[1]陈斯亮,石瑛,汤辰明,等.基于16S核糖体RNA测序研究健脾方对绝经后骨质疏松症小鼠肠道菌群的影响[J].中医正骨,2023,35(06):11-17,23.
　CHEN Siliang,SHI Ying,TANG Chenming,et al.A study of the effects of Jianpi Fang(健脾方)on intestinal flora of mice with postmenopausal osteoporosis based on 16S ribosomal RNA gene sequencing[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(06):11-17,23.
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基于16S核糖体RNA测序研究健脾方对绝经后骨质疏松症小鼠肠道菌群的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年06期
页码:
11-17,23
栏目:
基础研究
出版日期:
2023-06-20

文章信息/Info

Title:
A study of the effects of Jianpi Fang(健脾方)on intestinal flora of mice with postmenopausal osteoporosis based on 16S ribosomal RNA gene sequencing
作者:
陈斯亮; 石瑛; 汤辰明; 马威; 詹红生
(上海中医药大学附属曙光医院,上海 201203)
Author(s):
CHEN Siliang; SHI Ying; TANG Chenming; MA Wei; ZHAN Hongsheng
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China
关键词:
骨质疏松; 绝经后;  胃肠道微生物群;  RNA; 核糖体; 16S;  健脾方
Keywords:
osteoporosis; postmenopausal;  gastrointestinal microbiome;  RNA; Ribosomal; 16S;  Jianpi fang
摘要:
目的:探讨健脾方对绝经后骨质疏松症(osteoporosis,OP)小鼠肠道菌群的影响。方法:将30只雌性C57BL/6小鼠随机分为假手术组、模型组和健脾方组,每组10只。模型组和健脾方组小鼠通过双侧卵巢摘除术进行OP造模,假手术组小鼠打开腹腔后切除卵巢附近的一团脂肪组织,保留卵巢。造模后第7天,假手术组和模型组小鼠以生理盐水灌胃,健脾方组以健脾方药液灌胃(中药配方颗粒用量为54 mg·kg-1),3组小鼠均每天灌胃1次,每次0.2 mL,连续灌胃28 d。药物干预结束后,测定小鼠体质量,获取各组小鼠粪便样本、左侧股骨,切取子宫并称重,进行小鼠肠道菌群16S核糖体RNA测序分析及小鼠股骨病理学观察。结果:①一般情况。实验期间各组均无小鼠死亡。药物干预前,3组小鼠体质量的差异无统计学意义[(20.88±0.83)g,(20.65±0.74)g,(20.59±0.47)g,F=0.484,P=0.622]。药物干预结束后,模型组小鼠的体质量高于假手术组和健脾方组[(26.96±1.63)g,(23.42±0.84)g,P=0.000;(26.96±1.63)g,(24.62±1.55)g,P=0.001],假手术组和健脾方组小鼠体质量的差异无统计学意义(P=0.063)。假手术组小鼠的子宫质量高于模型组和健脾方组[(0.66±0.05)g,(0.24±0.05)g,P=0.000;(0.66±0.05)g,(0.28±0.06)g,P=0.000],模型组和健脾方组小鼠子宫质量的差异无统计学意义(P=0.053)。②小鼠肠道菌群丰度分析结果。假手术组与模型组含有499个共同操作分类单元(operational taxonomic units,OTUs)数据; 除共同OTUs数据外,假手术组有87个特异性OTUs数据,模型组有74个特异性OTUs数据。模型组与健脾方组含有489个共同OTUs数据; 除共同OTUs数据外,模型组有84个特异性OTUs数据,健脾方组有54个特异性OTUs数据。偏最小二乘法判别分析结果显示,3组小鼠肠道菌群丰度差异明显。③小鼠肠道菌群结构分析结果。物种累计曲线随样本量增加逐渐趋于平缓,说明样品中的物种已经基本被测序覆盖,测序数据量充足。3组小鼠肠道菌群相对丰度在门水平处于前4位的依次为拟杆菌门、厚壁菌门、放线菌门、脱硫菌门,相对丰度在属水平处于前5位的依次为Muri菌属、乳杆菌属、拟普雷沃菌属、梭菌属、粪杆菌属。3组小鼠厚壁菌门、脱硫菌门相对丰度比较,总体差异均无统计学意义。假手术组和健脾方组拟杆菌门相对丰度均高于模型组(23.19±2.09,17.12±3.87,P=0.016; 23.58±2.46,17.12±3.87,P=0.012)、放线菌门相对丰度低于模型组(0.32±0.29,0.79±0.31,P=0.027; 0.26±0.07,0.79±0.31,P=0.014); 其余各组间两两比较,组间差异均无统计学意义。3组小鼠Muri菌属、乳杆菌属、粪杆菌属相对丰度比较,总体差异均无统计学意义。假手术组拟普雷沃菌属相对丰度高于模型组(4.96±1.77,1.33±0.37,P=0.009)、梭菌属相对丰度低于模型组(0.45±0.17,1.41±0.18,P=0.000),健脾方组梭菌属相对丰度低于模型组(0.62±0.30,1.41±0.18,P=0.001); 其余各组间两两比较,组间差异均无统计学意义。④健脾方组与模型组差异菌群KEGG分析结果。健脾方组与模型组有明显差异的代谢途径有维生素B6代谢、硫化物代谢、硒化物代谢、硫氨基酸代谢、硫胺素代谢、钙离子通道。⑤小鼠股骨病理学观察结果。模型组小鼠的骨小梁厚度、骨小梁数量均低于假手术组[(50.13±7.72)mm,(71.87±6.20)mm,P=0.000;(1.87±0.92)个·mm-1,(4.40±1.24)个·mm-1,P=0.000],骨小梁分离度高于假手术组[(344.60±41.32)mm,(205.80±27.21)mm,P=0.000]。健脾方组小鼠的骨小梁厚度、骨小梁数量均低于假手术组[(60.53±6.38)mm,(71.87±6.20)mm,P=0.000;(3.13±0.92)个·mm-1,(4.40±1.24)个·mm-1,P=0.002],健脾方组与假手术组小鼠骨小梁分离度的差异无统计学意义。健脾方组小鼠的骨小梁厚度、骨小梁数量均高于模型组[(60.53±6.38)mm,(50.13±7.72)mm,P=0.000;(3.13±0.92)个·mm-1,(1.87±0.92)个·mm-1,P=0.002],骨小梁分离度低于模型组[(221.40±33.16)mm,(344.60±41.32)mm,P=0.000]。结论:健脾方能够调节绝经后OP小鼠肠道菌群结构,改变代谢途径,这可能是其治疗OP的作用机制之一。
Abstract:
Objective:To explore the effects of Jianpi Fang(健脾方,JPF)on intestinal flora of postmenopausal osteoporosis(PMOP)mice.Methods:Thirty female C57BL/6 mice were randomly divided into sham-operated group,model group and JPF group,10 mice in each group.The mice in model group and JPF group were subjected to bilateral ovariectomy for inducing PMOP; while the ones in sham-operated group underwent removal of peri-ovarian adipose tissues,with ovaries retained.On the 7th day after the modeling operation,the mice in sham-operated group and model group were intragastric administrated with normal saline,while the ones in JPF group with JPF decoction(the dosage of Chinese medicinal granules was 54 mg/kg).All mice in the 3 groups were intragastric administrated once a day,0.2 mL at a time for consecutive 28 days.After the end of drug intervention,the body mass of the mice was measured and their stool samples and left femurs were harvested,moreover their uteri were dissected and weighed,and analysis on 16S ribosomal RNA(rRNA)gene sequencing of intestinal flora and pathological observation on femurs of the mice were conducted.Results:①No dead mice were found during the experiment period.There was no statistical difference in body mass among the 3 groups before the drug intervention(20.88±0.83,20.65±0.74,20.59±0.47 g,F=0.484,P=0.622).After the end of drug intervention,the body mass was higher in model group compared with that of sham-operated group and JPF group(26.96±1.63 vs 23.42±0.84 g,P=0.000; 26.96±1.63 vs 24.62±1.55 g,P=0.001),while the difference in body mass between sham-operated group and JPF group was not statistically significant(P=0.063).The quality of uteri was higher in sham-operated group compared with that of model group and JPF group(0.66±0.05 vs 0.24±0.05 g,P=0.000; 0.66±0.05 vs 0.28±0.06 g,P=0.000),while there was no significant difference between model group and JPF group(P=0.053).②The sham-operated group and model group shared 499 common operational taxonomic units(OTUs).In addition to the common OTUs,87 specific OTUs were found in sham-operated group,and 74 ones were observed in model group.The model group and the JPF group shared 489 common OTUs.In addition to the common OTUs,84 specific OTUs were found in model group,and 54 ones were observed in JPF group.The results of partial least squares discriminant analysis(PLS-DA)showed that there were significant differences in the abundance of intestinal flora among the 3 groups.③The species accumulation curve gradually flattened out with the sample size increased,indicating that the species in the samples had been adequately covered by sequencing and the sequencing data were sufficient.At the phylum level,the top 4 abundant phyla in the mouse intestinal flora of the 3 groups were Bacteroidetes,Firmicutes,Actinobacteria and Desulfobacterota in turn; while,at the genus level,the top 5 abundant genera were Muri,Lactobacillus,Alloprevotella,Clostridium and Faecalibacterium in turn.There was no significant differences in the relative abundance of Firmicutes and Desulfobacterota among the 3 groups.The relative abundance of Bacteroidetes was higher,while the relative abundance of Actinobacteria was lower in sham-operated group and JPF group compared to model group(23.19±2.09 vs 17.12±3.87,P=0.016; 23.58±2.46 vs 17.12±3.87,P=0.012; 0.32±0.29 vs 0.79±0.31,P=0.027; 0.26±0.07 vs 0.79±0.31,P=0.014).Further pairwise comparison showed that there was no statistical difference in the relative abundance of Bacteroidetes and Actinobacteria between sham-operated group and JPF group.There was no significant differences in the relative abundance of Muri,Lactobacillus and Faecalibacterium among the 3 groups.The relative abundance of Alloprevotella was higher in sham-operated group compared to model group(4.96±1.77 vs 1.33±0.37,P=0.009),and the relative abundance of Clostridium was lower in sham-operated group and JPF group compared to model group(0.45±0.17 vs 1.41±0.18,P=0.000; 0.62±0.30 vs 1.41±0.18,P=0.001).Further pairwise comparison showed that there was no statistical difference in the relative abundance of Alloprevotella between sham-operated group and JPF group as well as between model group and JPF group,and no statistical difference in the relative abundance of Clostridium between sham-operated group and JPF group.④The significant differences in metabolic pathways,including vitamin B6 metabolism,sulfide metabolism,selenocompound metabolism,sulfur amino acid metabolism,thiamine metabolism and calcium ion channels,were found between JPF group and model group.⑤The trabecular thickness(Tb.Th)and trabecular number(Tb.N)in femurs were less,while the trabecular separation(Tb.Sp)was higher in model group compared to sham-operated group(50.13±7.72 vs 71.87±6.20 mm,P=0.000; 1.87±0.92 vs 4.40±1.24 trabeculae/mm,P=0.000; 344.60±41.32 vs 205.80±27.21 mm,P=0.000).The Tb.Th and Tb.N in femurs were less in JPF group compared to sham-operated group(60.53±6.38 vs 71.87±6.20 mm,P=0.000; 3.13±0.92 vs 4.40±1.24 trabeculae/mm,P=0.002),while there was no significant differences in Tb.Sp between JPF group and sham-operated group.The Tb.Th was greater,the Tb.N was more,while the Tb.Sp was lower in JPF group compared to model group(60.53±6.38 vs 50.13±7.72 mm,P=0.000; 3.13±0.92 vs 1.87±0.92 trabeculae/mm,P=0.002; 221.40±33.16 vs 344.60±41.32 mm,P=0.000).Conclusion:The JPF can modulate the intestinal flora structure and alter the metabolic pathways in PMOP mice,which may be one of its mechanisms of action in treatment of OP.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(81674003); “华东片区及市级中医专科筋骨病损专科联盟建设”项目 通讯作者:石瑛 E-mail:shiying1974@126.com
更新日期/Last Update: 1900-01-01