Protective Effect Of Tanshinone ⅡA On Apoptosis And Synaptic Plasticity Damage In Mg²⁺-Free-Induced Epileptic Hippocampal Neurons Via PI3K/Akt Signaling Pathway

Objective:Tanshinone ⅡA was selected as the treatment drug in this study.Firstly,lithium chloride-pilocarpine induced epilepsy rat model was established.Tanshinone ⅡA and sodium valproate were administered respectively.Potential mechanism of tanshinone ⅡA in the treatment of epilepsy was analyzed by RNA sequencing.Then magnesium-free-induced epileptic hippocampal neurons model was used.Through the intervention of LY294002 inhibitor,the protective effect of tanshinone ⅡA on apoptosis and synaptic plasticity damage in status epilepticus via PI3K/Akt signaling pathway was investigated,to explore the mechanism of tanshinone ⅡA in the treatment of epilepsy and to provide theoretical solution for anti-epileptic drug treatment.Methods:1.The epileptic SD rat model was induced by intraperitoneal injection with lithium chloride-pilocarpine.After successful modeling,rats were stochastically divided into model group（Model）,tanshinone ⅡA group(TSⅡA,20 mg·kg^-1)and sodium valproate group(VPA,200 mg·kg^-1).Rats in Blank group were given equal volume of normal saline.Rats in TSⅡA group and VPA group were given continuous intragastric administration for 60 days while the others were administrated the same amount of normal saline.Three rats were randomly selected from each group.After treated with anesthesia,hippocampus was taken and the left part of the tissue was collected.Total RNA was extracted and detected by RNA sequencing.Differential expression genes（DEGs）were used to analyze the potential mechanism of TSⅡA through KEGG and GO in the treatment of epilepsy.2.Primary hippocampal neurons were extracted from SD rats within 24 h and purity of hippocampal neurons was indentified by MAP2 immunofluorescence staining.After culturing 9 d,the magnesium-free external solution was intervened for3 h to establish epileptic hippocampal neurons while hippocampal neurons in Blank group were cultured in normal external solution for 3 h.The optimal dose of TSⅡA was screened by the CCK8 method.After that,the experimental groups were stochastically divided into blank group（Blank）,model group（Model）,tanshinone ⅡA group（TSⅡA,20μM）,LY294002 group（LY294002,25μM）and TSⅡA+LY294002group（TSⅡA+LY294002,20μM+25μM）.3.After the corresponding drug intervention and culturing for 24 h,the cell viability and apoptosis rate of hippocampal neuronal cells in each group were respectively evaluated by CCK8 and TUNEL staining.The relative expression of p-Akt/Akt,Bcl-2/Bax in hippocampal neuronal cells of each group was determined by Western Blot.4.After giving the corresponding drug intervention and culturing for 24 hours,the changes of neurite complexity,total lenghth of hippocampal neurons,number of primary dendrites and density of dentrtic spines were anaylized.The expression of Drebrin and BDNF was evaluated by immunofluorescence staining and the relative expression of p-Akt/Akt,BDNF,SYN and PSD-95 was determined by Western Blot.Results:1.The results of RNA sequencing showed that there were 255 common DEGs between Blank-vs-Model group and TSⅡA-vs-Model group,and there were 89common DEGs among Blank-vs-Model group,TSⅡA-vs-Model group,and VPA-vs-Model group.Excluding 89 differentially expressed genes,166 DEGs were obtained and were significantly enriched in 6 KEGG pathways（Pvalue<0.05）,including PI3K/Akt signaling pathway.At the same time,166 DEGs were significantly enriched in 441 GO terms（Pvalue<0.05）,including 44 cellular components terms,101 molecular functions terms and 296 biological processes terms.2.The primary hippocampal neuronal cells were successfully cultured and the purity was over 95%.After culturing with the optimal concentration of TSⅡA（20μM）,the cell viability was 90.7%±2.7%,which was significantly increased compared with Model group（P<0.01）.Compared with TSⅡA group,the cell viability of LY294002and TSⅡA+LY294002 groups was significantly decreased（P<0.01）.3.The results of TUNEL staining were obtained,indicating that apoptosis rate of TSⅡA group and Blank group showed no significant difference（P>0.05）,but the apoptosis rate of TSⅡA group was significantly lower than that of Model group（P<0.01）.The apoptosis rate of LY294002 group and TSⅡA+LY294002 group was significantly increased when making comparison with TSⅡA group（P<0.01）.4.Compared with Model group,the ratio of p-Akt/Akt and Bcl-2/Bax in TSⅡA group was both attenuated showed by the results of Western Blot（P<0.01）.Compared with TSⅡA group,LY294002 group and TSⅡA+LY294002 group had a much lower expression of p-Akt/Akt and Bcl-2/Bax（P<0.01）.5.Sholl analysis showed that TSⅡA could significantly increase the complexity of neurites,the number of primary dendrites,the total length of neurons and the density of dendritic spines（P<0.05）.The relative fluorescence intensity of Drebrin and BDNF in TSⅡA group was significantly increased（vs.Model,P<0.05）.Compared with TSⅡA group,the expression of Drebrin and BDNF in LY294002 group and TSⅡA+LY294002 group was significantly decreased（P<0.05）.6.The relative expression of BDNF,SYN,PSD-95 and p-Akt/Akt proteins in TSⅡA group was all significantly attenuated（vs.Model,P<0.01）.While the relatve expression of BDNF,SYN,PSD-95 and p-Akt/Akt in LY294002 group and TSⅡA+LY294002 group was significantly decreased（vs.TSⅡA,P<0.05）.Conclusion:The therapeutic effect of TSⅡA has the characteristics of multi-targets and the expression of genes can be regulated by TSⅡA via PI3K/Akt signaling pathway in lithium chloride-pilocarpine induced epilepsy rat model.In epileptic hippocampal neurons model induced by magnesium-free external fluid,TSⅡA can effectively alleviate apoptosis and synaptic plasticity damage caused by status epilepticus and its mechanism is related to the regulation of PI3K/Akt signaling pathway.