In addition to vomiting, GER is closely related to chronic cough, refractory asthma, recurrent pneumonia, malnutrition, and sudden infant death syndrome. However, its pathogenic mechanism is not fully understood [
1,
2]. Animal models are commonly used to investigate its underlying pathogenesis. In this study, we first established a GER model rat with reference to Erbil Y [
8]. Our results showed that the GER model group had significantly lower esophageal pH than the sham-operated control group and that RE occurred in 74.1% (20/27) in the GER model group and in 0% in the control group, indicating that GER and RE models were successfully established.
In general, acid reflux is thought to be one of the major factors causing esophageal mucosal injury. The severity of mucosal injury depends on the nature of reflux substances, clearance ability of the esophageal mucosa, and the resistance or sensitivity of the mucosal epithelium. The clearance ability of esophageal mucosa is regulated by esophageal motor function, i.e., coordination of esophageal smooth muscle contraction and relaxation. It has been suggested that esophageal inflammation may be associated with esophageal motility disorder. It was reported that neurotransmitters regulating gastrointestinal motility, secretion, and sensation play an important role in the RE pathogenesis. Rats with esophageal reflux have decreased smooth muscle contractile responses to carbachol (receptor mediated) and KCl (non-receptor mediated), as well as decreased relaxation to 5-HT (receptor mediated) [
9].
In this study, we found that the 5-HT concentrations in esophageal tissue were significantly higher in the RE group than in the controls, while the levels in the NERD group were not higher than in the controls, indicating that reflux can increase 5-HT levels in esophageal tissue. 5-HT is mainly synthesized in enterochromaffin cells through tryptophan metabolism. Synthesized 5-HT is stored as secretory granules in the basal membrane of secretory cells, functioning as paracrine molecules. Secretion of 5-HT is affected by many factors, such as local metabolites, hypertonic solution, chemical stimulation, and cell injury [
10]. The increased 5-HT levels in the RE group were possibly due to acid stimulation of the esophageal mucosa. In addition, the injured esophageal mucosa perhaps further promotes 5-HT release. Released 5-HT is rapidly transported by SERT and is taken up again by adjacent cells in which 5-HT is further oxidized by monoamine oxidase into 5-HIAA and is metabolized after binding to UDP-glucuronide. Our results indicate that SERT mRNA levels in both the RE and NERD groups were significantly higher than in the controls. The SERT mRNA level in the RE group was also significantly higher than in the NERD group, suggesting that the generation of esophagitis is related to an increased SERT level. SERT can quickly transport 5-HT from cell gaps into cells, where it is inactivated, leading to decreases in esophageal peristaltic function and reflux clearance ability.
The biological function of 5-HT is exerted by binding to its specific receptor, 5-HTR. Based on the pharmacological parameters, cDNA sequence, and signal transduction mechanisms, 5-HTRs are divided into seven categories: 5-HT
1A-E, 5-HT
2A-C, 5-HT
3, 5-HT
4, 5-HT
5, 5-HT
6, and 5-HT
7. Among these categories, 5-HT
1A, 5-HT
1B, 5-HT
1P, 5-HT
2A, 5-HT
2B, 5-HT
3, 5-HT
4, and 5-HT
7 are expressed in the gastrointestinal tract. 5-HT
4R has been shown to exist in the esophageal tissues of both humans and animals [
11,
12]. Furthermore, activation of 5-HT
4R can increase LES tension and promote esophageal submucosal gland (SMG) secretion [
9,
13]. We found that protein expression of 5-HT
4R was significantly decreased in the RE group compared to both the NERD and control groups. 5-HT
4R agonists, such as gastrointestinal prokinetic drugs, can promote gastrointestinal motility, accelerate clearance of reflux substrates, and reduce reflux symptoms [
14]. Many 5-HT
4R agonists can alleviate RE symptoms. For example, tegaserod, a partial 5-HT
4R agonist, can safely and effectively reduce postprandial esophageal acid exposure by enhancing the esophageal acid clearance capacity, accelerating gastric emptying, and reducing the occurrence of transit lower esophageal sphincter relaxation (TLESR) [
15], promoting bicarbonate and mucus secretion from the SMGs [
13] and adjusting visceral sensitivity, thus reducing reflux, lowering the pain threshold of the esophagus to mechanical expansion, improving esophageal peristalsis, and reducing functional heartburn symptoms [
14,
16,
17]. Another 5-HT
4R agonist, renzapride, can promote acetylcholine (Ach) release from parasympathetic axons, increasing LES contraction and LES tension [
9]. Cisapride, another 5-HT
4R agonist, can increase LES tension and esophageal peristalsis, promoting gastric emptying, reducing the contact of gastric acid and non-acid reflux substances with the esophageal mucosa and decreasing reflux symptoms [
14]. Decreased 5-HT
4R expression, together with increased SERT expression, synergistically weakens 5-HT receptor-mediated signaling, thereby promoting GER occurrence and leading to esophageal mucosal damage. It was also reported that the SERT inhibitor citalopram can reduce esophageal sensitivity to chemical and mechanical stimuli [
18].
A potential limitation of the study is related to the relatively small sample size, which did not allow us to identify the concentration of 5-HT in varying degrees of esophageal mucosal injury. Another limitation is that esophageal smooth muscle peristalsis in the RE group has not been detected. Therefore, further studies are needed to substantiate these findings.