Gastroesophageal reflux disease (GERD) is a common upper gastrointestinal disease in both Western countries and Asia, defined as a condition which develops when the reflux of stomach contents causes troublesome symptoms such as heartburn and regurgitation, and/or complications. Heartburn is defined as a burning sensation in the retrosternal area (behind the breastbone), while regurgitation is defined as the perception of flow of refluxed gastric content into the mouth or hypopharynx (1). As one subtype of GERD, non-erosive reflux disease (NERD) has been commonly defined as the presence of classic GERD symptoms caused by intraesophageally reflux (acidic or weakly acidic), in the absence of visible esophageal mucosal injury during upper endoscopy (2).

From precious studies, NERD has been reported in more than 50% of the Western population and the incidence of NERD has been increasing annually. In Asia, Rosaida and Goh have shown in a carefully studied prospective study that 65.5% of their patients had NERD (3, 4). Typical symptoms associated with NERD include heartburn and regurgitation, as well as extraesophageal manifestations such as nausea, chronic cough, asthma, and hoarseness that can affect the quality of their daily lives, including social functioning, sleeping, and work productivity (5, 6).

Many studies have revealed the relationship between described symptoms of NERD and emotional status. There is a large overlap in the symptomatic spectrum between NERD and somatoform disorders (7). A cross-sectional study revealed that anxiety and depression levels were significantly higher in subjects with GERD (notably in the NERD) than in controls (8). A case-control study also showed that mental factors (anxiety and depression) play important roles in the development of GERD, especially NERD (9). Most of NERD patients with nighttime heartburn have sleep disturbances. These subjects also had more depression, more anxiety, more severe gastrointestinal reflux disease symptoms, and higher prevalence of NERD (10, 11). Actually, psychotherapy combined with medication can relieve clinical symptoms and improve quality of life to varying degrees in patients with NERD (12). The factors that link NERD and psychology are not clear yet, but gut microbiome might play a role.

The numbers of bacteria in the Human Gut Microbiome are estimated to be close to equal to human cells (about 3.9 × 10¹³) (13). The complex bidirectional communication between the gut and the brain is finely orchestrated by different systems, including the endocrine, immune, autonomic, and enteric nervous systems (14). For example, in animal research conducted by Kelly et al., fecal microbiota transplantation from depressed patients to c-depleted rats can induce behavioral and physiological features of depression in the recipient animals, which suggests that the gut microbiome may play a causal role in the development of features of mental disorders (15). Stevens and Goel et al. got the results to support their hypothesis that anxiety and depressive disorders are associated with gut dysbiosis (16).

The underlying mechanisms associated with NERD and emotion were still unclear. Based on the above summary, we assumed that the gut microbiome may be an important part, so we designed this study to test this hypothesis.

A total of 48 subjects were enrolled: Group E: 24 patients with NERD (The duration of illness ranged from 1 month to 7 years), Group C: 24 healthy controls (HC).

GERD includes non-erosive reflux disease (NERD), reflux esophagitis (RE), and Barrett's esophagus, and foreign epidemiological survey data showed that NERD accounts for about 50–70% of GERD (28). Patients with RE and NERD were often combined with mental and psychological abnormalities (29), which have attracted the attention of domestic and foreign scholars in recent years. For this group of patients, psychotherapy combined with medication can relieve clinical symptoms and improve quality of life to varying degrees in patients with NERD (12). In our study, we used several different questionnaires to evaluate the emotional state of patients. Finally, the psychological assessment results showed that above 60% of the subjects had emotional disorders including somatoform disorders, anxiety, and depression, most of them were mild to moderate, but the proportion was significantly higher than that of the general population, which provides similar evidence about a strong connection between the symptoms of NRED and emotional disorders as previous study showed (9, 30, 31). Among them, patients with somatoform disorders were accounting for 50%. Actually, we could boldly assume that some NERD patients were somehow equivalent to be part of the patients with somatoform disorders.

Estimated by previous research, about 10¹⁴ microorganisms reside in the adult gastrointestinal tract, which amounts to 10 times the number of cells in the human body. Most of them consist of bacteria from 500 to 1,000 different species that vary in stability, diversity, and number across different human populations, showing that the gut microbiome is more complex than those found in the mouth, esophagus, stomach, colon, vagina, and skin (32). However, very little data was found in the literature on the association between NERD and the gut microbiome (33). The present study also has provided some new contributions to the field of microbiome in patients with NERD. We have performed a study of the human gut microbiome rather than esophagus microbiome in NERD patients in which little prior knowledge was available. More importantly, the results confirmed that the diversity of gut flora was significantly different between NERD patients and the healthy controls. The gut microbiome of NERD patients was more complex, which may be associated with dysbacteriosis.

Among the gut microbiome, the Firmicutes and Bacteroidetes were the two dominant phylum (34, 35). Our research also got the same result in Figure 3. We found that Firmicutes increased and Bacteroidetes decreased in NERD patients and those with emotional disorders compared with healthy controls. Data from other GI illnesses, such as IBS, which are often accompanied by depressive symptoms, have revealed reduced Bacteroidetes and increased Firmicutes content in the fecal samples of patients with these disorders (36–38). In other aspects, the patients who suffered from heartburn and regurgitation for a long time may had a long-term anti-acid therapy. Long-term PPI use had been shown to decrease Bacteroides and increase Firmicutes in the gut, and meanwhile increase the risk of SIBO (small intestinal bacterial overgrowth) (39). When SIBO happened, food starch, polysaccharides, and fibers fermented to produce a large amount of gas, which increased abdominal pressure and affected gastric emptying, then aggravated regurgitation (40).

Furthermore, the intestinal flora was divided into three categories: beneficial bacteria, conditional pathogenic bacteria, and pathogenic bacteria. Conditional pathogenic bacteria, such as Enterobacteriaceae and Enterococcus, played an adverse role when the homeostasis of the body was broken. The results showed that Unclassified.Ruminococcaceae, Unclassified.Enterobacteriaceae, Blautia, Parabacteroides, Streptococcus, Dorea, Clostridium, Collinsella, Unclassified.Mogibacteriaceae, Eubacterium, Unclassified.TM73, Eggerthella, Adlercreutzia, Escherichia, and Actinomyces were rich in NERD patients compared with healthy individuals. The patients with somatoform disorders also had more Streptococcus but less Synergistetes. To some extent, NERD was also considered as a kind of inflammatory diseases. Bacteria such as Streptococcus, which are present in the human oral cavity, throat, and nasal cavity, increased in the intestine, implying that bacterial translocation, as well as enteric infections, may have occurred (41). Synergistetes was widely found in nature and had a high detection rate in oral diseases. Though it was found in gut microbiome, its proportion was very small and was meaningless. An up-regulation of the abundance of Ruminococcaceae can lead to degradation of the intestinal mucosa, instability of the intestinal barrier, and mild mucosal immune disorders (42). TM7 possibly played a role of inflammatory promoters in inflammatory gastrointestinal diseases. As for other microflora, there were few studies to clarify its specific function and correlation with NERD.

Overall, in recent years, the microbiome-brain-gut axis is fast becoming a topic of interest among researchers, which is vital for maintaining homeostasis, containing neural, humoral, immune, and metabolic pathways. This complex network of communication between the gut microbiota and the brain encompasses the CNS, the autonomic nervous system (ANS), the enteric nervous system (ENS), and the neuroendocrine and neuroimmune systems (32, 43). The dysregulation of microbiome-brain-gut axis has been implicated in various disease states. Current studies have shown that the diversity and types of intestinal flora are associated with anxiety and depression. Our study indicates that NERD patients with psychiatric disorders, especially with somatization disorder, had association with gut microbiome, considering that the microbial community residing in the gut plays an important role in the development of various aspects of brain function including anxiety, mood, cognition, and more recently in sociability.

Exactly, we predicted that microbiome in NERD patients played a role in the pathways of multiple substances metabolism, energy metabolism, Transporters, oxidative phosphorylation, Citrate.cycle.TCA.cycle, and so on, which perhaps effected the function of microbiome-brain-gut axis. Moreover, it was reported ATP-binding cassette (ABC) transporters could confer multidrug resistance by active efflux of intracellular drugs (44). In contact with NERD patients, we could consider that flora disorder may affect drug efficacy.

While we have identified some significant results, our study has certain limitations. First, the composition of the changing microbiome is distinct at different milestones of life. We cannot precisely identify the relationship of early life changes in stool microbiome to what is found in these patients as adults. Second, middle-aged patients were more likely to be enrolled, while young people often refused to participate in our study because of their individual considerations. This led to fewer young patients in the study and the results may be biased while considering all ages. Third, clinical data is limited by the small sample size although the sequencing depth and data amount of the samples are sufficient. The difference of intestinal flora among the groups in our research did not fully represent the composition of intestinal flora in the NERD patients. As we got the results shown in the Figure 5, there was little difference between female and male, patients with or without sleep disorder, which was probably caused by inadequate sample size. In the future, we would expand the sample size for further confirmation. Fourth, we limited our analyses to stool samples as we were interested in the association of gut microbiome rather than the esophageal microbiome as the former has already been reported, which could be divided into two categories, while type II may affect the occurrence of NERD by changing the speed of gastric emptying (45). We would prefer to perform duplications on drug-naïve subjects and compare alterations in NERD patients before and after pharmacotherapy. This present study provides some crucial background knowledge. Finally, it still needs to be further assessed whether our data support the use of probiotics-based interventions as adjuncts to current therapeutic strategies in NERD (46).

The datasets presented in this article are not readily available because it contains confidential personal information and data. Requests to access the datasets should be directed to the corresponding author.

The study was approved by the Ethics Committees of Peking University Shenzhen Hospital, and all the patients signed the informed consent forms.

FY: conceptualization, methodology, investigation, formal analysis, visualization, writing original draft, and revising. X-hX: methodology, investigation, writing – review, and editing. XL: conceptualization, resources, data curation, and writing – review. H-nL: formal analysis and data curation. BZ: conceptualization, resources, supervision, validation, writing – review, and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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