| BackgroundWhen we faced to the complex and changing living environment, the body was always ina state of stress, and produced a non-specific reaction to adapt and defense to stressors. Whengiven any exogenous biological stress, the organism could produce non-specific reactions torestore stability in vivo environment. Glucocorticoids (GC) is an important stress hormone,when stress response occurs in the body, the hypothalamic-pituitary-adrenal axis (HPAaxis)could be activated, and the body secrets numerous hormones. In mice, the main stresshormone is corticosterone (CORT), when stress occurs in the body, a lot of the hormonesecrete from the adrenocortical cells.As a regulator of stress responses, Hippocampus is a critical brain area in the centralnervous system, and is closely related to learning and memory, on the other hand,hippocampus has a higher concentration of glucocorticoid receptors than the other brain areas,therefore it is particularly sensitive to stress and is susceptible to stress damage. Synapticvesicles gather large amounts of zinc ions in the hippocampal dentate gyrus granule cellsmossy fiber terminals, as a neuromodulator with the neurotransmitter cosecrets into thesynaptic cleft, regulates cellular signal recognition, second messenger metabolism, proteinkinases and protein phosphatase activity, and zinc ions can activate or inhibit activation oftranscription factors, which plays an important physiological function.Zinc is necessary for all organisms, and is detected in many enzymes and proteins due tothe function of catalytic and structure. Zinc has a broader function compared with othertransition metals, such as zinc ions have important signal regulation function. Zinc ions areone of the most abundant cations in brain, most of which are in combination form, only asmall portion existing in neurons are in free state, mainly in the axon terminals of zincexcitatory neuronal. Intracellular zinc homeostasis is very sensitive to pathophysiologicalenvironmental factors changes, under stress status. We detected a significant increase ofintracellular free zinc ions in a variety of cells. Zinc has so many important functions that itsdeficiency associates with a variety of disorders, but on the other hand an excess of zinc willalso have toxic effects on cells, an excessive increase of intracellular free zinc ions maychange oxidation state in the cells, but also may cause mitochondrial dysfunction, lead to cellnecrosis or apoptosis. We presume that microelement zinc may play an important role in stress process. Stressinduce intracellular zinc homeostasis change, and affect the redistribution of intracellular zinc,resulting in an increase of the concentration of intracellular free zinc, which may producetoxic effects in cells, causing changes of oxidation state in the cells.ObjectiveIn this study, we observe the changes of free zinc in mice hippocampal neuron cell(HT-22cell) which is induced with corticosterone, and explore the possible mechanisms ofthese changes, and detect effective on cell vialibility, ROS level andATP level. It will not onlyhelp us understand the effect of glucocorticoids in stress process on free zinc levels in neuroncells, but also provide experimental evidence and theoretical reference for the study ofstress-related diseases on this basis.Experimental Methods1. Cell culture and induceHT-22cells (Mouse hippocampal neuronal cells) were kindly provided by Dr. DavidSchubert at Salk Institute in Canada. HT-22cells were cultured in DMEM (Dulbecco’sModified Eagle’s Medium) supplemented with10%fetal bovine serum, in a5%CO2humidified atmosphere at37°C. Discard the above-mentioned culture solution when the cellsproceeding cell differentiation, and add culture medium containing1xN2Neuro Basal tocontinue culture24hours.Using10μΜ concentration of CORT for different time (0h,3h,6h,12h,24h) and usingdifferent concentrations of CORT (500nM,1μΜ,10μΜ,50μΜ) induced HT-22cells for6h.2. Determination of free zinc content in CORT induced HT-22cellsAfter cells were collected, they were incubated with HBSS concluding1mMFluoZin-3-AM for30min protected from light. Using flow cytometer determined thefluorescence intensity of Fluozin-3-AM in HT-22cells at the excitation wavelength of488nm.3. Determination of total zinc content in CORT induced HT-22cellsAfter cells were collected, added100μl concentrated nitric acid per cells to digest cells at80℃. Until the liquid evaporation to dryness, added50μl nitric acid to dissolve the residue and metered volume to2ml with Milli-Q water. Total zinc was measured by flame atomicabsorption spectrophotometer method.4. Determination of the mRNAexpression level of MT1, MT3, ZnT1,ZnT3and ZIP1in CORT induced HT-22cellsReal time PCR was performed to determine the mRNAexpression level of MT1, MT3,ZnT1, ZnT3and ZIP1in CORT induced HT-22cells.5. Determination of cell activity in CORT induced HT-22cellsCell Counting Kit-8was used to detect HT-22cell activity.6. Determination of reactive oxygen species in CORT induced HT-22cellsReactive Oxygen Species Assay Kit was used to detect ROS level in HT-22cells.7. Determination ofATPlevel in CORT induced HT-22cellsUsingATPAssay Kit detect ATP level in HT-22cells.8. Statistical MethodUsing statistical software SPSS16.0to carry out statistical analysis, using Excel2007togather data and plot, we choose test level α=0.05; two-sample T-test and one-way analysis ofvariance (ANOVA), Dunnett’s, Newman-Keuls were performed for the multiplicitycomparison.Results1. Concentration and time of CORT induced HT-22cellsExperimental result showed that, using10μΜ CORT induced HT-22cells for differenttime, free zinc content of6h,12h group were higher than the control group, and the differencehas statistical significance (P<0.05). With different concentrations of CORT induced HT-22cells for6hours, free zinc content of10μΜ CORT group was higher than the control group,and the difference has statistical significance (P<0.05). Half an hour before treating with CORT, glucocorticoid receptor antagonist RU486was added to induce HT-22cells, comparedwith the control group, cannot cause free zinc content increase.2. Effect of total zinc content in CORT induced HT-22cellsExperimental result showed that, compared with the control group, the concentration oftotal zinc of CORT group decreased, and the difference was statistical significance (P<0.05).Whereas RU486group showed no significantly change when compared with the controlgroup.3. The effect of zinc sulfate on intracellular free zinc content in CORTinduced HT-22cellsExperimental result showed that, compared with the control group, free zinc content wassignificantly increased after adding ZnSO4, and the difference has statistical significance(P<0.01). Compared with the CORT group, intracellular free zinc content increasedsignificantly at ZnSO4+CORT group, and the difference has statistical significance (P<0.01).4. Effect of zinc ion chelator on intracellular free zinc content in CORTinduced HT-22cells(1) The effect of DTPAon intracellular free zinc content in CORT induced HT-22cellsExperimental result showed that, compared with the CORT group, intracellular free zinccontent in DTPAgroup showed no significant change(P>0.05); Compared with the CORTgroup, intracellular free zinc content in the DTPA+CORT group decreased little, but thedifference has no statistical significance (P>0.05).(2) The effect of TPEN on intracellular free zinc content in CORT induced HT-22cellsExperimental result showed that, compared with the CORT group, intracellular free zinccontent decreased significantly in TPEN group, the difference has statistical significance(P<0.01), and compared with the CORT group, intracellular free zinc content decreasedsignificantly in TPEN+CORT group, the difference has statistical significance (P<0.01). 5. The effect of CORT on MT1, MT3, ZnT1, ZnT3and ZIP1mRNAlevels in CORT induced HT-22cellsReal-time PCR experimental results showed that compared with the control group,10μΜCORT induced HT-22for6h, MT1, MT3mRNAexpression levels increased in HT-22cells,and the difference has statistical significance (P<0.05); zinc transporters ZIP1, ZnT1andZnT3mRNAexpression levels increased, and the difference has statistical significance(P<0.05).6. The effect of CORT on cell viability in CORT induced HT-22cellsExperimental result showed that, compared with the control group, differentconcentrations CORT induced HT-22cells for6hours,10μΜ CORT group was notsignificantly changed (P>0.05),50μΜ CORT group cell viability showed decreased (P<0.05).Using the same concentration10μM of CORT induced HT-22cells for different time,6hourgroup showed no significantly changes (P>0.05), however,12h,24h group cell viabilityshowed decreased, and the difference has statistical significance (P<0.05).7. The effect of CORT on ROS levels in CORT induced HT-22cellsExperimental result showed that, compared with the control group, reactive oxygenspecies level was significantly higher after10μM CORT induced HT-22cells for6h, thedifference has statistical significance (P<0.01), compared with the CORT group, reactiveoxygen species level decreased in TPEN+CORT group, and the difference has statisticalsignificance (P<0.01); Reactive oxygen species level was significantly higher in ZnSO4groupwhen compared with the control group (P<0.01), Reactive oxygen species level wassignificantly higher in ZnSO4+CORT group when compared with the control group, and thedifference has statistical significance (P<0.01).8. The effect of CORT on theATPlevel in CORT induced HT-22cellsExperimental result showed that, compared with the control group, after10μM CORTinduced HT-22cells for6h, theATP level decreased in HT-22cells, and the difference hasstatistical significance (P<0.01), compared with the CORT group,ATP level increased inTPEN+CORT group, and the difference has statistical significance (P<0.01); ATP level wasdecreased in ZnSO4group when compared with the control group (P<0.01),ATP level was significantly decreased in ZnSO4+CORT group when compared with the control group, andthe difference has statistical significance (P<0.01).ConclusionsIn this study, we used CORT to stimulate HT-22cells, and observed intracellular freezinc changes in HT-22cells under stress circumstances, and then added zinc ion chelators andzinc sulfate to the culture medium, real-time PCR to explore the reasons for the changes ofintracellular free zinc, and further investigate the effect of CORT on HT-22cells viability,ROS level, andATP level. The main conclusions were as follows:1. CORT-stimulated HT-22cells induced intracellular free zinc concentrations increase.2. The reason for CORT induced intracellular free zinc concentrations increase in HT-22cells may be mainly the result of CORT promoted combination zinc release.3. CORT induced HT-22cells so that multiplicity free zinc released from combinationzinc, this may change MT and zinc transporters expression level, and increase zinc ions flowout of the HT-22cells, and induce intracellular total zinc content decrease.4. CORT induced HT-22cells caused intracellular free zinc concentrations increase, sothat reactive oxygen species level increased in HT-22cells, andATP level decreased, whichmay affect mitochondrial functions. |
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