Proteobacteria phylum belongs to Gram-negative bacteria, which is the largest phylum of bacteria and one of the most abundant phyla in the human gastrointestinal microbiota. Its name comes from Proteus, the god capable of shape-shifting in ancient Greek mythology. Proteobacteria phylum is divided into five classes, α, β, γ, δ, and ε, according to rRNA sequences, and the bacteria are highly heterogeneous and can present different morphologies. Within humans, Proteobacteria phylum exists not only in the gastrointestinal tract, but also in the skin, mouth, vagina and other parts, which can be symbiotic bacteria or pathogenic bacteria. An increased abundance of Proteobacteria is a sign of dysbiosis of the gastrointestinal microbiota and can be used as a potential diagnostic criterion for diseases (Shin et al., 2015). Most of the bacteria in Firmicutes phylum belongs to the Gram-positive bacteria, and it’s named for the thicker cell wall of most of the bacteria in the phylum, which is often spherical or rod-shaped under the microscope. Firmicutes phylum can be divided into three classes: anaerobic Clostridium, facultative or aerobic Bacillus, and non-cell wall Hymenomycetes. Actinobacteria phylum is also Gram-positive bacteria, named for the radiating growth of their colonies, which has a higher GC content than Firmicutes phylum. Bacteroidetes phylum is abundant in the gastrointestinal tract of humans and animals, accounting for more than 60% of the total gastrointestinal microbiota of the digestive tract. And it can be divided into three classes, Bacteroidetes, Flavobacteria and Sphingobacillaceae. Bacteroidetes phylum is involved in the fermentation of carbohydrate, the utilization of nitrogenous substances and the biotransformation of bile acids and other steroids in the human gastrointestinal tract.

Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes are known as the “four phyla” of the gastrointestinal microbiota in the human body (Rizzatti et al., 2017). Once their relative abundance or composition proportion in the gastrointestinal tract is abnormal, it will cause dysbiosis of the gastrointestinal microbiota and lead to diseases. In recent years, the development of research technologies such as analytical biology, 16SrDNA high-throughput sequencing, and gene sequencing has facilitated the study of the gastrointestinal microbiota and increased the heat on the correlation between the gastrointestinal microbiota and human health and disease. Table 1 summarizes data from studies investigating gastrointestinal microbiome alterations in FD.

In one study, sequencing of feces from rats in a liver-depression and spleen-deficiency model of FD using 16S rDNA high-throughput sequencing technology revealed that the relative abundance of the Bacteroidetes phylum in the feces of rats with FD was significantly decreased compared to healthy rats, whereas the relative abundance of the Proteobacteria and Firmicutes phylum was significantly increased (Qiu et al., 2017). In another experimental animal study, it was also found that the relative abundance of Bacteroidetes, Lactobacillus and Prevotellaceae in the gastrointestinal tract of FD mice decreased, while the relative abundances of Proteobacteria, Verrucomicrobia, Epsilonbacteraeota, Firmicutes, Lachnospiraceae NK4A136 group, and Lachnospiraceae increased (Zhang et al., 2020). In addition, 16SrRNA sequencing of duodenal mucosal flora of 9 FD patients and nine healthy subjects showed that Streptococcus was the most abundant in the duodenal mucosa of both FD patients and healthy subjects, but there was a significant decrease in the relative abundance of Actinomycete, Atopobium Collin, Leptotrichia Trevisan, Prevotella, and Veillonella in the duodenal mucosa of FD patients compared to normal subjects. Moreover, the total relative abundance of bacteria was positively correlated with the severity of clinical symptoms of the patients (Zhong et al., 2017). In one study, FD patients were found to have significantly increased the phylum Firmicutes and Streptococcus in the upper gastrointestinal tract compared to healthy subjects, and the relative abundance of Streptococcus was positively correlated with upper gastrointestinal symptoms (Fukui et al., 2020). In another study, it was shown that FD patients had reduced abundance of Prevotella and increased abundance of Bifidobacterium and Clostridium compared to healthy subjects, and that the relative abundance of Prevotella was negatively correlated with the severity of PDS symptoms (Nakae et al., 2016).

In conclusion, it is evident that the disturbance in the relative abundance and composition of the microbiota in the gastrointestinal tract is important for the process of FD occurrence and progression. In addition, different segments of gastrointestinal tract contain different microbiota. Studies have shown that human colon segments are dominated by anaerobic bacteria such as Bacteroidetes and Lachnospiraceae, while small intestine segments are dominated by parthenogenic anaerobic bacteria (Frank et al., 2007). Compared with healthy controls, FD patients not only had different gastrointestinal microbiota, but also had different oral microbiota abundance and composition. Proteobacteria were the dominant bacteria in FD patients’ saliva, while Bacteroidetes were the dominant bacteria in healthy controls. According to 16SrRNA sequencing of saliva, the abundance of Spirochaetes in FD patients was higher than that in healthy controls, while the abundance of Fusobacteria, TM7 and Proteobacteria was lower than that in healthy controls, and the levels of Kingella and Abiotrophia genus levels were also significantly different (Liu et al., 2021).

The gastrointestinal microbiota has a complex metabolic process in the human body. It not only provides itself with the necessary energy for growth and reproduction, but also can use the intestinal contents and the endogenous mucus secreted by the intestinal epithelium to produce a variety of metabolites, including short-chain fatty acids (ScFAs), cholic acid, choline metabolites, phenols, lipids, carbohydrates, etc. These metabolites may be harmful or beneficial to the human body, and are closely related to human health and the occurrence and progression of many diseases. Table 2 summarizes data from studies investigating metabolites of the gastrointestinal microbiota.

The metabolites of ScFAs include formic acid, acetic acid, propionic acid, butyric acid, etc. Formic acid is less abundant in the intestinal tract, while the content of the latter three accounts for more than 90% of the total ScFAs in the intestinal tract (Ríos-Covián et al., 2016). ScFAs are mainly produced by the Clostridium group of Firmicutes phylum, as well as by Lactobacillus, Bifidobacterium, Eubacteriaceae and Fecal bacteria using some dietary fiber, resistant starch, oligosaccharides and other compounds that are not easy to digest in the intestinal tract (Markowiak-Kopeć and Śliżewska, 2020). Acetic acid is the metabolite produced by fermentation of most bacteria, propionic acid is the main metabolite of Bacteroidetes, and butyric acid is the main metabolite of Firmicutes. Studies have shown that ScFAs can regulate the pH value in the intestine, promote the absorption of water, sodium, calcium, magnesium and other substances, and provide more than 70% of the energy for the intestinal epithelial cells, especially butyric acid (Serpa et al., 2010). ScFAs are also able to inhibit the multiplication and growth of pathogenic bacteria and the activity of intestinal inflammatory mediators, thus playing an anti-inflammatory role in the intestinal tract (Havenaar, 2011). In recent years, studies on the pathogenesis of FD have shown that the damage of duodenal mucosal barrier and immune activation played an important role in the pathogenesis of FD, and more than 40% of FD patients had microinflammatory cell infiltration in the duodenum (Nojkov et al., 2020). In addition, gastrointestinal motility disorders is an important pathogenesis of FD. And interstitial cells of Cajal (ICCs) are the pacemaker cells of gastrointestinal tract, which can form SIP syncytium structures with surrounding platelet-derived growth factor receptor α-positive cells (PDGFRα⁺) and smooth muscle cells (SMCs) through tight intercellular gap junctions (Baker et al., 2013). It can also form a nerve fibers-ICCs-SMCs network with the surrounding enteric nervous system and SMCs through tight intercellular gap junctions, which plays an important role in regulating the pacing and slow wave propagation of the gastrointestinal tract (Sanders et al., 2014). Studies have shown that ScFAs are able to maintain the integrity of gap junctions in the intestine.

Lipid metabolites include cholesterol, lipopolysaccharide (LPS), peptidoglycan, and sphingolipids, which are mainly produced by Bifidobacterium, Lactobacillus, Enterobacteriaceae and Clostridium. Studies have shown that lipid metabolites can affect intestinal permeability and intestinal immunity. LPS is generally released by the death lysis of Gram-negative bacteria, and it stimulates tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), interferon-gamma (IFNγ), interleukin-8 (IL-8) and other inflammatory factors are released to disrupt the body’s immune system and induce inflammatory responses (Alexandrov et al., 2020). Sphingolipids can be produced by the intestinal symbiotic bacteria Bacteroidetes and Prevotellaceae. It has been found in animal studies that sphingolipids can also aggravate intestinal inflammation (Brown et al., 2019). Indole-derived metabolites are produced by the fermentation of Clostridium sporogenes and Escherichia coli. Such metabolites are able to participate in the regulation of gastrointestinal disorders by regulating the brain-gut axis and protecting against stress-induced damage in the gastrointestinal tract. Tryptophan is a key monoamine neurotransmitter involved in the regulation of central neurotransmission and intestinal physiological functions, and studies have shown that the gastrointestinal microbiota can regulate the brai006E-gut axis through tryptophan metabolism (Gao et al., 2019; Gao et al., 2020).

The normal intestine is protected by a biological barrier that separates the intestinal luminal contents from the internal environment of the organism and prevents the invasion of foreign bacteria, which is called the intestinal mucosal barrier. It is a semi-permeable barrier that allows the absorption and transport of nutrients but not the entry of harmful substances, luminal antigens and pathogens, and plays an important role in a variety of physiological functions such as digestion, absorption and metabolism (Farré et al., 2020). The composition of the intestinal mucosal barrier includes the mucus layer of the intestine, intestinal epithelial cells, microbiota in the intestine and antimicrobial peptides secreted by intestinal epithelial cells, etc. The mucus layer is located in the innermost layer of the intestinal cavity, which on the one hand can facilitate the downward movement of the intestinal contents down the intestine, and on the other hand separates the intestinal contents from the intestinal epithelial cells, which can protect the intestinal epithelium from acids, digestive enzymes, and pathogenic bacteria in the intestinal lumen (Paone and Cani, 2020). When the abundance and composition of the gastrointestinal microbiota are altered, the number of beneficial bacteria in the intestine decreases and the number of pathogenic bacteria increases, and pathogenic bacteria, as well as the endotoxins released by them, can invade the intestinal mucosa, which can damage the intestinal mucosal barrier and cause increased permeability of the intestine. Studies have shown that the gastrointestinal microbiota can affect the barrier function of the intestinal mucosa directly by stimulating the proliferation of epithelial cells or inducing the secretion of cytokines by epithelial cells, and indirectly by synthesizing essential nutrients, vitamins and ScFAs, which are energy sources for intestinal epithelial cells.

Damage to the intestinal mucosal barrier can cause increased permeability of the intestinal mucosa and reduced blockage of harmful substances by the intestinal mucosal barrier, which is one of the important mechanisms in the pathogenesis of FD (Taki et al., 2019). In a pilot study using measurement of baseline impedance to assess the integrity of the small intestinal mucosa, baseline impedance was found to be significantly lower in the duodenum and jejunum of patients with dyspepsia compared to healthy controls, indicating that patients with dyspepsia have impaired small intestinal mucosal integrity and increased permeability (Nakagawa et al., 2020). In addition, tight junction proteins play an important role in the barrier function of the duodenal mucosa, and tight junction proteins such as ZO-1 and CX43 are commonly used as indicators to assess the barrier function and permeability function of the intestinal epithelial mucosa (Oshima and Miwa, 2016). Studies show that monobutyric acid and monovaleric acid in ScFAs can upregulate the expression of the tight junction protein ZO-1 and thus protect the intestinal mucosal barrier (Nguyen et al., 2020). Intestinal epithelial cells are an important component of the intestinal mucosal barrier and are differentiated from stem cells at the base of the intestinal mucosal crypts, and the maintenance and repair of the intestinal mucosal barrier depends on the normal proliferation and differentiation of these stem cells (You et al., 2021). The Wnt/β-catenin signaling pathway is a key regulator of intestinal epithelial stem cells and plays an important regulatory role in the proliferation and maintenance of intestinal epithelial stem cells (Koch, 2017). Studies have shown that Citro Bacter and Salmonella can cause excessive proliferation of stem cells at the base of the intestinal mucosal crypts in mice and instead disrupt the intestinal epithelial barrier, whereas Lactobacillus reuteri and Lactobacillus acidophilus can protect the integrity of the intestinal epithelial barrier and maintain the intestinal mucosal barrier function by modestly regulating the proliferation of intestinal epithelial cells and increasing the secretion of antimicrobial peptides through activation of the Wnt/β-catenin signaling pathway (Wu et al., 2020).

Another important role of the gastrointestinal microbiota is to shape the intestinal immune response as well as to initiate systemic innate immunity. Recent studies have confirmed that immune activation of the duodenal mucosa plays an important role in the development and progression of FD, with mild inflammatory cell infiltration of the duodenum present in upwards of 40% of FD patients (Nojkov et al., 2020). Dysbiosis of the gastrointestinal microbiota can cause abnormalities in the intestinal barrier and function, which can activate the intestinal immune response and trigger gastrointestinal diseases such as FD. A one-year follow-up of patients with gastroenteritis caused by Salmonella infection revealed a significant increase in the probability of FD (Mearin et al., 2005), and an increase in the number of eosinophils and mast cells was evident in the gastric mucosa and duodenal mucosa of FD patients after infection (Kindt et al., 2009). An animal study showed that gut flora feeding germ-free mice drove the development of B lymphocytes and T lymphocytes in mice, and also reduced interleukin-4 (IL-4) production and increased interleukin-12 (IL-12), IL-10 and IFNγ production (Hrncir et al., 2008). Another study showed that nuclear factor-κB (NF-κB) is a key regulator of immune function and has an important role in the pathogenesis of autoimmune diseases as well as inflammatory diseases (Sun, 2017). Activation of the Akt/NF-κB signaling pathway can lead to the release of inflammatory factors in the intestinal barrier, resulting in intestinal inflammation. In contrast, the intestinal microbial metabolites ScFAs can inhibit NF-κB transfer and suppress the secretion of inflammation-related factors interleukin-2 (IL-2), interleukin-6 (IL-6), and TNF-α.

In addition, small intestinal bacterial overgrowth (SIBO) characterized by an increased concentration of bacteria in the small intestine, is also an important mechanism in the pathogenesis of FD (Gurusamy et al., 2021). Usually, the bacterial concentration in the small intestine is lower than 103 colony forming units (CFU)/mL, and the bacterial species is predominantly Gram-positive. Bacterial concentrations higher than 105 CFU/ml in the small intestine are diagnosed as SIBO, and also in the intestine of patients with the presence of SIBO, bacterial species are found to be altered, often with large numbers of Gram-negative and anaerobic bacteria (Losurdo et al., 2020). Studies have shown that there are two possible mechanisms by which SIBO triggers FD: one may be the direct damage to the integrity of the intestinal mucosa by overgrown bacteria, which can lead to increased intestinal mucosal permeability, the other may be the activation of the intestinal immune response by metabolites produced by these bacteria, causing the release of inflammatory factors or immune mediators that trigger the intestinal inflammatory response (Gasbarrini et al., 2007; Lauritano et al., 2010).

The physiological activity of the gastrointestinal tract is regulated by the enteric nervous system, the central nervous system and the autonomic nervous system in multiple ways, among which there is a bidirectional regulation mechanism between the gut and the brain. The interaction between the microbiota in the intestine, the intestine and the central nervous system constitutes the microbial-gut-brain axis, which refers to the phenomenon that changes in the microbiota in the intestine can cause changes in various physiopathological activities in the intestine, and then transmit the stimuli to the central nervous system, which in turn can regulate various physiopathological activities in the intestine. Under stress conditions, alterations in central nervous system activity can also regulate gastrointestinal motility, immunity, secretion, and other functions as well as affect the composition of the gastrointestinal microbiota (Wang and Kasper, 2014). ScFAs, metabolites of the gastrointestinal microbiota, are often considered as key mediators of communication between the central nervous system and the intestine, and ScFAs induce intestinal secretion of glucagon-like peptide 1 (GLP1), γ- aminobutyric acid (GABA), and other hormones that can transmit stimuli to the central nervous system via the circulatory system or the vagal pathway (Silva et al., 2020). In addition, the gastrointestinal microbiota such as Bacteroides, Bifidobacterium, Escherichia coli can also overproduce neurotransmitters such as GABA (Strandwitz et al., 2019).

In one study, it was found that feeding with a high-fat diet can lead to dysbiosis of the gastrointestinal microbiota and stimulate Toll-like receptors 4 (TLR4) inflammation on microglia by increasing LPS ectopic and activating LPS-binding proteins (LBP) activation of the pathway (Jamar et al., 2021). In another animal study, TLRs expression was elevated and gastrointestinal motility was decreased after stimulation of mice using antimicrobial drugs, suggesting that the gastrointestinal microbiota can regulate gastrointestinal motility function stimulating neuroimmunity through activation of TLRs inflammatory pathway (Grasa et al., 2015).

The process of FD is closely related to factors such as gastrointestinal motility disorders and gastrointestinal hypersensitivity, which in turn are closely related to the gastrointestinal microbiota. 5-Hydroxytryptamine (5-HT), as an immunomodulatory factor, is present in large quantities in the gastrointestinal tract and is involved in the regulation of gastrointestinal motility and sensation (Fu et al., 2019). Dysbiosis of the gastrointestinal microbiota and some of the metabolites it produces can lead to an increase in the number of mast cells in the intestinal mucosa, thus causing the release of active substances such as 5-HT and histamine in the gastrointestinal tract, which are involved in the regulation of gastrointestinal dynamics and visceral sensitivity. It has been shown that the increased abundance of Prevotella, Lactobacillus and Alistipes in the intestine is positively correlated with the concentration of saturated long-chain fatty acids (SLCFAs), and the increased concentration of SLCFAs in the intestine can promote the contraction of intestinal smooth muscle, which causes increased intestinal motility (Zhao et al., 2018). The injection of bacteria into germ-free rats induces an increase in gastrointestinal slow-wave activity.

In addition, there was a correlation between the gastrointestinal microbiota and the visceral pain pathway, where the presence of inflammatory stimuli in germ-free mice was found to diminish pain perception, while the use of probiotics effectively improved the pain response in mice; indicating that the presence of the gastrointestinal microbiota can influence the onset and progression of gastrointestinal disease processes not only by affecting gastrointestinal myoelectric activity, but also by affecting visceral nociception, among other pathways (Chichlowski and Rudolph, 2015).