| Objective:In this study,Huazhuo Jiedu Shugan Decoction was used as a therapeutic drug to observe the behavioral performance,improvement of learning and memory,pathomorphological changes of hippocampal neurons,and expression of PI3K-AKT-GSK3β pathway-related proteins in pentylenetetrazol-induced epileptic rats after drug treatment,to explore the possible mechanism of Huazhuo Jiedu Shugan Decoction,and to provide new ideas and theoretical basis for the in-depth study of Huazhuo Jiedu Shugan Decoction and the improvement of cognitive function in epileptic patients.Methods:70 SPF SD rats were selected,10 rats in blank group were randomly selected,and the other60 rats were used to establish chronic epilepsy model(PTZ 35 mg/kg/every other day).After14 times of modeling,those with unsuccessful modeling were excluded.the remaining 50 epileptic rats were included in the experimental study and randomly divided into model group,sodium valproate group,Huazhuo low-dose group,Huazhuo medium-dose group,and Huazhuo high-dose group,with 10 rats in each group.On the next day after grouping,intragastric administration was started.The treatment group was intragastrically administered with high,medium and low doses of Huazhuo Jiedu Shugan Decoction and sodium valproate,and the model group and the blank group were given the same volume of saline gavage for 28 days.During the modeling and treatment period,the behavioral performance of rats in each group was observed,and the seizure grade and latency were recorded.On the next day after the end of treatment,the learning and memory of rats was tested by applying the Morris water maze,hematoxylin-eosin staining was used to observe the pathomorphological changes of hippocampal neurons,and immunohistochemistry and immunoblotting were used to detect the expression of PI3K-AKT-GSK3β pathway-related proteins.Result:1.Seizure grading and seizure latency results of rats before and after treatment(1)Seizure grade: The seizure grade of rats in each treatment group was not statistically different from that in the model group before treatment(P > 0.05).After 28 days of treatment,the seizure grade of each treatment group decreased,with statistically significant differences compared with the model group(P < 0.01).(2)Seizure latency: The seizure latency of rats in each treatment group was not statistically different from that in the model group before treatment(P > 0.05).After 28 days of treatment,the seizure latency was longer in the sodium valproate group,the medium and high dose groups of Huazhuo than in the model group(P < 0.01),longer in the high dose group of Huazhuo than in the low dose group of Huazhuo(P < 0.01),and there was no statistical difference between the high dose group of Huazhuo and the sodium valproate group(P > 0.05).2.Morris water maze results(1)Place navigation test: There was no difference in escape latency among the groups of rats on the first day(P > 0.05).The escape latency of rats was longer in the model group than in the blank group(second,third,and fourth days)(P < 0.05),was shorter in the sodium valproate group than in the model group(second and fourth days)(P < 0.05),was shorter in the Huazhuo high dose group than in the model group(second,third,and fourth days)(P < 0.05),and was shorter in the Huazhuo high dose group than in the Huazhuo low dose group(third and fourth days)(P < 0.05).(2)Spatial probe test: The number of rat platform crossings was less in the model group than in the blank group(P < 0.01),more in the sodium valproate group and the Huazhuo high dose group than in the model group(P < 0.01),more in the Huazhuo high dose group than in the Huazhuo low dose group(P < 0.01),and there was no difference between the sodium valproate group and the Huazhuo high dose group(P > 0.05).3.Pathomorphological changes of hippocampal neurons: In the blank group,the number of pyramidal cells in the CA1 and CA3 regions of the hippocampus was more,the cell morphology was full,and the cells were arranged regularly and densely between them.In the model group,the number of pyramidal cells in the CA1 and CA3 regions of the hippocampus was significantly reduced,the cell morphology was irregular,and significant neuronal cell body pyknosis and hyperchromasia were observed.The cell morphological changes in the CA1 and CA3 areas of the hippocampus of rats in each treatment group were improved compared with the model group,and the most obvious changes were found in the Huazhuo high dose group and the sodium valproate group.4.Immunohistochemistry results: The expression levels of p-AKT and p-GSK3β in CA1 and CA3 regions of the hippocampus in the model group were reduced,which were significantly different from those in the blank group(P < 0.01).The expression levels of pAKT and p-GSK3β in the CA1 and CA3 regions of the hippocampus were increased in rats in the Huazhuo high dose and sodium valproate groups,which were statistically different from those in the model group(P < 0.05).The expression levels of p-AKT and p-GSK3β in the CA1 and CA3 regions of the hippocampus were increased in rats in the Huazhuo high dose group,which were statistically different from those in the Huazhuo low dose group(P < 0.05).There was no significant difference in the expression levels of p-AKT and p-GSK3β in CA1 and CA3 regions of hippocampus in rats in the Huazhuo high dose group and sodium valproate group(P > 0.05).5.Western blot results: There was no statistically significant difference in the expression of AKT and GSK3β total protein in the hippocampal tissue of rats in each group(P > 0.05).The expression of p-AKT and p-GSK3β protein in hippocampus was less in the model group than in the blank group(P < 0.01).In the sodium valproate group,the medium and high dose groups of Huazhuo were more than those in the model group(P < 0.01).In the Huazhuo high dose group and sodium valproate group,it was more than that in the Huazhuo low dose group(P < 0.01).Conclusion:1.The rat model of chronic epilepsy can be successfully established by pentylenetetrazol chemical kindling,which leads to pathological changes in hippocampal neurons and causes learning and memory impairment.Huazhuo Jiedu Shugan Decoction can reduce the seizure grade and prolong the seizure latency period,reduce hippocampal neuronal injury in epileptic rats,and improve learning and memory,with the best effect in the Huazhuo high dose group,which is equivalent to the efficacy in the sodium valproate group.2.In pentetrazol-induced epilepsy model,the expression of AKT and GSK3β total protein did not change significantly,and the expression of activated form p-AKT and inactivated form p-GSK3β was significantly reduced,suggesting that recurrent seizures lead to cerebral ischemia and hypoxia and hippocampal neuronal injury,causing learning and memory impairment.Huazhuo Jiedu Shugan Decoction inactivates GSK3β by activating AKT,and the activated form of AKT may inhibit its activity and resist cerebral ischemia and hypoxia and hippocampal neuronal injury,thereby preventing neuronal apoptosis and playing an important role in neuronal protection and improving learning and memory. |
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