A Study On The Effect Of Zonisamide On Ferroptosis Of Hippocampal Neurons In Mouse Epileptic And Its Therapy In Patients With Epilepsy

Part I:A study on the effect of Zonisamide on ferroptosis of hippocampal neurons in mouse epilepticObjective:To construct a chronic epilepsy mice model of with lithium chloride-Pilocarpine,and to discuss the effect of zonisamide on ferroptosis of hippocampal neurons in mouse epilepsy model.Methods:A total of 63 C57BL/6 mice were divided into a control group and an experimental group(epilepsy model group,Zonisamide group,and Ferrostain-1 group).The epileptic model was established in the experimental group,and 0.3ml normal saline was administered intragastrically 12 hours after the onset of epilepsy in both the control group and the epilepsy model group;the Zonisamide group was administered intragastrically 100mg/kg/day;and Ferrostain-1 group was administered an intraperitoneal injection of 2.5umol/kg/day.After 21 consecutive days,the hippocampus tissue was taken for reserve.Western blot was used to detect the expression levels of acyl-CoA synthetase long-chain family member 4(ACSL4)and nuclear factor red series 2-related factor 2(Nrf2)in the hippocampus,and PCR was used to detect the mRNA content of prostaglandin peroxidase synthetase 2(PTGS2)in the hippocampus.GSH and MDA contents in hippocampal tissues were detected by glutathione(GSH)kit and malondialdehyde(MDA)kit,respectively.The neurons in hippocampal tissues were evaluated by nissl’s staining,and the mitochondria in hippocampal tissues were evaluated by transmission electron microscopy.Results:After modeling,11 of 63 C57BL/6 mice died,with a mortality rate of 22%.Western blot showed that ACSL4 decreased in zonisamide or Ferrostain-1 groups compared with the model group.Compared with the model group,Nrf2 showed slightly higher expression in Zonisamide or Ferrostain-1 groups.Q-PCR showed that compared with the model group,the expression of PTGS2 mRNA in Zonisamide or Ferrostain-1 groups showed a decreasing trend(P<0.05;P<0.01).GSH results showed that compared with the model group,GSH content decreased in the Zonisamide group and Ferrostain-1 group(P<0.05;P<0.01).The results of MDA detection showed that compared with the model group,MDA content in the Zonisamide group and the Ferrostain-1 group decreased(P<0.05;P<0.01).The results of nissl’s staining showed that the number of neurons in the model group was reduced,desaturated,and necrotic,and the nissl body were reduced or even disappeared.The Zonisamide group and Ferrostain-1 group showed a small amount of neuron loss and partial incomplete neurons.The results of projective electron microscopy showed that the mitochondrial bilayer membrane in the model group was blurred,dissolved,or broken,and disappeared with cavitation or partial cavitation.In the zonisamide group,mitochondrial volume increased,edema,partial ridge dissolved,disappeared,and partial cavitation.The mitochondrial volume of the Ferrostain-1 group was slightly increased,edema,bilayer membrane was blurred,and a small part of vacuolation occurred.Conclusion:In this study,we found that zonisamide can reduce ferroptosis in the Li-Pilocarpine epilepsy mouse model by regulating the expression of ACSL4 and Nrf2 proteins,increasing GSH content,decreasing PTGS2 mRNA level and MDA content.In addition,Zonisamide may acts as a mitochondrial protector.Part Ⅱ:Efficacy and safety of Zonisamide as the first additional therapy in patients with focal or secondary bilateral tonic-clonic seizuresObjective:To evaluate the efficacy and safety of zonisamide(ZNS)as the first additional treatment for focal or secondarily bilateral tonic-clonic seizure(sBTCS).Methods:A total of 118 patients aged 18-75 years were recruited from several hospitals in Shandong Province from May 13,2021,to February 16,2022.After clinical judgment,all patients received Zonisamide as the first additional therapy.The seizure frequency,retention rate,and Adverse effect(AE)were assessed at 2 and 5 months after Zonisamide was added.Results:Overall response rates at 2 and 5 months were 79.5%and 75.5%,respectively,whereas seizure-free rates at the same point were 53.6%and 51%,respectively.The review’s retention rates at 2 and 5 months were 95%and 86%,respectively.The most common AE were anorexia with weight loss(11.8%),dizziness(6.9%),and headache(3.9%).Conclusions:Our real-world study confirmed the efficacy and safety of ZNS as a first-additional treatment for focal or secondary bilateral tonic-clonic seizure in Chinese patients,with a high short-term retention rate.