Aβ _1-40 Neural Stem Cell Apoptosis Induced By Potassium Channel Change And JNK Signal Transduction Pathways

Objective: Investigate the role of potassium channels in Aβ_1-40induced Neural stem cellsthe process of apoptosis; Explore JNK signal transduction pathway play a role in Aβ_1-40inducedthe process of apoptotic Neural stem cells; And the relationship between potassium channel andJNK. To further understand the pathogenesis of alzheimer’s disease, which offers a new ideasand method for treating Alzheimer’s disease to develop medicines.Methods:1To separate and extract nascent Wistar rats’s Neural stem cells of hippo-campus area, Then they were subcultured. passing on the three generations after gaining stableNeural stem cells which were detected thought Nestin of immunocytochemistry method.2Previously added TEA5mM,30minutes later, the survival rate of Neural stem cells at differenttime points（0h、12h、24h、48h）by MTT assay. TEA intervene in Aβ_1-40-induced Neuralstem cells, Then record the change of the Neural stem cell survival rate.3Previously addedTEA5mM,30minutes later, Neural stem cells are chronic incubated with Aβ_1-405μm,observed morphological changes of Neural stem cell under a fluorescence microscope,apoptosis was detected by Hoechst33342staining method.4Previously added TEA5mM,30minutes later, Neural stem cells are chronic incubated with Aβ_1-405μm at each point in time（0h、12h、24h、48h）, Expression of Caspase-3specific activity calculated by colorimetricmethod. To detect the effects of TEA act on Aβ_1-40-induced Neural stem cells.5To examine theexpression of Aβ_1-40-induced Neural stem cells JNK phosphorylation by Western blot. Torecord the expression of JNK phosphorylation in Neural stem cells by Aβ_1-405μM incubated fordifferent time（0h、12h、24h、48h）.6Incubated30min before adding SP60012510μΜ,Aβ_1-405μM and Neural stem cells were incubated at each time point （0h、12h、24h、48h）,Detected SP60012510μM act on Aβ_1-40-induced Neural stem cells survival rate by MTTmethod.7Previously added SP60012510μM,30minutes later, Neural stem cells are chronicincubated with Aβ_1-405μΜ, observed morphological changes of Neural stem cell under afluorescence microscope, apoptosis was detected by Hoechst33342staining method.8Previously added SP60012510μM,30minutes later, Neural stem cells are chronic incubatedwith Aβ_1-405μm at each point in time（0h、12h、24h、48h）, Expression of Caspase-3specific activity calculated by colorimetric method. To detect the effects of TEA act on Aβ_1-40-induced Neural stem cells.9Because of Aβ_1-40-induced Neural stem cells JNK phosphorylation activated peaked at24h, Therefore, at this time point added TEA5mM, then using Western blotmethod to detect a status which is JNK phosphorylation of Aβ_1-40-induced Neural stem cells.Results:1Single neuron in hippocampus of neonatal rats in freshly isolated stem cellsshowed bright round, at the third passage visible culture larger neurosphere formation fluid, thepositive expression of Nestin protein marker of Neural stem.2Effect of TEA on Aβ_1-40-inducedNeural stem cells’s survival rate, apoptosis rate and Caspase-3activity.2.1The MTT assayTEA survival of Aβ_1-40-induced Neural stem cells. The results show: Aβ_1-40group of Neural stemcells at different time points, cell significantly reduced （p＜0.05）, and over time prolong cellsurvival decreased even more obvious. Aβ_1-40+TEA group compared with Aβ_1-40group of cellssurvival was significantly increased （p＜0.05）, the control group and the TEA Neural stem cellsdid not change significantly （p＞0.05）.2.2Hoechst33342method was used to observe theoccurrence of TEA on the apoptosis of Neural stem cells induced by Aβ_1-40. The results show:Under a fiuorescence microscope Neural stem cells of Aβ_1-40group which some nuclei werehyperchromatic chunky48h after, for the typical apoptotic morphological changes. Apoptosisrate is20.3%±0.6%,（p＜0.01）. Aβ_1-40+TEA group than Aβ_1-40group of apoptosismorphological changes. Apoptosis rate is11.6%±0.4%, The difference was significant （p＜0.01）.2.3Colorimetric detection TEA neural stem cells of Aβ_1-40-induced Caspase-3activitychanges. The results show: Aβ_1-40+TEA group at each time point can reduce the expression ofCaspase-3（p＜0.05）, special in24h, this trend is more obvious （p＜0.01）. Blank control groupand TEA control group at all time points Caspase-3specific activity did not change significantly.3JNK and SP600125of JNK selective blocking agent on Aβ_1-40-induced Neural stem cellsphosphory-lation changes.3.1Western blot method to detect Aβ_1-40-induced Neural stem cellsof JNK expression of phosphorylated.The results show: Neural stem cells were incubated Aβ_1-405μM48h, Different time points the phosphorylation of JNK were expressed. JNK phosphorylation level gradually increase with time prolonging, and reached a peak at24h （p＜0.01）, andJNK blocker SP600125can significantly interrupt the activation.3.2MTT assay survival ofSP600125on Aβ_1-40-induced Neural stem cells. The results show: Aβ_1-40+SP600125group cansignificantly reduce Neural stem cells death, the survival of neural stem cells from72.7%±3.2%,50.35%±2.7%,42.7%±7.3%, respectively increased to92.6%±0.70%,76.1%±0.73%,67.0%±0.64%,（p＜0.01）.3.3Using Hoechst33342method to detect SP600125Neural stem cells apoptosis of Aβ_1-40-induced. The results show:Aβ_1-40+SP600125group cansignificantly reduce the incidence of Neural stem cells after Aβ_1-405μM incubated apoptisis. Theapoptotic rate of SP600125from20.3%±0.6%to9.6%±1.3%（p＜0.01）, SP600125group nosignificant changes. SP600125itself does not affect cell apoptosis.3.4Colorimetry to detectSP600125on Aβ_1-40-induced Caspase3activity changes of neural stem cells. The results show:Examining the role of JNK inhibitor SP600125, Aβ_1-40+SP600125group can significantlyreduce the activation of caspase-3, Aβ_1-40group12.46±0.47)pmol/h/μg decreased to （5.09±0.40） pmol/h/μg （n=3，p＜0.01）.4TEA effect of stem cells on the expression of JNKphosphorylation of Aβ_1-40-induced. The results show: Aβ_1-40+TEA group was significantlylower （p＜0.01） compared with Aβ_1-40group of JNK phosphorylation expression.Blank controlgroup and the TEA group Neural stem cells JNK phoshorylation showed no significant change（p＞0.05）.Conclusion：1Potassium channel blocker TEA can significantly mitigate Aβ_1-40-inducedNeural stem cells death,increased the survival of neural stem cells, significantly decreased theincidence of apoptdsis.2JNK signal transduction pathways involved in apoptosis signaltransduction, has an important role in the Aβ_1-40-induced Neural stem cells damage.3JNKspecific inhibitor SP600125inhibit Aβ_1-40-induced Neural stem cells death,has a significantprotective effect of Aβ_1-40-induced Neural stem cells toxicity.4There are certain connectionbetween potassium channel Aβ_1-40induces the activation and phosphorylation of JNK in both,and induced the apoptosis related proteins downstream.