Endothelin-1 Signaling Mediates Hypoxia-Induced Microglial Activation Through Reactive Oxygen Species and Mitogen-Activated Protein Kinase Pathways.
Source: PubMed, NCBI / U.S. National Library of Medicine
Oxidative stress and neuroinflammation are critical contributors to hypoxic-ischemic brain injury. Microglia, the CNS-resident immune cells, undergo rapid activation in response to hypoxic stress. Endothelin-1 (ET-1), a vasoconstrictor implicated in cerebrovascular pathology, is upregulated by hypoxia; however, its role in microglial activation remains poorly understood. HMC3 human microglial cells were exposed to hypoxia (1% O) for 4 hours. Reactive oxygen species (ROS) were quantified by flow cytometry. ET-1 and interleukin-6 (IL-6) protein concentrations were measured by ELISA and mRNA levels by qPCR. Mitogen-activated protein kinase (MAPK) activation and ET-1 localization were assessed by flow cytometry and immunofluorescence, respectively. The endothelin B receptor (ETR) antagonist BQ788 was used to assess ET-1 signaling in hypoxia-induced responses. Hypoxia significantly upregulated ET-1 gene expression (5.0-fold increase, < 0.001, = 4) and elevated ET-1 protein production by 1.4-fold ( < 0.01, = 4). IL-6 expression and secretion increased 1.5-fold under hypoxic conditions ( < 0.01, = 4), an effect that was attenuated by BQ788 pretreatment. ROS levels increased 1.9-fold in hypoxic HMC3 cells ( < 0.01, = 4) but were significantly reduced by ETR inhibition. Additionally, hypoxia elevated the percentage of MAPK-activated cells compared to both normoxic and
Abstract
Oxidative stress and neuroinflammation are critical contributors to hypoxic-ischemic brain injury. Microglia, the CNS-resident immune cells, undergo rapid activation in response to hypoxic stress. Endothelin-1 (ET-1), a vasoconstrictor implicated in cerebrovascular pathology, is upregulated by hypoxia; however, its role in microglial activation remains poorly understood. HMC3 human microglial cells were exposed to hypoxia (1% O) for 4 hours. Reactive oxygen species (ROS) were quantified by flow cytometry. ET-1 and interleukin-6 (IL-6) protein concentrations were measured by ELISA and mRNA levels by qPCR. Mitogen-activated protein kinase (MAPK) activation and ET-1 localization were assessed by flow cytometry and immunofluorescence, respectively. The endothelin B receptor (ETR) antagonist BQ788 was used to assess ET-1 signaling in hypoxia-induced responses. Hypoxia significantly upregulated ET-1 gene expression (5.0-fold increase, < 0.001, = 4) and elevated ET-1 protein production by 1.4-fold ( < 0.01, = 4). IL-6 expression and secretion increased 1.5-fold under hypoxic conditions ( < 0.01, = 4), an effect that was attenuated by BQ788 pretreatment. ROS levels increased 1.9-fold in hypoxic HMC3 cells ( < 0.01, = 4) but were significantly reduced by ETR inhibition. Additionally, hypoxia elevated the percentage of MAPK-activated cells compared to both normoxic and BQ788-treated groups ( < 0.01). These findings demonstrate that hypoxia induces ET-1 overexpression, ROS generation, MAPK activation, and IL-6 production in microglia, establishing a self-perpetuating cycle of neuroinflammation. ETR blockade with BQ788 disrupts this cascade, suggesting that ET-1 signaling is a promising therapeutic target to mitigate secondary injury in hypoxic-ischemic brain conditions.