Calreticulin Ameliorates Hypoxia/Reoxygenation-Induced Human Microvascular Endothelial Cell Injury By Inhibiting Autophagy

Shock
You WangXiuhua Liu

Abstract

Autophagy has been found to be involved in myocardial ischemia/reperfusion injury. However, the underlying mechanism and significance of autophagy in reperfusion injury remain unclear. Herein, we evaluated the effects of exogenous calreticulin (CRT) on autophagy in hypoxia/reoxygenation (H/R)-treated human microvascular endothelial cells (MECs). Human MECs were pretreated with CRT (25 pg/mL) for 30 min, followed by exposure in an incubator filled with a gas mixture of 90% N2, 5% O2, and 5% CO2 for 8-h hypoxia. The cells were then placed back in the normoxic CO2 incubator for 16-h reoxygenation. Cell injury was assessed by the cell counting kit-8 assay. Autophagosomes were detected by transmission electron microscopy and immunofluorescence staining. Western blot analysis was performed to detect phosphorylated mammalian target of rapamycin (p-mTOR), Beclin 1, and microtubule-associated protein 1 light chain 3 (LC3). H/R induced marked autophagy through the mTOR pathway. CRT suppressed rapamycin- and H/R-induced autophagosome formation, the LC3-II/LC3-I ratio, and Beclin 1 expression in human MECs by upregulating mTOR phosphorylation, consequently attenuating H/R-induced human MEC injury. Exogenous CRT attenuated H/R-induced human...Continue Reading

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