Study On The Toxic Effect And Its Mechanism Induced By Arsenite In Astrocytes

IntroductionEnvironmental arsenic pollution is a serious public health problem, long-term oral intake of inorganic arsenic can cause systemic damage to multiple systems, including the nervous system, cardiovascular system and the typical skin lesions. At present, the population of high arsenic exposure of 300 million, is the world's worst affected by arsenic poisoning hazards, one of the countries. In recent years, the toxicity of arsenic on the role of the central nervous system caused widespread concern. Environmental arsenic exposure of children is susceptible population; the epidemiological surveys have found that long-term arsenic exposure can affect children's intellectual development. Animal studies have indicated that arsenic has a certain mental developmental toxicity. But the long-term arsenic exposure on how the mechanism of central nervous system injury, there is no clear conclusion.Astrocytes is the largest number of brain cell population, not just to support their functions, protection and nutrition neurons, it also involved the removal of synaptic cleft with neurotransmitter metabolism, promotion of synaptic synapse formation and maintenance of normal function. Astrocytes are the structural basis for blood-brain barrier, so the blood vessels and neurons, forming a barrier between, the neurons isolated with the external environment. Therefore, the arsenic poisoning of arsenic in the blood through the capillaries to be the first to enter after the astrocytes. In addition, astrocyte is within the glutamine synthetase (Glutamine Synthetase, GS), synaptic processes around the astrocyte on the expression of high affinity glutamate transporter GLAST (Glutamate/Aspartate transporter) and GLT-1 (Glutamate transporter-1) Therefore, astrocyte can rapidly clear the synaptic cleft of Glu, but also on the intake of Glu for metabolic conversion, the formation of glutamine (Glutamine, Gln).In conclusion, astrocytes is likely to inorganic arsenic in the blood into the brain after the initial damage of target cells. In this study, Wistar rats were cultured in vitro tests of astrocytes as the object of the toxic effect of arsenic on the astrocytes and its mechanism of arsenic on the central nervous system to clarify the mechanism of injury in experimental reference data.Materials and Methods1,Cell cultureCerebral cortex was dissected and trypsinized as well as mechanical method to make an initial cell suspension from 1 to 3 day-old Wistar rat pups. Use differential attachment to reduce the amount of contaminating fibroblasts. After an initial culture period between 9 to 12 day, the cultures were shaken on an orbital shaker, to reduce oligodendrocytes and microglial cells. Passaged cells were immunocytochemically stained with anti-GFAP antibody to identify astrocytes.2,Chemicals and groupsSodium arsenite was used to be the poison. The cultured cells were divided into 5 groups randomly, in which astrocytes were exposed to 0,5,10,20 and 30μmol/L arsenite for 24h.3,Methods(1) MTT was used to assess the effect of NaAsO2 on the cell proliferation capacity of astrocytes.(2) Cell morphological changes.(3) Flow cytometry was applied to measure the effect of NaAsO2 on the mitochondrial membrane potential of astrocytes.(4) Fluorescence microscope was applied to measure the effect of NaAsO2 on the mitochondrial membrane potential of astrocytes.(5) Protein expression of GLAST, GLT-1, and GS were detected with Western Blot.4,StatisticsThe data were input into computers, analyzed and treated by the SPSS 13.0. The significant difference was evaluated by analysis of variance (ANOVA). Student Newman-Keuls test was employed to analyze between two different groups. The statistical significance was defined as P<0.05.ResultsMTT results showed that with the increase of the dose NaAsO, cell viability decreased, and among the three groups were statistically significant. Cell morphology and control cells were polygonal, larger cell body, growth-intensive; cell morphology after 24h exposure has no significant change in 5μmol/L group; 10μmol/L, after exposure, can cause significant changes in cell morphology, cell body refraction, the cell gap increases, a small amount of cell swelling or shrinking circle off the wall; with the dose increasing,20μmol/L and 30μmol/L group increased the severity of injury, cells decreased significantly. Flow cytometry showed that all groups with the astrocytes of the mitochondrial membrane potential dye arsenic concentration increases. Which, 20μmol/L and 30μmol/L groups compared with the control group there were significant differences in meaning. Astrocytes in all groups within the GLAST, GLT-1 and GS protein decreased with the arsenic concentration increases. Compared with the control group, there were significant differences in experimental groups of GLAST, which 5μmol/L group and 10μmol/L,20μmol/L,30μmol/L group also showed a significant difference.20μmol/L and 30μmol/L groups of GLT-1 compared with the control group there were significant differences; compared with 30μmol/L group,5μmol/L group and 10μmol/L group also have significant differences. Both 20μmol/L group and 30μmol/L group of GS protein have significant differences compared with which of the control, 5μmol/L and 10μmol/L groups. There are also significant differences between 20μmol/L and 30μmol/L groups of the GS protein.Conclusions1,Dose-dependent toxic effects on primary cultured astrocytes could be induced by sodium arsenite, and in 5-30μmol/L concentration range of dye its toxicity with the increase of arsenic concentration.2,Sodium arsenite can damage mitochondrial function, resulting in decreased mitochondrial membrane potential within the astrocytes.3,With the increased concentration of sodium arsenite in vitro astrocytes, the GLAST, GLT-1 and GS protein expression decreased.