3.1. Overall structural changes in microbiota composition
Dilution curve is used to reflect the rationality of sequencing data and indirectly the species richness. As shown in Figure S1-2, the curve gradually flattens, indicating that the amount of sequencing data is sufficient. At the same time, we found that the good coverage of all experimental groups was greater than 99.0%, which indicated that the sequencing depth of this microbiome analysis was very deep (Fig. 1a). For alpha diversity analysis, Chao and Shannon indexes were used to evaluate the richness and diversity of flora. As shown Fig. 1B, the richness of the bacterial population (Chao1 indices) in the GA group was significantly lower than that in the normal group (p < 0.01), and there was no significant difference in the species richness of intestinal flora between KO, FC, MO and DI Group compared with normal rats. The species richness of constipation model, mosapride group and low- medium- dose Chinese medicine group are similar to that of normal healthy rats, and there were no significant difference. The observed species indicates that the number of OTU actually observed with the increase in sequencing depth. As shown Fig. 1C, the number of OTU actually observed in the MO group was the largest, which is significantly higher than that in the FC (p < 0.01) and the DI group (p < 0.05). There was no significant difference in KO between the FC group and the control group, and the low-medium-high-dose Chinese medicine group showed a gradually decreasing trend (p < 0.05). From Fig. 1D, the Shannon index of MO and DI group was significantly higher than that of normal group (p < 0.05), which were indicates that the diversity of intestinal flora of mosapride and low-dose Chinese medicine group was more than that of healthy rats, while the diversity index of FC were not significantly different from normal group, which is clearly indicating that the intestinal diversity of constipation group was not significantly different from that of healthy rats. The intestinal diversity of GA group was significantly lower than that of normal control group (p < 0.01) and ZH group (p < 0.01), which were indicating that Chinese medicine at a high dose reduced the intestinal flora diversity.
In order to further study, the similarity or difference of the composition of the intestinal flora of the sample, cluster, Non-metric multidimensional scaling method (NMDS) and principal co-ordinates analysis (PCoA) were performed. Cluster analysis uses tree structure to describe and compare similarities between multiple samples. It can be clearly seen from Fig. 1E that the microbiological composition of the samples in the group is similar, and the samples of the same treatment group was gathered together, indicating that the differences between the groups was small and the sample repeatability good. It can be seen from NMDS and PCoA analysis (Fig. 1F-G) that the microbial composition of the normal control group was different from that of DI, ZH and GA groups, indicating that the intake of low, medium and high doses of traditional Chinese medicine changes the composition of the whole intestinal flora, and the degree of change is also different under different doses. In addition, it can be found that the differences between FC group and MO and DI groups are not obvious, and the composition of the flora tends to be the same, indicating that the intestinal flora composition of the constipation group, the mosapride group and the low-dose Chinese medicine group similar.
[Figure 1]
3.2. Classification based comparison of phylum and genus levels
From the phylum-level analysis (Fig. 2A), we could clearly found that 90% of the intestinal microorganisms in six groups were mainly composed of Firmicutes and Bacteroides. The abundances of Firmicutes in KO, FC, MO, DI, ZH and GA groups were 76.0%, 86.7%, 85.1%, 81.0%, 83.4% and 87.8%, respectively. The relative abundances of Bacteroides in KO, FC, MO, DI, ZH and GA groups were 15.7%, 7.3%, 9.0%, 12.0%, 8.2% and 6.0%, respectively. It can be seen from Fig. 2B that the relative abundance of Firmicutes in the constipation model group, mosapride group and high-dose traditional Chinese medicine group were significantly higher than that in the control group (p < 0.01), and there were no significant difference between the three groups. Meanwhile, the abundance of Firmicutes in the DI group was significantly lower than that in the middle dose ZH and constipation group (p < 0.05). As can be seen from Fig. 2C, the relative abundance of Bacteroidetes in the constipation model group, the mosapride group and the different doses of the Chinese medicine group was significantly lower than the control group (p < 0.01), and there was no significant difference between the constipation model group and the mosapride group. At the same time, the relative abundance of Bacteroidetes in the DI group was significantly higher than that in the constipation group. At the same time, it showed a gradual decrease with the rising the dose of Chinese medicine (p < 0.05).
We could clearly found that the intestinal microorganisms in six groups were mainly composed of Lachnospiraceae, Lactobacillus and Ruminococcaceae (Fig. 2D). The abundances of Lachnospiraceae in KO, FC, MO, DI, ZH and GA groups were 19.5%, 16.9%, 10.8%, 15.7%, 11.7% and 8.8%, respectively. The abundances of Lactobacillus in KO, FC, MO, DI, ZH and GA groups were 12.3%, 8.3%, 6.1%, 6.3%, 9.4% and 23.1%, respectively. The abundances of Ruminococcaceae in KO, FC, MO, DI, ZH and GA groups were 8.3%, 11.4%, 12.9%, 11.0%, 12.5% and 11.7%. We found that there was no significant difference in the relative abundance of Ruminococcaceae among the six groups. The abundance of Lachnospiraceae decreased with the increasing of the dosage of Chinese medicine (p < 0.05), at the same time, the intake of Mosapride significantly reduced the relative abundance of Lachnospiraceae (p < 0.01) (Fig. 2E). The abundance of Lactobacillus increased with the increasing of the dosage of Chinese medicine, and the high-dose Chinese medicine group was significantly higher than the low-dose group, the constipation group and the normal control group (p < 0.01) (Fig. 2F). The results indicated that the abundance of Lactobacillus in the constipation group was significantly lower than KO (p < 0.05). Lactobacillus has a significant role in promoting intestinal peristalsis, so we speculate that the role of traditional Chinese medicine in the treatment of constipation may be related to promoting the proliferation of probiotics.
[Figure 2]
3.3. The differences in the dominant members of the microbiota
In order to verify and further determine the LEfSe was used to identify the specific phylotypes responding to FC and DI, ZH, GA groups. As shown Figs. 3A and B, the main differential microbial species between the constipation simulation group and other groups were Carnobacteriaceae and Clostridiales, the main differential microbial species between the DI group and other groups were Porphyromonadaceae and Lachnospiraceae, the main differential microbial species between the ZH group and other groups were Methylobacteriaceae, Christensenellaceae and Erysipelotrichaceae, the main differential microbial species between the GA group and other groups were Lactobacillaceae, Bacilli and Peptostreptococcaceae, the main differential microbial species between the MO group and other groups were Bifidobacteriaceae, Actinomycetaceae, Deferribacteraceae, Aerococcaceae, Clostridiaceae, Peptococcaceae and Ruminococcaceae.
[Figure 3]
3.4. Network relationship and functional prediction analysis
Faust et al. [17] proposed a network inference analysis based on the relationship between microbial members. The fundamental purpose of this analysis was to examine the interaction patterns between different microbial community members in a sample. The numbers of the nodes and links were counted through statistical network symbiosis, it can be seen from Fig. 4A that the dominant symbiotic dominant flora in the KO group is Firmicutes, Spirochaetae and Proteobacteria. At the same time, the genus level of Firmicutes, the dominant symbiotic flora includes Turibacharacter, Roseburia, Eubacterium, Ruminocaceae, Ruminostridium, Ruminocaccus, Allobaculum, Lactobacillus, Spirochaetae, Treponema, Proteobacteria and Desulfovibrio. In addition, 7 symbiotic relationships between Lactobacillus and other bacteria, 5 positive and 2 negative ones. From Fig. 4B that the dominant symbiotic dominant flora in the FC group is Firmicutes and Spirochaetae. At the genus level of Firmicutes, the dominant symbiotic flora includes Turicibacter, Eubacterium, Ruminocaceae, Ruminostridium, Ruminocaccus, Allobaculum, Christensenellaceae, Lactobacillus, Spirochaetae and Treponema. There are 7 symbiotic relationships between Lactobacillus and other bacteria, 2 positive and 5 negative ones. The above shows that in constipation group FC was significantly reduce the symbiotic relationship between Proteobacteria and other intestinal bacteria, increase symbiosis of Firmicutes and other flora, but it does not change and affect the symbiotic flora of Spirochaetae. Compared with control group KO, the negative symbiotic relationship of Lactobacillus was increased in the constipation group. From Fig. 4C that the dominant symbiotic flora in the GA group was Firmicutes and Spirochaetae. At the genus level of Firmicutes, the dominant symbiotic flora includes Lactobacillus, Turicibacter, Eubacterium, Ruminocaceae, Ruminostridium, Ruminocaccus, Allobaculum, Christensenellaceae, Lachnospiraceae, Spirochaetae and Treponema. There are 24 symbiotic relationships between Lactobacillus and other bacteria, 14 positive ones and 13 negative ones. In this study, we found that a certain dose of Chinese medicine significantly increased the symbiotic relationship between Lactobacillus and other intestinal flora, making Lactobacillus from the edge of the symbiotic relationship to the dominant dominant flora.
Changes in the symbiotic relationship of the gut microbiota often also indicate functional changes; thus, functional prediction analysis was also performed. From Fig. 4D, it can be seen that the distribution of functional genes in these six groups is mainly concentrated in glycan biosynthesis and betabolism, followed by Lipid metabolism, biosynthesis of other secondary metabolites and transport and catabolism. The less distributed ones were neurodegenerative diseases, and the other ones, such as Digest System, account for less than 1%. At the same time, the Fig. 4E also indicted that the expression of glycosaminoglycan degradation, apoptosis, G protein-coupled receptors, stilbenoid, diarylheptanoid and gingerol biosynthesis, protein digestion and absorption, glycosphingolipid biosynthesis - ganglio series and bill secretion were significantly up-regulated by Chinese medicine compared with FC group, significant down-regulation of glycosphingolipid biosynthesis - globo series, amyotrophic lateral sclerosis (ALS), amoebiasis, prion diseases, alpha-linolenic acid metabolism, chlorocyclohexane and chlorobenzene degradation and steroid hormone biosynthesis.
[Figure 4]
3.5. qRT-PCR analysis
The total copy number range of 16S rDNA gene of bacteria was from 8.4 × 107 to 8.2 × 108, 2.1 × 108 to 1.1 × 1010, 1.4 × 1010 to 9.0 × 1013, 4.1 × 1010 to 5.9 × 1012, 3.2 × 106 to 1.4 × 109, 2.5 × 106 to 1.3 × 108 copies per gram of tissue content in the KO, FC, MO, DI, ZH and GA samples, respectively. We found that the total copy number of bacteria in the constipation group was not significantly different from that in the control group, and that in the mosapride group and the low-dose traditional Chinese medicine group was significantly higher than that KO (p < 0.05). The difference between the medium dose traditional Chinese medicine group and the control group was not significant, while the copy number of bacteria in the high-dose group was significantly lower than that KO (Table 1).
Table 1
Total copies of intestinal bacteria in each group
Total bacteria (copy number/g) | FC | MO | DI | ZH | GA | KO |
1 | 1482131000 | 14424390000 | 1.30734E + 11 | 877964800 | 2532737 | 821453400 |
2 | 530056400 | 66255020000 | 5.94403E + 12 | 1375961000 | 45190660 | 84881970 |
3 | 476350900 | 14133200000 | 4.09941E + 12 | 65151450 | 69905180 | 84399230 |
4 | 3238729000 | 1.35541E + 12 | 41278390000 | 16213720 | 144515900 | 311785000 |
5 | 216104600 | 2.33782E + 11 | 52287600000 | 17307860 | 84531920 | 178919400 |
6 | 241080800 | 9.06385E + 13 | 54849440000 | 10236970 | 107940800 | 168030700 |
7 | 4593153000 | 65143480000 | 5.61591E + 11 | 3292147 | 120057200 | 820202500 |
8 | 11431060000 | 63403100000 | 1.96913E + 11 | 5663928 | 133493100 | 490321300 |
FC: constipation model group, MO: mosapride group was given 2mg· (kg·d)−1 gavage, DI: low dose group with 5.15g· (kg·d)−1, ZH: medium dose group with 10.3G·(kg·d)−1, GA: high dose group with 20.6g·(kg·d)−1 doses, KO: control group. |
[Table 1]