口服肝素与小鼠肠道菌群的相互作用

Interaction between orally administrated heparin and intestinal microbiota in mice

摘要:

口服肝素药物的开发需要系统地理解口服肝素与肠道菌群之间的互作过程。通过荧光体视镜观察荧光素标记的肝素经小鼠口服后在体内的分布情况，利用高效液相色谱法检测肝素在模拟胃肠液中的稳定性和体外培养肠道菌群模拟肠道菌对肝素的降解作用，发现口服肝素主要分布在小鼠胃肠道内，在体外模拟胃肠液条件下肝素结构稳定，但能够被添加肝素的厌氧培养基培养后的肠道菌群降解。为了进一步揭示口服肝素对健康小鼠肠道菌群的影响，利用Illumina MiSeq高通量测序技术测定口服肝素后C57BL/6J小鼠粪便菌群的16S rRNA序列，与口服生理盐水的小鼠粪便菌群进行对比，发现口服肝素的小鼠粪便菌群的生物多样性降低；在门水平上，菌群结构差异不显著；而在属水平上，别样杆菌属Alistipes、副萨特氏菌属Parasutterella和艾克曼菌属Akkermansia相对丰度增高，而嗜胆菌属Bilophila、肠杆菌属Enterorhabdus、瘤胃梭菌属Ruminiclostridium、普雷沃氏菌科Prevotellaceae_UCG_001、瘤胃梭菌属Ruminiclostridium-9、拟杆菌属Bacteroides、Lachnoclostridium、Candidatus_ Saccharimonas、Intestinimonas和Dubosiella的相对丰度减少，表明口服肝素能够影响小鼠肠道菌群结构。此外，实验发现口服肝素对小鼠无明显毒副作用，具有较高安全性。研究结果将为开发肝素口服递送策略提供新的思路，为口服肝素类药物的开发提供参考。

Abstract:

The development of orally administrated heparin drugs requires a systematic understanding of the interaction between heparin and gut flora. The in vivo distribution of fluorescein-labeled heparin that is orally administrated by mice was observed using fluorescein microscopy. In addition, the stability of heparin in simulated gastric and intestinal fluids, as well as the in vitro degradation of heparin by gut flora were detected by HPLC. The results show that orally administrated heparin was mainly distributed in the gastrointestinal tract of mice, and exerted structural stability under the condition of simulated gastric and intestinal fluids in vitro. However, heparin could be degraded by intestinal flora cultured in medium containing heparin. In order to further study the effect of orally administrated heparin on intestinal flora in mice, the fecal microbiota 16S rRNA fragment of C57BL/6J mice was tested by the Illumina Mi-Seq high-throughput sequencing technology. Compared with the gut flora of mice that orally administrated by saline, the biodiversity of gut flora in mice with orally administrated heparin was decreased. The difference of microflora structure was not significant at the phylum level, and the relative abundance of Alistipes, Parasutterella and Akkermansia was increased at the genus level, and the relative abundance of Bilophila, Enterorhabdus, Ruminiclostridium, Prevotellaceae_UCG_001, Ruminiclostridium-9, Bacteroides, Lachnoclostridium, Candidatus, Saccharimonas, Intestinimonas and Dubosiella was reduced. These findings indicate that heparin could influence the gut flora of mice. In addition, no obvious toxic and side effects were found in mice that orally administrated heparin, suggesting the safety of orally administrated heparin.