基于16S rRNA技术分析柴胡龙骨牡蛎汤对精神分裂症大鼠肠道菌群多样性的影响
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摘要
目的:基于16S rRNA技术研究柴胡龙骨牡蛎汤对精神分裂症模型大鼠肠道菌群多样性的影响,并探讨该复方治疗精神分裂症的作用机制。方法:雄性SD大鼠除正常组外,每日按剂量0. 1 mg·kg~(-1)腹腔注射地卓西平马来酸盐进行造模,模型成功后,柴胡龙骨牡蛎汤高、中、低剂量组给予实验大鼠柴胡龙骨牡蛎汤(11. 2,5. 6,2. 8 g·kg~(-1)),利培酮组按0. 4 mg·kg~(-1)灌胃给予利培酮片,正常组与模型组灌胃给予蒸馏水,给药体积均为10 mL·kg~(-1),连续给药14 d,将大鼠麻醉后取大鼠盲肠内容物,以Illumina MiSeq为测序平台,对其16S rRNA V4区的肠道菌群可操作分类单元(OTUs)数量,alpha与beta多样性,丰富度指数与多样性指数以及差异菌门与菌属进行综合性分析与评价。结果:柴胡龙骨牡蛎汤可改善精神分裂模型大鼠OTUs数量及alpha与beta多样性的失调,降低菌群生物丰富度指数与多样性指数,对精神分裂症模型大鼠的5种差异菌门(上调拟杆菌门、酸杆菌门、变形菌门和TM7菌门,下调厚壁菌门)与20种差异菌属的分布结构均产生回调作用。结论:柴胡龙骨牡蛎汤对精神分裂症模型大鼠的异常菌群多样性起到治疗作用,并通过16S rRNA技术揭示了肠道菌群在精神分裂状态下的相关病理机制。
Objective: To explore the effect of Chaihu Longgu Mulitang on intestinal microflora diversity of schizophrenic model rats,and further reveal its therapeutic characteristics and mechanisms based on the 16 S rRNA technique. Method: Except the normal group,male SD rats were intraperitoneally injected dizocilpine maleate with daily dose of 0. 1 mg·kg~(-1). After the success of the model,Chaihu Longgu Mulitang high,middle and low dose groups were converted into the human clinical upper limit daily,and the experimental rats were given Chaihu Longgu Mulitang with doses of 11. 2,5. 6,2. 8 g·kg~(-1),respectively. And the positive drug group was treated with 0. 4 mg·kg~(-1) of risperidone tablets. The normal group and model group was treated with water. The rats were continuous administrated 14 days with dosing volume of 10 mL·kg~(-1),the contents in caecum of rats were taken after anesthesia. Illumina Mi Seq was used as the sequencing platform,the number of operational taxonomic units( OTUs),richness and diversity indexes, diversity of alpha and beta, differential phylum and genus of intestinal flora in V4 zone of 16 S rRNA were comprehensively analyzed and evaluated. Result: Chaihu Longgu Mulitang could improve the number of OTUs,richness and diversity indexes of intestinal flora,imbalance of alpha and beta diversity of schizophrenic model rats. And this formula had a callback effect on 5 differential phyla of bacteria( Bacteroidetes, Acidobacteria, Proteobacteria, Firmicutes and TM7) and 20 genera of bacteria in schizophrenic model rats. Conclusion: Chaihu Longgu Mulitang plays an therapeutic effect on diversity of abnormal microflora in schizophrenic model rats,and this paper reveals the pathological mechanism of intestinal microflora in the state of schizophrenia by 16 S rRNA technique.
Objective: To explore the effect of Chaihu Longgu Mulitang on intestinal microflora diversity of schizophrenic model rats,and further reveal its therapeutic characteristics and mechanisms based on the 16 S rRNA technique. Method: Except the normal group,male SD rats were intraperitoneally injected dizocilpine maleate with daily dose of 0. 1 mg·kg~(-1). After the success of the model,Chaihu Longgu Mulitang high,middle and low dose groups were converted into the human clinical upper limit daily,and the experimental rats were given Chaihu Longgu Mulitang with doses of 11. 2,5. 6,2. 8 g·kg~(-1),respectively. And the positive drug group was treated with 0. 4 mg·kg~(-1) of risperidone tablets. The normal group and model group was treated with water. The rats were continuous administrated 14 days with dosing volume of 10 mL·kg~(-1),the contents in caecum of rats were taken after anesthesia. Illumina Mi Seq was used as the sequencing platform,the number of operational taxonomic units( OTUs),richness and diversity indexes, diversity of alpha and beta, differential phylum and genus of intestinal flora in V4 zone of 16 S rRNA were comprehensively analyzed and evaluated. Result: Chaihu Longgu Mulitang could improve the number of OTUs,richness and diversity indexes of intestinal flora,imbalance of alpha and beta diversity of schizophrenic model rats. And this formula had a callback effect on 5 differential phyla of bacteria( Bacteroidetes, Acidobacteria, Proteobacteria, Firmicutes and TM7) and 20 genera of bacteria in schizophrenic model rats. Conclusion: Chaihu Longgu Mulitang plays an therapeutic effect on diversity of abnormal microflora in schizophrenic model rats,and this paper reveals the pathological mechanism of intestinal microflora in the state of schizophrenia by 16 S rRNA technique.
引文
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[19]张宁,李自辉,赵洪伟,等.寒凝血瘀证大鼠的肠道菌群变化与粪便代谢特征分析[J].中国实验方剂学杂志,2018,24(2):79-85.
[20] Severance E G,Gressitt K L,Stallings C R,et al.Probiotic normalizationof Candida albicans in schizophrenia:a randomized, placebo-controlled,longitudinal pilot study[J]. Brain Behav Immun,2017,62:41-45.
[21] Sampson T R,Mazmanian S K. Control of brain development,function,and behavior by the microbiome[J]. Cell Host Microbe,2015,17(5):565-576.
[22] Schwarz E,Maukonen J,Hyytiainen T,et al. Analysis of microbiota in first episode psychosis identifies preliminary associations with symptom severity and treatment response[J]. Schizophr Res,2018,192:398-403.
[2]魏妍妍,刘丹丹,戎文娟,等.温胆汤对精神分裂症模型鼠海马PKC,p38MAPK及P-Cx43的影响[J].中国实验方剂学杂志,2015,21(11):103-106.
[3] Rebollo B,Maria P Z,Marcel R M,et al. Beta and gamma oscillations in prefrontal cortex during nmda hypofunction:an in vitro model of schizophrenia features[J]. Neuroscience,2018,43(6):1324-1333.
[4] Pyndt J B,Krych L,Pedersen T B,et al. Investigating the longterm effect of subchronic phencyclidine-treatment on novel object recognition and the association between the gut microbiota and behavior in the animal model of schizophrenia[J]. Physiol Behav,2015,141:32-39.
[5]陈琪,杨德爽,李诗梦,等.柴胡加龙骨牡蛎汤研究进展[J].医学综述,2016,22(17):3441-3443.
[6]李东阳,周秀芳,孙晓,等.柴胡龙牡汤对精神分裂症大鼠模型刻板行为和胶质细胞纤维酸性蛋白(GFAP)表达的影响[J].现代预防医学,2011,38(7):1296-1297.
[7] McGovern E,Kenny D A,McCabe M S,et al. 16S rRNA sequencing reveals relationship between potent cellulolytic genera and feed efficiency in the rumen of bulls[J]. Front Microbiol,2018,doi:10. 3389/fmicb. 2018. 01842.
[8]顾宁宁,张兴德,郁红礼,等.基于16S rRNA基因测序的黄连对2型糖尿病大鼠肠道微生物多样性影响研究[J].中草药,2018,48(19):3998-4004.
[9]徐航宇,王彦礼,王敦方,等.高通量测序技术研究黄芩汤对溃疡性结肠炎大鼠肠道菌群的影响[J].药学学报,2017,52(11):1673-1681.
[10]张春娟.十味温胆汤对MK-801诱导精神分裂症模型大鼠神经递质及免疫功能影响的研究[D].哈尔滨:黑龙江省中医药科学院,2016.
[11]王超,杜泓森,张秀静,等.柴胡龙骨牡蛎汤对心肌梗死大鼠骨髓c-kit+干细胞动员的影响[J].北京中医药大学学报,2018,41(3):203-209.
[12] SHENG Y,ZHENG S,MA T,et al. Mulberry leaf alleviates streptozotocin-induced diabetic rats by attenuating NEFA signaling and modulating intestinal microflora[J]. Sci Rep,2018,21(9):12041-12051.
[13]张宏耕,王杨,范荣,等.精神分裂症的中医证候规范化研究[J].国际精神病学杂志,2018,45(2):271-274.
[14] Dinan T G,Cryan J F. Gut-brain axis in 2016:brain-gutmicrobiota axis-mood,metabolism and behaviour[J]. Nat Rev Gastroenterol Hepatol,2017,14(2):69-70.
[15] Galland L. The gut microbiome and the brain[J]. J Med Food,2014,17(12):1261-1272.
[16] Hsiao E Y,McBride S W,Hsien S,et al. Microbiota modulate behavioral and physiological ahnormalities associated with neurodevelopmental disorders[J]. Cell,2013,155(7):1451-1463.
[17] Fiorentino M,Sapone A,Senger S,et al. Blood-brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders[J]. Mol Autism,2016,doi:10. 1186/s13229-016-0110-z.
[18]梁惠,吕锐,傅泳,等.益生菌对大鼠酒精性肝损伤的保护作用及机制研究[J].中国药理学通报,2016,32(7):991-997.
[19]张宁,李自辉,赵洪伟,等.寒凝血瘀证大鼠的肠道菌群变化与粪便代谢特征分析[J].中国实验方剂学杂志,2018,24(2):79-85.
[20] Severance E G,Gressitt K L,Stallings C R,et al.Probiotic normalizationof Candida albicans in schizophrenia:a randomized, placebo-controlled,longitudinal pilot study[J]. Brain Behav Immun,2017,62:41-45.
[21] Sampson T R,Mazmanian S K. Control of brain development,function,and behavior by the microbiome[J]. Cell Host Microbe,2015,17(5):565-576.
[22] Schwarz E,Maukonen J,Hyytiainen T,et al. Analysis of microbiota in first episode psychosis identifies preliminary associations with symptom severity and treatment response[J]. Schizophr Res,2018,192:398-403.