An integrated metabolomics and 16S rRNA gene sequencing approach exploring the molecular pathways and potential targets behind the effects of Radix Scrophulariae

Abstract

To assess the impact of the caecal microbiota on faecal metabolic phenotypes in the presence of Radix Scrophulariae (Chinese name: Xuanshen), an integrated approach involving 16S rRNA gene sequencing combined with ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF-MS)-based faecal metabolomics was applied to Radix Scrophulariae-treated rats. Interestingly, Radix Scrophulariae led to significant gut microbiota changes at the phylum and genus levels in treated rats compared to control rats. Additionally, distinct changes in faecal metabolites, including linoleic acid (LA), guanosine, inosine, hypoxanthine, xanthine, 4-hydroxycinnamic acid, cholic acid, N-acetyl-d-glucosamine, l-urobilinogen and uridine, were observed in Radix Scrophulariae-treated rats. Of these, seven metabolites were up-regulated, and the remaining three metabolites were down-regulated. Moreover, there were substantial associations between altered levels of gut microbiota genera and discrepant levels of faecal metabolites, particularly for compounds involved in LA and purine metabolism. These results demonstrated that the gut microbiota is altered in association with faecal metabolism following treatment with Radix Scrophulariae. Our findings suggest that further application of this 16S rRNA gene sequencing and UHPLC/TOF-MS-based metabolomics approach will facilitate the assessment of the pharmacological action of Radix Scrophulariae and thus expand the scope of this herb.

Fig. 1. Fingerprint chromatograms of the aqueous extract of Radix Scrophulariae, which include (A), angoroside C (B), harpagoside (C) and cinnamic acid (D).

Fig. 2. (A) PCA plot of the faecal metabolic trajectory of rats at different time points before and after the administration of Radix Scrophulariae. A 2D-PCA plot based on data from the control and Radix Scrophulariae groups at day 11 in positive (B) and negative (C) ion mode. S and VIP plots of faecal samples from the control and Radix Scrophulariae groups in both positive (D and F) and negative (E and G) ion modes. K represents the control group; g represents the Radix Scrophulariae group. The points in Fig. 2A represent control and Radix Scrophulariae group samples. Q and g in Fig. 2B represent the Radix Scrophulariae group, and k in Fig. 2B represents the control group.

Fig. 3. Ion intensity of potential biomarkers in different faecal sample groups (A and B). Data represent the mean ± SE of each group (n = 10 in each group). *Altered trend compared to the control group, p < 0.05; **altered trend compared to the control group, p < 0.01.

Fig. 4. Pathway analysis of metabolites regulating metabolism and metabolic pathway analysis of potential intervention targets of Radix Scrophulariae. Elliptical nodes represent pathways, rectangle nodes represent metabolites (red: upregulation, green: downregulation), and diamond nodes represent Radix Scrophulariae. Black edges represent the relationships between the metabolites and pathways, and red edges represent possible relationships between Radix Scrophulariae and metabolites.

Fig. 5. OTUs of all levels (A and B) are represented by a Venn diagram of the OTUs at the phylum level and a bar chart of the taxon abundance at the phylum level (C). Each ellipse represents a group, and overlapping regions of the ellipses indicate that OTUs are shared by groups. The number in each block indicates the number of OTUs contained in each group or the number of unique OTUs. k represents the control group, and SQ represents the Radix Scrophulariae group.

Fig. 6. (A) Correlation of specific gut bacteria and gut microbiota-related faecal metabolites. (B) Metabolic pathway analysis of metabolites and genera in the Radix Scrophulariae-treated group. Diamond nodes represent metabolites, ellipse nodes represent pathways and round rectangles represent genera (red: upregulation, green: downregulation). Red edges represent positive correlations between metabolites and genera, while green edges represent negative correlations between metabolites and genera. (C) Correlation between gut microbiota genera, perturbed metabolites and the most relevant metabolic pathways. Pink rectangle nodes represent metabolites. The levels of potential biomarkers in the Radix Scrophulariae group compared to the control group were labelled with (↓) for downregulation and (↑) for upregulation.