Intestinal microbes affect bile acid metabolism involved in gallstone formation

Background: The gut microbiota participates in the metabolism of substances and energy, promotes the development and maturation of the immune system, forms the mucosal barrier, and protects the host from pathogen attacks. Although the pathogenesis of cholesterol gallstones is still not clear, studies have suggested that gut microbiota dysbiosis plays an important role in their formation. Methods: Microbial DNA from faeces of normal control patients and those of patients with calculi was subjected to 16S rRNA gene sequencing to detect gene expression changes in intestinal microbes. ELISA kits were used to measure free bile acids, secondary bile acids and coprostanol according to the manufacturer’s instructions. The relationship between flora and their metabolites was then analysed. Results: In the gallstone group, the diversity of intestinal bacteria and the abundances of certain phylogroups were significantly decreased (p<0.05), especially Firmicutes (p<0.05), the largest phylum represented by the gut microbiota. This study found an increase in free bile acids (p<0.001) and secondary bile acids (p<0.01) in the enterohepatic circulation. Bile salt hydrolase activity was not related to the abundances of BSH-active bacteria. 7a-dehydroxylating gut bacteria were significantly increased (p<0.01), whereas cholesterol-lowering bacteria were significantly reduced (p<0.05). The Ruminococcus gnavus group could be used as a biomarker to distinguish the gallstone group from the control group. Conclusion: Substantial changes in the intestinal flora of patients with gallstones were observed, which affect cholesterol and bile acid metabolism and can lead to

of adults have gallstones, which cause great suffering [1]. Most (90%) gallstones are cholesterol gallstones [2]. The pathogenesis of cholesterol gallstones is still not clear, though studies suggest that gut microbiota dysbiosis plays an important role in their formation [3]. The gut microbiota participates in the metabolism of substances and energy, promotes the development and maturation of the immune system, forms the mucosal barrier, and protects the host from pathogen attacks [4].
Under normal circumstances, gut microbes form a mutual symbiotic relationship with the host, and maintaining the homeostasis of the intestinal microenvironment is conducive to homeostasis regulation in the human body [5]. If this balance is disrupted, it may lead to the development of chronic conditions, including colonic inflammation, colon cancer, gallstones [6], and such metabolic diseases as obesity and diabetes [7].
Current studies have shown that changes in gut microbiota are associated with the formation of cholesterol gallstones. Wang et al. [3] investigated the faeces of mice with cholesterol gallstones using 16S ribosomal RNA (16s rRNA) gene sequencing and found that in the intestinal tract of mice, the Firmicutes content and the Firmicutes/Bacteroidetes (F/B) ratio decreased, the abundances and diversity of the microbiota also significantly decreased, and a lithogenic diet (LD) reshaped the abundances of gut microbiota at different levels. Keren et al. found that in patients with gallstones, the microbial diversity was reduced and the total bile acid concentration in faeces was increased [8]. Bile acid is not only a simple fat emulsion but also an important signalling regulator molecule, as it can activate nuclear farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR), and G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5); it can affect host metabolism of energy, lipids, and glucose; and it can regulate its own synthesis and cholesterol degradation [9]. The gut microbiota can convert cholesterol into coprostanol, which is not easily absorbed or esterified; therefore, it is excreted from the body [10]. The gut microbiota can also shape the development of the intestinal immune system [11,12].
This study analysed the changes in the structure and function of the gut microbiota and the relationships between the gut microbiota and bile acids and cholesterol in patients with gallstones to provide evidence for the correlation between gut microbiota dysbiosis and gallstone formation. as follows. The length of the window was 50 bp. If the mean quality value within the window was lower than 20, the back bases were truncated from the window, and the tags whose quality control was shorter than 75% of the length of the tags were filtered to obtain high-quality tag data, i.e., clean tags. The principle of UCHIME is shown in the figure below. In the first step, the query sequence is split into chunks that do not overlap, and then the chunks are compared with the database. The second step is to select the best match for each chunk in the database and ultimately select the 2 best parent sequences. In the third step, the query sequence to be detected is compared with those 2 parent sequences. If there is a sequence in each of the 2 parent sequences whose similarity with the query sequence is over 80%, the query is determined to be a chimaera.
Data analysis: Operational taxonomic unit (OTU) analysis clustered sequences at the 8 97% similarity level (USEARCH [16], version 10.0), and 0.005% of all sequences was used as the threshold to filter OTUs [17]. Species annotation and taxonomic analysis: the 16S:Silva rRNA gene database was selected [18]

Statistics
Data are expressed as the mean ± standard deviation (SD). If the data were normally distributed, the differences between 2 groups were compared using the t test. If the data were not normally distributed, the difference was compared using the Mann-Whitney U test. P<0.05 was considered statistically significant. The relationship between tree bile acids, secondary bile acids, and coprostanol with bacteria was separately analysed by Spearman's correlation.

Results from the OTU-based analysis
In all, 336 public OTUs from two groups of intersections are publicly available. The gallstone group had 8 unique bacteria, while the control group had 6 unique bacteria ( Figure 1A). The bacterial species of each sample in the gallstone group were essentially the same, and 24 OTUs were publicly available ( Figure 1B). The OTU of each sample in the control group was also basically the same, and 19 OTUs were in the public database ( Figure 1C). The results showed that the individual differences within the group were small.
Decreased abundance and diversity of intestinal bacteria in the gallstone group The rarefaction curve was used to verify that the amount of sequencing data was sufficient to reflect the species diversity in the sample and that the data could indirectly reflect the abundances of the species in the sample. The results showed that as the number of sequences increased, the curve tended to be flat, indicating that the sequencing of each sample was sufficient to reflect the species diversity in the sample ( Figure 2A) and that the species richness in the gallstone group was lower than that in the control group using the same number of sequences ( Figure   2B). The rank abundance curve was mainly used to explain the abundances and uniformity of the species contained in the sample. The results showed that the rank abundance curve of most samples in the gallstone group was shorter than that in the control group in the horizontal direction, and the shape of the curve was steeper ( Figure 2C), indicating that the species richness in the gallstone group was reduced and that the species uniformity was low ( Figure 2D). The Shannon index was used to reflect the microbial diversity of each sample using different numbers of sequences.
As the number of sequences increased, the curve tended to be flat, indicating that the amount of sequencing data was large enough to reflect the biological diversity of the sample ( Figure 2E). Interestingly, the Shannon index of the gallstone group was significantly smaller than that of the control group (3.04±0.38<3.28±0.31, p = 0.012) ( Figure 2F,G), while the Simpson index was significantly greater in the gallstone group than in the control group (0.11±0.04>0.08±0.03, p = 0.015), demonstrating a significant reduction in species diversity in the gallstone group ( Figure 2H).
β-Diversity analysis of the microbial composition in the 2 groups The β-diversity of the gut microbiota in the two groups was measured by principal coordinates analysis (PCoA) and non-metric multi-dimensional scaling (NMDS) analysis, which show the degree of similarity between the two microbial communities. The results revealed significant differences in species presence and evolution between the two groups ( Figure 3).

Abundance and structural changes in gut microbiota at different levels in the gallstone group
At the phylum level, the major bacterial phyla in both groups were Firmicutes, Bacteroidetes, and Proteobacteria. These 3 major phyla accounted for 95.6% of the bacteria in the control group and 92.0% in the gallstone group, and the most abundant phylum in both groups was Firmicutes. However, compared with the control group, the number of Firmicutes was significantly reduced in the gallstone group. In the gallstone group, the Firmicutes/Bacteroidetes (F/B) ratio was also significantly decreased. Cyanobacteria, Fusobacteria, and Spirochaetes were significantly increased in the gallstone group ( Figure 4A). The structure and relative abundance of the flora in the two groups were shown at the class, order, family, genus, and species levels ( Figure 4B). At the genus level, the gallstone and control groups were compared, and the bacteria with significantly increased abundance ( Figure 4C) and the bacteria with significantly reduced abundance ( Figure 4D) were determined and are shown in the figures.

LEfSe analysis between groups
LEfSe analysis aided in determining the biomarkers that were significantly differentially present among the different groups. The figure generated by the LEfSe analysis showed the taxonomic groups with the largest difference between the two groups at different levels ( Figure 5A). The histogram showed the observed differences in the 7 phylotypes between the gallstone and control groups ( Figure   5B). At the genus level, 3 genera could be used as biomarkers to distinguish between the control group and the gallstone group. In the gallstone group, Ruminococcus gnavus could be used as a biomarker, while in the control group, Prevotella 9 and Faecalibacterium could be used as biomarkers.
Abundance changes in gut microbiota with BSH activity at different levels in the gallstone group Using the literature and databases, the gut microbiota with bile salt hydrolase (BSH) activity were screened at different levels. The results showed that at the phylum, class, order, family and genus levels, the abundance of bacteria with BSH activity was reduced in the gallstone group compared with the control group, but this difference was not statistically significant ( Figure 6A). At these different levels, the abundance of bacteria with BSH activity in the gallstone group was also not correlated with free bile acids ( Figure 6B). Interestingly, a significant increase in intestinal free bile acids was observed in the gallstone group ( Figure 7H).Bile salt hydrolase activity was not related to the abundances of BSH-active bacteria.
Relationship between abundance changes in 7α-dehydroxylating gut 13 bacteria and secondary bile acids Enzymes involved in 7α-dehydroxylation are encoded by the bile acid-inducible gene (bai).. Currently only the Clostridium genus and some strains of Lachnospiraceae and Peptostreptococcaceae are known to have 7α-dehydroxylation activity, and these microbes account for only a small portion of the gut microbiota. This study demonstrated a significant increase in Clostridium in the gallstone group ( Figure   7A), while the abundance of Lachnospiraceae and Peptostreptococcaceae was not significantly increased ( Figure 7B, C). The results also showed a significant increase in secondary bile acids in the gallstone group ( Figure 7I), and Clostridium was positively correlated with secondary bile acids ( Figure 7D).

Abundance changes in intestinal bacteria with cholesterol-lowering effects
Small amounts of cholesterol-lowering strains can be isolated from animal faeces (7,12). All these strains exhibit similar properties and are currently attributed to the Eubacterium genus. In this study, compared with the control group, the abundance of Eubacterium was significantly decreased in the gallstone group. At the species level, the relative abundance of coprostanoligenic species ( Figure 7F) was significantly reduced. Moreover, coprostanol levels were significantly higher in the gallstone group than in the control group ( Figure 7J), and Eubacterium was positively correlated with coprostanol ( Figure 7G). Coprostanol is a conversion product of cholesterol. The abundance of these cholesterol-catalysed bacteria may result in reduced cholesterol conversion and increased cholesterol absorption. 14 Wang et al. [3] showed that gut microbiota at different levels changed with gallstone formation, indicating that gut microbiota dysbiosis plays an important role in the promotion of gallstone formation. Fremont-Rahl et al. found that compared to the control group, the incidence rate of cholesterol gallstones in sterile mice fed LD significantly increased. They believed that changes in gut microbiota could increase the occurrence of cholesterol gallstones by changes in gallbladder movement, local inflammation, and increases in mucin gene expression and mucin gel accumulation [22]. Our data also indicated that gallstone formation was associated with gut microbiota dysbiosis. In the gallstone group, the diversity of intestinal bacteria and the abundances of certain phylogroups significantly decreased ( Figure), especially could inhibit CYP7A1 through the FFX-FGF19 pathway. Since CYP7A1 is a key enzyme in cholesterol elimination, the inhibition of CYP7A1 could cause the accumulation of cholesterol in the liver [28]. Decreases in gut microbiota in patients with gallstones can increase the hepatic cholesterol secretion [24], which may lead to oversaturation of bile cholesterol and induction of cholesterol stone formation. Of course, this hypothesis has not been confirmed, and further validation is needed.

DISCUSSION
The intestinal bacteria uncouple the bound bile acid into free bile acid through BSH and then convert the free bile acids into secondary bile acids through 7αdehydroxylation [29]. Currently, only the Clostridium genus [30] and some strains of Lachnospiraceae and Peptostreptococcaceae [31] in intestinal bacteria are known to have 7α-dehydroxylation activity, and these bacteria are known as 7αdehydroxylating bacteria [32]. 7α-Dehydroxylating bacteria have bile acid-inducible (bai) genes, which encode 7α-dehydroxylase enzymes. There are 3 key enzymes in this pathway, which are encoded by baiA2, baiB, and baiE and produce the bile acid 3α-hydroxysteroid dehydrogenase, bile acid coenzyme A-linked enzyme, and bile acid 7-alpha dehydrase, respectively [33]. In the presence of cholic acids, the expression of bai can be upregulated [32].
The abundances and diversity of 7α-dehydroxylating bacteria in the intestinal tract of patients with gallstones are poorly understood, and these bacteria account for a small portion (<1%) of the total gut microbiota. However, the effects of these lowabundance gut microbiota on the host may be enormous, and they can increase the secondary bile acids in the intestinal tract, such as deoxycholic acid (DCA) and lithocholic acid (LCA). These secondary bile acids return to the liver through the enterohepatic circulation. Because 7α-hydroxylation does not occur with secondary bile acids in the human liver, bile acids can accumulate to a very high level in 16 human bile [34]. In contrast, 7α-hydroxylation can occur on secondary bile acids in rodents to maintain the hydrophilic bile acid pool size [35].
The formation of secondary bile acids by gut microbes has been a topic of great biomedical significance. 7α-Dehydroxylating bacteria convert primary biliary acids to secondary bile acids to inhibit the growth of Clostridium difficile in the body.
Monocytes/macrophages, dendritic cells, and natural killer T cells express the functional bile acid receptor FXR. Under physiological concentrations, bile acids, especially hydrophilic secondary bile acids, play a role in the assembly of NLRP3 inflammasomes as negative regulators, and this effect requires functional FXR [36,37]. However, in the presence of CDCA, DCA and LCA can inhibit FXR [38]. Our study found an increase in free bile acids and secondary bile acids in the enterohepatic circulation, which may lead to the inhibition of functional FXR in immune cells. This will promote the release of inflammatory cytokines, which will cause local inflammation.
GPBAR1 is a bile acid receptor that is expressed by innate immune cells, macrophages, and natural killer T cells and is considered necessary to maintain immune homeostasis in the intestine and liver [39,40,41]. In mice and humans, GPBAR1 is also abundant in epithelial cells of the gallbladder and gallbladder smooth muscle. The activation of GPBAR1 in epithelial cells promotes bile secretion, but the activation of the GPBAR1 receptor in smooth muscle cells leads to relaxation of smooth muscle, resulting in gallbladder filling [42]. Application of an endogenous or synthetic TGR5 ligand significantly increases gallbladder volume by more than two-fold [43]. The secondary bile acids DCA and LCA are potent GPBAR1 agonists.
The increase in the concentration of secondary bile acids promotes gallstone formation. Our data showed that the abundances of 7α-dehydroxylating bacteria, such as Clostridium, Lachnospiraceae and Peptostreptococcaceae all increased, especially Clostridium. We detected significant increases in secondary bile acids in patients with gallstones. The secondary bile acid content was positively correlated with the relative abundance of Clostridium. Berr et al. showed that the increase in secondary bile acids promoted the formation of cholesterol gallstones [44,45,46].
Our finding of a significantly increased abundance of Clostridium is consistent with the study by FRIEDER et al. [44]. However, the increases in Lachnospiraceae and Peptostreptococcaceae were not significant, which may be related to the increase in free bile acids in the intestine.
BSH-active bacteria can convert bound bile acids into free bile acids. Our study showed that the intestinal free bile acids significantly increased in the gallstone group ( Figure), indicating that intestinal BSH activity was significantly increased.
We analysed the abundances of BSH-active bacteria and found that the abundances of BSH-active bacteria at different levels were not significantly different between the control group and the gallstone group, indicating that BSH activity was not related to the abundances of BSH-active bacteria [47]. Ziwei et al. detected the enzyme activity of different BSH systems and found that BSH-T3 showed the highest enzyme activity, which was only found in Lactobacillus [21].
Our study also indicated that the Lactobacillus abundance was positively correlated with free bile acid concentration ( Figure 7K). Ridlon  This indicates that gallstone formation may be related to the reduction in  A At the phylum, class, order, family and genus levels, the abundance of bacteria with BSH a 31 Figure 7 A The relative abundance of Clostridium was significantly increased. B The abundance of Lac