Correlation Between The Changes Of Na~+-K~+-ATPase And Calponin-3 In Epileptic Brain Tissues

Objectives:Na+-K+-ATPase is the most representative type of ATPase,controls the transmembrane transport of sodium and potassium ions,and is one of the main factor regulating the excitability in the brain.However,the change of Na+-K+-ATPase in the development of epilepsy is undetermined.The previous study found that calponin-3 was abnormally expressed in astrocytes and neurons in the brain tissue of patients with refractory seizures.And it participates in the regulation of epilepsy susceptibility,but the specific action is still unclear.Calponin-3 can regulate ATPase activity,so this study is to clarify the changes of Na+-K+-ATPase after seizure,and to explore whether its changes are related to calponin-3 and their significance.Methods:Male adult C57BL/6 mice were given 290mg/kg pilocarpine by intraperitoneal injection to induce epilepticus,and then they were randomly divided into 1 hour group(1H),6 hours group(6H),24 hours group(24H),3 days group(3D),1 week group(1W),2 weeks group(2W),1 month group(1M),2 months group(2M).The hippocampus was collected at the specified time points,and the activities of ATPase and Na+-K+-ATPase were detected by the method of phosphorus determination with ATPase activity kit,the mRNA levels of Na+-K+-ATPase subtypes(ATP1?1,ATP1?2,ATP1?3 and ATP1?2)and CNN3(encoding calponin-3 protein)were detected by RT-qPCR.The protein expressions of calponin-3,Na+-K+-ATPase and ATPlal were detected by Western blot,and immunofluorescence was used to analyze the expression and cell-level localization of ATPlal and calponin-3.The mRNA levels of CNN3,ATP1?2,ATP1?3 and ATP1?2 was quantitatively detected by RT-qPCR in the temporal lobe brain tissue from the age and gender matched refractory temporal lobe epilepsy patients and non-epileptic control patients.Pearson linear correlation analysis was used to analyze the correlation between CNN3 and Na+-K+-ATPase and its subtypes.Results:Among the pilocarpine injected 138 C57BL/6 mice,about 127(92.03%)had seizures above grade IV or higher,106 mice were terminated with the onset of diazepam after lasted 1 hour,and then 22 epileptic mice died,with the highest mortality rate of 14.43%at 24H and no death at 2M.After the epileptic ignited,the activity of ATPase in hippocampus of epilepsy mice began to decrease,dropping to the lowest at 24H,and then gradually rising.The Na+-K+-ATPase activity also had similar changes,but only the increase in 1M was more statistically significant than 24H.Pearson analysis showed that the change of ATPase activity was negatively correlated with mortality in epilepsy mice but positively correlated with Na+-K+-ATPase activity.At the transcriptional level,compared with non-epileptic temporal lobe tissue,CNN3 mRNA expression was significantly up-regulated in temporal lobe brain tissue of refractory epileptic patients,and ATP1?1 mRNA expression was significantly decreased,the Pearson correlation coefficient between them was-0.379(P<0.05).The mRNA levels in hippocampal tissue of epilepsy mice:CNN3 was significantly increased in 24H,3D,1W and 2W groups compared with the blank control group;ATPlal was decreased in the 3D group compared with the control group,while increased in 2W,1M,2M groups compared with the 3D group;ATP1?2 and ATP1?2 were increased in the 1M group compared with the control,ATP1?3 in 1H and 1M groups was significantly higher than the control group.However,Pearson correlation analysis showed that there only had a negative correlation between CNN3 and ATPlal in hippocampus of epileptic mice(P<0.05).At protein level,calponin-3 increased in 24H,3D,1W,2W and 1M groups compared with the control group;Na+-K+-ATPase decreased at 24H compared with the control,while its expression in 3D,1M and 2M was higher than the 24H group,and the differences were statistically significant;ATPlal increased in the 1M group compared with the control.Pearson analysis suggests that three of they have no correlation at the protein level.Immunofluorescence showed that ATP1?1 and calponin-3 were co-stained in astrocytes in the hippocampus and temporal cortex of mouse brain tissue at 3 days of seizure.Conclusions:After seizures,ATPase,Na+-K+-ATPase and calponin-3 in hippocampus of epilepsy mice were altered.ATPase activity was reduced to a minimum at 24H in epileptic seizures,while epileptic rats had the highest mortality.At Status epilepticus of 24H,ATPase activity was minimized,while epileptic rats had the highest mortality.The change of Na+-K+-ATPase activity was not completely consistent with the change of ATPase activity,because the enzyme activity,transcription level and cell localization indicated that the change of Na+-K+-ATPase was related to caiponin-3.However,the different subtypes of Na+-K+-ATPase have different correlation with calponin-3.The al subtype of Na+-K+-ATPase is co-expressed with calponin-3 in astrocytes,suggesting that calponin-3 is most likely to affect epileptic vulnerability by participating in triple synapses by regulating al subunit Na+-K+-ATPase,which provides new ideas and targets for the prevention and treatment of drug-resistant epilepsy.