高通量测序研究补肾化痰方对去势骨质疏松大鼠肠道菌群结构与多样性的影响
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摘要
目的:观察补肾化痰方对去势骨质疏松大鼠肠道菌群结构与多样性的影响。方法:6月龄SD大鼠随机分为假手术组、模型组、补肾化痰组和补肾组,以去卵巢(OVX)建立去势骨质疏松大鼠模型。造模后第5周开始分组灌胃,12周末处死取材。每组选取3只大鼠,取结肠粪便样本并提取总DNA,利用Illumina Miseq平台进行高通量测序。采用micro CT检测大鼠的骨密度及骨微结构,比较分析各组间各个指标的差异。结果:与假手术组相比,模型组大鼠的BMD、Tb.N、Tb.Th均显著降低(P<0.05),Tb.Sp显著升高(P<0.05);与模型组相比,各中药组BMD、Tb.N显著升高(P<0.05)。与假手术组相比,模型组大鼠肠道菌群多样性均降低,厚壁菌门丰度降低,而拟杆菌门丰度增加;与模型组相比,各中药组厚壁菌门丰度均增加,拟杆菌门丰度减少,而补肾化痰组的样品多样性最高。结论:补肾化痰方能够通过改善骨微结构,升高骨密度,其作用机制可能同增加肠道菌群的多样性、调节肠道菌群结构有关。
Objective: To study the effect of Bushen Huatan Fromula on the structure and diversity of intestinal microflora in ovariectomized rats. Methods: Six-month-old SD rats were randomly divided into 4 groups, namely the sham operation group, the model group, the Bushen Huatan group and the Bushen group. Postmenopausal osteoporosis rats were established by ovariectomy(OVX). After fifth weeks of modeling, each group was intragastric separately and were executed at the end of the 12 weeks. Three rats in each group were selected, and fecal samples from the colon were taken to extract the total DNA of fecal samples. The Illumina Miseq platform was used to carry out high-throughput sequencing. The BMD and the microstructure of bone were detected by micro CT. Results: Compared with sham operation group, the level of BMD, Tb.N, Tb.Th were decreased in the model group(P<0.05), while Tb.Sp was significantly increased in the model group(P<0.05). Compared with model group, the level of BMD, Tb.N were significantly increased in Bushen Huatan group and Bushen group(P<0.05).Compared with sham operation group, the intestinal flora diversity of OVX rats decreased, the flora abundance of Firmicutes was decreased, while the Bacteroidetes abundance was increased. Compared with model group, the Firmicutes abundance increased and Bacteroidetes abundance decreased in Bushen Huatan group and Bushen group, while the samples of Bushen Huatan group had the highest diversity. Conclusion: Bushen Huatan Fromula can improve bone mineral density and bone mineral density through improving bone microstructure, and its mechanism may be related to increasing the diversity of intestinal flora and regulating the structure of intestinal flora.
Objective: To study the effect of Bushen Huatan Fromula on the structure and diversity of intestinal microflora in ovariectomized rats. Methods: Six-month-old SD rats were randomly divided into 4 groups, namely the sham operation group, the model group, the Bushen Huatan group and the Bushen group. Postmenopausal osteoporosis rats were established by ovariectomy(OVX). After fifth weeks of modeling, each group was intragastric separately and were executed at the end of the 12 weeks. Three rats in each group were selected, and fecal samples from the colon were taken to extract the total DNA of fecal samples. The Illumina Miseq platform was used to carry out high-throughput sequencing. The BMD and the microstructure of bone were detected by micro CT. Results: Compared with sham operation group, the level of BMD, Tb.N, Tb.Th were decreased in the model group(P<0.05), while Tb.Sp was significantly increased in the model group(P<0.05). Compared with model group, the level of BMD, Tb.N were significantly increased in Bushen Huatan group and Bushen group(P<0.05).Compared with sham operation group, the intestinal flora diversity of OVX rats decreased, the flora abundance of Firmicutes was decreased, while the Bacteroidetes abundance was increased. Compared with model group, the Firmicutes abundance increased and Bacteroidetes abundance decreased in Bushen Huatan group and Bushen group, while the samples of Bushen Huatan group had the highest diversity. Conclusion: Bushen Huatan Fromula can improve bone mineral density and bone mineral density through improving bone microstructure, and its mechanism may be related to increasing the diversity of intestinal flora and regulating the structure of intestinal flora.
引文
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[16]邓昶,周明旺,付志斌.骨质疏松症的中医病因病机及其治疗进展.中国骨质疏松杂志,2017,23(8):1105-1111
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[21]周亚娜,向楠.从“痰“论治OP.湖北中医杂志,2013,35(12):36-38
[22]饶翀,肖新华.肠道菌群和脂代谢异常.中华临床医师杂志,2016,10(8):1053-1055
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[27]Nagano Y,Itoh K,Honda K.The induction of Treg cells by gutindigenous clostridium.Current Opinion in Immunology,2012,24(4):392-397
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[31]丁文鸽.卵巢切除骨质疏松小鼠骨折愈合不同时期骨微结构及力学性能变化.中国组织工程研究与临床康复,2008,12(42):8247-8250
[32]Davison K S,Kendler D L,Ammann P,et al.Assessing fracture risk and effects of osteoporosis drugs:Bone mineral density and beyond.Am J Med,2009,122(11):992-997
[2]贾小玥,郑黎薇,袁泉,等.肠道菌群:绝经后骨质疏松防治新靶点.中国骨质疏松杂志,2017,23(3):392-396
[3]周广文,向楠,周亚娜.补肾化痰方对去势骨质疏松大鼠钙沉积的影响.中国骨质疏松杂志,2018,24(3):366-370
[4]周亚娜,陈辉,向楠.补肾化痰法影响骨髓间充质干细胞成脂分化的实验研究.时珍国医国药,2015,26(10):2325-2327
[5]周亚娜,向楠,陈辉.补肾化痰法影响骨髓间充质干细胞成骨分化的实验研究.中国中医基础医学杂志,2015,21(3):275-277
[6]胡娅,唐曦,向楠.补肾化痰方对去卵巢大鼠骨组织TNF-α和IL-6的影响.中国实验方剂学杂志,2014,20(6):687-689,710
[7]郭文清,何保丽.中医药防治骨质疏松症动物模型研究进展.中华中医药杂志,2017,32(2):666-668
[8]Caporaso J G,Kuczynski J,Bittinger K,et al.QIIME allows analysis of high-throughput community sequencing data.Nature Methods,2010,7(5):335-336
[9]Edgar R C.Search and clustering orders of magnitude faster than BLAST.Bioinformatics,2010,26(19):2460-2461
[10]Hanbo C,Paul C B.Venn Diagram:A package for the generation of highly-customizable Venn and Euler diagrams in R.BMC Bioinformatics,2011,12(1):35
[11]Grice E A,Kong H H,Conlan S,et al.Topographical and temporal diversity of the human skin microbiome.Science,2009,324(5931):1190-1192
[12]Mc Cabe L,Britton R A,Parameswaran N.Prebiotic and probiotic regulation of bone health:Role of the intestine and its microbiome.Current Osteoporosis Reports,2015,13(6):363-371
[13]Espinoza J L,Elbadry M I,Nakao S.An altered gut microbiota Maytrigger autoimmune-mediated acquired bone marrow failure syndromes.Clin Immunol,2016,171:62-64
[14]Villa C R,Ward W E,Comelli E M.Gut microbiota-bone axis.Crit Rev Food Sci Nutr,2017,54(8):1664-1672
[15]刘洲君,胡咏新,刘超.肠道益生菌对骨代谢的影响.国际内分泌代谢杂志,2016,36(1):55-57
[16]邓昶,周明旺,付志斌.骨质疏松症的中医病因病机及其治疗进展.中国骨质疏松杂志,2017,23(8):1105-1111
[17]丁维俊,高峰,杨杰,等.肾阳虚证患者肠道菌群失调状况的临床研究.新中医,2007,39(1):9-10
[18]王洋,孟胜男,张鑫.中药治疗肠道菌群失调研究进展.环球中医药,2015,8(5):620-623
[19]李维娜,冯玲,隋歌川.从痰浊论治高脂血症研究进展.山东中医杂志,2017,36(5):430-433
[20]张冰雨,曾瑞翔,雷涛.脂代谢与骨代谢相关机制的研究进展.中国骨质疏松杂志,2015,21(10):1263-1266
[21]周亚娜,向楠.从“痰“论治OP.湖北中医杂志,2013,35(12):36-38
[22]饶翀,肖新华.肠道菌群和脂代谢异常.中华临床医师杂志,2016,10(8):1053-1055
[23]易园骊,姚萍.脂代谢与肠道菌群关系的研究进展.医学综述,2013,19(6):991-994
[24]Tumbaugh P J,Hamady M,Yatsunenko T,et al.A core gut microbiome in obese and lean twins.Nature,2009,457(7228):480-484
[25]Benson A K,Kelly S A,Legge R,et al.Individuality ingut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors.Proc NatlAcad Sci USA,2010,107(44):18933-18938
[26]Fuhrman B J,Feigelson H S,Flores R,et al.Associations of the fecal microbiome with urinary estrogens and estrogen metabolitesin postmenopausal women.The Journal of Clinical Endocrinology and Metabolism,2014,99(12):4632-4640
[27]Nagano Y,Itoh K,Honda K.The induction of Treg cells by gutindigenous clostridium.Current Opinion in Immunology,2012,24(4):392-397
[28]Atarashi K,Tanoue T,Shima T,et al.Induction of colonic regulatory T cells by indigenous clostridium species.Science,2011,331(6015):337-341
[29]Galdeano C M,Perdigon G.The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity.Clin Vaccine Immunol,2006,13:219-226
[30]曾艳华,陈玛宁,张宁,等.大蒜多糖对人体肠道中拟杆菌生长的影响.现代食品科技,2010,26(5):445-447,489
[31]丁文鸽.卵巢切除骨质疏松小鼠骨折愈合不同时期骨微结构及力学性能变化.中国组织工程研究与临床康复,2008,12(42):8247-8250
[32]Davison K S,Kendler D L,Ammann P,et al.Assessing fracture risk and effects of osteoporosis drugs:Bone mineral density and beyond.Am J Med,2009,122(11):992-997