类风湿关节炎与功能便秘患者肠道菌群差异及血清5-羟色胺分析

Difference of Intestinal Flora and Analysis of Serum 5-Hydroxytryptamine in Patients with Rheumatoid Arthritis and Functional Constipation

目的:分析类风湿关节炎(RA)患者、功能便秘(FC)患者、健康人群(HC)肠道菌群结构及血清5-羟色胺(5-HT)水平差异,初步探讨肠道菌群在RA合并FC发病中的作用。方法:选取90例研究对象(其中类风湿关节炎患者组——RA组30例,功能性便秘患者组——FC组30例、健康对照组——HC组30例),并收集三组新鲜粪便标本,然后利用Illumina MiSeq平台对三组样本16SrRNA基因序列V3~V4高变区进行PCR扩增及测序,进行Alpha多样性分析、Beta多样性分析及门属水平上的菌群差异性分析,用LEfSe聚类分析法得到各组代表性差异肠道菌群。应用ELISA法检测各组血清5-HT水平,对连续变量资料采用方差分析。结果:属水平,FC与RA组Prevotella_9较HC增多,拟杆菌属(Bacteroides)、(Agethobacter)均减少。PCoA分析提示RA组菌群差异明显大于FC及HC组。LEfSe分析寻找组间代表性菌属,RA组g_Prevotella_9、s_uncultureda-bacterium_g_Prevotella_9、f_Prevotellaceae、o-Enterobacteriales、f-Enterobacteriaceae;FC组为p_Firmicutes、s_Megasphaera_micronuciformis,差异有统计学意义(LDA值均大于4)。PICRUSt软件进行功能基因预测分析得到,RA组在淀粉和蔗糖代谢、二级胆汁酸生物合成、RNA转运等方面均低于HC组。FC与HC组无明显比较无明显差异。ELISA法检测分析,FC与RA组5-HT水平较HC组明显降低,差异有统计学意义(P 【0.0001)。结论:RA可通过肠道菌群调节肠道神经递质水平从而影响胃肠道功能,可能与5-HT依赖途径作用于c-kit及其配体干细胞因子途径(SCF/C-kit通路)、肠道菌群代谢产物作用等方面相关。

Objective: To analyze the structural differences of intestinal flora and 5-hydroxytryptamine (5-HT) level in patients with rheumatoid arthritis (RA), patients with functional constipation (FC), and healthy people. And to explore the role of intestinal flora in the pathogenesis of RA combined with FC. Method: Collecting 90 subjects (rheumatoid arthritis patients group—RA group 30 cases, functional constipation patients group—FC group 30 cases, healthy control group—HC group 30 cases), and their fecal samples. And then use Illumina MiSeq platform to amplify and sequence the V3~V4 hypervariable region of 16SrRNA gene sequence using PCR, and analyze Alpha diversity, Beta diversity and the difference of intestinal flora at phylum and genus level. LEfSe clustering analysis was used to find the representative intestinal flora of each group. ELISA was used to detect the levels of serum 5-HT in each group, and analysis of variance was used for constinuous variable data. Results: at the level of genus, Prevotella_9 increased, Bacteroides and Agethobacter decreased in RA and FC groups compared with HC group. PCoA analysis showed the difference of intestinal flora in RA group was significantly greater than that in FC and HC groups. LEfSe analysis was performed to find the representative bacteria in different groups: g_Prevotella_9, s_uncultureda-bacterium_g_Pr- evotella_9, f_Prevotellaceae, o-Enterobacteriales, f-Enterobacteriaceae in RA group. p_Firmicutes, s_Megasphaera_micronuciformis in FC group. The difference was statistically significant (LDA values all greater than 4). PICRUSt software was used for functional gene prediction analysis, and it was found that RA group was lower than HC group in starch and sucrose metabolism, secondary bile acid biosynthesis, RNA transport and other aspects. There was no difference between FC and HC groups. ELISA method detected that the level of 5-HT in RA and HC groups was significantly lower than that in HC group (P <0.0001). Conclusion: RA can regulate the level of intestinal neurotransmitters and affect gastrointestinal function through intestinal microflora, which may be related to the action on c-kit and its ligand stem cell factor pathway (SCF/C-kit pathway) and intestinal microflora metabolites and so on, through 5-HT dependent pathway.