资源环境科技发展态势分析平台

BACKGROUND: As a ubiquitous environmental pollutant, methylmercury (MeHg) induces toxic effects in the nervous system, one of its main targets. However, the exact mechanisms of its neurotoxicity have not been fully elucidated. Hypoxia-inducible factor-1 alpha (HIF-1 alpha), a transcription factor, plays a crucial role in adaptive and cytoprotective responses in cells and is involved in cell survival, proliferation, apoptosis, inflammation, angiogenesis, glucose metabolism, erythropoiesis, and other physiological activities.

OBJECTIVES: The aim of this study was to explore the role of HIF-1 alpha in response to acute MeHg exposure in rat brain and primary cultured astrocytes to improve understanding of the mechanisms of MeHg-induced neurotoxicity and the development of effective neuroprotective strategies.

METHODS: Primary rat astrocytes were treated with MeHg (0-10 mu M) for 0.5 h. Cell proliferation and cytotoxicity were assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide (MU) assay and a lactate dehydrogenase (LDH) release assay, respectively. Reactive oxygen species (ROS) levels were analyzed to assess the level of oxidative stress using 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescence. HIF-l alpha, and its downstream proteins, glucose transporter 1 (GLUT-1), erythropoietin (EPO), and vascular endothelial growth factor A (VEGF-A) were analyzed by means of Western blotting. Real-time PCR was used to detect the expression of HIF-la mRNA. Pretreatment with protein synthesis inhibitor (CHX), proteasome inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of HIF-1 alpha inhibition by MeHg. To investigate the role of HIF-1 alpha in MeHg-induced neurotoxicity, cobalt chloride (CoCl2), 2-methoxyestradiol (2-MeOE2), small interfering RNA (siRNA) transfection and adenovirus overexpression were used. Pretreatment with N-acetyl-L-cysteine (NAC) and vitamin E (Trolox) were used to investigate the putative role of oxidative stress in MeHg-induced alterations in HIP-1 alpha levels. The expression of HIF-1 alpha and related downstream proteins was detected in adult rat brain exposed to MeHg (0-10 mg/kg) for 0.5 h in vivo.

RESULTS: MeHg caused lower cell proliferation and higher cytotoxicity in primary rat astrocytes in a time- and concentration-dependent manner. In comparison with the control cells, exposure to 10 mu M MeHg for 0.5 h significantly inhibited the expression of astrocytic HIF-1 alpha, and the downstream genes GLUT-1, EPO, and VEGF-A (p < 0.05), in the absence of a significant decrease in HIF-1 alpha mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of HIF-1 alpha. MG132 and DHB significantly blocked the MeHg-induced decrease in HIF-1 alpha expression (p < 0.05). Overexpression of HIF-1 alpha significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of HIF-1 alpha significantly increased the decline in cell proliferation (p <0.05). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in HIF-1 alpha protein levels and the decrease in cell proliferation (p < 0.05). MeHg suppressed the expression of HIF-1 alpha and related downstream target proteins in adult rat brain.

DISCUSSION: MeHg induced a significant reduction in HIF-1 alpha protein by activating proline hydroxylase (PHD) and the ubiquitin proteasome system (UPS) in primary rat astrocytes. Additionally, ROS scavenging by antioxidants played a neuroprotective role via increasing HIF-1 alpha expression in response to MeHg toxicity. Moreover, we established that up-regulation of HIF-1 alpha might serve to mitigate the acute toxicity of MeHg in astrocytes, affording a novel therapeutic target for future exploration.