基于16S rDNA序列分析骨质疏松大鼠肠道菌群结构变化及固本增骨方的调控机制
|
摘要
目的:通过16S rDNA序列分析探讨骨质疏松模型大鼠肠道菌群结构的多样性特征及固本增骨方作用的靶点菌种。方法:将48只Wistar雌性大鼠,随机分为造模组(40只)和空白对照组(8只),造模组采用去卵巢法复制骨质疏松模型,造模成功后随即分为模型对照组、戊酸雌二醇组、固本增骨方(高、中、低剂量)组,每组8只。固本增骨方高、中、低剂量组给予固本增骨方(9.2、4.6、2.3g/kg)灌胃,空白对照组和模型对照组以等剂量0.9%氯化钠溶液灌胃,戊酸雌二醇组按90μg/kg给予戊酸雌二醇灌胃,16周后收集大鼠粪便,提取并检测粪菌总DNA,16sRNA扩增子测序,对标本中的细菌16S rRNA V3-V4进行定性分析、OUT分析、16S功能基因预测分析。结果:与空白对照组比较,模型对照组大鼠肠道菌群的多样性增加;与模型对照组比较,戊酸雌二醇组和固本增骨方高、中、低浓度组可调节厚壁菌门、螺旋菌门及Candiadatus Soleaferrea的相对丰度;固本增骨方高剂量组在翻译、细胞壁的生物合成、细胞运动、复制和修复、能源生产与转化等功能方面显著性增高。结论:卵巢切除大鼠诱导骨质疏松模型中,肠道微生态失衡可能为绝经后骨质疏松症的发病机制之一;固本增骨方对肠道菌群具有调节作用,通过"健脾补肾"之效应机制发挥对PMOP的防治作用。
Objective: To analyze the diversity of intestinal flora structure in osteoporosis model rats and the bacterial species of target of Guben Zenggu Formula with the sequence of 16 S rDNA. Methods: Forty-eight female Wistar rats were randomly divided into model group(40 rats) and blank control group(8 rats), the model group was used to replicate the osteoporosis model by ovariectomy, and then divided into model control group, estradiol valerate group, and Guben Zenggu Formula(high, medium and low) group, with 8 in each group. The high, medium and low dose groups of Guben Zenggu Formula were given corresponding concentration by intragastric administration, while the blank control group and the model control group were given isodose normal saline by intragastric administration, the estradiol valerate group was given estradiol valerate with90μg/kg by intragastric administration. After 16 weeks, the feces of rats were collected, and extracted and detected total DNA of the fecal bacteria. The 16 S rRNA amplicon was sequenced, the 16 S rRNA V3-V4 of bacteria in specimens were qualitatively analysis, OUT analysis and 16 S functional genes prediction analysis. Results: Compared with the blank control group, the diversity of rats' intestinal flora increased in the model control group; Compared with the model control group, the estradiol valerate group and the high, medium and low dose groups of the Guben-Zenggu Formula could adjust the relative abundance of phylum firmicutes, spiral phylum and Candiadatus Soleaferrea; In the high dose group of the Guben Zenggu Formula, the function of translation, cell wall biosynthesis, cell movement, replication and repair, energy production and transformation were significantly increased. Conclusion: In the model of osteoporosis induced by ovariectomized rats, the imbalance of intestinal microecology may be one of the pathogenesis of postmenopausal osteoporosis. The Guben Zenggu Formula has the effect of regulating intestinal flora and exerting its preventive and therapeutic effect on PMOP through the effective mechanism of strengthening the spleen and tonifying the kidney.
Objective: To analyze the diversity of intestinal flora structure in osteoporosis model rats and the bacterial species of target of Guben Zenggu Formula with the sequence of 16 S rDNA. Methods: Forty-eight female Wistar rats were randomly divided into model group(40 rats) and blank control group(8 rats), the model group was used to replicate the osteoporosis model by ovariectomy, and then divided into model control group, estradiol valerate group, and Guben Zenggu Formula(high, medium and low) group, with 8 in each group. The high, medium and low dose groups of Guben Zenggu Formula were given corresponding concentration by intragastric administration, while the blank control group and the model control group were given isodose normal saline by intragastric administration, the estradiol valerate group was given estradiol valerate with90μg/kg by intragastric administration. After 16 weeks, the feces of rats were collected, and extracted and detected total DNA of the fecal bacteria. The 16 S rRNA amplicon was sequenced, the 16 S rRNA V3-V4 of bacteria in specimens were qualitatively analysis, OUT analysis and 16 S functional genes prediction analysis. Results: Compared with the blank control group, the diversity of rats' intestinal flora increased in the model control group; Compared with the model control group, the estradiol valerate group and the high, medium and low dose groups of the Guben-Zenggu Formula could adjust the relative abundance of phylum firmicutes, spiral phylum and Candiadatus Soleaferrea; In the high dose group of the Guben Zenggu Formula, the function of translation, cell wall biosynthesis, cell movement, replication and repair, energy production and transformation were significantly increased. Conclusion: In the model of osteoporosis induced by ovariectomized rats, the imbalance of intestinal microecology may be one of the pathogenesis of postmenopausal osteoporosis. The Guben Zenggu Formula has the effect of regulating intestinal flora and exerting its preventive and therapeutic effect on PMOP through the effective mechanism of strengthening the spleen and tonifying the kidney.
引文
[1]申浩,魏戌,谢雁鸣,等.绝经后骨质疏松症骨折危险因素及中医症状相关性研究.中国中西医结合杂志,2017,37(1):50-56
[2]Steves C J,Bird S,Williams F M,et al.Themicrobiomeand musculoskeletal conditions of aging:A review of evidence for impact and potential therapeutics.J Bone Miner Res,2016,31(2):261-269
[3]Ibá?ez L,Rouleau M,Wakkach A,et al.Gutmicrobiomeand bone.Joint Bone Spine,2018,pii:S1297-319X(18)30061-7.doi:10.1016
[4]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):392-401
[5]Lambert M N T,Thybo C B,Lykkeboe S,et al.Combined bioavailable isoflavones and probiotics improve bone status and estrogen metabolism in postmenopausal osteopenic women:A randomized controlled trial.Am J Clin Nutr,2017,106(3):909-920
[6]董万涛,周灵通,宋敏,等.固本增骨方对去卵巢大鼠血清骨钙素和NEI网络组织中游离[Ca~(2+)]i的影响.中药药理与临床,2018,34(1):121-127
[7]Fang S,Xiong X,Su Y,et al.16S rRNA gene-based association study identified microbial taxa associated with porkintramuscular fat content in feces and cecum lumen.BMC Microbiol,2017,17(1):162-171
[8]Tabatabaei-Malazy O,Norani M,Heshmat R,et al.Efficacy and safety of the biosimilar recombinant human parathyroid hormone cinnopar?in postmenopausal osteoporotic women:A randomized double-blind clinical trial.Iran J Public Health,2018,47(9):1336-1344
[9]Liu C,Gao X,Liu Y,et al.Icariin combined with snailase shows improved intestinal hydrolysis and absorption in osteoporosis rats.Biomed Pharmacother,2017,94(9):1048-1056
[10]Macari S,Madeira M F M,Lima ILA,et al.ST2 regulates bone loss in a site-dependent andestrogen-dependent manner.J Cell Biochem,2018,119(10):8511-8521
[11]Zhang J,Lu Y,Wang Y,et al.The impact of the intestinal microbiome on bone health.Intractable Rare Dis Res,2018,7(3):148-155
[12]Srivastava R K,Dar H Y,Mishra P K.Immunoporosis:Immunology ofosteoporosis-role of T cells.Front Immunol,2018,9:657
[13]Zhao H,Zhao N,Zheng P,et al.Prevention and treatment of osteoporosis using chinese medicinal plants:Special emphasis on mechanisms of immune modulation.J Immunol Res,2018,2018:6345857
[14]Collins F L,Rios-Arce N D,Schepper J D,et al.The potential of probiotics as a therapy for osteoporosis.Microbiol Spectr,2017,5(4):doi:10.1128
[15]Sj?gren K,Engdahl C,Henning P,et al.The gut microbiota regulates bone mass in mice.Journal of Bone&Mineral Research,2012,50(6):S91-S92
[16]Yan J,Charles J F.Gut microbiome and bone:To build,destroy,or both?.Curr Osteoporos Rep,2017,15(4):376-384
[17]彭颖,李晓波.脾虚证与肠道微生态.世界华人消化杂志,2012,20(34):3287-3291
[18]Collins F L,Irwin R,Bierhalter H,et al.Lactobacillus reuteri6475 increases bone density in intact females only under an inflammatory setting.PLoS One,2016,11(4):e0153180
[19]韩基红.黄芪多糖的研究进展.世界最新医学信息文摘,2017,17(20):93-94
[20]旭云,贺姣姣,郑宁宁,等.黄芪多糖对肥胖小鼠的减肥作用与调节肠道菌群的关系研究.世界中医药,2016,11(11):2379-2384
[21]Zhang P,Hu L,Bai R,et al.Structural characterization of a pectic polysaccharide from Codonopsis pilosula and its immunomodulatory activities in vivo and in vitro.Int J Biol Macromol,2017,doi:10.1016
[22]王广.党参多糖对肠道菌群失调小鼠的调整作用机制的初步研究.佳木斯:佳木斯大学,2010:36-39
[23]刘伟伟,史丽强,万强,等.基于NLRP3炎性体探究黄芪桂枝五物汤对IgA肾病小鼠肾脏保护机制.中华中医药杂志,2018,33(5):1746-1751
[2]Steves C J,Bird S,Williams F M,et al.Themicrobiomeand musculoskeletal conditions of aging:A review of evidence for impact and potential therapeutics.J Bone Miner Res,2016,31(2):261-269
[3]Ibá?ez L,Rouleau M,Wakkach A,et al.Gutmicrobiomeand bone.Joint Bone Spine,2018,pii:S1297-319X(18)30061-7.doi:10.1016
[4]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):392-401
[5]Lambert M N T,Thybo C B,Lykkeboe S,et al.Combined bioavailable isoflavones and probiotics improve bone status and estrogen metabolism in postmenopausal osteopenic women:A randomized controlled trial.Am J Clin Nutr,2017,106(3):909-920
[6]董万涛,周灵通,宋敏,等.固本增骨方对去卵巢大鼠血清骨钙素和NEI网络组织中游离[Ca~(2+)]i的影响.中药药理与临床,2018,34(1):121-127
[7]Fang S,Xiong X,Su Y,et al.16S rRNA gene-based association study identified microbial taxa associated with porkintramuscular fat content in feces and cecum lumen.BMC Microbiol,2017,17(1):162-171
[8]Tabatabaei-Malazy O,Norani M,Heshmat R,et al.Efficacy and safety of the biosimilar recombinant human parathyroid hormone cinnopar?in postmenopausal osteoporotic women:A randomized double-blind clinical trial.Iran J Public Health,2018,47(9):1336-1344
[9]Liu C,Gao X,Liu Y,et al.Icariin combined with snailase shows improved intestinal hydrolysis and absorption in osteoporosis rats.Biomed Pharmacother,2017,94(9):1048-1056
[10]Macari S,Madeira M F M,Lima ILA,et al.ST2 regulates bone loss in a site-dependent andestrogen-dependent manner.J Cell Biochem,2018,119(10):8511-8521
[11]Zhang J,Lu Y,Wang Y,et al.The impact of the intestinal microbiome on bone health.Intractable Rare Dis Res,2018,7(3):148-155
[12]Srivastava R K,Dar H Y,Mishra P K.Immunoporosis:Immunology ofosteoporosis-role of T cells.Front Immunol,2018,9:657
[13]Zhao H,Zhao N,Zheng P,et al.Prevention and treatment of osteoporosis using chinese medicinal plants:Special emphasis on mechanisms of immune modulation.J Immunol Res,2018,2018:6345857
[14]Collins F L,Rios-Arce N D,Schepper J D,et al.The potential of probiotics as a therapy for osteoporosis.Microbiol Spectr,2017,5(4):doi:10.1128
[15]Sj?gren K,Engdahl C,Henning P,et al.The gut microbiota regulates bone mass in mice.Journal of Bone&Mineral Research,2012,50(6):S91-S92
[16]Yan J,Charles J F.Gut microbiome and bone:To build,destroy,or both?.Curr Osteoporos Rep,2017,15(4):376-384
[17]彭颖,李晓波.脾虚证与肠道微生态.世界华人消化杂志,2012,20(34):3287-3291
[18]Collins F L,Irwin R,Bierhalter H,et al.Lactobacillus reuteri6475 increases bone density in intact females only under an inflammatory setting.PLoS One,2016,11(4):e0153180
[19]韩基红.黄芪多糖的研究进展.世界最新医学信息文摘,2017,17(20):93-94
[20]旭云,贺姣姣,郑宁宁,等.黄芪多糖对肥胖小鼠的减肥作用与调节肠道菌群的关系研究.世界中医药,2016,11(11):2379-2384
[21]Zhang P,Hu L,Bai R,et al.Structural characterization of a pectic polysaccharide from Codonopsis pilosula and its immunomodulatory activities in vivo and in vitro.Int J Biol Macromol,2017,doi:10.1016
[22]王广.党参多糖对肠道菌群失调小鼠的调整作用机制的初步研究.佳木斯:佳木斯大学,2010:36-39
[23]刘伟伟,史丽强,万强,等.基于NLRP3炎性体探究黄芪桂枝五物汤对IgA肾病小鼠肾脏保护机制.中华中医药杂志,2018,33(5):1746-1751