糖尿病前期痰证患者肠道菌群及血清代谢组学分析

Analysis of Intestinal Flora and Serum Metabolomics in Patients withPre-diabetic Sputum Syndrome

目的:探讨糖尿病前期痰证患者的肠道菌群及血清代谢产物的差异性表达与潜在生物标志物。方法:共纳入34例糖尿病前期痰证患者为痰证组,并选取同期体检的37例健康人为正常组,留取2组研究对象的血清和粪便标本,采用液相色谱-质谱法(LC-MS)非靶向代谢组学及16S rRNA高通量测序技术,来检测两者的血清代谢物和差异肠道菌群,探讨糖尿病前期痰证与血清代谢物、肠道菌群之间的关联性。结果:痰证组证素分布情况中,病性证素按照频数和占比前5位兼杂证素为湿(73.53%)、气滞(58.82%)、阴虚(50.00%)、血瘀(41.18%)、热(35.29%);病位证素按照频数和占比前3位主要病位证素为脾(100.00%)、肝(41.18%)、肾(23.53%)。16S rRNA高通量测序结果显示,两组有44种差异肠道菌群,在目属上,双歧杆菌、韦荣氏球菌、罗氏菌两组间差异具有统计学意义(P<0.05)。痰证组在肠道菌群多样性、丰富度和均匀度较正常组减少,但差异无统计学意义,群落结构上两组亦差异无统计学意义。血清代谢组学结果显示,两组共同拥有13种差异代谢物,主要富集在氨基酸代谢、胆汁分泌和胆汁酸生物合成、脂质代谢等通路(P<0.05)。将差异代谢物、肠道菌群和证素进行相关性分析,(1)赖氨酸和脾、阴虚、血瘀呈正相关;牛磺胆酸与肝、肾、血瘀、湿呈正相关,与阴虚、热呈正相关;牛磺鹅去氧胆酸与肝、湿呈正相关;花生四烯酸与湿呈负相关;葡萄糖与脾、血瘀呈正相关。(2)梭杆菌属和脾、肾、阴虚、气滞呈正相关;罗氏菌属与脾呈负相关;萨特氏菌属与湿呈负相关;拟杆菌属与脾、肾呈负相关;双歧杆菌与湿、脾呈负相关。(3)双歧杆菌与甘氨酸、苏氨酸、赖氨酸、鹅去氧胆酸呈正相关,与胆酸呈负相关,与牛磺鹅去氧胆酸、丙酮酸呈正相关;梭杆菌属与甘氨酸呈正相关,与苏氨酸、赖氨酸呈正相关;毛螺菌属与甘氨酸、苏氨酸、丙酮酸呈负相关;赖氨酸还与粪杆菌属、凸腹真杆菌呈负相关,与巨单胞菌属呈正相关;牛磺胆酸、甘氨胆汁酸与弯曲杆菌属,牛磺鹅去氧胆酸与韦荣氏球菌属,葡糖糖与罗氏菌属、凸腹真杆菌呈正相关;丙酮酸和埃希氏-志贺氏菌属,牛磺鹅去氧胆酸与普雷沃氏菌属呈负相关。结论:糖尿病前期痰证患者与健康者之间存在肠道菌群和血清代谢物差异,肠道菌群及代谢产物在糖尿病前期这一疾病阶段已然发生紊乱,双歧杆菌、梭杆菌属、毛螺菌属、甘氨酸、苏氨酸、赖氨酸可能是探索糖尿病前期痰证发展的突破口。

Objective to investigate the differential expression of intestinal flora and serum metabolites and potential biomarkers in patients with pre-diabetic sputum syndrome.Method A total of 34 patients with pre-diabetic sputum syndrome were included as the phlegm syndrome group,and 37 healthy people were selected as the normal group.Serum and fecal samples of the two groups were collected,and liquid chromatography-mass spectrometry(LC-MS)non-targeted metabolomics and 16S rRNA high-throughput sequencing technology were used to detect serum metabolites and different intestinal flora of the two groups and explore the relationship among pre-diabetic sputum syndrome,serum metabolites,and intestinal flora.Result In the distribution of disease syndrome elements in the phlegm syndrome group,the first five disease syndrome elements in terms of frequency and proportion were dampness(73.53%),Qi stagnation(58.82%),Yin deficiency(50.00%),blood stasis(41.18%),and heat(35.29%).According to the frequency and proportion of disease location syndrome elements,the first three main disease location syndrome elements were spleen(100.00%),liver(41.18%),and kidney(23.53%).The results of 16S rRNA high-throughput sequencing showed that there were 44 different intestinal flora between the two groups.In order genus,there were significant differences in Bifidobacterium,Veillonococcus,and Roseococcus between the two groups(P<0.05).The diversity,abundance,and evenness of intestinal flora in the phlegm syndrome group were lower than those in the normal group,with the difference not statistically significant.There was no significant difference in the community structure between two groups.The results of serum metabolomics showed that there were 13 differential metabolites in the two groups,which were mainly concentrated in amino acid metabolism,bile secretion,bile acid biosynthesis,and lipid metabolism(P<0.05).The correlation among differential metabolites,intestinal flora,and syndrome elements was analyzed,and the results showed that①lysine was positively correlated with spleen,Yin deficiency,and blood stasis,while taurocholic acid was positively correlated with liver,kidney,blood stasis,and dampness,and there was a positive correlation between taurocholic acid and yin deficiency and heat.The taurochenodeoxycholic acid was positively correlated with liver and dampness,and there was a negative correlation between arachidonic acid and dampness,as well as a positive correlation between glucose and spleen and blood stasis.②Clostridium was positively correlated with spleen,kidney,Yin deficiency,and Qi stagnation.Rosepiella was negatively correlated with spleen,and Sutterella was negatively correlated with dampness.Bacteroides was negatively correlated with the spleen and kidney,and Bifidobacterium was negatively correlated with the spleen and dampness.③Bifidobacterium was positively correlated with glycine,threonine,lysine,and deoxycholic acid significantly,negatively correlated with cholic acid significantly,and positively correlated with taurochenodeoxycholic acid and pyruvic acid.Clostridium was positively correlated with glycine significantly and positively correlated with threonine and lysine.Lachnospira was negatively correlated with glycine,threonine,and pyruvic acid.Lysine was also negatively correlated with Faecalibacterium and Eubacterium ventriosum and positively correlated with Megamonas.There was a positive correlation between taurocholic acid and glycine bile acid and Campylobacter,between taurochenodeoxycholic acid and Veillonococcus,and between glucose and Rosepiella and Eubacterium ventriosum.There was a negative correlation between pyruvic acid and Escheria-Shigella and between taurochenodeoxycholic acid and Prevotella.Conclusion there are differences in intestinal flora and serum metabolites between patients with pre-diabetic sputum syndrome and healthy people.The intestinal flora and metabolites have been disturbed in the stage of pre-diabetes,Bifidobacterium,Clostridium,Lachnospira,glycine,threonine,and lysine may be the breakthrough to explore the development of pre-diabetic sputum syndrome.