RESUMO
Objective To explore the alteration of ATP-sensitive K~+(K_(ATP))channel expression in hippocampal neurons after severe chronic hypoxia. Methods The hippocampal neurons from 1-week-old rat were incubated and divided into normal control(incubated under 5%CO_2 and 95%air for 8h),hypoxia(incubated under 5%CO2 and 95%N_2 for 8h),hypoxia combined with diazoxide-treated (incubated with 100 μmol/L diazoxide under 5%CO_2 and 95%N_2 for 8h)and hypoxia combined with tolbutamide-treated groups(incubated with 100 μmol/L tolbutamide under 5%CO_2 and 95%N_2 for 8h).Cell apopmsis was identified by MTT.And the mRNA and protein expressions of K_(ATP) channel were estimated by RT-PCR and Western blot analysis.respectively. Results Hypoxia combined with diazoxide-treated group showed a significantly decreased apoptosis rate of neuron as compare with the normal control group 8h after hypoxia(P<0.05);while hypoxia combined with tolbutamide-treated group showed a significantly increased apoptosis rate of neuron compared with the normal control group(P<0.05).The expression of SUR1 in the three hypoxia groups significantly increased as compared with that in the normal control group(P<0.05);however,the expression of Kir6.2 in the three hypoxia groups did not change as compared with that in the normal control group(P>0.05).Conclusion K(ATP),channels can protect the hippocampal neurons under severe chronic hypoxia through the activation of KATP channels and upregulation of expression of KATP channels SUP1 subunit.
RESUMO
Objective To evaluate the influence of Eag-1 channel blocking on bioactivity of glioma cells in vitro. Methods Different small interfering RNAs (siRNAs) targeting for Eag-1 channel were designed and transfected to the U87 cells, and the blocking effects of those siRNAs were further confirmed on mRNA and protein levels by RT-PCR and Western blotting. The 50 nmol/l siRNAs (siRNA1 and siRNA2) and quinidine (5, 10, 20, 30 and 40 mmol/l) were used to block the activity of Eag-1 channel, respectively; and blank control group was also established. The proliferation of U87 cells 24, 48 and 72 h after the treatments was detected by MTT method; the changes of generation cycle,apoptosis ratio and intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Results High mRNA and protein levels of Eag-1 channel on glioma cell line U87 were confirmed in the blank control group, however, siRNA1 and siRNA2 transfection groups showed significantly lower mRNA and protein levels of Eag-1 channel on glioma cell line U87. MTT method indicated that, 24, 48 and 72 h after the treatments, the proliferation of U87 cells in the siRNA1 and siRNA2 transfection groups, and quinidine treatment groups (10, 20, 30 and 40 mmol/l) was significantly inhibited as compared with that in the blank control group (P＜0.05). The IC50 value of quinidine is33.7mmol/l. As compared with the blank control group, 50 nmol/L siRNA1 and siRNA2 transfection groups, and 33.7 mmol/l quinidine treatment group enjoyed a significantly increased cell percentage at G1 stage, cell apoptosis ratio and intracellular ROS level (P＜0.05). Conclusion Eag-1 channel blocking can obviously inhibit the proliferation of glioma cells, increase the cell percentage at G1 stage and intracellular ROS level, and induce apoptosis of glioma cells.