM by improving cell migration, alleviating apoptosis, and reducing endothelial permeability and the release of pro-inflammatory factors in endothelial cells. The mechanism is partly related to AKT/GSK3 beta pathway activation after BMSCs-(ghrelin-pretreated)-CM treatment. There were five upregulated candidate genes (Wnt5 alpha [i.e., Wnt Family Member 5A], S100b [i.e., S100 Calcium-Binding Protein B], Bmp2 [i.e., Bone Morphogenetic Protein 2], Id4 [i.e., Inhibitor Of DNA Binding 4], and PTHLH [i.e., Parathyroid Hormone Like Hormone]) in BMSCs after ghrelin treatment, and all were associated with AKT pathway activation and endothelial function. Conclusions: Ghrelin pretreatment enhanced the protective effect of BMSCs-CM on LPS-induced endothelial cell injury, partly by activating the AKT/GSK3 beta pathway.