| BackgroundEpilepsy is a chronic brain disease caused by a variety of diseases.It is haracterized by epileptic,recurrent and transient central nervous system dysfunction caused by excessive discharge of brain neurons.According to the World Health Organization(WHO)estimates,there are about 70 million people with epilepsy worldwide.According to the epidemiological data in China,the incidence of epilepsy is about 0.4-0.7%.Most epilepsy patients start in childhood,especially between birth and 3 years of age.It seriously affects the life and health of children and brings huge economic burden to their families.The pathogenesis of epilepsy is very complex,the cause has not been fully clarified,and the generally accepted view is imbalance of inhibition and excitation in the central nervous system.In recent years,a large number of studies have confirmed the interaction between the central nervous system(CNS)and intestinal flora(GM),and preliminatively described the factor pathway of interaction between the two through immune,nervous,endocrine and other systems,namely microbial-brain-gut axis.The incidence of epilepsy may be related to the imbalance of brain-gut axis.Another way gut bacteria affect brain function is through the immune system.The immune system is an important mediator between the central nervous system and intestinal flora.It has been established that a large number of epilepsy cases have an autoimmune basis for which adjuvant immunotherapy can effectively slow,stop or even reverse the progression of epilepsy.Antiepileptic drugs(AED)are currently the main known treatment for epilepsy,but AED is only effective in 60 to 70 percent of patients with epilepsy.Considering the limited therapeutic efficacy and severe adverse reactions among existing antiepileptics,it is urgent to further elucidate the underlying etiology of epilepsy in order to develop new therapeutic strategies.Through clinical observation and literature study,it is found that many children with epilepsy are phlegm-eclampsia syndrome based on TCM syndrome differentiation.This study intends to investigate the changes of related symptoms in epilepsy patients after treatment with "Ditan Decoction",as well as the effects on Th17 cells,IL-17 A and IL-6 cytokines.Meanwhile,16 S r DNA high-throughput sequencing was used to analyze the intestinal flora structure of patients before and after treatment with Ditan Decoction,and to compare the intestinal flora structure with that of normal healthy people.In order to find the changes of cytokines and intestinal microflora of patients after treatment with Ditan decoction,prove that ditan decoction intervention can improve the immune function and intestinal microecology of patients with epilepsy while improving related symptoms,and further demonstrate that there is a correlation between the therapeutic effect of "Ditan decoction" intervention and cytokines and intestinal microecology.It is proved that the curative effect of traditional Chinese medicine on children epilepsy may play a role by affecting the intestinal flora and immune function of children.Thus provide new evidence and new method for the study of TCM treatment of epilepsy.ObjectiveThis study effectively combined 16 S r RNA Amplicon with immune indicators such as IL-17 A and IL-6 cytokines secreted by Th17 cells,and applied multivariate analysis method to study the clinical efficacy of Ditan decoction in the treatment of children with phlegm-turbidity and obstruction of orifice epilepsy.The changes of intestinal flora,Th17 cells,IL-17 A and IL-6 cytokines were observed before and 12 weeks after treatment.To observe the clinical effect of "ditan decoction" on preventing and treating epilepsy in children.It was further confirmed that "cleanser phlegm decoction" combined with sodium valproate,an antiepileptic drug,could maintain the balance of immune system and inhibit immune-mediated inflammation.The difference of intestinal flora between epileptic children and normal infants was confirmed by intestinal flora detection.It was further verified that the compound Chinese medicine "di Tan Tang" has a certain effect on the intestinal microecology of children with epilepsy,and some key flora is the basis of the curative effect of Chinese medicine.These results provide a preliminary theoretical basis for the study of intestinal microbiota as a biomarker for the therapeutic effect of Ditan Decoction and a new target for the precise treatment of drug-resistant epilepsy.MethodsA total of 60 children with epilepsy who were treated in the pediatric neurology outpatient department and ward of Liuzhou Maternal and Child Health Hospital from November 2017 to September 2019 and met the inclusion criteria were included in this study.A prospective cohort study was conducted.According to the principle of patients’ voluntary,the children with epilepsy were divided into experimental group and control group(30 cases in each group).The experimental group was given "decontamination and phlegm decoction" combined with oral solution of sodium valproate,while the control group was given oral solution of sodium valproate alone.Thirty healthy children of the same age in the same area were recruited as blank control.At the same time,the difference of clinical efficacy between the experimental group and the control group after 12 weeks of treatment was observed.The levels of Th17 cells(by flow cytometry(FCM)),IL-17 A and IL-6 cytokines(by ELISA)in experimental group and control group were detected before and 12 weeks after treatment.Children with epilepsy in the experimental group who were treated with Ditan decoction combined with sodium valproate,aged from 12 months to 36 months,had stopped breastfeeding,had no recent gastrointestinal symptoms such as constipation and diarrhea,and agreed to participate in the clinical study and signed informed consent.Finally,8 eligible children with epilepsy were screened out.At the same time,8 healthy children at the same age and in the same area were selected as blank control.16 S r DNA Amplicon sequencing was used to observe the changes of intestinal microflora structure in the intestinal contents of the experimental group before and 12 weeks after treatment,and to compare the intestinal microflora with 8 healthy children of the same age in the same area.Then a series of in-depth statistical analysis of community structure was conducted to compare the changes of intestinal microflora structure in the three groups.Results1.After 12 weeks of treatment,the total effective rate of the experimental group was93.10%(27/29)and that of the control group was 57.14%(16/28),the difference was statistically significant(P<0.05).In addition,the experimental group was better than the control group in reducing seizure frequency,epilepsy syndrome score and eeg change(P<0.05).The comparison of the incidence of adverse reactions between the two groups after12 weeks of treatment showed that the incidence of adverse reactions in the experimental group was lower.Statistical analysis showed that the comparison of the incidence of total adverse reactions between the two groups after treatment was P<0.05.2.The percentage of Th17 cells in peripheral blood of children with epilepsy was significantly higher than that of blank control group before treatment(P<0.05).Compared with blank control group,serum LEVELS of IL-17 A and IL-6 cytokines in epileptic children were significantly increased(P<0.05).3.Compared with before treatment,the percentage of Th17,IL-17 A and IL-6cytokines in experimental group and control group were significantly decreased after treatment(P<0.05),while there were no significant changes in control group(P> 0.05).After 12 weeks of treatment,compared with the experimental group,the percentage of Th17,IL-17 A and IL-6 cytokines in the experimental group were significantly decreased(P<0.05).4.Compared with the blank control group,there were significant differences in intestinal microbial diversity in epileptic children before treatment(P<0.05).At phylum level,the proportion of bacteroides and actinomycetes in the experimental group was significantly lower than that in the healthy group,while the proportion of Proteus and firmicutes in the healthy group was significantly higher.After 12 weeks of treatment with Ditan decoction,the proportion of Bacteroides in the experimental group was significantly higher,while the proportion of Firmicutes in the experimental group was significantly lower.For the dominant bacteria at the genus level,the relative abundance of bifidobacteria and Purple omonas in the experimental group was significantly higher than that in the healthy group,while the relative abundance of Faecalis,Bacteroidetes and Prevotella in the experimental group was significantly lower than that in the healthy group.5.The results of species analysis showed that,compared with the blank control group,the proportion of actinomycetes and Bacteroides significantly decreased,and the proportion of proteobacteria and firmicutes significantly increased due to epilepsy at phylum level.The proportion of bacteroides and actinomycetes was significantly increased after treatment with Ditan decoction.Among the dominant bacteria at the genus level,the relative abundance of bifidobacteria in the experimental group was significantly higher than that in the healthy group,but the relative abundance of Bacteroides and lactic acid bacteria in the experimental group was significantly lower than that in the healthy group.The dominant species of intestinal flora in experimental groups were highly similar before and after treatment,but their proportions were different.6.LEf Se difference analysis was conducted for three groups of children before treatment,after treatment and blank control group.The results showed that the comparison between blank control group and blank control group before treatment was as follows:Before treatment,the abundant bacteria in the experimental group were g＿Bifidobacterium,g＿Prevotella and g＿Akkermansia.The bacteria with high abundance in blank control group were g＿Bacteroides,g＿Lactobacillus,g＿Prevotella and G Akkermansia.After intervention,the bacteria with high abundance in the experimental group were g＿Bacteroides and g＿Lactobacillus.The results showed that ditan decoction did not significantly change the diversity of intestinal flora in epileptic children,but it could increase the levels of Bacteroidetes and lactobacillus.ConclusionsDitan decoction combined with sodium valproate can significantly reduce the frequency of seizures,shorten the duration of seizures,improve the electroencephalogram,and has a good clinical effect.Compared with healthy children,the percentage of Th17,IL-17 A and IL-6 cytokines in epileptic children were higher than those in healthy children,suggesting that the autoimmune function of epileptic children was in an overactive state.After 12 weeks of treatment,the experimental group could significantly down-regulate the percentage of Th17 cells,IL-17 A and IL-6 levels in epileptic children.In the control group,the percentage of Th17 cells,IL-17 A and IL-6 levels were not significantly affected after treatment.These results indicated that Ditan decoction could significantly reduce the percentage of Th17 cells and the expression levels of related cytokines IL-17 A and IL-6.Compared with healthy children of the same age,the intestinal flora of epileptic children changed significantly,and there were significant differences in the level of phylum and genus.In addition,ditan decoction can change the composition and function of intestinal microbiome in patients with epilepsy.It can help the intestinal flora structure of epileptic children to return to normal and healthy state. |
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