Abstract: BACKGROUND: Drawing on the characteristics of the in vivo ischemic and hypoxic microenvironment, hypoxic preconditioning, as an active strategy that simulates physiological and pathological conditions, can significantly enhance the paracrine function of stem cells. However, the specific effects and underlying mechanisms of hypoxic preconditioning on the angiogenic behavior of damaged endothelial cells remain to be further elucidated.
OBJECTIVE: To investigate the regulatory effect of hypoxic preconditioning on the paracrine function of bone marrow mesenchymal stem cells, aiming to quantitatively explore the effects and mechanisms of conditioned medium from hypoxic preconditioned bone marrow mesenchymal stem cells on the angiogenic capacity of damaged endothelial cells.
METHODS: Human umbilical vein endothelial cells were divided into control group, model group, normoxic group, and hypoxic group. The control group consisted of human umbilical vein endothelial cells cultured under normal conditions. The model group was treated with 1 μg/mL lipopolysaccharide for 24 hours. The normoxic and hypoxic groups were treated with supernatant from bone marrow mesenchymal stem cells cultured under normoxic and hypoxic conditions for 48 hours, respectively, for 1 hour, followed by treatment with 1 μg/mL lipopolysaccharide for 24 hours. Cell proliferation was detected by CCK-8 assay. Cell apoptosis rate was determined by flow cytometry. Cell angiogenesis capacity was measured by tube formation assay. The levels of tumor necrosis factor-α and interleukin-6 in the cell supernatant were detected by ELISA. The mRNA and protein expression levels of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and vascular endothelial growth factor in cells were detected by RT-qPCR and western blot assay, respectively. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the model group showed significantly lower cell survival rate, tube formation ability, and mRNA and protein expression levels of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and vascular endothelial growth factor (P < 0.01), while the apoptosis rate and levels of tumor necrosis factor α and interleukin 6 in the cell supernatant were significantly higher (P < 0.01). (2) Compared with the model group, there were no significant differences in the above indicators in the normoxic group (P > 0.05), while the hypoxic group showed significantly higher cell survival rate, tube formation ability, and mRNA and protein expression levels of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and vascular endothelial growth factor (P < 0.01), and significantly lower apoptosis rate and levels of tumor necrosis factor α and interleukin 6 in the cell supernatant (P < 0.01). The results indicate that paracrine cytokines from hypoxic preconditioned bone marrow mesenchymal stem cells can inhibit the inflammatory response of vascular endothelial cells and promote angiogenesis by upregulating angiogenic factors. 

Key words: fracture healing, hypoxia pretreatment, bone marrow mesenchymal stem cells, angiogenesis, paracrine cytokines, inflammation