ENaCδ aggravates gastroesophageal reflux disease via PI3K/AKT/mTOR: Human umbilical cord mesenchymal stem cells restore barrier function

Abstract

BACKGROUND

Gastroesophageal reflux disease (GERD) is a widely occurring digestive disorder associated with persistent esophageal inflammation and compromised epithelial barrier integrity. Increasing evidence suggests that ion channels, particularly the epithelial sodium channel (ENaC), play a role in mucosal homeostasis and inflammatory regulation. The δ subunit of ENaC (ENaCδ), which is expressed in human tissues but absent in conventional rodent models, may contribute to disease pathogenesis. However, its role in GERD remains unclear, and current treatments such as proton pump inhibitors are often insufficient in addressing inflammation and barrier dysfunction. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have emerged as a promising therapeutic strategy due to their anti-inflammatory and tissue-repair properties.

AIM

To investigate the role of human ENaCδ in the development and progression of GERD using a humanized ENaCδ (hENaCδ) mouse model, and to evaluate the therapeutic potential of hUC-MSCs in alleviating esophageal inflammation and restoring epithelial barrier function.

METHODS

In the present study, a total of 48 mice were randomly assigned to eight experimental groups to investigate the role of hENaCδ in GERD and to evaluate the therapeutic potential of hUC-MSCs. A hENaCδ mouse model was established using CRISPR/Cas9 technology, followed by esophageal acid perfusion to induce GERD.

RESULTS

hENaCδ exacerbated inflammatory infiltration in the submucosa of the esophagus in GERD mice, promoted the expression of interleukin (IL)-1, IL-1β, IL-17 and inducible nitric oxide synthase in mice (P < 0.001), upregulated the relative expression and phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) (P < 0.05), and downregulated the expression of myosin phosphatase target subunit 1 (MYPT1) and claudin-1 (P < 0.001). hUC-MSCs could alleviate the pathological changes of GERD mice with hENaCδ, inhibit the expression of IL-1, IL-1β, IL-17 and inducible nitric oxide synthase in mice (P < 0.001), downregulate the expression of PI3K, AKT and mTOR in esophageal tissue (P < 0.001), and upregulate the expression of MYPT1 and claudin-1 (P < 0.001). Although both hUC-MSCs and proton pump inhibitor treatments could effectively alleviate the inflammatory response of GERD, hUC-MSCs had a more significant effect in inhibiting IL-17 and restoring the expression of MYPT1 and claudin-1.

CONCLUSION

hENaCδ exacerbates GERD-related inflammation and barrier injury in mice by activating the PI3K/AKT/mTOR pathway and downregulating MYPT1 and claudin-1, while hUC-MSCs treatment can effectively block this pathway and reduce inflammation and barrier injury.

Key Words: Human umbilical cord mesenchymal stem cells; Gastroesophageal reflux disease; Humanized epithelial sodium channel δ mouse model; Epithelial sodium channel; Proton pump inhibitor

Core Tip: We developed a CRISPR/Cas9-generated humanized epithelial sodium channel δ mouse model to better mimic human gastroesophageal reflux disease. Humanized epithelial sodium channel δ aggravated acid-induced esophageal inflammation by activating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway, increasing interleukin 1 (IL-1), IL-1β, IL-17 and inducible nitric oxide synthase, and impairing barrier and contractile regulators (claudin-1 and myosin phosphatase target subunit 1). Human umbilical cord mesenchymal stem cells reversed these changes and outperformed proton pump inhibitor in suppressing IL-17 and restoring claudin-1/myosin phosphatase target subunit 1.

INTRODUCTION

Gastroesophageal reflux disease (GERD) is a chronic disorder defined by the retrograde flow of gastric contents into the esophagus and, in some cases, into extra-esophageal sites such as the oral cavity, larynx, or lungs. It is a common gastrointestinal disorder that primarily leads to inflammation and damage of the esophageal mucosa[1]. GERD presents with a broad spectrum of clinical features, including typical esophageal symptoms such as heartburn and regurgitation, complications like erosive esophagitis, Barrett’s esophagus, and esophageal adenocarcinoma, as well as extra-esophageal manifestations, including hoarseness, chronic cough, asthma, and sore throat[2].

The global burden of GERD has increased markedly over recent decades. The number of affected individuals rose from 450.76 million in 1990 to 825.60 million in 2021. The highest case burden is observed in individuals aged 35-39 years, and projections indicate that the total number of cases may exceed 1.2 billion by 2050[3]. The disease burden is higher in women than in men[3]. The burden of GERD among women of reproductive age has risen considerably over the past decades, with incident and prevalent cases increasing by 64.09% and 66.44%, respectively, and reaching approximately 99.1 million and 245.2 million in 2021[4]. Given the continued rise in prevalence, incidence, and disability-adjusted life years, GERD is expected to remain a significant global public health challenge[5].

The esophageal epithelium can resist acid damage during the passage of gastric contents. These protective mechanisms play a dual role by maintaining epithelial integrity and regulating both inflammatory responses and injury repair[6]. Epithelial sodium channel (ENaC), which has been recognized as a potential therapeutic target in various diseases[7], is a multimeric ion channel typically composed of either α, β, and γ subunits or a δ, β, and γ configuration[8,9]. Members of the ENaC/Degenerin superfamily include four homologous subunits (α, β, γ, and δ), which are encoded by the genes SCNN1A, SCNN1B, SCNN1G, and SCNN1D, respectively[8]. ENaC is usually a heterotrimer (αβγ or δβγ), mainly 1α:1β:1γ heterotrimer in kidney and colon tissues, while in tissues such as brain, testis and ovary, the δ subunit can replace the α subunit to form a δβγ channel[8-10]. δβγ-humanized ENaC (hENaC) exhibits proton sensitivity that may play a critical role in coordinating ischemic signal responses in inflamed and hypoxic tissues[11]. The activity of δ subunit of ENaC (ENaCδ) in humans is affected by changes in environmental pH, which may be involved in pH sensing and/or pH regulation in the human brain[12]. ENaCδ has been implicated in sour and salty taste sensing and may function as a terminal acid receptor in human skin and the gastrointestinal tract, with additional roles in promoting ATP release[13]. ENaCδ is a candidate molecule sensitive to gastrointestinal pH and may become a target for the treatment of GERD[6]. ENaCδ located on the esophageal epithelium can detect refluxed gastric acid and subsequently initiate signal transduction through terminal nerve receptors[13]. The human esophageal surface has ENaCδ, β and γ subunits as well as possible α subunits. Functionally, ENaCδ forms an acidic pH-activated channel in the human esophageal epithelium. Enhancing the defense function of the esophageal epithelium by activating ENaCδ may prevent esophageal mucosal damage under gastric acid reflux conditions[6].

Although ENaCδ mRNA and protein have been detected in mouse sperm[14], subsequent genetic analyses have demonstrated that SCNN1D is a pseudogene in mice[15]. In the mouse and rat lineage, a functional SCNN1D gene is absent; among commonly used experimental rodents, only guinea pigs possess a δβγ-ENaC channel functionally comparable to that of humans[16,17]. Humanized mouse models, generated by introducing functional human genes, cells, or tissues, provide a strategy to overcome the lack of corresponding human targets in conventional mouse models[18]. For example, insertion of the human δ802-ENaC coding sequence, tagged with HA at the N-terminus and His at the C-terminus, into the Rosa26 locus, combined with conditional expression via the Cre-loxP system, enables the generation of humanized transgenic mice expressing human δ802-ENaC[19].

In this study, a humanized ENaCδ (hENaCδ) mouse model was constructed using CRISPR/Cas9 gene-editing technology. A GERD model was subsequently induced through ovalbumin/aluminum hydroxide sensitization combined with esophageal acid perfusion, followed by treatment with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and proton pump inhibitors (PPIs). This study aimed to recapitulate the pathological features of human GERD using a hENaCδ mouse model and to evaluate the therapeutic effects of hUC-MSCs, along with their potential mechanisms of action.

MATERIALS AND METHODS

Cells and animals

hUC-MSCs (Cat. # CP-CL11) and the corresponding complete culture medium (Cat. # CM-H165) were obtained from Wuhan Procell Life Science & Technology Co., Ltd. Male C57BL/6 mice (6-8 weeks old, 20 ± 2 g) were provided by Beijing SPF Biotechnology Co., Ltd. [license No. SCXK (Jing)-2024-0001]. Animals were maintained under controlled environmental conditions (22 ± 4 °C, 50%-60% humidity) with a 12 hours light/dark cycle and had unrestricted access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the First Medical Center of the Chinese PLA General Hospital (Ethics No. IACUC-SR20250327).

Construction of hENaCδ mouse model

A hENaCδ mouse model was established using CRISPR/Cas9-based genome editing. Briefly, an sgRNA targeting the mouse Rosa26 locus was designed and synthesized, and a homologous recombination donor plasmid was constructed containing the full-length human SCNN1D coding sequence (NM_001130413.4) driven by the CAG promoter. Cas9 mRNA, sgRNA, and the donor plasmid were co-injected into fertilized zygotes, which were then transferred into recipient females to obtain founder mice. Founder animals were screened using polymerase chain reaction with human SCNN1D-specific primers, followed by sequencing to confirm correct knock-in events. Positive founders were subsequently crossed with wild-type mice to establish the line. Homozygous F1 mice with site-specific integration at the Rosa26 locus and stable expression of hENaCδ were ultimately obtained and verified by gap-polymerase chain reaction analysis.

Establishment of mouse model of GERD

A mouse model of GERD was established using ovalbumin/aluminum hydroxide adjuvant sensitization combined with esophageal acid perfusion. Ovalbumin (Shanghai Yuanye Biotechnology Co., Ltd., CAS 9006-59-1, Shanghai, China) and aluminum hydroxide adjuvant (InvivoGen, CAS 21645-51-2, CA, United States) were mixed and emulsified at a mass ratio of 1:2. On days 0 and 14, mice received intraperitoneal injections of the ovalbumin/aluminum hydroxide mixture at a dose of 0.1 mL per 10 g body weight for sensitization. On day 21, minimally invasive esophageal catheterization was performed. One end of the catheter was positioned in the mid-esophagus, while the other end was tunneled subcutaneously to the back of the neck and secured in place. The general condition of the mice was monitored daily, and body weight was recorded throughout the experiment. Starting on day 28, mice were subjected to esophageal perfusion with 0.1 mol/L hydrochloric acid solution (Sinopharm Chemical Reagent Co., Ltd., CAS 7647-01-0, Shanghai, China) for 20 minutes once daily, for four consecutive weeks.

Grouping and treatment

Mice (n = 48) were randomly distributed into eight groups (n = 6 per group): Control group (C), hENaCδ subunit group, GERD model group (GERD), humanized GERD model group (hENaCδ + GERD), PPI treatment for GERD group (GERD + PPI), PPI treatment for humanized GERD group (hENaCδ + GERD + PPI), hUC-MSCs treatment for GERD group (GERD + hUC-MSCs), and hUC-MSCs treatment for humanized GERD group (hENaCδ + GERD + hUC-MSCs).

The specific grouping and intervention methods were as follows: (1) Group C: Wild-type C57BL/6 mice; (2) Group hENaCδ: HENaCδ mice; (3) Group GERD: Wild-type C57BL/6 mice with GERD modeling; (4) Group hENaCδ + GERD: HENaCδ mice with GERD modeling; (5) Group GERD + PPI: Wild-type C57BL/6 mice with GERD modeling followed by PPI treatment; (6) Group hENaCδ + GERD + PPI: HENaCδ mice with GERD modeling followed by PPI treatment; (7) Group GERD + hUC-MSCs: Wild-type C57BL/6 mice with GERD modeling followed by hUC-MSCs treatment; and (8) Group hENaCδ + GERD + hUC-MSCs: HENaCδ mice with GERD modeling followed by hUC-MSCs treatment. PPI treatment: 20 mg/kg rabeprazole (MedChemExpress, Cat. # LY307640, NJ, United States) was intragastrically administered daily for 3 consecutive weeks. Human umbilical cord mesenchymal stem cells treatment: 100 μL of hUC-MSCs (1 × 10⁶) was injected via the tail vein weekly for 3 consecutive weeks. Mice in groups C, hENaCδ, GERD and hENaCδ + GERD received no drug/cell treatment and were given the same volume of normal saline.

Enzyme-linked immunosorbent assay

Mice were anesthetized by intraperitoneal injection of tribromoethanol (0.2 mL/10 g; Nanjing Aibei Biotechnology Co., Ltd., Cat. # M2910, Nanjing, Jiangsu Province, China). Blood samples were collected and allowed to clot at room temperature for 40 minutes, followed by centrifugation at 4000 rpm for 20 minutes at 4 °C to obtain serum. Serum concentrations of interleukin (IL)-1, IL-1β, IL-17, and inducible nitric oxide synthase (iNOS) were quantified using commercially available ELISA kits (Quanzhou Ruixin Biotechnology Co., Ltd., Cat. # RXW203076M, RXW203063M, RX203067M, and RXW201999M, Quanzhou, Fujian Province, China) in accordance with the manufacturers’ protocols. Absorbance was measured at 450 nm using a microplate reader (RT-6100, Shenzhen Rayto Life Science Co., Ltd., Shenzhen, Guangdong Province, China).

Histopathology

The mouse esophagus was isolated and removed, gently rinsed with pre-cooled normal saline, and immediately immersed in paraformaldehyde fixative for fixation. The fixed tissues were dehydrated, cleared, wax-embedded, sectioned, baked and dewaxed, then stained with hematoxylin-eosin. After mounting, the esophageal tissue structure was observed under a microscope.

Immunohistochemistry

Esophageal tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at a thickness of 4 μm. After deparaffinization and rehydration, antigen retrieval was performed, followed by quenching of endogenous peroxidase activity. Sections were then blocked with 5% normal goat serum to minimize nonspecific binding. Primary antibodies against claudin-1 (Proteintech, Cat. # 13050-1-AP, IL, United States) and myosin phosphatase target subunit 1 (MYPT1) (Proteintech, Cat. # 22117-1-AP, IL, United States) were applied and incubated overnight at 4 °C. After washing, sections were incubated with HRP-conjugated secondary antibody (iVision™, Tongling Biomedical Technology Co., Ltd., Cat. # DD13, Anhui Province, China) at room temperature for 1 hour. Immunoreactivity was visualized using a DAB substrate kit (Servicebio, Cat. # G1212-200T, Wuhan, Hubei Province, China), followed by hematoxylin counterstaining. Finally, sections were dehydrated, cleared, mounted, and examined under a light microscope.

Western blot

Esophageal tissues were snap-frozen in liquid nitrogen, pulverized, and transferred into microcentrifuge tubes. Pre-chilled RIPA lysis buffer supplemented with a phosphatase inhibitor (Cell Signaling Technology, Cat. #9806, MA, United States) was added, and samples were incubated on ice for 10 minutes with intermittent vortexing. Lysates were then centrifuged at 12000 × g for 10 minutes at 4 °C, and the supernatant containing total protein was collected. Protein concentrations were determined using a BCA assay kit (Cell Signaling Technology, Cat. #7780, MA, United States). Equal amounts of protein were mixed with loading buffer at a 4:1 ratio, denatured by boiling for 10 minutes, and separated by sodium-dodecyl sulfate gel electrophoresis. Proteins were subsequently transferred onto membranes, which were then blocked and incubated overnight at 4 °C with primary antibodies against GAPDH (Abcam, Cat. #ab181602, Cambridge, United Kingdom), phospho-phosphoinositide 3-kinase (PI3K) p85α (Tyr607) (Affinity, Cat. #AF3241, OH, United States), PI3K p85α (Proteintech, Cat. #60225-1-Ig, IL, United States), phospho-protein kinase B (AKT) (Ser473) (Proteintech, Cat. #28731-1-AP, IL, United States), AKT (Proteintech, Cat. #10176-2-AP, IL, United States), phospho-mammalian target of rapamycin (mTOR) (Ser2481) (CST, Cat. #2974, MA, United States), mTOR (Abcam, Cat. #ab134903, Cambridge, United Kingdom), and MYPT1 (Proteintech, Cat. #22117-1-AP, IL, United States). After washing, membranes were incubated with appropriate secondary antibodies (1:10000) at room temperature for 2 hours. Protein bands were visualized using a chemiluminescent substrate and detected with an imaging system (OI-X6Touch, Guangzhou Guangyi Biotechnology Co., Ltd., Guangzhou, Guangdong Province, China). Band intensities were quantified using ImageJ software and normalized to the internal control.

Statistical analysis

All statistical analyses were conducted using SPSS 20.0 software. Group comparisons were carried out using one-way ANOVA, with the LSD method applied for post hoc pairwise analysis. Results are expressed as mean ± SD, and statistical significance was defined as P < 0.05.

RESULTS

hENaCδ exacerbates inflammatory infiltration in the esophageal submucosa of GERD mice, while hUC-MSCs alleviate this pathological change

Hematoxylin and eosin staining results showed that there were no obvious lesions in the esophageal submucosa and muscular layer of mice in group C. Mice in groups GERD and hENaCδ showed mild inflammatory cell infiltration (neutrophils, lymphocytes, macrophages), mild vascular proliferation, mild fibrosis, obvious collagen fiber deposition and a small number of multinucleated giant cells in the esophageal submucosa. Mice in group hENaCδ + GERD showed severe inflammatory cell infiltration (neutrophils) accompanied by mild edema in the esophageal submucosa, with a large amount of pink exudate (Figure 1).

Figure 1 Pathology of mouse esophageal tissue (hematoxylin and eosin staining, × 200). C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Mice in group GERD + PPI showed a small number of inflammatory cell infiltration (lymphocytes), mild vascular proliferation and mild fibrosis in the esophageal submucosa. Mice in group hENaCδ + GERD + PPI showed mild inflammatory cell infiltration (neutrophils), mild vascular proliferation and mild fibrosis in the esophageal submucosa. Mice in group GERD + hUC-MSCs showed no obvious lesions or edema in the esophageal submucosa and muscular layer. Mice in group hENaCδ + GERD + hUC-MSCs showed a small number of inflammatory cell infiltration (neutrophils, lymphocytes) and mild edema in the esophageal submucosa (Figure 1).

hENaCδ promotes the expression of inflammatory factors in the esophageal tissue of GERD mice, and hUC-MSCs inhibits this effect

To investigate the roles of hENaCδ and hUC-MSCs in GERD-associated esophageal inflammation and to further validate the histopathological findings, inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. As shown in Figure 2, serum concentrations of IL-1, IL-1β, IL-17, and iNOS were markedly elevated in both the GERD and hENaCδ groups relative to the control group (P < 0.001). Notably, the hENaCδ + GERD group exhibited a further increase in these inflammatory markers compared with either the hENaCδ or GERD group (P < 0.001). In contrast, treatment with PPI or hUC-MSCs significantly reduced the levels of these cytokines in GERD mice (P < 0.01), with a more pronounced reduction observed in the hENaCδ + GERD + PPI and hENaCδ + GERD + hUC-MSCs groups compared with the untreated hENaCδ + GERD group (P < 0.001).

Figure 2 Serum levels of interleukin-1, interleukin-1β, interleukin-17 and inducible nitric oxide synthase in mice detected by enzyme-linked immunosorbent assay. A: Interleukin-1; B: Interleukin-1β; C: Interleukin-17; D: Inducible nitric oxide synthase. ^aP < 0.05, compared with group C; ^bP < 0.05, compared with group hENaCδ; ^cP < 0.05, compared with group GERD; ^dP < 0.05, compared with group hENaCδ + GERD; ^eP < 0.05, compared with group GERD + PPI; ^fP < 0.05, compared with group hENaCδ + GERD + PPI; ^gP < 0.05, compared with group GERD + hUC-MSCs. IL: Interleukin; INOS: Inducible nitric oxide synthase; C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Overall, hENaCδ enhanced the expression of IL-1, IL-1β, IL-17, and iNOS, whereas both hUC-MSCs and PPI effectively suppressed inflammatory responses in GERD mice. Furthermore, hUC-MSC treatment resulted in a greater reduction in IL-17 levels than PPI treatment, as evidenced by significantly lower IL-17 concentrations in the hENaCδ + GERD + hUC-MSCs group compared with the hENaCδ + GERD + PPI group (P < 0.001), and in the GERD + hUC-MSCs group compared with the GERD + PPI group (P < 0.01) (Figure 2).

hENaCδ-mediated activation of PI3K/AKT/mTOR signaling pathway in GERD mouse esophageal tissue is inhibited by hUC-MSCs

To explore the molecular mechanisms underlying hENaCδ-mediated regulation of esophageal inflammation in GERD, the activation status of the PI3K/AKT/mTOR signaling pathway was assessed by measuring the expression and phosphorylation levels of its key components. As shown in Figures 3 and 4, phosphorylation of PI3K, AKT, and mTOR in esophageal tissues was markedly increased in the GERD group compared with controls (P < 0.01). Notably, AKT phosphorylation was also significantly elevated in the hENaCδ group (P < 0.05). Furthermore, the hENaCδ + GERD group exhibited a further increase in both total expression and phosphorylation levels of these signaling molecules relative to the hENaCδ group (P < 0.05). Collectively, these results indicate that activation of the PI3K/AKT/mTOR signaling pathway may contribute to hENaCδ-mediated aggravation of esophageal inflammation in GERD.

Figure 3 Expression of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin detected by western blot in mouse esophageal tissue. PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; mTOR: Mammalian target of rapamycin; C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Figure 4 Relative expression levels and phosphorylation levels of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin detected by western blot in mouse esophageal tissue. Abscissa: Grouping. A-C: Relative expression levels of phosphorylated phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) proteins; D-F: Relative expression levels of PI3K/AKT/mTOR proteins; G-I: Ratio of relative expression levels of phosphorylated PI3K/AKT/mTOR proteins to relative expression levels of PI3K/AKT/mTOR proteins. ^aP < 0.05, compared with group C; ^bP < 0.05, compared with group hENaCδ; ^cP < 0.05, compared with group GERD; ^dP < 0.05, compared with group hENaCδ + GERD; ^eP < 0.05, compared with group GERD + PPI; ^fP < 0.05, compared with group hENaCδ + GERD + PPI; ^gP < 0.05, compared with group GERD + hUC-MSCs. PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; mTOR: Mammalian target of rapamycin; C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Compared with the GERD group, the hENaCδ + GERD group exhibited significantly higher expression levels of PI3K, AKT, and mTOR in esophageal tissues (P < 0.001). In contrast, treatment with PPI markedly reduced the phosphorylation levels of these signaling molecules in GERD mice (P < 0.01), while hUC-MSC administration led to a significant decrease in AKT phosphorylation (P < 0.001) (Figures 3 and 4). Furthermore, both expression and phosphorylation levels of PI3K, AKT, and mTOR were significantly reduced in the hENaCδ + GERD + PPI group compared with the untreated hENaCδ + GERD group (P < 0.05). A more pronounced reduction in the expression of these pathway components was observed in the hENaCδ + GERD + hUC-MSCs group (P < 0.001) (Figures 3 and 4). Overall, hENaCδ enhanced AKT phosphorylation in wild-type mice and upregulated the expression of PI3K, AKT, and mTOR under GERD conditions, whereas hUC-MSC treatment effectively attenuated the overactivation of the PI3K/AKT/mTOR signaling pathway.

Compared with group hENaCδ + GERD + PPI, the phosphorylation level of AKT in the esophageal tissue of group hENaCδ + GERD + hUC-MSCs was significantly increased (P < 0.01), and compared with group GERD + PPI, the phosphorylation level of AKT in the esophageal tissue of group GERD + hUC-MSCs was significantly increased (P < 0.05) (Figures 3 and 4). Compared with hUC-MSCs treatment, PPI showed a more significant effect in inhibiting AKT phosphorylation level.

hUC-MSCs improve hENaCδ-aggravated GERD esophageal smooth muscle contraction dysfunction by upregulating MYPT1

To evaluate the effects of hENaCδ and hUC-MSCs on esophageal smooth muscle contractile function, MYPT1 expression was assessed in esophageal tissues. As a regulatory subunit of myosin light chain (MLC) phosphatase, MYPT1 plays a key role in modulating smooth muscle contraction and cytoskeletal stability. Immunohistochemical analysis revealed that MYPT1 was predominantly localized in the esophageal muscular and mucosal layers. As shown in Figure 5, MYPT1 expression was significantly reduced in both the GERD and hENaCδ groups compared with controls (P < 0.05), with a further decrease observed in the hENaCδ + GERD group (P < 0.01). In contrast, treatment with PPI or hUC-MSCs markedly increased MYPT1 expression in GERD mice (P < 0.001), and similar restorative effects were observed in the hENaCδ + GERD + PPI and hENaCδ + GERD + hUC-MSCs groups relative to the untreated hENaCδ + GERD group (P < 0.001).

Figure 5 Immunohistochemical staining of myosin phosphatase target subunit 1 and its positive area percentage. A: Immunohistochemical staining; B: The positive area percentage of myosin phosphatase target subunit 1. ^aP < 0.05, compared with group C; ^bP < 0.05, compared with group hENaCδ; ^cP < 0.05, compared with group GERD; ^dP < 0.05, compared with group hENaCδ + GERD; ^eP < 0.05, compared with group GERD + PPI; ^fP < 0.05, compared with group hENaCδ + GERD + PPI; ^gP < 0.05, compared with group GERD + hUC-MSCs. MYPT1: Myosin phosphatase target subunit 1; C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

Consistent with the immunohistochemistry findings, western blot analysis (Figure 6) demonstrated a significant reduction in MYPT1 expression in the GERD and hENaCδ groups (P < 0.001), with a further decline in the hENaCδ + GERD group. Both PPI and hUC-MSC treatments significantly restored MYPT1 expression, particularly in the hENaCδ + GERD + PPI and hENaCδ + GERD + hUC-MSCs groups (P < 0.001). Notably, hUC-MSC treatment resulted in a greater increase in MYPT1 expression compared with PPI treatment, as evidenced by higher MYPT1 levels in the hENaCδ + GERD + hUC-MSCs group relative to the hENaCδ + GERD + PPI group (P < 0.001), and in the GERD + hUC-MSCs group compared with the GERD + PPI group (P < 0.05). Overall, MYPT1 expression was downregulated under GERD conditions and further suppressed by hENaCδ, whereas both PPI and hUC-MSCs effectively restored its expression, with hUC-MSCs exhibiting a more pronounced regulatory effect.

Figure 6 Expression of myosin phosphatase target subunit 1 in mouse esophageal tissue detected by western blot. ^aP < 0.05, compared with group C; ^bP < 0.05, compared with group hENaCδ; ^cP < 0.05, compared with group GERD; ^dP < 0.05, compared with group hENaCδ + GERD; ^eP < 0.05, compared with group GERD + PPI; ^fP < 0.05, compared with group hENaCδ + GERD + PPI; ^gP < 0.05, compared with group GERD + hUC-MSCs. MYPT1: Myosin phosphatase target subunit 1; C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

hUC-MSCs improve hENaCδ-aggravated GERD esophageal epithelial tight junction injury by upregulating claudin-1

To evaluate the effects of hENaCδ and hUC-MSCs on esophageal epithelial barrier integrity in GERD, the expression of the tight junction protein claudin-1 was assessed in esophageal tissues. Immunohistochemical analysis showed that claudin-1 was predominantly localized in the esophageal mucosal layer (Figure 7). As shown in Figure 7, claudin-1 expression was significantly reduced in both the GERD and hENaCδ groups compared with controls (P < 0.05), with a more pronounced decrease observed in the hENaCδ + GERD group (P < 0.001). In contrast, hUC-MSC treatment markedly increased claudin-1 levels in GERD mice (P < 0.05), while both PPI and hUC-MSC interventions significantly restored its expression in the hENaCδ + GERD model (P < 0.001). Notably, hUC-MSC treatment resulted in higher claudin-1 expression than PPI treatment in the hENaCδ + GERD background (P < 0.05). Collectively, these findings suggest that hENaCδ exacerbates esophageal epithelial barrier disruption in GERD by downregulating claudin-1, whereas hUC-MSCs promote barrier repair through upregulation of this tight junction protein.

Figure 7 Immunohistochemical staining of claudin-1 and its positive area percentage. ^aP < 0.05, compared with group C; ^bP < 0.05, compared with group hENaCδ; ^cP < 0.05, compared with group GERD; ^dP < 0.05, compared with group hENaCδ + GERD; ^eP < 0.05, compared with group GERD + PPI; ^fP < 0.05, compared with group hENaCδ + GERD + PPI; ^gP < 0.05, compared with group GERD + hUC-MSCs. C: Wild-type C57BL/6 mic; hENaCδ: Humanized δ subunit of epithelial sodium channel; GERD: Gastroesophageal reflux disease; PPI: Proton pump inhibitor; hUC-MSCs: Human umbilical cord mesenchymal stem cells.

DISCUSSION

In this study, a hENaCδ mouse model was established, and we found that expression of human ENaCδ exacerbated inflammatory infiltration in the esophageal submucosa of GERD mice. This may be attributed to the absence of functional ENaCδ in mice[13], which limits their ability to sense luminal protons. In contrast, hENaCδ mice possess a functional proton-sensing pathway, resulting in more severe esophageal inflammation under acid stimulation. Histopathological analysis indicated that PPI treatment had limited efficacy in alleviating esophageal inflammation in GERD model mice, particularly in those expressing hENaCδ. By comparison, hUC-MSC treatment demonstrated a more pronounced anti-inflammatory effect. ENaCδ has been identified as an acid sensor in the human esophagus[6], and its functional expression may enhance esophageal hypersensitivity to residual acid reflux in GERD mice, thereby diminishing the therapeutic efficacy of PPI.

Our previous work has shown that IL-1β, IL-17, and iNOS levels are elevated in wild-type mice with GERD. Building on these observations, the present study demonstrates that hENaCδ further enhances the expression of multiple inflammatory mediators, including IL-1, IL-1β, IL-17, and iNOS, indicating that ENaCδ may function as an upstream regulator of inflammatory activation. In contrast, administration of hUC-MSCs significantly reduced the levels of these cytokines, which is consistent with the observed histopathological improvements. Notably, hUC-MSCs exerted a stronger inhibitory effect on IL-17 compared with PPI treatment. IL-17, a key cytokine produced by T helper 17 cells, is critically involved in the amplification and progression of inflammatory responses[20]. Previous studies have demonstrated that IL-17A can activate the PI3K/AKT/mTOR signaling pathway[21], and elevated IL-17 levels are closely associated with increased expression and phosphorylation of its downstream components[20]. Consistent with this mechanism, our data showed that hENaCδ enhanced the activation of the PI3K/AKT/mTOR pathway in esophageal tissues, whereas hUC-MSC treatment effectively attenuated its overactivation. Taken together, these findings suggest that hENaCδ may aggravate esophageal inflammation through IL-17-dependent activation of the PI3K/AKT/mTOR signaling cascade, while hUC-MSCs confer protective effects by suppressing this pathway.

On the one hand, PI3K signal transduction can inhibit the phosphorylation of MYPT1, thereby maintaining the activity of MLC phosphatase, and ultimately downregulating actomyosin contractility. Decreased PI3K activity will enhance actomyosin contractility[22]. Suppression of the PI3K/AKT signaling pathway has been reported to activate Rho kinase, resulting in increased phosphorylation of MLC and MYPT1 and consequent hypercontraction of vascular smooth muscle[23]. As a key component of the myosin phosphatase target subunit family, MYPT1 is predominantly expressed in smooth muscle cells[24]. MYPT1 may be phosphorylated by ILK and ZIP-like kinase, and also by Rho kinase, thereby inhibiting phosphatase activity and promoting muscle contraction[25]. The traditional Chinese medicine Tongjiang Hewei decoction can inhibit the expression of MYPT1, thereby alleviating airway hyperresponsiveness caused by gastroesophageal reflux[26]. The PPI pantoprazole may act as a ROCK-2 inhibitor, inhibiting the phosphorylation of MYPT1, reducing the tension of the lower esophageal sphincter in the stomach and esophagus of rats, thereby exacerbating esophageal reflux[27]. Our findings demonstrated that MYPT1 expression was downregulated in the esophagus of GERD mice, and hENaCδ further inhibited the expression of MYPT1, but both hUC-MSCs and PPI treatments could upregulate the expression of MYPT1 in esophageal tissue. This suggests that GERD mice and GERD mice with hENaCδ may have esophageal smooth muscle contraction dysfunction, which can be improved by hUC-MSCs and PPI treatments. On the other hand, the damaged epithelial barrier function can be restored by regulating the PI3K/AKT pathway and enhancing claudin-1 expression[28]. Claudin-1 is an important member of the tight junction protein family, which can tighten the expansion of cell gaps caused by epithelial damage[29]. In some GERD patients, claudin-1 expression is upregulated, but the identified molecular changes are generally not related to tissue morphological changes[30]. In contrast, claudin-1 expression is significantly decreased in GERD patients[31,32]. The reason for the different trends of claudin-1 expression in GERD patients may be related to different subtypes and disease stages of GERD, suggesting that claudin-1 not only plays a key role in esophageal mucosal barrier function but also has a complex regulatory mechanism. The results of this study showed that hENaCδ can aggravate GERD esophageal epithelial tight junction injury by downregulating claudin-1 expression, while hUC-MSCs treatment can promote the repair of esophageal epithelial barrier injury by upregulating claudin-1.

Despite these findings, several limitations should be acknowledged. The relatively small sample size may restrict the generalizability of the results. In addition, although the mouse model offers valuable mechanistic insights, interspecies differences may limit direct clinical translation. Furthermore, the long-term therapeutic efficacy of hUC-MSCs was not assessed in the present study. Future investigations incorporating larger sample sizes and extended follow-up periods are warranted to further validate these observations.

CONCLUSION

By constructing a hENaCδ mouse model, this study first confirmed that ENaCδ humanization can significantly increase the susceptibility to GERD. Mice in the hENaCδ + GERD model group had increased IL-1, IL-1β, IL-17 and iNOS in vivo, significantly upregulated PI3K/AKT/mTOR in esophageal tissue, accompanied by downregulated expression of MYPT1 (related to esophageal smooth muscle contraction function) and claudin-1 (related to epithelial barrier function), suggesting that ENaCδ humanization exacerbates the pathological process of GERD by activating inflammatory signals and destroying esophageal epithelial barrier function. hUC-MSCs treatment can effectively reverse the above molecular changes: On the one hand, it inhibits the excessive activation of the PI3K/AKT/mTOR pathway, and on the other hand, it restores the expression levels of MYPT1 and claudin-1, thereby reducing inflammatory response and repairing the barrier. This study reveals a new role of ENaCδ in the pathogenesis of GERD and provides experimental basis for the therapeutic strategy of hUC-MSCs based on PI3K/AKT/mTOR pathway regulation.