Detection results of cytokines in soft, hard feces and cecal contents
The levels of cytokines TNF-α, IL-1β, IL-6, TGF-β, IL-4, IL-10 and IL-13 in cecum contents, soft feces and hard feces were measured using ELISA kits, respectively, and the results are shown in Fig. 1. TNF-α, IL-1β, IL-6, TGF-β, IL-4, IL-10 and IL-13 were significantly higher in both soft and hard feces groups than in the cecum contents group; IL-1β, TGF-β, IL-4 and IL-13 contents were higher in the hard feces group than in the soft feces group, and the data were statistically significant between the two groups; however, TNF-α, IL-6 and IL-10 levels were higher in the soft feces group than in the hard feces group, but the difference between them was not significant (P-value>0.05) (Fig. 1A-G). In addition, three-dimensional PCA plots (Fig. 1H) were normalized according to the cytokines content of each sample and plotted by R software (v3.6.0), and principal component analysis (PCA) was used to perform a natural decomposition of the community data structure and to rank (sort) the samples to observe the differences between samples. The results showed that there was less dispersion between samples within groups in the cecum contents group, and the dispersion between samples within groups in the soft feces and hard feces groups was greater than that in the cecum contents group; while the samples between the cecum contents group, soft feces group and hard feces group were each independently distributed, with the samples in the soft and hard feces groups being closer together and farther away from the samples in the cecum contents group.
Detection results of SCFAs in soft, hard feces and cecal contents
The GC/MS method was used to determine the content of volatile short-chain fatty acids in the three samples, and the results are shown in Fig. 2. The contents of isobutyric acid, butyric acid, valeric acid and isohexanoic acid in the cecal content group were significantly higher than those in the soft stool group and hard stool group (P-value <0.05). The contents of acetic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid and caproic acid in the soft feces group were significantly higher than those in the hard feces group (P-value <0.05). The content of formic acid in the soft feces group and the hard feces group was significantly higher than that of the cecal content group (P-value <0.05), and the content of the hard feces group was higher than that of the soft feces group, the difference was statistically significant (P-value <0.05). There was no significant difference in the content of enanthic acid in the cecal contents group, soft stool group and hard stool group (P-value >0.05).
Species composition among groups soft, hard feces and cecal contents
Using QIIME software, the composition and abundance distribution of each sample at two levels of phylum and genera were obtained, and the analysis results were expressed in the form of a histogram. At the phylum classification level (Fig. 3A), Firmicutes and Bacteroides are the main phyla of the three groups of samples. The relative abundance of Firmicutes (84.48%) in the soft feces group is significantly higher than that in the hard feces group. (69.66%) and cecal contents group (56.82%), but the relative abundance of Bacteroides in the soft stool group (8.30%) was significantly lower than that in the hard stool group (26.67%) and cecal contents group (38.31%). At the genus level (Fig. 3B), the relative abundance of Muribculaceae in the cecal content group (31.17%) and the hard feces group (23.74%) was significantly higher than that of the soft feces group (3.78%); Ruminococcaceae_NK4A214_group in the soft feces group, The relative abundances of Lachnospiraceae_NK4A136_group, Christensenellaceae_R-7_group and Subdoligranulum (21.73%, 8.18%, 8.13%, and 7.93%, respectively) were significantly higher than those of the cecal contents group (5.71%, 2.30%, 1.39%, and 1.56%, respectively) and hard Fecal group (5.07%, 4.76%, 2.92% and 1.34%, respectively); the relative abundance of Ruminococcaceae_UCG-014 (12.84%) in the hard feces group was significantly higher than that of the cecal contents group (2.80%) and soft feces group (3.39) %). Then use ggtree in R software as evolutionary tree to display the position of each ASV/OTU in the evolutionary tree, and the evolutionary distance between each other, and reflect their composition, abundance, taxonomy and other information through heat maps and histograms. The result is shown in Fig. 3C. Among them, Ruminococcaceae_NK4A214_group, Ruminococcaceae_UCG-014, Ruminococcaceae_1, Ruminiclostridium_6, Muribaculaceae, Clostridiales_vadinBB60_group, Christensenellaceae_R-7_group and other bacterial genera are closely related. Ruminococcaceae_UCG-013 and Subdoligranulum are closely related, but farther than the other genus mentioned above. It can be seen from the heat map that Ruminococcaceae_NK4A214_group and Christensenellaceae_R-7_group have higher relative abundance in the soft feces group. The relative abundance of bacteria such as Ruminiclostridium_6 and Ruminococcaceae_UCG-014 in the HF group was higher; the relative abundance of bacteria such as Muribaculaceae and Ruminococcaceae_1 in the cecal content group was higher. In addition, the abundance histogram results show that Firmicutes and Bacteroides are the main two phyla.
Alpha and beta diversity in microbiota of groups soft, hard feces and cecal contents
Alpha diversity and beta diversity index were used to characterize the diversity of species within and between habitats respectively, so as to comprehensively evaluate their overall diversity. In this study, Chao1 and observed species indexes are used to represent richness, Shannon and Simpson indexes are used to represent diversity, faith's PD index is used to represent evolution based diversity, Pielou's evenness index is used to represent evenness, and good's coverage index is used to represent coverage. Alpha diversity results are shown in Fig. 4A. The indexes of Chao1, faith's PD, Shannon, Pielou's evenness and observed species in cecal content group are significantly higher than those in soft feces group and hard feces group. There was no significant difference in all the above indexes between soft feces group and hard feces group (P-value > 0.05). The sparse curve results of good's coverage are shown in Fig. 4B. The smoothness of the curves of the three groups of samples reflects the impact of sequencing depth on the diversity of observed samples. The curves of the three groups of samples tend to be gentle, indicating that the sequencing results have enough to reflect the diversity contained in the current samples. If the sequencing depth continues to increase, a large number of new ASV/OTUs that have not been found. Then, the dimension of multidimensional microbial data is reduced by Principal Coordinate Analysis (PCoA), and the main trend of data change is displayed by the distribution of samples on the continuous sorting axis. At the same time, permutational multivariate analysis of variance (PERMANOVA) was used to identify whether there were significant differences among the three groups of samples. The beta diversity of the three groups of samples was analyzed by the above methods. The results showed that in the PCoA diagram (Fig. 4C), there was an obvious separation between the samples of cecal content group, soft feces group and hard feces group, and the dispersion between the samples of cecal content group was less than that between the samples of soft feces group and hard feces group. The results of difference analysis between groups are shown in Fig. 4C. There is a very significant difference between cecal contents group and soft and hard feces group (P-value < 0.05), but there is no significant difference between soft and hard feces groups (P-value > 0.05).
Species differences and marker analysis
The results of LEfSe analysis showed that p_Bacteroidetes, c_Bacteroidia, o_Bacteroidales, f_Muribaculaceae and g_Muribaculaceae were the most significant bacterial genera in the Con group. g_Ruminococcaceae_UCG-014, g_Lachnospiraceae_UCG_001 and g_Izimaplasmatales are the most significant bacterial genera in the HF group. p_Firmicutes, c_Clostridia and o_Clostridiales are the most significant differences in the SF group (Fig. 5A). Use the ASV/OTU abundance table (Supplementary Table S1) to make a Venn diagram, the Con group, the soft feces group and the HF group. The three groups of samples have 13765, 5442 and 7344 ASV/OTUs respectively. Three groups of samples There are a total of 623 ASV/OUT (Fig. 5B). RDA analysis results show that SCFAs are mainly related to the Con group and SF group, and Cytokines were mainly correlated with fecal microbiome (HF and SF groups) (Fig. 5C). The relative abundance of the top 15 bacterial genera in the three groups of samples was drawn using software as shown in Fig. 5D. The relative abundance of Muribaculaceae and Ruminococcus_1 in the Muribaculaceae Con group and the HF group was higher than that of the SF group; Ruminococcaceae_NK4A214_group in the SF group The relative abundances of, Christensenellaceae_R-7_group, Subdoligranulum and Candidatus_Saccharimonas were higher than the Con group and the HF group.
Functional potential predictive analysis
The relative abundance results of primary and secondary metabolic pathways in metacyc database are shown in Fig. 6A. The relative abundance of pathways related to biosynthesis is high, mainly including amino acid biosynthesis, carbohydrate biosynthesis, coenzyme factor and vitamin biosynthesis. The relative abundances of enzymes related to the synthesis of acetic acid, propionic acid and butyrate in the three groups were further analyzed. It was found that the relative abundances of acetic acid synthase such as EC: 6.3.4.3, EC: 1.5.1.20, EC: 2.3.1.54 and EC: 2.3.1.169 in the soft feces group were significantly higher than those in the Con group (Fig. 6B). In propionate synthase, the relative abundance of EC: 2.7.1.1 enzyme in soft feces group was significantly higher than that in Con group (Fig. 6C). Among butyrate synthase, the relative abundances of EC: 2.3.1.9, EC: 4.2.1.17, EC: 1.3.8.1 and EC: 2.8.3.8 in soft feces group were significantly higher than those in Con group (Fig. 6D). After obtaining the abundance data of metabolic pathways, Metagenome-seq was used to find out the metabolic pathways with significant differences among the three groups. As shown in Fig. 6E and F, the up-regulated metabolic pathways in the hard fecal group were PWY-7198 and PWY-7210 compared with the cecal content group, and the up-regulated metabolic pathways in the soft fecal group were PWY-7198, PWY -7210, p341- PWY and P123-PWY compared with the cecal content group, that is, P341-PWY and P123-PWY were up-regulated in soft feces group compared with hard feces group. Finally, according to the significantly different metabolic pathways, the hierarchical sample metabolic pathway abundance table (Supplementary Table S2) is used to analyze the species composition of different pathways. The results are shown in Fig. 6G and H. The P125-PWY pathway is superpathway of (R, R)-butanediol biosynthesis, in which the relative abundance of Christensenellaceae_R-7_group in the soft stool group is higher than that of the cecal content group and the hard stool group; the P341-PWY pathway is glycolysis V (Pyrococcus), of which the soft stool group and the hard stool group The relative abundance of Lachnoclostridium in the group was higher than that of the cecal content group.
Correlation analysis of cytokines, SCFAs and microbiota
The correlation analysis results of SCFAs and the microbiota are shown in Fig. 7A. Among them, Ruminococcaceae_NK4A214_group has significant positive correlation with acetic acid, butyric acid, isobutyric acid, isovaleric acid and hexanoic acid; Christensenellaceae_R-7_group has positive correlation with acetic acid, butyric acid, isobutyric acid, isovaleric acid and hexanoic acid; Muribaculaceae has a significant positive correlation with isocaproic acid. Ruminococcaceae_UCG-014 has a significant negative correlation with acetic acid, propionic acid, isobutyric acid, butyric acid, valeric acid and caproic acid. The results of correlation analysis between cytokines and The top 15 relative abundance bacteria in the three groups of samples are shown in Fig. 7B. Muribaculaceae has a significant positive correlation with IL-1β, IL-4 and TGF-β, respectively; On the contrary, Candidatus_Saccharimonas has a significant negative correlation with IL-1β, IL-4 and TGF-β.
Effect of fasting soft feces on cecal immune microenvironment
After fasting soft feces, the cytokines TNF-α and IL-1β Significantly increased, while cytokines IL-4 and IL-10 decreased significantly (Fig. 8A). In addition, the expression levels of tight junction related proteins in CN group and CP group were measured by Western blotting and multiple immunofluorescence staining respectively. The results of Western blotting showed that the expression levels of tight junction proteins such as ZO-1, Claudin-1 and Occludin increased significantly after fasting soft feces (Fig. 8B). The results of immunofluorescence staining were consistent with those of Western blotting. The staining degree of ZO-1 protein (purple), Claudin-1 protein (green) and Occludin (red) protein in CP Group was deeper (compared with CN group), indicating that the contents of Claudin-1 and Occludin protein in CP group were higher (Fig. 8C). Meanwhile, after fasting soft feces, the cecal mucosa was damaged (Fig. 8D, the purple part indicated by the arrow), while the integrity of cecal mucosa in the CN group was better.