The Study Of Hypertonic Saline Influence The AQP4Internalization In Plasma Membrane Of Human Astrocytoma U251

Background Aquaporins(AQPs) are water channel protein families on the plasma membrane that play critical roles in distribution of water molecule and it were widespread in mammals. AQP4is the main water channel protein of the central nervous system. The occurrence of cerebral edema and intracranial hypertension are closely related with AQP4.There were a large number of studies shown that AQP4is rate limiting component in astrocyte edema of cytotoxic brain edema. Hypertonic saline is another kind of osmotic dehydrant, studies have shown that hypertonic saline could reduce the expression of AQP4on astrocytes to treat cerebral edema and intracranial hypertension. Our previous research has found that3%hypertonic saline could reduce AQP4content on the cell membrane surface when directly affect the primary astrocyte for15min. Further research suggests that3%hypertonic saline caused the decrease of AQP4in astrocytoma U251plasma membrane due to AQP4internalization.Objective To investigate the optimal osmotic concentration and affect time of hypertonic saline expose could processing the maximum AQP4internalization. Also, which iron cause this change, sodium or chloridion?Methods1. PEGFP-AQP4-M23recombinant plasmid transfected U251cells to obtained the positive AQP4expression.2. MTT colorimetric method is used to observe the different processing methods on U251cell damage.3. The optimal osmotic concentration and affect time of3%hypertonic saline expose could processing the maximum AQP4internalization was determined by automatic microplate reader. Compare the different sodium liquid with chlorine liquid to determine which ions cause this phenomenon.4. Using flow cytometry and laser scanning confocal microscope observation method to further confirm the above conclusion.Results1. We successfully established AQP4expression of U251cells.2. When the osmotic concentration of Mannitol group increased to920mOsm/L, there had no significant effect on the cell survival rate. In the contrast, when the osmotic concentration of hypertonic saline group reaches820mOsm/L, the cell survival rate decreased obviously (P<0.05). processing cell infiltration in the nutrient solution concentration Mannitol group under the condition of different concentrations of mannitol permeation of AQP4green fluorescent protein (reflect U251cells in total AQP4protein) has no obvious influence, on the surface of membrane AQP4-PE antibody red fluorescent (reflect membrane AQP4in U251cell) also have no obvious impacts. But, hypertonic saline group under the condition of different concentrations of sodium chloride, AQP4green fluorescent protein (reflect total AQP4protein in U251cells) has no obvious effect, but when the concentration of sodium chloride reaches720mosm/L AQP4-PE antibody red fluorescent on the cell membrane surface (reflect U251cell membrane membrane AQP4) appeared decreased significantly (P<0.05), although along with the increase of the concentration of sodium chloride penetration decreases, but compared with the720mosm/L sodium chloride penetrating concentration has no obvious difference. These results prompt that720mosm/L is the best osmotic concentration of hypertonic saline to promote AQP4internalization.3. Exposed to720mosm/L mannitol osmotic concentration for30min had no obvious effect on the cell survival rate, but exposed to720mosm/L hypertonic saline osmotic concentration for30min has obvious decrease on the survival rate. These results prompt that expose to best osmotic concentration of hypertonic saline less than30min is safety. Mannitol group in720mosm/L mannitol osmotic concentration under the condition of different treatment time had no obvious effect on AQP4protein green fluorescence, also has no obvious influence on the surface of the red fluorescence (P>0.05). In contrast, concentration of hypertonic saline group in720mosm/L sodium chloride permeability under the condition of different processing time of AQP4green fluorescent protein had no obvious effect, but the AQP4antibody red fluorescence on the surface of the cell membrane significantly decreased (P<0.05) when expose to720mosm/L hypertonic saline osmotic concentration for15min. Delay the expose time AQP4antibody of red fluorescence strength rebound rise, prompt that processing15min is the best processing time to promote AQP4protein internalized.4. The different treatment groups of U251cells were no obvious effects of AQP4green fluorescent, compared with control group, Sodium nitrate, sulfur sodium cyanide group performance no obvious difference in AQP4-PE antibody red fluorescence intensity. But lithium chloride group and choline chloride group compared with control group, with its AQP4-PE antibody red fluorescence intensity is decreased obviously, and the difference compare with sodium chloride group were similar, prompt that 3%hypertonic saline promote the AQP4protein on U251cell membrane surface internalized is mediated by the chloride ion, rather than the sodium ions. Further flow cytometry and laser confocal microscopy confirmed the results.Conclusion1. Successfully established AQP4expression U251cells.2. Hypertonic saline can promote AQP4on the U251cell membrane internalization. And the optimal osmotic concentration of3%hypertonic saline promote AQP4on the U251cell membrane internalized is720mOsm/L, the optimal time is15min.3. Hypertonic saline to promote U251cell membrane internalization is mediated by the chloride ion, rather than the sodium ions.