1 Praxiology Change of Mice with Stomach Perfusion of the Water Extract of Folium sennae
During gastric administration with sterile saline, the mice in pfck group had dense and glossy back hair with bright eyes; diet water quantity, stools and mental state were normal. Diarrhea symptoms appeared in the afternoon of the first day of Folium sennae extracts given by gavage in pfm group mice, and feces began to become thinner and softer. The feces of pfm group mice became thinner and perianal contamination on the second day of modeling since Day 2. Mice in pfm group began to show hairy back, sleepiness, arched back and dry back; and diarrhea symptoms in the afternoon were more serious than those in the morning on the third day of modeling. On the 8th day, it was found that the stool in pfck group was water-like after dissecting the mice, while in pfm group, the stool was granular, moist, dark, normal in size and shape. There was no significant difference in body weight change and body weight change rate between pfm group and pfck group [(9.97 + 4.48) g vs (10.26 + 5.00) g, p-value = 0.06; (29.23 + 9.87)% vs (28.74 + 10.86%), p-value-=0.06].
2 Intestinal Microbial Diversity in Diarrhea Mice Caused by Water Extract of Folium sennae
2.1 Sample sequences and operational taxonomic units
The number of ERIC-PCR sample sequences is positively correlated with the number of microbial populations, and the intensity of sample sequences indicate the number of DNA fragments, hence the size of a certain microbial population. There were a total of 475,973 effective sequences and 439,518 high quality sequences obtained by sequencing mostly between 150 bp and 300 bp in this study for subsequent analysis.
According to sequence homogeneity, Qiime soft was used in clustering 97% similarity DNA sequences into a same operational taxonomic unit (OTU). As shown in Figs. 1, 288 and 202 OTUs were uniquely identified from two groups, respectively; and the number of OTUs coincided between the two groups was 749. The result suggested that the OTU of bacterial gene had decreased after diarrhea caused by Folium sennae.
2.2 Bacterial diversity in intestinal contents of diarrhea mice caused by Folium sennae
In order to illustrate the diversity of intestinal microflora in two group of mice, we calculated Alpha diversity and Beta diversity[23]. The Alpha diversity index includes Chao1 and Shannon: the Chao index (showing the richness of bacteria) and the Shannon index (showing the diversity of bacteria) are demonstrated in our study. The higher the Alpha diversity, the better the richness of the bacterial species, the more uniform the number of intestinal bacteria, and the more stable the flora. The results showed that both the Chao1 index curve (Fig. 2A1) and Shannon index curve (Fig. 2B1) of the sample had entered the plateau and reached saturation, which suggested that the amount of sequencing data of our study is large enough to reflect the vast majority of microbial species information in the sample. As shown in Fig. 2, within all the range of gene sequences pfm group mice showed lower Chao1 index and Shannon index. Overall, Chao1 index (Fig. 2A2) and Shannon index (Fig. 2B2) decreased after Folium sennae intervention, and Shannon index decreased with statistical significance (p-value < 0.05). It suggested that Folium sennae could affect intestinal flora diversity in mice.
Beta diversity is a comparative analysis of the microbial community composition of different samples. PCA and NMDS analysis was conducted to measure differences in communities of bacteria at genus level to illustrate the Beta diversity. PCA of log2-transformed normalized abundance for OTUs with normalized total abundance. Unifrac analysis was carried out to obtain the distance matrix of the differences between samples by using the evolutionary information of species and the abundance information of species. The distance matrix information between the samples was analyzed by NMDS. As shown in Fig. 3, from PCA (Fig. 3A) or NMDS (Fig. 3B) analysis of OTU, OTUs of pfm group were mainly concentrated in the right quadrant. Thus it can be inferred that the intestinal microbial diversity of normal mice could be changed by Folium sennae.
3 Active Ingredients of Folium Sennae Regulate the Key Protein CYP3A4 and Influence Tryptophan Metabolic Pathways Associated with Diarrhea
3.1 Filtration of active ingredients and potential target genes associated with diarrhea of Folium sennae
Total 53 chemical constituents of Folium sennae were retrieved from TCMSP. All compounds were subjected to Active Perl 5.26 screening, and a total of 10 active compounds had OB ≥ 30% and DL ≥ 0.18. Therefore, the selected 10 compounds (Table 1) from Folium sennae were subjected to further analysis. After removing the redundancy, total of 370 potential targets (Table S1) were obtained from the 10 active ingredients. Genecard database was used to establish a new diarrhea-related gene database, which was docked with the selected 370 target genes of Folium sennae. Finally, 51 potential target genes of Folium sennae-Diarrhea were filtered out (Table S2).
3.2 Compounds-Targets-Disease (C-T-D) network construction and analysis
After removing the redundancy lacking target protein gene information, we further eliminated the compounds whose target genes did not intersect with the diarrhea-related gene database. A total of 4 compounds were incorporated into the C-T-D network construction, including rhein, kaempferol, sitosterol and stigmasterol. As shown in Fig. 4, the 4 active compounds and related 51 target genes constructed the network schematic diagram. Totally, this C-T-D network is composed of 57 nodes (4 active compounds and 51 potential targets) and 333 edges. In this picture, the edges indicated an association between the active ingredients, target genes and diarrhea. The degree values indicated the intensity of the interaction among the ingredients of Folium sennae, target genes and diarrhea. We found that kaempferol in Folium sennae was the main active ingredient that could regulate the CYP3A4 protein target gene, which had an effect on diarrhea.
3.3 PPI network construction and analysis
We constructed functional protein association PPI network to screen target protein genes that play a key role in diarrhea caused by Folium sennae. 49 target genes (2 disconnected nodes of genes, F7 and SCN5A, were hid) associated with active ingredients and diarrhea were imported into the STRING database for PPI network construction and analysis. There are 49 interacting targets in the network, resulting in 278 edges representing the interaction between proteins. As shown in Fig. 5, the 49 protein genes were clustered into three groups according to the interaction relationship, showing three different node colors; in the PPI network, the line color indicates 7 different type of interaction evidence: gene neighborhood, gene fusions, gene co-occurrence, co-expression, protein homology, texting and experimented.
Next, according to the protein relationship of PPI network, the protein relationship is further analyzed by the number of protein links. Combining the disease score of genes (the higher the score, the more evidence is available for disease regulation), and the disease score originates in the diarrhea-related gene database (Table S2), the key proteins are selected. As shown in Fig. 6, the color of protein dots represents the counting of PPI network protein connections. There were 21 proteins with a count greater than 10, of which the count of CYP3A4 protein was 12. The horizontal ordinate represents the disease score of genes; and except for the more recognized AKT1 and TNF proteins associated with diarrhea, only CYP3A4 protein scores were greater than 15. Therefore, we inferred that CYP3A4 protein was one of the main target protein in both C-T-D network and PPI network.
3.4 Kyoto encyclopedia of genes and genomes (KEGG) metabolic signaling pathway analysis
Intestinal flora could synthesize vitamins and amino acids necessary for human growth and development through bacterial metabolism, and participated in the metabolism of sugars and proteins. In the process of triggering the mechanism of intestinal diseases changes in gastrointestinal microbial and bile acid profiles wide impact on sensitization of extrinsic, intrinsic gut neurons and immune activation, in which metabolism of substance and energy in human body by intestinal microbial community and metabolites of intestinal flora community plays an important role. Therefore, in this study, we choose the perspective of metabolic pathway to explore the mechanism of diarrhea caused by Folium sennae and intestinal flora disorder. To determine the relevant metabolic signaling pathways involved in diarrhea effect of Folium sennae and intestinal flora, we conducted pathway enrichment analysis using KEGG metabolic pathways. A total of 51 targets obtained 80 KEGG signaling pathways, and 57 channels were significantly enriched (p-value < 0.05). As shown in Fig. 7, among the 80 KEGG signaling pathways there are 8 KEGG metabolic signaling pathways of gene enrichment. The color of the bars in the graph were decided the p-values, and the first 6 metabolic signaling pathways have statistical significance (p-value < 0.05). The abscissa indicates the count of proteins enriched in the pathway. In the picture, the pathway enriched more protein, the more evidence suggests that this metabolic pathway is the main mechanism of diarrhea caused by Folium sennae. Because their protein enrichment counts are 7 and 6, the drug metabolism - cytochrome P450 (CYP450) and metabolism of xenobiotics by CYP450 are the main pathways drug action for diarrhea, which is consistent with the analysis of PPI network protein interaction in this study. In addition, the results of this study suggested that tryptophan metabolism is also one of the main pathways of diarrhea caused by Folium sennae.
Previous studies have suggested that the amino acid tryptophan played a key role in the agonistic binding of an inducer of CYP450 by activation of the constitutive androstane receptor and confirmed the structural impact of mutations of tryptophan on CYP450[24, 25]. Therefore, we hypothesized that diarrhea caused by Folium sennae could affect tryptophan metabolism by diversity disorder of intestinal flora. Based on the sequencing results of microbial taxonomy, we validated the hypothesis by simulating intestinal microbial metabolic activities with VHM.
4 Diarrhea Caused by Folium sennae Affects Intestinal Bacterial Characteristic and Tryptophan Metabolism
4.1 Diarrhea caused by Folium sennae affects intestinal bacterial characteristic in mice
According to the OTU annotation results, the abundance level and composition ratio of each sample in different taxonomic levels had been obtained, which reflects the community structure of pfck group and pfm group in different taxonomic levels. By comparing the community structure of the two groups in different taxonomic levels, the effects of Folium sennae on the community structure of intestinal flora in mice were analyzed. In this study, abundance of bacteria of OTU in genus level of single sample in pfck group and pfm group was obtained. Based on the information of microflora and its abundance, the structure map of microflora community could reflect comprehensively the distribution of microflora and abundance in samples, and the microflora with higher abundance (top 20) could be found (Fig. 8). The picture had reflected the overall difference in the intestinal bacterial characteristic of the two groups of mice.
Lactobacillus had high abundance in pfck group (57%) and pfm group (69%) on phylum level. And as shown in Fig. 9, comparing the OTU abundance of the two groups, there were 33 bacterial flora communities (p-value < 0.05) with significant difference in OTU abundance between the two groups. Comparing the OTU abundance of the two groups, there were 10 bacterial flora communities with OTU abundance greater than 0.1% and q-value less than 0.05 on genus level. Paraprevotella, streptococcus, Epulopiscium, Sutterella and Mycoplasma increased significantly in the genus level of intestinal flora in mice with Folium sennae intervention (q-value < 0.05); the abundance of Adlercreutzia, Lactobacillus, Dehalobacterium, Dorea and Oscillospira were reduced in the pfm group (q-value < 0.05).
4.2 VMH simulation of tryptophan metabolism in intestinal flora
Bacterial flora communities labeled by OTU which its abundance changed with the intervention of diarrhea caused by Folium sennae were selected to simulate tryptophan metabolism of intestinal bacteria. The intestinal metabolic function of the 10 microflora from the above results was simulated, 7 of which were related to tryptophan metabolism through the synthesis of functional enzymes (Table. 2). In the experiment, we found that the intestinal flora communities of Folium sennae intervention were mainly influence the production of Exchange of L-Tryptophan enzyme and L-tryptophan reversible transport via proton symport enzyme to complete the metabolism of tryptophan, and L-Tryptophan was produced by the intestinal flora enzymatic reaction and completes amino tryptophan metabolism. We therefore conclude that diarrhea caused by Folium sennae could affect tryptophan metabolism of intestinal flora.