Oxidative stress and inflammatory responses play critical roles in hypoxic-ischemic brain injury. Microglial cells are rapidly activated in response to injury and stressful stimuli, including hypoxia. Endothelin-1 (ET-1) is a potent vasoconstrictor that has been associated with cerebrovascular diseases. Hypoxia stimulates endothelial ET-1 production. However, the role of ET-1 in microglia under hypoxia is not clear. The aim of this project was to characterize the effect of hypoxia in a human microglial cell line, HMC3. We induced hypoxia using a chamber (1% O2, 5% CO2, and 92 % N2) at 37 °C for 4h, 24h, and 48h. MUSE Oxidative Stress Assay was performed to measure reactive oxygen species (ROS) formation, ELISA to determine TNF, IL-6, and ET-1 levels, qPCR to measure gene expression of ET-1, and immunofluorescence staining to visualize and compare the presence of ET-1 in hypoxic and control group cells. It was observed that, when compared to normoxic HMC3 cells, hypoxic HMC3 exposure significantly increased the ROS by a factor of 2.5 (p<0.001, n=3), the pro-inflammatory cytokine TNF-α increased 3.8 times (p<0.01, n=4), and IL-6 increased by a factor of 1.6 (p<0.01, n=4). In addition, hypoxia stimulates ET-1 gene expression 5.0-fold (p<0.001, n=4) and increased protein production 1.3 times (p<0.01, n=4). Consequently, treatment with ET-1 increased the amount of ROS, TNF-α, and IL-6 in HMC3 cells by a factor of 1.4 (p<0.05, n=4), 1.6 (p<0.001, n=4), and 1.9 (p<0.05, n=4), respectively. All these events were blocked by ET-1 receptor A (ETRA) antagonist, BQ123. Our results suggest that hypoxic conditions create a cycle of microglial cell activation leading to increased ROS and ET-1 production that further stimulate microglial cells. Thus, we posit that the ET-1 receptor blockade represents a promising therapeutic approach to regulate microglial cell responses in hypoxic-ischemic brain injury.
