An increase in reactive oxygen spieces and the downregulation of thrombospondin-1 in primary rat astrocytes under CoCl2-induced oxidative stress

• Main Authors: Yan-Jie Zhan, 詹焱捷
• Other Authors: Shun-Fen Tzeng
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/59501968906154710401

碩士 === 國立成功大學 === 生命科學系碩博士班 === 96 === Oxidative stress induced by cerebral ischemia along with a mass of the generation of reactive oxygen species (ROS) contribute to neuronal degeneration. Astrocytes, the major glial population in the central nervous system (CNS), mediate neuronal maturation, survival, and transmission. Thrombospondin (TSP)-1, a multidomain glycoprotein, can be secreted by astrocytes and has the role in brain functional repair because of its importance in synaptogenesis, synaptic plasticity and axonal sprouting. However, little was known about the regulation of TSP-1 expression in astrocytes under oxidative stress, so we wanted to figure out that TSP-1 expression in primary rat astrocytes under oxidative stress. In this study, the culture of astrocytes isolated from neonatal rat cortical tissues was exposed to a hypoxic mimetic reagent, cobalt chloride (CoCl2), which is often used as stimulus for chemically induced oxidative stress. Cell viability assay indicated that CoCl2 at the concentrations of 0.1-0.5 mM did not cause significant cell death in astrocytic cultures. However, ROS levels in astrocytes were dose- and time-dependently elevated by CoCl2. Exposure to CoCl2 at 0.3 mM and 0.5 mM also induced an increase in gene expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is known to be sensitive to oxidative damage. Since ROS-triggered oxidative stress can lead to c-Jun N-terminal Kinase (JNK) activation, we examined whether CoCl2 can induce c-Jun phosphorylation in astrocytes. Indeed, Western Blot analysis showed that treatment with a brief increase in the phosphorylation of c-Jun was observed at 5 min after treatment with CoCl2. Based on these observations, CoCl2-induced oxidative stress in astrocytes was verified. Furthermore, real-time quantitative PCR analysis showed that TSP-1 mRNA expression in astrocytes was slightly upregulated at 2 h after exposure to CoCl2, followed by a significant downregulation at 12 h and at 24 h. After a 2 h-treatment with CoCl2 following cell recovery in the absence of CoCl2, the reduction in TSP-1 mRNA expression was continuously observed. However, GAPDH mRNA expression after cell recovery was increased to a lesser degree than that observed in the culture by a 2-h treatment with CoCl2. It was noticed that no significant difference in the cellular levels of ROS was observed in CoCl2-treated groups after cell recovery when compared to that in controls. Immunofluorescence indicated that treatment with CoCl2 resulted in the nuclear translocation of ATF-1 in astrocytes. Electrophoresis motility shift assay (EMSA) further showed that the DNA binding of ATF-1 was increased in astrocytes after a 2-h treatment with CoCl2. Based on our results, we conclude that CoCl2 induces oxidative stress in astrocytes, and subsequently reduces TSP-1 mRNA expression and its protein production. Moreover, CoCl2-induced inhibition of TSP-1 mRNA is irreversible, and is involved in ATF-1 that could act as the negative regulator for TSP-1 mRNA expression.