生酮饮食对于婴儿痉挛症患儿肠道微生态及代谢改变研究

Study of changes of gastrointestinal microbiota and metabolome in children with infantile spasms after receiving KD treatment

目的 研究生酮饮食(KD)治疗对婴儿痉挛症患儿肠道菌群结构及代谢组的改变,探讨KD治疗婴儿痉挛症微生态机制。方法 2015年7月至2020年4月在深圳市儿童医院神经内科确诊的婴儿痉挛症患儿16例,纳入年龄6月龄至3岁,并选取30名年龄匹配的健康儿童作为对照。分别采集婴儿痉挛症患儿KD治疗前粪便样本、KD治疗1周后的粪便样本和健康儿童的粪便样本,通过宏基因组和宏代谢组技术对粪便样本中的微生物组成和代谢物成分进行检测,探索治疗前、治疗后及相对健康儿童的菌群组成差异和代谢差异。结果 婴儿痉挛症患儿肠道微生态的组成模式与健康儿童存在不同,其中占主导地位的有益菌拟杆菌属占比为22.62%,明显低于健康儿童(38.86%);多氏拟杆菌Bacteroides dorei两组差异有统计学意义[对照组(3.24±7.14),pre-KD组(0.19±0.40);P<0.001]。KD治疗前、后与对照组比较:埃希氏菌属丰度在KD治疗前、后均比对照组增加,差异有统计学意义(均P<0.001)。种水平统计分析显示,KD治疗后多种拟杆菌如脆弱拟杆菌、多形拟杆菌、普通拟杆菌、多氏拟杆菌等增加,普氏栖粪杆菌、嗜黏蛋白阿克曼菌也有随KD治疗增加。KD治疗后长双歧杆菌、短双歧杆菌下降。与对照组比较,婴儿痉挛症患儿大肠杆菌明显富集,随着KD治疗回落[对照组(0.31±0.66),KD治疗前(7.41±9.03),KD治疗后(3.61±4.59);P<0.001]。从KEGG基因功能预测结果分析,可见肠道微生物功能在3组中存在丰度差异,尤其是涉及到碳水化合物代谢、基因转录翻译、核酸代谢等功能通路,差异有统计学意义(P<0.05)。在宏代谢组学差异分析中,肠道微生态的代谢物发生了显著改变,KD治疗后显著富集了与抗癫痫相关的癸酸、姜黄素等代谢物,差异有统计学意义(P<0.05)。结论 婴儿痉挛症患儿的肠道菌群组成与健康儿童存在差异,多氏拟杆菌在婴儿痉挛症患者中显著减少,大肠杆菌明显富集,经KD治疗后拟杆菌、普氏栖粪杆菌等有益菌增多。同时检测到多种与神经系统疾病相关的代谢物。这可能和KD改善婴儿痉挛症患儿临床症状的机制密切相关。

Objective To study the changes of gastrointestinal microbiota(GM)and metabolome after receiving ketogenic diet(KD)and explore the microecological etiology of children with infantile spasms(IS).Methods We selected sixteen children with IS,who were diagnosed in the Department of Neurology,Shenzhen Children′s Hospital between July 2015 and April 2020,aged 6 months to 3 years old.Healthy children(n=30)were enrolled as controls.We collected stool samples from patients at 2 time points,including before KD treatment and 1-week after KD treatment,and fecal samples from healthy children were also collected.The microbial composition and metabolite components were detected by metagenomic and metabolome methods to explore the changes of microbiota composition and metabolism after KD treatment.Results Principal componentanalysis(PCA)suggests that the GM pattern of ISpatients is significantly different from that of healthychildren,but it tends to that of healthy children'salong the KD treatment.The dominant genus isBacteroides,and the proportion in IS children (22.62%)is lower than that in healthy children(38.86%),and the relative abundance of Bacteroides dorei also reducedsignificantly in IS children[Healthy group:(3.24±7.14),pre-KD group:(0.19±0.40);P<0.001].On the other hand,theEscherichia genus was significantly enriched in patients(P<0.001),and the proportion of corresponding species,Escherichia coli,was also significant enriched in patients' GM(Health group:0.31%,pre-KD group:7.41%;P<0.01).After KD treatment,the statistical analysis at the species level showed that a variety of Bacteroides increased,such asBacteroides fragilis,Bacteroides thetaiotaomicron,B.vulgatus,and B.dorei,as well as Faecalibacterium prausnitzii,Akkermansia muciniphila.On the other hand,Bifidobacterium longum and Bifidobacterium breve decreased significantlyafter KD treatment,as well as E.coli.Based on KEGG analysis,we identified significant abundance differences ofmultiple metabolic pathways of GM among the three groups,especial for carbohydrate metabolism,gene transcription andtranslation,and nucleic acid metabolism.The analysis of metabolomics demonstrated that various metabolites,such ascapric acid and curcumin,which was related to anti-epileptic effect,were significantly enriched after KD treatment.These differentially abundant metabolites are the key compound to the various KEGG pathway,including metabolicpathways(such as galactose metabolism,phenylalanine metabolism,taurine metabolism),ABC transporters,digestionand absorption of carbohydrates and proteins,etc.Conclusion There are significant differences in the composition ofGM between IS patients and healthy children.B.dorei significantly reduced in IS patients' GM,but E.coli significantlyenriched.After KD treatment,Bacteroides,F.prausnitzii and other beneficial bacteria increased.Various neurologicaldiseases related metabolites were detected.These findings provide a new insight to the mechanism about how ketogenicdiet improves the clinical symptoms of IS patients.