ABSTRACT
Ischemia/reperfusion (I/R) is a pathological process that occurs in numerous organs and is often associated with severe cellular damage and death. Ectodysplasin-A2 receptor (EDA2R) is a member of the TNF receptor family that has anti-inflammatory and antioxidant effects. However, to the best of our knowledge, its role in the progression of myocardial I/R injury remains unclear. The present study aimed to investigate the role of EDA2R during myocardial I/R injury and the molecular mechanisms involved. In vitro, dexmedetomidine (DEX) exhibited a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and downregulated EDA2R expression. Subsequently, EDA2R silencing enhanced cell viability and reduced the apoptosis of cardiomyocytes. Furthermore, knockdown of EDA2R led to an elevated mitochondrial membrane potential (MMP), repressed the release of Cytochrome C and upregulated Bcl-2 expression. EDA2R knockdown also resulted in downregulated expression of Bax, and decreased activity of Caspase-3 and Caspase-9 in cardiomyocytes, reversing the effects of H/R on mitochondria-mediated apoptosis. In addition, knockdown of EDA2R suppressed H/R-induced oxidative stress. Mechanistically, EDA2R knockdown inactivated the NF-κB signaling pathway. Additionally, downregulation of EDA2R weakened myocardial I/R injury in mice, as reflected by improved left ventricular function and reduced infarct size, as well as suppressed apoptosis and oxidative stress. Additionally, EDA2R knockdown repressed the activation of NF-κB signal in vivo. Collectively, knockdown of EDA2R exerted anti-apoptotic and antioxidant effects against I/R injury in vivo and in vitro by suppressing the NF-κB signaling pathway.
ABSTRACT
OBJECTIVE: To establish a stable method for obtaining large quantity of highly purified immature dendritic cells (imDCs) in vitro, and identify the morphology, function and surface markers of the cells. METHODS: CD117(+) hemopoietic stem cells (HSCs) were isolated and purified from the bone marrow of healthy C57 mice by magnetic affinity cell sorting. After cell expansion by treatment with stem cell factor (SCF) and interleukin-3 (IL-3), the HSCs were induced for directional differentiation into imDCs by treatment with GM-CSF, IL-4 and IL-10. The imDCs obtained were identified by morphological and functional observation under inverted microscope, scanning electron microscope and transmission electron microscope, followed by detection of the expressions of the surface markers using flow cytometry. RESULTS: After 3, 5 and 7 days of culture in the presence of SCF+IL-3, the cells were expanded by 10.34-/+1.43, 22.65-/+2.71 and 54.39-/+3.08 folds, respectively. The HSCs were successfully induced to differentiate into imDCs with phagocytotic activity. The dendrites of the imDCs were short small, and appearing spinous. The expressions of surface markers were detected from the cells showing the phenotype of CD11c(+), I-A/I-E(low), CD40(-), CD80(-), CD86(-). CONCLUSION: The method described allows steadily acquisition of large quanty of highly purified imDCs and of their effective identification in vitro.