| Ischemic cerebrovascular diseases is common and frequently-occuring, its mortality and disability rate is still on high level, and brain edema is an important pathologic procedure of following this disease, which influences prognosis seriously. Discovery of aquaporins makes people realize to water transportation by plasma membrane not merely trans-phospholipid membrane by simple diffusion, a kind of more important way is express water transportation via idiosyncratic aquaporins in case of pathology. Among them AQP4 is distributed in the central nervous system mainly, especially the foot process of the astrocyte in the area closely related to water metabolism. So, investigating the expression change of AQP4 in astrocytes influenced singly by hypoxia/reoxygenation and intervened by puerarin would provid the new thinking in treating the cerebral edema clinically. [objective]: Observe the change of morphology of astrocytes and the expression of AQP4 and influenced by puerarin under the condition of hypoxia/reoxygenation. Probe into the relation of the ischemia/reperfusion damage of brain and AQP4 and the intervention effect of puerarin. [methods]: The pro-material came from fresh cerebral constitution of newly born Wistar rats (less than 24 hours). We began to make primary culture after machinery detachment and chemical digestion. After incubation for 8~9 days, the cells were trypsinized and subcultured. It would take 4 progress of subculture to get the stable cells. Homogeneity of the cell populations was evaluated by immunosytochemical examinations with anti-glia fibrillary acid protein (GFAP), the number of GFAP positive cells exceeded 95%. The cultured astrocytes were divided into three groups: normal contrast group; hypoxia/reoxygenation group; puerarin intervention group. The astrocytes were placed in a controlled atmosphere culture chamber, into which a mixture of 95%N2 and 5%CO2 to result in cell hypoxia, regarded spilling rate of LDH and degrading rate of MTT as cellular damaged index, carried on morphology observation to the cells by inverted phase contrast microscope, used RT-PCR semiquantification and Western-blot technique to examine the expression change of AQP4 in the astrocytes at some indicated time points after hypoxia/reoxygenation and intervention effect of puerarin. [results]: 1.The cultured astrocytes after the four generations in vitro,who's cell bodys were maximus and flat, the form was irregular, cell processes were more and relatively long, they appear radially. GFAP immunocytochemical stain evaluated that the astrocyte plasm is buffy. the cell morphology, LDH, MTT weren't impacted by hypoxia for 8 hours, some cells creased on the edge, however, reoxygenation made a damage obviously. The longer time, the greater damage to theshape of astrocytes correspondingly, the LDH changed little in hypoxia and made great transform in reoxygenation. The MTT was observed as the same. 2. The expression of AQP4 mRNA and protein didn't have obvious difference to normal contrast group after 8 hours of hypoxia, but expressed rising after reoxygenation and as time lengthens and showed a tendency to increase. 3. A few cells were swelling and most cells'outline was relatively clear, process was still in presence after H8/R12 in puerarin intervention group. But the expression degree of AQP4 mRNA and protein at some indicated time points after hypoxia/reoxygenation didn't have obvious difference to hypoxia/reoxygenation group. [discussion and conclusion]: Under the condition of hypoxia, the cell metabolism transfers from normal metabolism to anaerobic glycolysis mostly. And astrocytes have better tolerance to hypoxia, the damage caused by hypoxia is relatively little, so cells'shape and LDH, MTT don't have obvious change. When recovers oxygen supply abruptly after 8 hours of hypoxia, cellular oxidative metabolism strengthens compensatly for a fixed period of time, produces a large number of oxygen-derived free radicals and attacks the cell menbrane, causes to cellular damage, so LDH and MTT presents the obvious change. Meanwhile, after 8 hours of hypoxia, the expression of AQP4 mRNA and protein in astrocytes dosen't have obvious difference to normal contrast group, with the extension of time after reoxygenation,...
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