The Protective Effects And Mechanism Of L-23 On Pentylenetetrazole-induced Acute And Chronic Epilepsy Model Mice

At present,the number of people with epilepsy in my country is as high as 90 million,and it continues to increase at a rate of 600,000 per year,which seriously endangers people’s lives and health.Because the specific mechanism of epileptic seizures has not been fully elucidated,and existing antiepileptic drugs are ineffective for some patients.Therefore,it is of great significance to explore the pathogenesis of epilepsy and develop and produce safe and effective antiepileptic drugs.As first-line antiepileptic drugs,carbamazepine and oxcarbazepine mainly exert their antiepileptic effects by metabolizing into the active metabolite S-licarbazepine.However,because S-licarbazepine is difficult to penetrate the blood-brain barrier,it is difficult for this type of drug to reach the brain tissue,so it has a low bioavailability.At the same time,these drugs will also accumulate in the liver,affecting the normal physiological function of the liver.In view of the above drawbacks,our research group designed and synthesized a series of ester prodrugs with ricarbazepine as the lead compound,in order to find an antiepileptic drug with strong ability to penetrate the blood brain barrier and high bioavailability.Preliminary pharmacokinetic studies have shown that L-23 has the best brain uptake properties among many synthetic drugs.Purpose: Based on the previous work on pharmacokinetics,this paper studied the antiepileptic effect and mechanism of L-23 to provide experimental basis for the design and development of new antiepileptic drugs.Methods: 1.Establishment of pentylenetetrazole-induced acute convulsion model to investigate the antiepileptic effect of L-23 72 male Kunming mice were randomly divided into the following 6 groups: control group,model group,L-23 low-dose group（150 mg/kg）,L-23 medium-dose group（300 mg/kg）,L-23 High-dose group（600 mg/kg）and oxcarbazepine group（OXC,positive control group,200 mg/kg）.The control group and the model control group were given 0.5% CMC-Na solution by gavage,and the other groups were given corresponding drugs by gavage.After 1 h of gavage,except for the control group,mice in other groups were intraperitoneally injected with 80 mg/kg pentetrazole to induce acute convulsion model.After the intraperitoneal injection,the epileptic seizures of the mice in each group were recorded within 30 minutes according to the Racine’s scale.2.Establishment of pentylenetetrazole-induced chronic kindling model to investigate the antiepileptic effect and mechanism of L-23 84 male Kunming mice were randomly divided into the following 6 groups: control group,model group,L-23 low-dose group（150 mg/kg）,L-23 medium-dose group（300 mg/kg）,L-23 High-dose group（600 mg/kg）and OXC group（positive control group,200 mg/kg）.The control group and the model group were given 0.5% CMC-Na solution by gavage,and the other groups were given corresponding drugs by gavage,for 21 days in total.Except for the control group,mice in other groups were intraperitoneally injected with 35 mg/kg pentetrazole 1 h after gavage on odd days,and the seizures within 30 minutes were observed and recorded,and the antiepileptic effect of L-23 was evaluated;The effect of L-23 on learning and memory in PTZ-kindling epilepsy mice was investigated by passive avoidance experiment and Morris water maze test.The effect of-23 on the inflammation of PTZ-kindling epileptic mice was investigated by determinating the levels of TNF-α and IL-1β in mouse serum;The effect of L-23 on oxidative stress in PTZ-kindling epileptic mice was investigated by detecting the contents of SOD,MDA and GSH-Px in the mouse hippocampus;The expression of apoptosis-related proteins and BDNF were detected by immunohistochemistry to test the effect of L-23 on cell apoptosis and neuronal activity in PTZ-kindling epilepsy model.The survival,growth and development of neuronal cells and Nissl bodies in the hippocampus of mice were observed by HE staining and Nissl staining.Results:（1）Compared with the PTZ group,the L-23 medium and high-dose groups and the OXC group reduced the seizure grade of mice with acute convulsions induced by pentylenetetrazole（P < 0.01）;compared with the PTZ group,the L-23 high-dose group and OXC group prolonged the seizure latency（P < 0.01）and duration of GTCS（P < 0.01）.（2）On the 21 st day of the pentylenetetrazole-kindling epilepsy experiment,compared with the PTZ group,L-23 and OXC reduced the seizure grade of the epileptic mice（P < 0.01）and prolonged the epileptic seizure latency（P < 0.01）,latency to clonic seizure（P < 0.01）and latency to GTCS（P < 0.01）.（3）Compared with the PTZ group,the L-23 low,medium and high-dose groups and the OXC group prolonged the latency on the second day of the passive avoidance experiment in the pentylenetetrazole-kindling epilepsy mice（P < 0.01）and prolonged the distance in the target quadrant（P < 0.01）and time in the platform（P < 0.01）in Morris water maze experiment.（4）Compared with the PTZ group,the L-23 medium and high-dose groups and the OXC group reduced the serum levels of TNF-α（P < 0.01）and IL-1β（P < 0.01）in pentylenetetrazole-kindling epilepsy mice.（5）Compared with the PTZ group,the L-23 medium and high-dose groups and the OXC group increased the activities of SOD and GSH-Px in the hippocampus of pentylenetetrazole-kindling epilepsy mice（P < 0.01）and decreased the MDA content（P < 0.01）.（6）Compared with the PTZ group,both the L-23 high-dose group and the OXC group reduced the expression of Bax and Caspase-3 in the hippocampus of pentylenetetrazole-kindling epilepsy mice（P < 0.01）and increased the expression of Bcl-2（P < 0.01）.（7）Compared with the PTZ group,the L-23 medium and high dose groups and the OXC group improved the damage of hippocampal neurons and increased the expression of BDNF（P < 0.01）.Conclusion:L-23 can effectively inhibit the severity of epileptic seizures in mice,reduce the levels of inflammation,oxidative stress and apoptosis in mice during seizures,and improve the learning and memory abilities of epileptic mice,with anticonvulsant and neurological protective effects.Therefore,L-23 may be a potential drug for the treatment of epilepsy and a protective cure to prevent cognitive impairment caused by epilepsy.