DataSheet4_Rhubarb Enema Decreases Circulating Trimethylamine N-Oxide Level and Improves Renal Fibrosis Accompanied With Gut Microbiota Change in Chronic Kidney Disease Rats.docx

Objectives: Trimethylamine N-oxide (TMAO), a metabolic product of gut flora, is increased in chronic kidney disease (CKD) subjects and is recognized as one type of uremic toxins which is associated with poor cardiovascular outcomes and kidney function loss. Previous studies have suggested that rhubarb enema could reduce circulating uremic toxins such as urea, creatinine, and indoxyl sulfate and also regulate the intestinal microbiota. However, whether rhubarb enema retards kidney dysfunction by reducing circulating TMAO and its underlying mechanism, are still unclear. The present study aims to investigate the impact of rhubarb enema on TMAO and its precursors, as well as on the intestinal microbiota in 5/6 nephrectomized (5/6Nx) CKD rats.
Design: Rats in the treatment groups were given rhubarb enema after modeling. At the end of the study, blood, feces, and kidney tissues were collected and processed for biochemical analyses, histological and western blot analyses, 16S rRNA sequence and untargeted metabolomic analyses.
Results: Rhubarb enema reduced serum TMAO and trimethylamine (TMA) levels, inhibited the expression of inflammatory markers (interleukin-6, tumor necrosis factor α and Interferon-γ) and alleviated tubular atrophy, monocyte infiltration and interstitial fibrosis in 5/6Nx CKD rats. Moreover, rhubarb enema significantly increased the abundance of some symbiotic bacteria and probiotics, while reduced the abundance of some potential pathogens at the genus level. In addition, Spearman’s correlation analysis revealed that lachnospiraceae and romboutsia were positively correlated with TMAO.
Conclusion: Rhubarb enema decreases circulating TMAO level and improves renal fibrosis in 5/6Nx CKD rats, which may be related to the regulation of intestinal microbial community.

研究背景与目的：三甲胺N-氧化物（Trimethylamine N-oxide, TMAO）是肠道菌群的代谢产物，在慢性肾脏病（chronic kidney disease, CKD）患者体内水平升高，被认为是一类与不良心血管结局及肾功能下降相关的尿毒症毒素。既往研究表明，大黄灌肠疗法可降低循环中的尿素、肌酐、硫酸吲哚酚等尿毒症毒素水平，并可调节肠道菌群组成。然而，大黄灌肠疗法是否通过降低循环TMAO水平延缓肾功能减退，及其潜在作用机制尚未明确。本研究旨在探究大黄灌肠疗法对5/6肾切除（5/6 nephrectomized, 5/6Nx）慢性肾脏病大鼠体内TMAO及其前体物质、肠道菌群的影响。
实验设计：造模成功后，治疗组大鼠予以大黄灌肠疗法干预。本研究结束时，采集血液、粪便及肾脏组织，分别进行生化分析、组织学与蛋白质印迹（Western Blot）分析、16S核糖体RNA（16S rRNA）测序及非靶向代谢组学分析。
实验结果：大黄灌肠疗法可降低5/6Nx慢性肾脏病大鼠血清TMAO及三甲胺（trimethylamine, TMA）水平，抑制炎症标志物（白细胞介素-6、肿瘤坏死因子-α及干扰素-γ）的表达，并改善肾小管萎缩、单核细胞浸润及间质纤维化。此外，大黄灌肠疗法可显著增加部分共生菌及益生菌的丰度，同时降低属水平部分潜在致病菌的丰度。进一步的斯皮尔曼相关性分析显示，毛螺菌科（Lachnospiraceae）与罗姆布茨菌属（Romboutsia）的丰度与TMAO水平呈正相关。
研究结论：大黄灌肠疗法可降低5/6Nx慢性肾脏病大鼠的循环TMAO水平，改善肾纤维化，其作用机制可能与调控肠道微生物群落组成相关。