| ObjectiveIn this study,TanshinoneⅡA was selected as the target drug,and the epileptic model was established by intraperitoneal injection of lithium chloride and pilocarpine.Sodium valproate and TanshinoneⅡA(low,middle and high dose)were given respectively to the epileptic rats.This study aimed to investigate from epileptic behavior,brain histopathology,protein and gene expression of synaptic remodeling related proteins SYN,PSD-95 and BDNF-TrkB signaling pathway in rats,to explore effect of improving epileptic seizure,cognitive function and the proble mechanism of regulating synaptic plasticity by TanshinoneⅡA,thus providing new ideas and theoretical basis for further study on the pharmacological effect of TanshinoneⅡA and improvement of cognitive function of epileptic patients.Methods1.Healthy Wistar rats were randomly divided into blank group(Blank),model group(Model),sodium valproate group(VPA),TanshinoneⅡA low dose group(TSⅡA-L),TanshinoneⅡA dose group(TSⅡA-M)and TanshinoneⅡA high dose group(TSⅡA-H).The epileptic rat model was established by intraperitoneal injection of lithium chloride and pilocarpine(blank group was injected with the same amount of normal saline).After successful modeling,each group had twenty rats that treated with medicine via continuous gavage respectively for sixty days,while blank and model group were treated with the same amount of saline.2.Ten rats were randomly selected from each group,according to Racine grading standard,epileptic seizures were recorded in each group.Morris water maze experiment and eight-arm maze experiment were conducted on rats in each group.The escape latency of rats and the number of times they crossed the original platform within 120 s in the water maze were recorded.In the eight-arm maze experiment,the number of reference memory and working memory errors,as well as the time to complete eating food from the food arms were recorded.3.After the maze tests,rats were treated with anesthesia and perfusion fixation.Then the heads of rats were severed and their brains were taken out to prepare brain tissue sections.Histopathological changes of CA3 area in hippocampal were observed by staining with 1% toluidine blue.The sprouting of hippocampal mossy fibers was observed by staining with Timm dye.After staining with uranium acetate and lead citrate,the ultrastructural changes in the CA3 area of the hippocampus were observed under the transmission electron microscope.4.After modeling and sixty days of drug administration,four rats were selected from each group randomly.After anesthesia and perfusion fixation,heads of rats were severed and their brains were taken out to prepare frozen brain tissue sections.The expression of synaptic remodeling related proteins SYN,PSD-95 and BDNF-TrkB signaling pathway in frozen sections was investigated by immunofluorescence staining.5.After modeling and 60 days of drug administration,three rats were selected from each group randomly.After anesthesia,heads of rats were severed and hippocampal tissue was removed on the ice.The gene expressions of SYN,PSD-95,BDNF and TrkB were detected by real-time fluorescence quantitative PCR.6.After modeling and 60 days of drug administration,three rats were selected from each group randomly.After anesthesia,heads of rats were severed and hippocampal tissue was removed on the ice.The protein expression of SYN,PSD-95,BDNF and TrkB was investigated by Western Blot.Results1.After the successful establishment of the epileptic model with pirocarpine,compared with model group,the seizure stage and frequency all showed a trend of decline in medicaine treated groups.Among them,the seizure stage in VPA group,TSⅡA-M group and TSⅡA-H group were significantly declined(P < 0.01).The seizure frequency in VPA group,TSⅡA-L group,TSⅡA-M group and TSⅡA-H group were decreased significantly(P < 0.05,P < 0.01).2.In the navigation experiment,compared with model group,the navigation time was decreased significantly(P < 0.01)and the time of crossing the platform were increased significantly(P < 0.01)in TSⅡA-L group,TSⅡA-M group and TSⅡA-H group.Compared with VPA group,the navigation time was decreased significantly(P < 0.01)and the time of crossing the platform was significantly increased(P < 0.01)in TSⅡA-M group and TSⅡA-H group.In the eight arm maze experiment,compared with model group,the number of reference memory and working memory errors were significantly decreased in TSⅡA-L group,TSⅡA-M group and TSⅡA-H group(P < 0.01).Compared with VPA group,the number of reference memory and working memory errors were significantly decreased in TSⅡA-M group and TSⅡA-H group(P < 0.05,P <0.01).The total time required to finish eating food in TSⅡA-L group,the TSⅡA-M group and TSⅡA-H group showed a significant decline compared with model group(P < 0.05,P < 0.01).3.Pathological experiments showed that,compared with model group,after drug administration with TanshinoneⅡA,the morphology of neurons gradually tended to be normal,the number of neurons was increased(P < 0.05,P < 0.01),mossy fiber sprouting in hippocampal CA3 area was decreased(P < 0.05,P < 0.01),the disordered arrangement of ultrastructure,fuzzy morphology and vacuoles degeneration were obviously improved in TSⅡA-L group,TSⅡA-M group and TSⅡA-H group.The morphology and ultrastructure of neurons in VPA group were slightly better than that in model group,but the number of neurons and mossy fiber germination were not significantly improved(P>0.05).4.Immunofluorescence staining results showed that,the expression of SYN,PSD-95,BDNF and TrkB in model group were significantly decreased compared with blank group(P < 0.05,P < 0.01).Compared with model group,the expression of SYN,PSD-95,BDNF and TrkB in VPA group was slightly raised,however,there was no statistical difference(P>0.05).The protein expression of SYN,PSD-95(P < 0.01)and BDNF,TrkB(P < 0.05)were significantly increased in TSⅡA-L group.The expression of SYN,PSD-95,BDNF and TrkB protein was significantly increased in TSⅡA-M group and TSⅡA-H group(P < 0.01).5.Real-time fluorescence quantitative PCR experiment results showed that,the expression of SYN mRNA,PSD-95 mRNA,BDNF mRNA and TrkB mRNA in model group were significantly decreased compared with blank group(P < 0.01).Compared with model group,the expressions of SYN mRNA,PSD-95 mRNA,BDNF mRNA and TrkB mRNA in VPA group were slightly raised,however,there was no statistical difference(P>0.05).The expression of SYN mRNA(P < 0.05)was increased,PSD-95 mRNA,BDNF mRNA,TrkB mRNA were significantly increased in TSⅡA-L group(P < 0.01).The expression of SYN mRNA,PSD-95 mRNA,BDNF mRNA and TrkB mRNA was significantly increased in TSⅡA-M group and TSⅡA-H group(P < 0.01).6.Western Blot results showed that,the expression of SYN,PSD-95,BDNF and TrkB in model group were significantly decreased compared with blank group(P < 0.01).Compared with model group,the expression of SYN,PSD-95,BDNF and TrkB in VPA group were slightly raised,however,there was no statistical difference(P>0.05).The protein expression of SYN(P < 0.05)was increased,the expression of PSD-95,BDNF and TrkB were significantly increased in TSⅡA-L group(P < 0.01).The expression of SYN,PSD-95,BDNF and TrkB protein was significantly increased in TSⅡA-M group and TSⅡA-H group(P < 0.01).ConclusionTanshinoneⅡA could effectively inhibit epileptic seizures,improve the learning and memory abilities on epileptic rats,alleviate neuronal injuries and ultrastructure damages in hippocampal CA3 area,and inhibit the mossy fiber sprouting in hippocampus.TanshinoneⅡA could regulate synaptic plasticity and improve cognitive impairment of epileptic rats via increasing the expression of synaptic remodeling related proteins SYN and PSD-95,and up-regulating hippocampal BDNF-TrkB signaling pathway. |
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