基于16S rDNA和GC-MS技术研究胡黄连治疗便秘小鼠的作用机制

Mechanism of Picrorhizae Rhizoma against functional constipation in mice:an exploration based on 16S rDNA and GC-MS

研究胡黄连对便秘模型小鼠肠道菌群和代谢产物的影响。将ICR小鼠分为空白组、模型组以及胡黄连高、中、低剂量组,应用盐酸洛哌丁胺对模型组和胡黄连各组造模,造模成功后,胡黄连高、中、低剂量组给予相应剂量药物灌胃给药7 d,测量各组小鼠首次黑便时间、3 h粪便总量、粪便含水率及肠道推进率;HE染色观察肠道黏膜情况;16S rDNA检测结肠内容物菌群变化;气相色谱-质谱(GC-MS)技术检测粪便代谢物变化。结果显示,模型组小鼠首次黑便时间显著延长,3 h粪便质量、粪便含水率及肠道推进率均显著减少,结肠组织可见明显炎性细胞浸润,肠道菌群及粪便代谢物呈现显著变化。胡黄连干预可显著改善便秘症状,修复肠道黏膜,部分恢复便秘小鼠肠道菌群及代谢物的组成。肠道菌群测序显示,胡黄连可以显著降低厚壁菌门/拟杆菌门比值,降低Lactobacillus相对丰度,增加肠道Muribaculaceae、Enterorhabdus、Eggerthellaceae相对丰度,线性判别分析(linear discriminant analysis effect size,LEfSe)软件测得胡黄连组优势菌种包括Muribaculaceae、Dubosiella、Akkermansia等。小鼠粪便共筛查出43种差异代谢物,涉及D-谷氨酰胺和D-谷氨酸代谢,谷胱甘肽代谢,精氨酸生物合成,丙氨酸、天冬氨酸和谷氨酸代谢,嘌呤代谢,嘧啶代谢等6条特异性代谢通路。表明胡黄连可能通过调节肠道菌群结构及代谢产物,并影响相关代谢通路来增强胃肠道动力,保护胃肠道黏膜,缓解便秘症状。

The study was designed to determine the influences of Picrorhizae Rhizoma on gut microbiota and metabolites in mice with functional constipation(FC).ICR mice were divided into the blank control group,model group,and the low-,middle-,and high-dose Picrorhizae Rhizoma groups.Mice in the model and low-,middle-,and high-dose Picrorhizae Rhizoma groups were modeled with loperamide hydrochloride.After successful modeling,the ones in the low-,middle-,and high-dose Picrorhizae Rhizoma groups were gavaged with Picrorhizae Rhizoma at the corresponding doses for seven days.The first appearance time of tarry stool,the total fecal volume within 3 h,the fecal moisture content,and the intestinal transit rate were observed in each group.The pathological changes in intestinal mucosa were detected by HE staining.The flora dynamics in colon content were measured by 16 S rDNA sequencing,followed by the examination of fecal metabolomic profiles by gas chromatography-mass spectrometry(GC-MS).The results showed that the first appearance time of tarry stool in the model group was prolonged.The total fecal volume within 3 h,the fecal moisture content,and the intestinal transit rate were significantly reduced.The colon tissue showed inflammatory cell infiltration.Gut microflora and fecal metabolites changed dramatically.Picrorhizae Rhizoma alleviated the constipation symptoms,repaired intestinal mucosa,and partially restored the gut microbiota and metabolite compositions in mice with constipation.As demonstrated by intestinal microbiota sequencing,Picrorhizae Rhizoma remarkably reduced the Firmicutes/Bacteroidetes ratio and the relative abundance of Lactobacillus,but increased the relative abundance of Muribaculaceae,Enterorhabdus,and Eggerthellaceae.According to the linear discriminant analysis effect size(LefSe),the dominant bacterial species in the Picrorhizae Rhizoma groups were Muribaculaceae,Dubosiella,and Akkermansia.A total of 43 differential metabolites were detected in the feces of mice,involving the D-glutamine and D-glutamate metabolism,glutathione metabolism,arginine biosynthesis,alanine,aspartate and glutamate metabolism,purine metabolism,and pyrimidine metabolism.All these have demonstrated that Picrorhizae Rhizoma enhanced gastrointestinal motility,protectd gastrointestinal mucosa,and alleviated constipation symptoms possibly by regulating the intestinal microbial communities and metabolites and affecting the related metabolic pathways.