Mechanism Study Of ALDOA Succinylation Involved In Epileptic Seizures In Rats

Objective:To observe the effect of succinylation site of fructose diphosphate aldolase A(ALDOA)on epileptic seizure and the change of ATP content in hippocampus of rats,and to explore the possible mechanism of ALDOA succinylation involved in epileptic seizure.Methods:1.The rats were divided into normal control group(Con)and epilepsy group(time points:seizure5min,15min,30min,45min).Western blotting(WB)technique was used to compare the expression of ALDOA in hippocampus.2.The succinylation level of ALDOA in hippocampus was compared by immunoprecipitation(IP).3.Cell intervention experiment:the plasmids were constructed and divided into four groups:Con group,WT group,ALDOA succinylated non modified group(succ~-),ALDOA succinylated modified group(succ~+).The plasmids were transfected into HEK293 cells for expression,and the protein was extracted.The expression of ALDOA in each group was detected by WB technique.The succinylation level of ALDOA in each group was compared by IP.Use succinylated ALDOA(K13 site)antibody to detect the expression of ALDOA succinylation level in each group of cells by WB technology.The ATP content of each group was detected by ATP test kit.4.Animal intervention experiment:the lentivirus was constructed and divided into four groups:epilepsy control group(EP),wild type group(WT),ALDOA succinylated non modified group(succ~-),ALDOA succinylated modified group(succ~+).The degree of epileptic seizure:seizure latency and seizure frequency were evaluated by behavior.ATP test kit was used to detect the content of ATP in hippocampus.Results:1.The results showed that there was no difference in the total protein expression of aldoa between normal and epileptic rats(P>0.05).2.Expression of ALDOA succinylation in hippocampus of normal and epileptic rats.The results showed that compared with normal rats,the succinylation of ALDOA was down regulated after seizures,and the difference was statistically significant(P<0.05).3.Cell intervention experiment:WB technology was used to detect the expression of ALDOA in each group,and the results showed that there was no difference in the expression of ALDOA in each group(P>0.05);IP technology was used to detect the expression of succinylation of ALDOA in each group,and the results showed that the succinylation of ALDOA in succinylated non modified group(Succ~-)was down regulated,and the difference was statistically significant(P<0.05);The site antibody was used to detect the expression of ALDOA succinylation level in each group of cells by WB technology.The results showed that the succinylation expression of ALDOA in the succinylation non-modified group was reduced,and the succinylation expression of ALDOA was increased in the succinylation modified group.Compared with Con group,statistically significant(P<0.05).ATP test kit was used to monitor the succinylation of ALDOA The results showed that compared with Con group,the ATP content of ALDOA succinylated non modified group(Succ~-)was decreased,and the ATP content of ALDOA succinylated modified group(Succ~+)was increased(P<0.05).4.Animal intervention experiment:behavioral results:the latency of epileptic seizure in ALDOA succinylated non modified group(Succ~-)was significantly shortened,and that in ALDOA succinylated modified group(Succ~+)was significantly delayed,compared with the epilepsy model group(EP)and wild type(WT)group,the difference was statistically significant(P<0.05).The number of seizures per unit time(1 hour)in Succ~-group was significantly increased,while the number of seizures per unit time in Succ~+group was significantly decreased.Compared with EP group and WT group,the difference was statistically significant(P<0.05).The results showed that compared with EP group,the ATP content of Succ~-group was decreased,and the ATP content of Succ~+group was increased(P<0.05).Conclusion:The change of ALDOA succinylation level may be involved in the regulation of epileptic seizures,and its mechanism may be related to energy metabolism.