| BackgroundEpilepsy is the fourth most common nervous system disease.There are more than 9 million patients with epilepsy in China,and about 30%of the patients take the current anti epileptic drugs,which can not effectively control seizures,so it is called refractory epilepsy.The pathogenesis of refractory epilepsy is complex.It is believed that both neuroinflammation and gut microbiota are involved in the pathogenesis of refractory epilepsy.Chaibei Zhixian Decoction is an experienced prescription for the treatment of refractory epilepsy.It has good efficacy and high safety for refractory epilepsy.However,the research on its antiepileptic mechanism is still insufficient.It was found that Chaihu,Fritillaria thunbergii,Gastrodia elata,Pinellia ternata and Acorus tatarinowii in Chaibei Zhixian Decoction had certain anti-inflammatory and inhibitory effects on the proliferation of glial cells.Therefore,it is speculated that Chaibei Zhixian Decoction may play an antiepileptic role by inhibiting the neuroinflammation.As the first barrier for drugs to enter the body,gut microbiota can regulate the inflammatory response of nerve center through immune pathway.Whether gut microbiota is involved in the regulation of Chaibei Zhixian Decoction on neuroinflammation has not been discussed.Traditional Chinese medicine has the characteristics of multi-component,multi-target and multi-channel.Network pharmacology is a new pharmacology research method,which integrates the methods of systems biology and biological network,breaking through the traditional single drug,single target,single disease research mode.Therefore,the network pharmacological analysis of Chaibei Zhixian Decoction's blood components will help to more accurately explore its possible mechanism of action,lay the foundation for subsequent experimental research,and enrich the anti epileptic mechanism of Chaibei Zhixian Decoction.ObjectivesThe aim of this study is to explore the anti epileptic mechanism of Chaibei Zhixian Decoction.The effects of Chaibei Zhixian Decoction on gut microbiota and neuroinflammation in rats with epilepsy induced by Kainic acid were studied by animal experiments.Secondly,with the help of Ultra Performance Liquid Chromatography(UPLC)-Tandem mass spectrometry(MS/MS)technology and gas chromatography(GC)-MS/MS technology,the main components absorbed into the blood of Chaibei Zhixian Decoction were analyzed.The network pharmacology analysis of the components absorbed into the blood of Chaibei Zhixian Decoction was carried out,and the results were verified by animal experiments.Methods1.Wistar rats were randomly divided into sham operation group and model group.The model rats were injected kainic acid into lateral ventricle to induce chronic spontaneous epilepsy.The successful model rats were randomly divided into model group and traditional Chinese medicine group.Rats in the traditional Chinese medicine group were given Chaibei Zhixian Decoction formula granules by gavage,while rats in the model group and sham operation group were given the same amount of drinking water by gavage for 4 weeks.Fresh fecal tissues were collected for gut microbiota detection,and fresh hippocampal tissues for cytokine detection Brain tissues were perfused and fixed.Paraffin sections of hippocampus were made for Nissl staining,GFAP and Iba-1 immunohistochemical staining2.After intragastric administration of 17 kinds of traditional Chinese medicine,the rats were divided into three groups:Bupleurum saponin,ethanol,palmatine and D,respectively The concentrations of aldehydes,anethole,?-asarone,?-asarone and asarone in rat blood at different time points were calculated by Das 2.0 software3.By searching the relevant databases,we collected the targets for the treatment of epilepsy and the 15 targets identified by Chaibei Zhixian decoction,constructed the target network of Chaibei Zhixian Decoction for the treatment of epilepsy,and searched the protein-protein interaction between the targets.Use Cytoscape to build network diagram and analyze network.Cluster profiler package was used for go enrichment analysis and KEGG enrichment analysis.4.Rats were randomly divided into sham operation group and model group.Kainic acid was injected into the lateral ventricle of model group.The rats were randomly divided into model group and traditional Chinese medicine group.0.9%normal saline was injected into the lateral ventricle of sham operation group.The traditional Chinese medicine group was given Chaibei Zhixian Decoction granules by gavage,and the other two groups were given the same amount of drinking water by gavage.After 5 weeks of intervention,the learning and memory abilities of rats were detected,and the expression of PI3K/Akt signaling pathway,p-tau and tau protein in hippocampus were detected.Results1.Compared with the model group,the expression of Iba-1 was significantly decreased(P<0.01),and the expression of G-CSF,GM-CSF,IFN-?,IL-1?,IL-1?,IL-18,IL-6,M-CSF,mip-3 ?,TNF-? was significantly decreased(P<0.05),while the expression of IL-13,IL-4,IL-5 was up-regulated(P<0.05).The diversity of gut microbiota alpha in kainic acid-induced epileptic rats was lower than that in normal rats.The composition and abundance of gut microbiota in the traditional Chinese medicine group and the sham operation group were close,and there was a big difference with the model group.2.15 kinds of monomers were detected in rat plasma,and their contents were in the order of gastrodin,p-hydroxybenzaldehyde,trigonelline,gastrodin,protocatechuic aldehyde,saikosaponin a,vanillin,asaronal,fritillarin B,?-asarone,anethole,?-asarone,saikosaponin C,fritillarin a,fritillarin,citral and saikosaponin D.3.There are 125 targets in Chaibei Zhixian decoction.Akt is one of the key targets in the treatment of epilepsy.The possible mechanism of Chaibei Zhixian decoction is to regulate the synapse and soma of neurons,and play a therapeutic role in oxidative stress,nerve cell death and neurotransmitter response.4.Compared with the sham operation group,the escape latency of the model group was significantly prolonged(P<0.01),the number of crossing platform was decreased(P<0.05),the expression of PI3K and Akt protein was decreased,and the expression of p-tau was significantly increased(P<0.01).Compared with the model group,the spatial learning and memory ability of the traditional Chinese medicine group was significantly improved(P<0.01,P<0.05),the hippocampal tissue was relatively intact,the loss of neurons was reduced,the expression of PI3K and Akt protein was increased,and the expression of p-tau was decreased(P<0.05).Conclusions1.The anti epileptic mechanisms of Chaibei Zhixian decoction may inhibit the activation of microglia,reduce the level of pro-inflammatory cytokines,reduce the neuroinflammation,thus reduce the death of neurons and inhibit seizures.2.Chaibei Zhixian decoction can also help to restore the gut microbiota of kainic acid-induced epileptic rats,and its contribution to the treatment of epilepsy remains to be further studied.3.The components of Chaibei Zhixian decoction are complex,including gastrodin,p-hydroxybenzaldehyde,trigonelline,gastrodin,protocatechuic aldehyde,saikosaponin a,vanillin,asaronal,fritillarin B,?-asarone,anethole,?-asarone,saikosaponin C,fritillarin A and fritillarin.4.Chaibei Zhixian decoction can also up regulate PI3K/Akt signaling pathway,down regulate p-tau protein expression,and improve learning and memory ability of rats. |
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