基于16SrRNA序列分析脾虚证骨质疏松症患者肠道菌群分布特点

Analysis of the Distribution of Intestinal Flora in Osteoporosis Patients with Spleen Deficiency Syndrome Based on 16SrRNA Sequence

【目的】研究脾虚证骨质疏松症患者肠道菌群的分布特点。【方法】根据世界卫生组织(WHO)关于骨质疏松症的诊断标准以及中医脾虚证的辨证分型标准,于2022年1月至2023年9月从广州中医药大学顺德医院就诊人群中,选择26例骨量正常且无脾虚证的健康人为正常骨量组,23例骨量减少且符合脾虚证诊断的患者为脾虚证骨量减少组,69例骨质疏松且符合脾虚证诊断的患者为脾虚证骨质疏松组,共计118例。收集各组受试者的性别、年龄、身高、体质量、体质量指数(BMI)等相关信息,测定其骨密度及血清钙和碱性磷酸酶(ALP)水平,收集其粪便,测定粪便16SrRNA V3~V4区序列,并对测序结果进行物种注释,分析组间群落差异及骨密度与肠道菌群之间的相关性。【结果】(1)正常骨量组的肠道菌群相对丰度与脾虚证骨质疏松组存在显著性差异的有:厚壁门菌(t=2.490,P=0.016)、疣微菌门(t=2.180,P=0.003)、梭杆菌门(t=2.270,P=0.026),酸氨基球菌目(t=3.003,P=0.003)、乳杆菌目(t=3.150,P=0.002)、双歧杆菌目(t=7.248,P=0.001),粪杆菌属(t=2.810,P=0.006)、罗氏菌属(t=2.810,P=0.006)。(2)正常骨量组的肠道菌群相对丰度与脾虚证骨量减少组存在显著性差异的有:乳杆菌目(t=3.841,P=0.001)、双歧杆菌目(t=2.712,P=0.01),粪杆菌属(t=2.466,P=0.017)。(3)脾虚证骨质疏松组的肠道菌群相对丰度与脾虚证骨量减少组存在显著性差异的有:厚壁门菌(t=2.321,P=0.025)、拟杆菌门(t=2.393,P=0.020)、疣微菌门(t=3.109,P=0.031)。(4)Logistic回归分析结果显示:在脾虚证骨质疏松组中,乳杆菌目与腰椎骨密度呈显著负相关(R=0.355,P=0.003),双歧杆菌目与腰椎骨密度呈显著负相关(R=0.366,P=0.002);拟杆菌属与腰椎骨密度呈显著正相关(R=0.245,P=0.042),罗氏菌属与腰椎骨密度呈显著负相关(R=0.330,P=0.006)。【结论】肠道菌群中某些菌与脾虚证骨质疏松症患者骨密度有相关性,正常骨量组与脾虚证骨质疏松组的肠道菌群分布存在显著性差异,这为基于肠道菌群变化进行预防和治疗脾虚证骨质疏松症提供了一定理论依据。

Objective To study the distribution of intestinal flora in osteoporosis patients with spleen deficiency syndrome.Methods According to the diagnostic criteria of osteoporosis of the World Health Organization(WHO)and the syndrome differentiation criteria of spleen deficiency syndrome in traditional Chinese medicine(TCM),26 healthy attenders with normal bone mass while without spleen deficiency were selected from the population visited Shunde Hospital of Guangzhou University of Chinese Medicine from January 2022 to September 2023 as the normal bone mass group,23 patients with bone mass reduction and spleen deficiency syndrome served as decreased bone mass with spleen deficiency group(shorten as DBM-SD group),and 69 patients with osteoporosis and spleen deficiency syndrome diagnosis were osteoporosis with spleen deficiency group(shorten as OS-SD group).A total of 118 attenders were enrolled in the analysis.The gender,age,body height,body weight,and body mass index(BMI)of the subjects were collected.The bone mineral density(BMD)and serum levels of calcium and alkaline phosphatase(ALP)of the subjects were measured.Stools were collected for the detection of the sequence of the 16SrRNA V3-V4 region,and the sequencing results were given species annotation,and then the correlation of community difference between groups and BMD with the intestinal flora was explored.Results(1)There were significant differences in the relative abundance of intestinal flora between the normal bone mass group and the OS-SD group:differences were shown in Firmicutes(t=2.490,P=0.016),Verrucomicrobia(t=2.180,P=0.003)and Fusobacteria(t=2.270,P=0.026),in Acidobacteria(t=3.003,P=0.003),Lactobacillus(t=3.150,P=0.002)and Bifidobacterium(t=7.248,P=0.001),and in Fecalibacterium(t=2.810,P=0.006)and Rothia(t=2.810,P=0.006).(2)There were significant differences in the relative abundance of intestinal flora between the normal bone mass group and the DBM-SD group:differences were shown in Lactobacillus(t=3.841,P=0.001)and Bifidobacterium(t=2.712,P=0.01),and in Faecalibacterium(t=2.466,P=0.017).(3)There were significant differences in the relative abundance of intestinal flora between the OS-SD group and DBM-SD group:differences were shown in Firmicutes(t=2.321,P=0.025),Bacteroidetes(t=0.393,P=0.020)and Verrucomicrobia(t=3.109,P=0.031).(4)The results of logistic regression analysis showed that in the OS-SD group,lumbar BMD was negatively correlated with Lactobacillus(R=0.355,P=0.003)and Bifidobacterium(R=0.366,P=0.002),positively correlated with Bacteroides(R=0.245,P=0.042),and was negatively correlated Rothia(R=0.330,P=0.006).Conclusion Some bacteria in the intestinal flora are related to the BMD of osteoporosis patients with spleen deficiency syndrome,and significant difference exists in the distribution of intestinal flora between the normal bone mass group and the OS-SD group.The results will provide a theoretical basis for the prevention and treatment of osteoporosis patients with spleen deficiency syndrome from the perspective of the changes of intestinal flora.