| Epilepsy is one of the three common chronic diseases of the brain,which characterized by chronic recurrent spontaneous seizures,and often accompanied by emotional and cognitive dysfunction.At present,the drug treatment is still the main treatment for epilepsy,but there is about 20% to 30% of patients with episodes which is called refractory epilepsy.Temporal lobe epilepsy is the most common refractory epilepsy in adults,often secondary to a variety of primitive brain damage,these brain damage induced brain cascade incident,eventually leading to increased susceptibility to epilepsy and repeated spontaneous seizures,which process is called epileptogenesis.With the development of science and technology,the concept of epilepsy has been constantly updated,the latest consensus shows that the definition of epilepsy is able to produce spontaneous seizures and accompanied by brain tissue growth and development of an inflammatory response.Experimental evidence shows that epilepsy is a dynamic process,especially in the brain epilepsy prone area,the relevant molecular structure and function of the pathophysiology will continue to change,leading to the new pathology of the brain to produce,and ultimately induced epilepsy.Although the current prevention and treatment of epilepsy is still controversial,and the effect is questionable,but the relationship between epilepsy and inflammatory response and the mechanism is still the focus of clinical and basic research.Amentoflavone(AF)is a natural polyphenolic compound with a variety of biological activities,which plays an important role in anti-inflammatory,anti-microbial,antioxidant,anti-tumor and neuroprotection.Studies have shown that AF can play an antiinflammatory role by inhibiting many inflammatory factors,which inhibits TNF-?-induced expression of COX-2 and PGE2 by inhibiting the activation of NF-?B in tumor cells;AF in the nervoussystem disease modulates the expression of cytokines such as IL-1 [beta] and ILInflammation reaction.AF can significantly down-regulate the expression of COX-2 protein and inhibit the synthesis of PGE2.In addition,AF can block apoptosis by interfering with Caspase activation.Therefore,we speculated that AF can inhibit the regulation of epilepsy in the brain by the relevant factors in the inflammatory pathway,reduce neuronal damage to prevent the occurrence of epilepsy.In the present study,we focus on the model of epilepsy induced by lithium chloride-pilocarpine,and the following hypothesis was applied by electrophysiology,behavior,histology and molecular biology: AF could inhibit inflammation by inhibiting glial cell activation Pathway related factors to regulate the level of brain inflammation,and thus play a neuroprotective role in the prevention of epilepsy,which was discussed in our study.Part 1 The effect of Amentoflavone on epilepsy susceptibility and cognitive function in lithium chloride-pilocarpine ratObjective: To investigate the effect of pretreatment of AF on epilepsy susceptibility and cognitive function in SD rats with lithium chloride-pilocarpine.Method: 100 rats were randomly divided into 5 groups: pretreatment of AF group(pre-AF group),treatment with AF group(AF group),pretreatment of VPA groups(pre-VPA group),epilepsy group(EP group),blank control group(Control group).Establishment of rat model of lithium chloride-pilocarpine epilepsy,racine score was used to assess the severity of epileptic seizures,erythrocytes were used to monitor the electrophysiological changes of rats in each group.Morris water maze and Intelli Cage analysis system were used to evaluate the learning and memory ability of rats.The effects of AF pretreatment on epilepsy persistence and spontaneous seizure susceptibility in epileptic rats were evaluated.Explored the preventive effect of AF on epilepsy in epilepsy rats.Result: The pre-AF group had few seizures after modeling,and the seizure level was significantly reduced and the onset time was shortened.EEG signals showed that the frequency and amplitude of epileptic wave were significantly decreased.The behavioral and EEG signals were significantly different from those in the control group There was no significant difference.(P <0.05).In the space exploration experiment,the number of pre-AF rats in the pre-AF group was shorter than that in the AF group,pre-VPA group and EP group(P <0.05)Group,pre-VPA group and EP group were significantly increased(P <0.05).Intelli Cage behavioral examination showed that the number of visits in the pre-AF group,the AF group,the pre-VPA group,and the EP group was significantly lower than that in the control group(P <0.05),and the difference was statistically significant(P <0.05).Compared with EP group,the number of access visits in pre-AF group,AF group and pre-VPA group was significantly increased(P <0.05),and the number of pre-AF group was the most significant.At the same time,the results of the correct number of nasal poke(nosepoke)were consistent with the visit;in the position learning test,compared with the control group,the pre-AF group,the AF group,the pre-VPA group,the EP group(P <0.05).Compared with EP group,the number of correct visits in pre-AF group,AF group and pre-VPA group was significantly increased(P <0.05),and the difference was statistically significant(P <0.05)In the intensive learning test,compared with the control group,the pre-AF group,the AF group,the pre-VPA group,the EP group,the pre-AF group,the pre-AF group,the pre-AF group,the pre-The number of correct nasal poke significantly reduced,the difference was statistically significant(P <0.05).Compared with EP group,the number of correct nasal poke in pre-AF group,AF group and pre-VPA group was significantly improved(P <0.05),and the improvement of pre-AF group was the most obvious Consistent with the enhanced learning test results.Conclusion: The preventive treatment of AF can reduce the seizure level of epilepsy rats,shorten the onset time,improve the ability of space learning and memory decline,reduce the susceptibility to epilepsy.Part 2 Effects of Amentoflavone on hippocampal neuron injury and oxidative stress in lithium chloride-pilocarpine epilepsy ratsObjective: To investigate the effect of AF on the injury and oxidative stress of hippocampal neurons induced by lithium chloride-pilocarpine in epilepsy rats,and to clarify its role in the prevention of epilepsy and its possible mechanism.Methods: Eight male SD rats were divided into the same group.The neuronal injury and apoptosis of hippocampal neurons in epilepsy rats were observed by Nissel staining and TUNEL staining.Immunohistochemistry and Western-Blot technique were used to detect the expression of caspase(MDA),NO,GSH and catalase(CAT)and SOD in the hippocampus of rats in each group.At the same time,the levels of malondialdehyde(MDA),NO,GSH and catalase(CAT)and SOD were detected in the hippocampus of each group.Results: Compared with control group,Nissel staining showed that there was no significant difference(P> 0.05)between the CA1 and CA3 regions in the hippocampus of the pre-AF group(P <0.05).Compared with control group,TUNEL staining in pre-AF group showed no significant difference(P> 0.05)in the hippocampal CA1 and CA3 regions(P> 0.05),which was significantly lower than that in EP group(P <0.05).Compared with the control group,the number of caspase-3 positive cells in the hippocampal CA1 and CA3 regions of the pre-AF group was not significantly different(P> 0.05),which was significantly lower than that in the EP group(P <0.05)There was no significant difference in the expression of caspase-3 protein between pre-AF group and control group(P> 0.05),but decreased significantly compared with other groups(P <0.01).The levels of GSH and SOD in the pre-AF group were significantly different(P> 0.05)compared with the control group at 24 h and 36 h after SE(P> 0.05),which was significantly higher than that in the EP group(P <0.05).There was no significant difference in CAT,MDA and NO between pre-AF group and control group(P> 0.05),which was significantly lower than that in EP group(P <0.05).Conclusion: pretreatment of AF can inhibit epilepsy-induced neuronal injury and apoptosis by inhibiting hippocampal oxidative stress levels and prevent epilepsy.Part 3 Effects of amantadine flavonoids on glial cell activation and hippocampal inflammatory response in lithium chloride-pilocarpine epilepsy ratsObjective: To explore the effect of AF on glial cell activation and hippocampal oxidative stress in rats with epilepsy induced by lithium chloride-pilocarpine and to explore its mechanism of prevention of epilepsy and the possibility of glial cells in the process of epilepsy.Methods: 60 male SD rats,the grouping is the same as the first part..The expression and distribution of Iba-1 and GFAP in hippocampus of rats were observed by immunohistochemistry.And the hippocampus tissue of rats was extracted at 24 h,48h,72 h and 96 h after SE.The expression of NF-?B and COX-2 protein in inflammatory pathways were detected by Western-Blot technique.ELISA were used to detect the levels of IL-1?,TNF-? and PGE2.Results: Immunohistochemistry showed that Iba-1 positive cells were found in AF group,pre-VPA group and EP group,and there was significant difference between control group and control group(P <0.05),while pre-AF(P> 0.05).There were more GFAP-positive cells in the hippocampal CA1 and CA3 regions of AF group,pre-VPA group and EP group,and there was no significant difference between the control group and the control group(P> 0.05)(P <0.05).The number of GFAP-positive cells in pre-AF group was significantly lower than that in control group(P> 0.05).Western Blot showed that there was no significant difference between control group and control group(P <0.05).Compared with the control group,the levels of COX-2 and NF-?B p65 protein in hippocampus were significantly higher than those in control group(P <0.05),and the difference was statistically significant(P <0.05).The expression of COX-2 and NF-?B p65 protein in the control group was higher than that in the control group(P <0.05).The expression of COX-2 and NF-?B p65 protein in the control group was significantly higher than that in the control group Trend.The levels of IL-1?,PGE2 and TNF-? in the hippocampus of the hippocampus were significantly increased at 24 h,48 h,72 h and 96 h,respectively(P <0.05)(P <0.05).Compared with EP group,the levels of inflammatory factors in pre-AF group were significantly lower than those in EP group(P <0.05).Conclusion: AF can inhibit the expression of COX-2 and NF-?B p65 in hippocampus of rats with epilepsy induced by pilocarpine and decrease the levels of IL-1?,TNF-? and PGE2,inhibit the activation of microglia and the response of astrocytes hyperplasia play an important role in the prevention of epilepsy and neuroprotective effects. |
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