Ginsenoside Rh2 Alleviating Epileptic Related Oxidative Stress Injury

Objective:Epilepsy is a common chronic neurological disorder affecting nearly 50million people worldwide.To date,one-third of patients have seizures that cannot be controlled with medication.Currently,clinical antiepileptic drugs only target symptoms without changing the course of the disease.In fact,the pathophysiology of epilepsy is affected by many factors.The development of drugs to delay the course of epilepsy may have a positive impact on clinical treatment.Since natural plant extracts have multiple pharmacological effects and few side effects,more and more studies have focused on their effects in resisting the damage caused by epilepsy.The pathogenesis of epilepsy is still unclear,but oxidative stress has been proved to play an important role in the development of epilepsy.Epileptic seizures can lead to the production of reactive oxygen species（ROS）,which can induce further oxidative damage of neurons.In recent years,many studies have been devoted to the treatment of epilepsy from the perspective of reducing reactive oxygen species,and the significant effect of antioxidant therapy has been verified.Ginsenoside is the active component of ginseng C.A.Mey,which is divided into several subtypes,such as Rb1,Rb2,Rc,Rh2 and Rg3.It has been proven to have neuroprotective,cardiovascular protective,immunomodulatory and antibacterial activities.Rh2 can protect neurons by acting as an antioxidant against nerve damage induced by acute exposure to trimethyltin（TMT）,but whether it has a therapeutic effect on epilepsy remains unknown.The purpose of this study was to explore whether ginsenoside Rh2 can resist the oxidative stress damage induced by epileptic activity in vitro and in vivo epilepsy models,and to further study the internal mechanism of this antioxidant effect,which provides a new idea for the search for natural drugs to assist the treatment of epilepsy and the research on the antioxidant effects of ginsenoside Rh2.Methods:1.SH-SY5Y cells were pretreated with different concentrations of ginsenoside Rh2and cultured without Mg2+for 3h to induce seizure-like activity to construct epileptic cell model.To investigate the protective effect of ginsenoside Rh2 on SH-SY5Y cells in vitro epilepsy model,the cell activity was detected by MTT assay,the degree of apoptosis was detected by flow cytometry,the fluorescence intensity of ROS and Rh123were observed by fluorescence microscopy,and the generation level of reactive oxygen species and mitochondrial membrane potential were measured by semi-quantitative analysis.2.Eligible epilepsy tissue sequencing data GSE31718 were selected from GEO database,and differential genes were screened by R language analysis.The potential target genes of ginsenoside Rh2 were predicted by online database and intersected with previously screened differential genes.The expression of some genes in the intersection was detected in tissues and cells and in the model after medication,and the possible functions of these genes were analyzed and studied.Then the binding model of ginsenoside Rh2 and Fox O3a protein was constructed to explore the possible mechanism of their interaction.3.The CO-IP experiment was used to compare the difference between ordinary SH-SY5Y cells and ginsenoside RH2-treated SH-SY5Y cells.Degree of Fox O3a acetylation among ordinary SH-SY5Y cells,SH-SY5Y cells cultured without Mg2+for3h,and SH-SY5Y cells pretreated with different concentrations of ginsenoside Rh2without Mg²⁺for 3h.The expression of FOXO3a-KEAP1-NRF2 pathway protein in SH-SY5Y cells after 3h culture without Mg²⁺and in SH-SY5Y cells pretreated with ginsenoside Rh2 were extracted from normal and drug-treated SH-SY5Y cells.SH-SY5Y cells transfected with p Fox O3-luc plasmid were also divided into blank control groups,which were cultured without Mg²⁺for 3h and pretreated with different concentrations of ginsenoside Rh2 without Mg²⁺for 3h.After the pretreatment,the differences of Fox O3a transcriptional activity in the three groups were detected with dual luciferase kit.4.SH-SY5Y cells were transfected with the plasmid,and Fox O3a silenced and overexpressed cell lines were constructed.The transfection efficiency was tested by WB assay,and the regulation effect of Fox O3a on KEAP1-NRF2 pathway proteins was verified.Mitochondrial membrane potential and ROS production levels were detected in normal SH-SY5Y cells,SH-SY5Y cells with sh FOXO3a and SH-SY5Y cells with FOXO3a overexpression after 3h culture without Mg²⁺.After the three kinds of cells were pretreated with ginsenoside Rh2 for 24h,the epileptic cell model was cultured without Mg²⁺for 3h.Mitochondrial membrane potential and ROS generation levels were compared between the groups.5.Intracranial injection of amygdaloid acid（KA）with stereotaxic instrument was used to construct rat epilepsy model ignited by amygdaloid acid（KA）,and appropriate drug concentration was selected for animal experiments through pre-experiments.The rats were divided into blank control group,carrier control group and drug treatment group.After isoflurane anesthesia,experimental reagent was injected into the lateral ventricle of the rats by stereotactic method.Two rats in the three groups were randomly selected to monitor the EEG after the end of the injection,and the EEG of the rats was observed when they had seizures after waking up.The remaining rats were killed one week later.The apoptosis of hippocampal neurons was detected by TUNEL staining,ROS accumulation was detected by DHE staining,and the expression of FOXO3a-KEAP1-NRF2 pathway protein in hippocampi was detected by WB assay.TAC levels in hippocampal homogenate and plasma and GSH/GSSG levels in hippocampal extract were determined.Results:1.Ginsenoside Rh2 can reduce the activity of SH-SY5Y cells,increase ROS production and increase mitochondrial depolarization caused by epilepsy,and alleviate the oxidative stress damage of SH-SY5Y cells under epilepsy.2.Ginsenoside Rh2 reduces the binding degree of FOXO3a to CBP/p300 in SH-SY5Y cells by binding to the binding site of FOXO3a and CBP/p300,and thus reduces the acetylation level of FOXO3a,so that FOXO3a cannot be transferred into the nucleus,and the transcriptional activity of Fox O3A is lost and finally degraded.3.FOXO3a RNA and protein levels showed significant increases in brain tissue and in vitro epilepsy models from focal human TLE patients compared with controls.4.Ginsenoside Rh2 regulates FOXO3a-KEAP1-NRF2 pathway by affecting the transcriptional activity of FOXO3a,and increases the expression of important antioxidant enzyme NQO1.5.SH-SY5Y cell lines overexpressing Fox O3a were more vulnerable to oxidative stress during epileptiform activity;SH-SY5Y cell lines with Fox O3a knockdown showed a certain resistance to oxidative stress damage caused by epileptic activity,but showed a weak response to ginsenoside Rh2 therapy.6.The administration of ginsenoside Rh2 can reduce the severity of epileptic seizure and the apoptosis of nerve cells in hippocampal CA1 region of rats in epileptic state induced by sea human acid.7.Ginsenoside Rh2 increased TAC and GSH levels in hippocampus and plasma of chronic epileptic rats.Conclusions:1.FOXO3a expression was significantly increased in epileptogenic focus tissue of acquired epilepsy patients,epileptic cell model constructed by SH-SY5Y cells,and hippocampus tissue of Ka-lit rat epileptic model.In addition,overexpression of FOXO3a will aggravate oxidative damage of SH-SY5Y cells in vitro epilepsy model.Conversely,knockdown of FOXO3a has a certain protective effect on such damage,suggesting that the high expression of FOXO3a may be an important factor aggravating oxidative stress damage of neurons in epilepsy patients.2.Overexpression of FOXO3a activated the negative NRF2 regulator KEAP1 and decreased the protein levels of NRF2 and NQO1.This relationship partially explains the decrease in endogenous antioxidant defense induced by epilepsy.3.Ginsenoside Rh2 showed a significant neuroprotective effect against oxidative stress in both in vivo and in vitro epilepsy models.In vitro,inhibition of seizures-like activity induced mitochondrial depolarization,ROS production,and cell death.Increased endogenous GSH levels and total antioxidant capacity in vivo,and significantly reduced seizures-like activity induced hippocampal neuronal cell death.4.The neuroprotective effect of ginsenoside Rh2 may reduce the transcriptional activity of FOXO3a by inhibiting the acetylation of FOXO3a,thus inhibiting the activation of Fox O3a-Keap1-Nr F2 pathway,and ultimately increasing the expression of Nr F2-related antioxidant enzymes.