Complex microbes inhabit the human intestine, and the group maintains the stability of the intestinal environment and protects the health of the human body. These microbes participate in defence and immunity against pathogens, development of intestinal microvilli, fermentation of nondigestible dietary fibres, and anaerobic metabolism of peptides and proteins, providing energy to the host. A number of studies have shown that the gut microbiome is associated with the occurrence of CAPs. However, most intestinal bacteria cannot be cultured in vitro, and the application of high-throughput sequencing technology helped us fully understand how the gut microbiome changed during the development process from healthy status to CAP and CRC.
In the 16S rRNA sequence analysis of faeces, the α diversity index was not significantly different between the two groups. Goedert et al. reported similar results for the faecal microbiota in CAP patients[14], but a reduction in the abundance of the faecal microbiota was observed in CRC patients[15]. PCoA of weighted UniFrac distance revealed no obvious aggregation in the CAP group and HC group. The changes in genera in both groups were analysed, and Weissella and Lactobacillus were present in the HC group; although their relative contents were not high, Weissella and Lactobacillus are probiotics[16]. The difference in the two groups indicated that the two genera might have a protective effect on the intestine. In addition, the abundances of Bacteroides and Citrobacter in the CAP group were higher than those in the HC group, which indicated that these two species might play an important role in the pathological process of CAP. Studies have found that Citrobacter can take over the cell-cell communication system to trigger colitis in mice[17], and the elevation of Bacteroides abundance in the faeces of CAP patients has been confirmed [5]. Compared with the bacteria in faeces, the bacteria attached to the colonic mucosa are more likely to affect the gene expression of colonic mucosa cells. Based on high-throughput sequencing of biopsy tissue[18], the α diversity of polyps was higher than that of healthy tissue, which indicated that polyps have higher within-habitat diversity than healthy tissue. This phenomenon of increased diversity also appeared in studies on CRC[19], which might suggest that increased diversity of the gut microbiome is not a sign of healthy intestines but rather the excessive growth of various harmful bacteria or archaea in adenoma and cancer development[3]. Studies on the faeces and tumour tissues of CRC patients have shown different results for Bacteroides[20-22]. Yu et al.[23] found that the abundances of Proteobacteria and Fusobacteria were high in tumour tissues of CRC patients, but in this study, there were no significant differences in the two groups of bacteria. Many studies have found that Fusobacteria were enriched in the faeces and tumour tissues of patients with CRC[24], but there were no significant differences in the abundance of Fusobacteria in faeces or adenoma tissues between CAP patients and HCs. The relative content of Fusobacteria in faeces and polyps was low, and it was speculated that the enrichment of Proteobacteria may be related to the degree of tissue abnormality.
Studies have indicated the relationship between the gut microbiota and metabolites in the intestinal tract. The ability of the gut microbiota to produce metabolites, such as butyrate, secondary bile acid and CLA, can vary with gut environment modulation, as has been shown in response to diet. This study aimed to investigate whether there were gene expression differences between CAP patients and healthy volunteers, and the acetic acid and butyric acid contents in the faeces were higher in the CAP group than in the HC group. Butyrate, as a major source of energy for intestinal epithelial cells, can reduce colonic inflammation, induce apoptosis, inhibit tumour cells and prevent CRC development. The anti-proliferative and anti-cancer properties of butyrate have been demonstrated and are probably attributable to the effect of high concentrations of butyrate as a histone deacetylase inhibitor (HDACi)[25]. However, Bultman et al. [26] believed that butyrate is a causative factor of CRC, and a study on APCMin/+MSH2-/- mice fed butyrate showed that the amount of butyrate administered was positively correlated with polyp formation in mice, which might be due to the stimulation of gut microbiota hyperproliferation and mouse intestinal epithelial cell transformation through metabolites. Polyp formation at low concentrations stimulates colonic epithelial cell proliferation[27]. These opposing effects of butyrate have been called the "butyrate paradox". Although the propionic acid content was not significantly different between the two groups, the propionic acid content in the CAP group was increased, and Bacteroides, which is a major contributor to propionate synthesis, was significantly more abundant in the CAP group. In the analysis of DNA from faeces, the expression of butyrate-producing bacterial genes in the CAP group was significantly lower than that in the HC group, but there was no significant difference in DNA between the two groups. The results indicated that the abundance of butyrate-producing bacteria in the faeces of CAP patients was decreased, while the butyric acid content in the CAP group was higher than that in the HC group. Ferrer-Picón Elena et al. observed that a lower stool content of butyrate-producing bacteria was not correlated with the butyrate concentration in IBD patients[28]. The faecal acetate and butyrate concentrations were positively correlated with supplementation with resistant starch and non-starch, which indicated that diet composition and intake influenced the actual SCFA concentrations in the gut[12]. Therefore, the faecal SCFA concentrations does not fully reflect the concentration of SCFAs produced by gut microbiota fermentation; thus, the intestinal health effects need to be carefully considered[29]. SCFAs can effectively reduce the intestine pH, promote glycolysis of food in the intestine and reduce carcinogen absorption, which can reduce CRC risk[30].
Due to the different positions and conformations of the conjugated double bonds, there are multiple isomers of conjugated linoleic acid, and the main isomers are c9,t11-CLA and t10,c12-CLA [31]. These two isomers play different roles in anti-cancer and anti-cardiovascular disease activity. Here, t10,c12-CLA content was found to be increased in the faeces of the HC group, but the difference in c9,t11-CLA content between groups was not statistically significant. CLA has functions such as reducing body fat, restricting tumour development, preventing cardiovascular disease and improving immunity[32]. As a fatty acid that protects the intestine, its anti-tumour properties in vitro and in vivo have been widely recognized[13]. Among the isomers, t10,c12-CLA has functions of reducing body fat, lowering triglyceride content and inhibiting adipocyte differentiation, and t10,c12-CLA was found to be more effective than other isomers in inhibiting tumours. The tumour cell growth inhibition effects were positively correlated with its concentration, and c9,t11-CLA played an important role in immune regulation[33-35]. In addition, t10,c12-CLA content showed a significant decrease in the CAP group, and the decrease in faecal c9,t11-CLA content might increase the risk of intestinal adenomatous polyps. Certain Bifidobacteria species in the gut, as natural colonizers, are capable of converting linoleic acid to c9,t11-CLA, t10,c12-CLA and small amounts of t9,t11-CLA [36]. There was no statistically significant difference in faecal c9,t11-CLA content between the two groups, and the abundance of Bifidobacteria that produce c9,t11-CLA was not significantly different between the two groups.
A high-fat diet strongly stimulates bile acid production, and bile acids are converted to secondary bile acids, deoxycholic acid(DCA) and lithocholic acid(LCA) , after structural modification of bacteria with 7α-dehydroxylating activity in the gut. DCA is the most typical secondary bile acid[15]. Secondary bile acids promote the proliferation of intestinal epithelial cells, induce apoptosis and mutation, and promote cancer progression[37]. There were no significant differences in the levels of any bile acids in our study, but the DCA content was higher in the CAP group than in the HC group. Lu et al.[38] found that faecal chenodeoxycholic acid(CDCA) and DCA contents were significantly increased only in CRC patients, but no significant differences were observed in CAP patients. The gut microbiota converts primary bile acids into secondary bile acids, suggesting that the gut microbiota can affect the composition of secondary bile acids, while changing the secondary bile acid profile could reshape the intestinal bacterial composition[39]. Although there was no significant difference in DCA or CDCA content between groups, the abundance of Bacteroides, which has bile acid-resistant characteristics, was positively correlated with fat and protein intake[26, 40, 41], and the Bacteroides abundance showed a significant increase in the CAP group. The differences in secondary bile acid-producing bacteria were not statistically significant in this study. Mullish et al. [42] found that the BaiCD operon was not present in all bacteria with 7α-dehydroxylating ability, which has been considered an important process for secondary bile acid formation in faeces[13].
Metabolomics provides a qualitative and quantitative method of metabolite in analysis that can complete analysis along with microbiology. Metabolites (small molecules<1500 Da) are cellular metabolism intermediates or end products, that can be produced directly by the host organism or can be derived from various other external sources, such as the diet, microbes, or xenobiotic sources[43]. Biological systems display complex and analytical limitations, and it is not possible to identify all the metabolites present in a specimen. Studies on metabolites and diseases indicate changes in diabetes[44], cardiovascular disease and heart failure[45, 46], autism[47] and anxiety[48]. As research progresses, metagenomic markers can be utilized for early disease diagnosis or cancer screening, and gut microbiota biology can indicate the effectiveness of cancer therapies and has predictive potential[49].