Abstract

Objective. Ischemia-reperfusion injury refers to the exacerbated and irreversible tissue damage caused by blood flow restoration after a period of ischemia. The hypoxia-reoxygenation (H/R) model in vitro is ideal for studying ischemia-reperfusion injury at the cellular level. We employed this model and investigated the effects of cobalt chloride- (CoCl2-) induced H/R in cells derived from mouse digital flexor tendons. Materials and Methods. Various H/R conditions were simulated via treatment of tendon-derived cells with different concentrations of CoCl2 for 24 h, followed by removal of CoCl2 to restore a normal oxygen state for up to 96 h. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Cell growth was determined via observation of cell morphology and proliferation. Oxidative stress markers and mitochondrial activity were detected. The expression levels of hypoxia-inducible factor- (HIF-) 1α, vascular endothelial growth factor-A (VEGF-A), collagen I, and collagen III were determined using Western blot (WB), real-time PCR, and immunofluorescence staining. Cellular apoptosis was analyzed via flow cytometry, and the expression of apoptosis-related proteins Bax and bcl-2 was examined using WB. Results. The cells treated with low concentrations of CoCl2 showed significantly increased cell viability after reoxygenation. The increase in cell viability was even more pronounced in cells that had been treated with high concentrations of CoCl2. Under H/R conditions, cell morphology and growth were unchanged, while oxidative stress reaction was induced and mitochondrial activity was increased. H/R exerted opposite effects on the expression of HIF-1α mRNA and protein. Meanwhile, the expression of VEGF-A was upregulated, whereas collagen type I and type III were significantly downregulated. The level of cellular apoptosis did not show significant changes during H/R, despite the significantly increased Bax protein and reduced bcl-2 protein levels that led