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标题：Acute Methylmercury Exposure and the Hypoxia-Inducible [... formula ...] Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro
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作者：Jie Chang ; Bobo Yang ; Yun Zhou 等
期刊名称：Environmental Health Perspectives
印刷版ISSN：0091-6765
电子版ISSN：1552-9924
出版年度：2019
卷号：127
期号：12
页码：1-12
DOI：10.1289/EHP5139
出版社：OCR Subscription Services Inc
摘要：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 α ( HIF- 1 α ), 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 α 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 μ M ) for 0.5 h . Cell proliferation and cytotoxicity were assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide (MTT) 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- 1 α , 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- 1 α 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 α inhibition by MeHg. To investigate the role of HIF- 1 α in MeHg-induced neurotoxicity, cobalt chloride ( CoC l 2 ), 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 HIF- 1 α levels. The expression of HIF- 1 α 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 μ M MeHg for 0.5 h significantly inhibited the expression of astrocytic HIF- 1 α , and the downstream genes GLUT-1, EPO, and VEGF-A ( p < 0.05 ), in the absence of a significant decrease in HIF- 1 α mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of HIF- 1 α . MG132 and DHB significantly blocked the MeHg-induced decrease in HIF- 1 α expression ( p < 0.05 ). Overexpression of HIF- 1 α significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of HIF- 1 α significantly increased the decline in cell proliferation ( p < 0.05 ). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in HIF- 1 α protein levels and the decrease in cell proliferation ( p < 0.05 ). MeHg suppressed the expression of HIF- 1 α and related downstream target proteins in adult rat brain. Discussion: MeHg induced a significant reduction in HIF- 1 α 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 α expression in response to MeHg toxicity. Moreover, we established that up-regulation of HIF- 1 α might serve to mitigate the acute toxicity of MeHg in astrocytes, affording a novel therapeutic target for future exploration.