Mesenchymal stem cells therapy ameliorates esophageal dysmotility in gastroesophageal reflux disease by modulating smooth muscle contractility, alleviating senescence by targeting the DNMT3a-HIF1α axis

Abstract

BACKGROUND

Gastroesophageal reflux disease (GERD) is associated with esophageal dysmotility, leading to impaired acid clearance and persistent symptoms. Mesenchymal stem cell (MSC) therapy holds promise for tissue regeneration and functional restoration.

AIM

To investigate the therapeutic efficacy and underlying molecular mechanisms of MSC treatment on esophageal dysmotility in a rat model of GERD and in human esophageal smooth muscle cells (HESMCs).

METHODS

A rat model of chronic GERD was established surgically. Rats were treated with human umbilical cord-derived MSCs via combined intravenous and local injection. Esophageal contractility was measured ex vivo. HESMCs were used for in vitro studies, including a senescence model induced by tumor necrosis factor (TNF)-α and MSC co-culture. Assessments included histology, senescence-associated β-galactosidase staining, immunohistochemistry, western blotting, intracellular Ca²⁺ imaging, small interfering RNA-mediated knockdown, and transcriptomic sequencing.

RESULTS

In vivo, GERD rats exhibited significantly reduced esophageal contractility, increased cellular senescence (β-galactosidase positivity), elevated TNF-α, decreased interleukin-10, and dysregulated expression of smooth muscle contraction-related proteins (decreased myosin light chain kinase, increased myosin phosphatase target subunit 1) and Ca²⁺ channels (decreased CACNA1C). MSC treatment restored esophageal contractility, mitigated senescence, normalized inflammatory markers, and reversed the protein dysregulation. In vitro, MSC co-culture effectively counteracted TNF-α-accelerated senescence in HESMCs, upregulated contractile proteins and ion channels (anoctamin 1, calcium voltage-gated channel subunit alpha1 C), and enhanced intracellular Ca²⁺ concentration. Mechanistically, MSCs modulated DNA methyltransferase 3a (DNMT3a) and hypoxia-inducible factor 1 alpha (HIF1α) pathways; inhibition of DNMT3a suppressed HIF1α, while HIF1α overexpression upregulated contractile proteins, establishing an MSC-DNMT3a-HIF1α axis. Transcriptomic analysis confirmed global gene expression changes in GERD and highlighted MSC-mediated modulation of inflammatory, signaling, and muscle-related pathways.

CONCLUSION

In conclusion, MSCs offer a potent therapeutic strategy for GERD-associated esophageal dysmotility by restoring smooth muscle function, alleviating senescence, and remodeling molecular pathways, primarily via a novel DNMT3a-HIF1α signaling cascade.

Key Words: Mesenchymal stem cells; Gastroesophageal reflux disease; Esophageal dysmotility; DNA methyltransferase 3a/hypoxia-inducible factor 1 alpha axis

Core Tip: This work shows mesenchymal stem cells (MSCs) restore impaired esophageal contractility and reduce inflammation in gastroesophageal reflux disease by regulating ion channels and smooth muscle proteins. MSCs counteract tumor necrosis factor-α-accelerated cell senescence and dysfunction, with DNA methyltransferase 3a and hypoxia-inducible factor 1 alpha pathways implicated. These results uncover critical mechanisms for MSC-mediated regenerative effects, proposing a promising cell-based therapeutic approach for restoring physiological function in gastroesophageal reflux disease.

INTRODUCTION

Gastroesophageal reflux disease (GERD) is a prevalent chronic disorder characterized by significant morbidity arising from gastric reflux into the esophagus[1]. Despite established acid suppression therapies, a substantial proportion of patients experience persistent symptoms, largely due to underlying esophageal dysfunction, notably esophageal dysmotility (ED)[2,3]. ED, involving both the lower esophageal sphincter and esophageal body, impairs acid clearance and is driven by complex factors including smooth muscle dysfunction, nerve damage, and cellular alterations[4]. Emerging evidence further implicates cellular senescence and chronic inflammation as critical contributors to GERD progression, exacerbating esophageal degeneration and dysfunction[5-8]. Addressing these intertwined pathological mechanisms is crucial for improved therapeutic outcomes.

Mesenchymal stem cells (MSCs) offer a compelling therapeutic strategy across various chronic diseases due to their potent regenerative, immunomodulatory, and anti-inflammatory attributes[9,10]. While MSCs have shown promise in other gastrointestinal disorders by mitigating inflammation and promoting tissue repair[11,12], their specific mechanisms in ameliorating ED in GERD, particularly concerning smooth muscle function, cellular senescence, and inflammatory resolution, remain largely unexplored.

This study investigated the therapeutic efficacy of MSCs on ED in GERD using both in vivo rat models and in vitro human esophageal smooth muscle cell (HESMC) experiments. We hypothesized that MSC treatment would restore esophageal contractility by modulating smooth muscle function, alleviating cellular senescence, and mitigating inflammation. Furthermore, we sought to elucidate the underlying molecular mechanisms, including changes in muscle contraction-related proteins, calcium-regulating ion channels, and key signaling pathways such as DNA methyltransferase 3a (DNMT3a) and hypoxia-inducible factor 1 alpha (HIF1α), within both animal tissues and cellular models. Through an integrated analysis encompassing functional, histological, molecular, and transcriptomic methodologies, this research endeavors to provide a comprehensive understanding of MSCs’ multifaceted impact on GERD pathophysiology, paving the way for novel translational strategies.

MATERIALS AND METHODS

Animals and establishment of the GERD model

Six-week-old male Sprague-Dawley rats (250 ± 20 g) were obtained from Si Pei Fu (Beijing, China). Animals were housed under controlled environmental conditions (temperature: 25 ± 1 °C, humidity: 35%-60%, 12-hour light-dark cycle from 07:00 to 19:00) with ad libitum access to standard rodent chow (5 L0D Rodent Diet, KeAoXieLi Co., Ltd., Beijing, China) and tap water. A total of fifteen rats were used and were processed in two batches (batch 1: n = 9; batch 2: n = 6). All rats were randomly assigned to three groups (n = 5/group): Normal, GERD, and MSC. All animal experiments were approved by the Experimental Animal Management Group of Beijing Shenrui Biotechnology Co., Ltd (No. SRSW-DWLL-2022-0820) and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with efforts made to minimize suffering and animal numbers. Surgical procedures were performed under 3%-4% isoflurane. In the normal group, rats underwent a sham operation. For the GERD and MSC groups, a GERD model was established. A 2-cm midline abdominal incision exposed the esophagogastric junction (EGJ) and duodenum. A 0.5-cm longitudinal incision was made in the outer muscle layer of the EGJ. The duodenum was partially ligated 1 cm distal to the pylorus to achieve approximately one-third luminal narrowing. Postoperatively, all animals were fasted for 2 days with supportive care, consisting of intraperitoneal injections of glucose solution and gentamicin to prevent dehydration and infection. Models were allowed to develop for 8 weeks before further intervention.

MSC culture and identification

Primary MSCs [human, derived from umbilical cord, batch number HF-P6-20220327-01, male donor, passage 3 (P3)] were obtained from Beijing RM Bio-tech Co., Ltd. (Beijing, China) and cultured in α-MEM medium (Cat.No.SH30265, Hyclone, Logan, UT, United States) supplemented with UltraGRO MSC nutrient supplement (Lot. No.69HF10. RM Bio-tech, China). MSCs at P3 were characterized by flow cytometry to confirm the expression of surface markers CD90 (Cat. No. 560847, BioLegend, San Diego, CA, United States) CD73 (Cat. No. 344006, BioLegend, San Diego, CA, United States), CD105 (Cat. No. 323220, BioLegend, San Diego, CA, United States) and absence of marker CD45 (Cat. No. 555483, BD Biosciences, San Jose, CA, United States) (Supplementary Figure 1A). Multi-potency was validated via in vitro osteogenic (Supplementary Figure 1B) and adipogenic (Supplementary Figure 1C) differentiation assays using MSC-specific differentiation media (Cat. No. 555483, BD Biosciences, San Jose, CA, United States). Cells at P3-5 were used for all in vitro and in vivo experiments. The study was approved by the Institutional Review Board of the Chinese PLA General Hospital (No. S2021-052-02).

MSC treatment and tissue sample collection

Eight weeks after GERD model induction, the duodenum ligature was removed from rats. Immediately thereafter, MSC group rats received a total dose of 1 × 10⁷ MSCs suspended in 200 μL phosphate buffered saline (PBS) intravenously via tail vein injection. An additional local injection of 20 μL of MSCs (1 × 10⁷ cells/mL) was administered locally into the EGJ muscle. The GERD and normal groups received equivalent volumes of PBS as vehicle control. Five days post-treatment, all rats were euthanized by cervical vertebrae luxation under isoflurane anesthesia. The distal 1 cm esophageal segment was immediately excised for ex vivo pressure measurements. Following measurements, the remaining esophageal tissue was washed with PBS. Portions were snap-frozen in liquid nitrogen and stored at -80 °C for transcriptome sequencing and western blotting. Other portions were fixed in 4% paraformaldehyde for 24 hours at 4 °C, then processed and embedded in paraffin for histological analyses.

Ex vivo esophageal pressure measurement

Esophageal contractility was assessed using a custom-constructed esophageal contractility pressure detection system. This system measured acute contractile responses of isolated esophageal segments under controlled thermal and chemical conditions.

Organ bath chambers and temperature control: Each esophageal segment was mounted in a sterile 5 cm diameter tissue culture dish, serving as the organ bath. Dishes were partially submerged in a pre-equilibrated 37 °C water bath. Physiological saline and potassium chloride (KCl) solutions were pre-warmed to 37 °C, and bath temperature was verified at 37 ± 0.5 °C before each experiment.

Oxygenation and solution exchange: For each measurement, 5 mL of pre-warmed physiological saline was added to the bath. After a 1-minute equilibration, this was quickly replaced with 5 mL of pre-warmed 60 mmol/L KCl solution. Continuous external oxygenation was not employed due to rapid solution exchange and the short assay duration (20 seconds).

Inflatable probe and pressure transducer: An inflatable probe, comprising a thin-walled latex balloon mounted to a flexible polyethylene catheter, was connected via silicone tubing to a calibrated physiological pressure transducer. The transducer was calibrated daily against a standard mercury manometer.

Signal acquisition and analysis: Analog output from the pressure transducer was amplified and digitized via a data acquisition system (ATK XCOM V2.0, www.openedv.com). Pressure values (mmHg) were recorded at 1000 Hz and displayed in real-time using LabChart Pro software (ADInstruments). After a 1-minute equilibration with physiological saline, the solution was rapidly exchanged for the 60 mmol/L KCl solution. Pressure responses were recorded for 20 seconds following KCl addition. Average contraction force was calculated.

Treatment of HESMCs

HESMCs (P4) were obtained from Zhongke Quality Inspection Biotechnology Co., Ltd. (Beijing, China, Cat. No. D002). HESMCs were cultured in RPMI 1640 medium (Cat. No. 11875085, Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% foetal bovine serum (Cat. No. 10082147, Thermo Fisher Scientific, Waltham, MA, United States) at 37 °C in a humidified atmosphere with 5% CO₂. HESMCs at P6 were used for all in vitro experiments. Cells were divided into three treatment groups. The control group was cultured in normal RPMI 1640 medium. The tumor necrosis factor-alpha (TNF-α) group was stimulated with 1 ng/mL TNF-α (Cat. No. 300-01A, PeproTech, Cranbury, NJ, United States) for 96 hours. The MSC group underwent TNF-α pre-treatment (1 ng/mL, 96 hours) followed by a 48-hour co-culture with MSCs using a Transwell system (0.4 μm pore size, Cat. No. 3413, Corning Inc., Corning, NY, United States), with HESMCs seeded in the lower chamber (1.5 × 10⁵ cells/well) and MSCs (3 × 10⁴ cells) in the upper chamber. After the respective treatments, all cells were collected for subsequent analysis.

Regulation of intracellular calcium in HESMCs by MSCs via anoctamin 1 and calcium voltage-gated channel subunit alpha1 C

To investigate the regulatory effects of MSCs on intracellular Ca²⁺ levels in HESMCs and the specific roles of the ion channels anoctamin 1 (ANO1) and calcium voltage-gated channel subunit alpha1 C (CACNA1C), a series of co-culture and gene knockdown experiments were performed. HESMCs at P6 were seeded on coverslips in 12-well plates at a density of 1 × 10⁵ cells per well. Cells were allocated into eight experimental groups: (1) Control: Untreated HESMCs; (2) GERD model (GM): HESMCs treated to mimic a GERD-like condition; (3) GM + negative control small interfering RNA (si-NC): GM HESMCs transfected with a negative control small interfering RNA (siRNA); (4) MSC: GM HESMCs co-cultured with MSCs in a Transwell system; (5) MSC + si-NC: GM HESMCs transfected with negative control siRNA and then co-cultured with MSCs; (6) MSC + si-ANO1: GM HESMCs transfected with ANO1-specific siRNA and co-cultured with MSCs; (7) MSC + si-CACNA1C: GM HESMCs transfected with CACNA1C-specific siRNA and co-cultured with MSCs; and (8) MSC + si-ANO1 + si-CACNA1C: GM HESMCs co-transfected with both ANO1 and CACNA1C siRNAs and co-cultured with MSCs.

For siRNA transfection, HESMCs were transfected with 20 μM of target-specific siRNA (specified sequences for ANO1-Homo-1732, CACNA1C-Homo-1054 from GenePharma, Shanghai, China) or a negative control sequence using Lipofectamine™ 2000 reagent (Cat. No.11668019, Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. Briefly, siRNA and Lipofectamine™ 2000 were separately diluted in Opti-MEM^® I Reduced Serum Medium (No. 31985070, GIBCO, Thermo Fisher Scientific, Waltham, MA, United States), incubated for 5 minutes at room temperature, combined, and then added to the cells for 5 hours. Subsequently, the transfection medium was replaced with normal RPMI 1640 medium. Following transfection, the designated groups were co-cultured with MSCs using a Transwell system for an additional 48 hours.

After treatments, intracellular Ca²⁺ levels were assessed. Cells were loaded with 5 μmol/L Fluo-3 AM (No. S1056, Beyotime Biotechnology, Shanghai, China) in serum-free medium at 37 °C for 20 minutes in the dark. After three washes with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes, counterstained with DAPI (Cat. No. C1002, Beyotime Biotechnology, Shanghai, China) for nucleus visualization, and mounted with an anti-fade mounting medium (Cat. No. 0100-01, Beyotime Biotechnology, Shanghai, China). Fluorescence images were acquired using a Nikon ECLIPSE Ts2 microscope. The fluorescence intensity of Fluo-3, corresponding to intracellular Ca²⁺ concentration, was quantified using ImageJ software.

MSCs modulate muscle contraction-related proteins via DNMT3a and HIF1α

To investigate the MSC-mediated regulation of muscle contraction-associated proteins via DNMT3a and HIF1α (Supplementary Figure 2), HESMCs (P6) were treated as follows. For DNMT3a inhibition, cells were treated with 10 μM SGI-1027 (Cat. No. HY-13962, MedChemExpress, Monmouth Junction, NJ, United States) or vehicle control (DMSO) for 24 hours. For HIF1α overexpression, HESMCs pre-treated with TNF-α were transfected at approximately 50% confluence with a pcDNA3.1-HIF1α plasmid (GeneChem, Shanghai, China, Cat. No. SC7001-C) using Lipofectamine 3000 reagent (Cat. No. L3000008, Thermo Fisher Scientific, Waltham, MA, United States) and harvested 24 hours after transfection. For MSC co-culture, HESMCs were seeded in Transwell lower chambers, with MSCs in upper chambers. The co-culture medium was a mixture of one-third DMEM and two-thirds RPMI 1640. Cells were collected after 12 hours, 24 hours, and 48 hours. All collected cells from these experiments were subjected to western blotting for protein expression analysis. All recombinant DNA research involving these plasmids was conducted in strict adherence to the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules.

Transcriptome sequencing and bioinformatics analysis

Transcriptome library construction and sequencing were performed by Bestnovo (Beijing, China) Medical Technology, Co., Ltd. Total RNA was extracted from each sample using the QIAamp RNA Blood Mini Kit (Cat. No. 51104, Qiagen, Hilden, Germany). RNA integrity and concentration were assessed using a 2100 Bioanalyzer System and a NanoPhotometer^® NP80 spectrophotometer, respectively.

RNA libraries were prepared using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina (Cat. No. E7770 L, Illumina, San Diego, CA, United States). This involved cDNA synthesis from fragmented mRNA, end-repair, A-tail addition, and sequencing adapter ligation. cDNA fragments (250-300 bp) were size-selected and purified using AMPure XP beads (Cat. No. A63881; Beckman Coulter, Brea, CA, United States) before PCR amplification and further purification. Libraries were quality-controlled with a Qubit^® 2.0 Fluorometer and a 2100 Bioanalyzer System, then sequenced on an Illumina HiSeq 2500 System.

For bioinformatics analysis, gene abundances were quantified by calculating transcripts per million using RSEM v1.3.1 (http://deweylab.biostat.wisc.edu/rsem/). Differential expression analysis between samples was performed using the DESeq2 package in R statistical software (v4.0.3). Differentially expressed genes (DEGs) were identified as those with |log2FC| > 1 and a false discovery rate adjusted P value (Q value) ≤ 0.005. Functional enrichment analyses were conducted with Gene Ontology (GO) using Goatools v1.2.9 (https://github.com/tanghaibao/Goatools) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using KOBAS v3.0 (http://kobas.cbi.pku.edu.cn/home.do). Significant enrichment was determined by a Bonferroni-corrected P-value < 0.05.

Western blotting

Esophageal tissue samples were homogenized in RIPA lysis buffer (Cat. No. P0013B, Beyotime, Shanghai, China) containing protease and phosphatase inhibitor cocktails. Protein concentrations were determined using a BCA Protein Assay Kit (Cat. No. P0010, Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% sodium-dodecyl sulfate gel electrophoresis and transferred onto polyvinylidene fluoride membranes using a TE77XP Semi-Dry Transfer Unit at 25 V for 30 minutes. Membranes were blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature, then incubated with primary antibodies overnight at 4 °C: Myosin phosphatase target subunit 1 (MYPT1) (rabbit polyclonal, 1:5000, No. 22117-1-AP; ProteinTech, Wuhan, Hubei Province, China); p-MYPT1 (rabbit polyclonal, 1:1000, at Thr853, No. bs-3288R, Thermo Fisher Scientific, Waltham, MA, United States); DNMT3a (rabbit polyclonal, 1:2000, No. Ab188470; Abcam, United Kingdom); interleukin-10 (IL-10) (rabbit polyclonal, 1:100, Cat. No. A2171; ABclonal, Wuhan, Hubei Province, China); TNF-α (rabbit polyclonal, 1:100, Cat. No. bs-0078R; Bioss, Inc., Beijing, China); MLCK (rabbit polyclonal, 1:1000, Cat. No. 21642-1-AP; ProteinTech, Wuhan, Hubei Province, China); HIF1α (rabbit polyclonal, 1:2000, No. 20960-1-AP; ProteinTech, Wuhan, Hubei Province, China); alpha-smooth muscle actin (α-SMA) (mouse monoclonal, 1:200, No. BM0002; Boster, Wuhan, Hubei Province, China); ANO1 (rabbit polyclonal, 1:1000, Cat. No. A10498; ABclonal, Wuhan, Hubei Province, China); CACNA1C (rabbit polyclonal, 1:1000, Cat. No. EPR24535-131; Abcam, United Kingdom); After five 5-minute washes in TBST, membranes were incubated with goat anti-rabbit IgG-HRP (Cat. No. BA1054; Boster, Wuhan, Hubei Province, China). Immunoreactive bands were detected using ECL Western Blotting Substrate and visualized with a 5200 imaging system. Densitometric analysis was performed using ImageJ software.

Immunohistochemistry, hematoxylin and eosin, and β-galactosidase staining

Paraffin-embedded esophageal sections were deparaffinized and rehydrated. Hematoxylin and eosin (H&E) staining was performed using a H&E staining kit (Cat. No. C0105, Beyotime, Shanghai, China). Primary antibodies, diluted in PBS, were incubated overnight at 4 °C: ANO1 (polyclonal rabbit anti-rat, 1:1000, Cat. No. A10498; ABclonal, Wuhan, Hubei Province, China); α-SMA (mouse monoclonal, 1:5000, Cat. No. BM0002; Boster, Wuhan, Hubei Province, China); MLCK (rabbit polyclonal, 1:500, Cat. No. 21642-1-AP; ProteinTech, Wuhan, Hubei Province, China); MYPT1 (rabbit polyclonal, 1:200, Cat. No. 22117-1-AP; ProteinTech, Wuhan, Hubei Province, China); CACNA1C (rabbit polyclonal, 1:100, Cat. No. EPR24535-131; Abcam, United Kingdom); TNF-α (rabbit polyclonal, 1:100, Cat. No. bs-0078R; Bioss, Inc., Beijing, China). Following primary antibody incubation, sections were washed three times with PBS and then incubated for 30 minutes at 37 °C with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Visualization was achieved using DAB with 0.05% H₂O₂ for 3 minutes. Protein expression and localization (brown staining) were examined under a microscope, and images were analyzed using Image-Pro Plus 6.0. For β-galactosidase (β-gal) staining: Frozen sections were prepared. Senescence-associated β-gal activity was detected using a β-Gal staining solution (Senescence β-Galactosidase Staining Kit, Cat. No. C0602, Beyotime, Shanghai, China) at 37 °C overnight, following the manufacturer’s instructions.

Morphometric analysis of esophageal muscularis propria thickness

For quantitative assessment of esophageal wall remodeling, the thickness of the muscularis propria layer was measured on H&E-stained cross-sections. One representative esophageal cross-section from each rat was analyzed. Using Image-Pro Plus 6.0 software, the thickness was measured at four standardized positions corresponding to 3, 6, 9, and 12 o’clock on the esophageal circumference. The average of these four measurements was calculated and recorded as the muscularis propria thickness for that individual animal.

Statistical analysis

All data are presented as the mean ± SD. All in vitro experiments were independently repeated three times to ensure reproducibility. Esophageal pressure was measured at 20 time points. Statistical analyses were performed using GraphPad Prism (version 9.0). Data are presented as mean values at each time point within groups. To assess the dynamic changes in esophageal contractility over the 20-second recording period and compare these responses between different experimental groups, a Two-Way Repeated Measures Analysis of Variance (ANOVA) was employed. The two factors were “Group” and “Time Point”. The dependent variable was the calculated “Pressure Change” at each time point. All bioinformatic analyses of transcriptome sequencing data were conducted using three biological samples per group. Statistical comparisons between two groups were performed using unpaired Student’s t-tests. For comparisons involving three or more groups, one-way ANOVA followed by Tukey’s Honestly Significant Difference post-hoc test was used to identify specific group differences. All statistical analyses were performed using Prism 9.0 software. Differential expression analysis for transcriptomic data was specifically conducted using DESeq2 package. Differences were considered statistically significant at a two-sided P-value < 0.05.

RESULTS

MSC treatment restores esophageal contractility in vivo

To assess the functional impact of MSC therapy on esophageal dysfunction, ex vivo esophageal pressure measurements were performed. Rats in the GERD group exhibited a significant reduction in esophageal contractility compared to the normal group after 8 weeks of model establishment. Specifically, the average contraction force over 20 seconds in GERD rats was significantly decreased by 25.65 mmHg compared to normal controls (P < 0.001). MSC treatment remarkably improved this impaired contractility. The MSC group showed a substantial increase in average contraction force by 16.53 mmHg compared to the GERD group (P < 0.001). Representative recordings of the esophageal pressure dynamics are presented in Figure 1, showing the baseline contractions in physiological saline during the initial 20 seconds, followed by the KCl-stimulated contraction responses.

Figure 1 Mesenchymal stem cells restore impaired esophageal contractility in gastroesophageal reflux disease rats. This figure displays representative recordings of esophageal pressure dynamics, visually comparing baseline and potassium chloride-stimulated contractions in normal, gastroesophageal reflux disease, and mesenchymal stem cell-treated rat groups. Error bars represent SEM. ^cP < 0.001 for mesenchymal stem cell group vs gastroesophageal reflux disease group. KCl: Potassium chloride; GERD: Gastroesophageal reflux disease; MSC: Mesenchymal stem cell.

MSCs ameliorate esophageal cell senescence and modulate inflammatory markers in GERD rats

Histological examination by H&E staining (Figure 2A) revealed hyperproliferation and hypertrophy of muscle cells in both the GERD and MSC groups, leading to a noticeable thickening of the esophageal wall. Quantitative morphometric (Figure 2A) analysis confirmed this observation. The average thickness of the muscularis propria was significantly increased in GERD rats (749 ± 166 μm) compared to the normal group (379 ± 69 μm; P < 0.001). MSC treatment did not significantly reverse this structural hypertrophy, with the MSC group (703 ± 177 μm) showing a comparable thickness to the GERD group (P > 0.05), indicating that the therapeutic effect of MSCs on contractility is primarily functional and molecular rather than through regression of muscular hyperplasia. Notably, despite these structural changes, no overt inflammatory cell infiltration was observed in either group. Immunohistochemical analyses were performed to investigate the cellular and molecular changes in the esophagus of GERD rats and the effects of MSC treatment. β-gal staining, a marker for cellular senescence, showed that senescent cells were more prevalent in the esophageal mucosa and muscle layers of GERD rats compared to normal controls (Figure 2B). MSC administration substantially reduced β-gal positive staining, indicating an alleviation of cellular senescence in the esophageal tissue (Figure 2B). Immunohistochemistry also demonstrated that the GERD group exhibited elevated expression levels of the pro-inflammatory cytokine TNF-α (P < 0.001) and decreased levels of the anti-inflammatory cytokine IL-10 (P = 0.018) in whole esophageal tissue compared to the normal group (Figure 2C and D). The expression trends of these inflammatory factors were consistent across the muscularis mucosa, inner muscularis propria, and outer muscularis propria layers, with the exception of IL-10 in the mucosa, where its level in the GERD group did not differ significantly from that in the control group. Following MSC treatment, TNF-α expression was significantly downregulated (P = 0.012), while IL-10 expression was markedly upregulated (P = 0.046) compared to the GERD group (Figure 2C and D). Similar to the GERD group, these restorative trends for TNF-α and IL-10 expression were generally observed across all esophageal muscle layers, although statistical significance was not reached for TNF-α in the inner muscularis propria and mucosa, or for IL-10 in the mucosa.

Figure 2 Mesenchymal stem cells mitigate esophageal aging and inflammation in gastroesophageal reflux disease rats. This figure illustrates mesenchymal stem cell effects on esophageal aging and inflammation in gastroesophageal reflux disease rats. A: Hematoxylin and eosin staining histological sections; B: Β-galactosidase staining images indicating cellular senescence; C and D: Immunohistochemical images for tumor necrosis factor-α (pro-inflammatory marker) and interleukin-10 (anti-inflammatory marker), respectively. Error bars represent SEM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 for gastroesophageal reflux disease group vs normal group; ^dP < 0.05, ^eP < 0.01 for mesenchymal stem cell group vs gastroesophageal reflux disease group. NS: Not significant; Nor: Normal; GERD: Gastroesophageal reflux disease; MSC: Mesenchymal stem cell; β-gal: Β-galactosidase; TNF: Tumor necrosis factor; IL: Interleukin.

MSC treatment modulates smooth muscle contraction-related proteins and ion channels in vivo

We further investigated the expression of key proteins involved in smooth muscle contraction and Ca²⁺ handling within esophageal tissue. α-SMA, a marker for smooth muscle cells, was analyzed via immunostaining and western blot. We observed that α-SMA was predominantly expressed in the muscularis mucosae, with lower expression in the muscularis propria of normal esophageal tissue. In GERD rats, α-SMA expression was generally increased compared to controls (Figure 3A), and MSC treatment further augmented α-SMA levels in the esophageal muscular layers compared to the GERD group (P < 0.001) (Figure 3A). Western blot analysis of key proteins in the myosin light chain phosphorylation pathway revealed distinct patterns. In GERD rats, the expression of myosin light chain kinase (MYLK) and its phosphorylated form, phosphorylated MYLK (p-MYLK), as well as the p-MYLK/MYLK ratio, were significantly decreased compared to the normal group (P < 0.001) (Figure 3B). Conversely, MSC treatment led to a significant increase in MYLK, p-MYLK, and the p-MYLK/MYLK ratio compared to the GERD group (P < 0.001) (Figure 3B). For MYPT1, the GERD group showed a modest but significant increase in the levels of MYPT1 (P = 0.003), phosphorylated MYPT1 (p-MYPT1) (P = 0.002), and the p-MYPT1/MYPT1 (P = 0.039) ratio compared to the normal group (P < 0.001) (Figure 3B). MSC group showed the similar trend compared to GERD group in the levels of MYPT1 (P = 0.023), p-MYPT1 (P = 0.005) (Figure 3B).

Figure 3 Mesenchymal stem cells modulate smooth muscle contraction-related proteins in gastroesophageal reflux disease rat esophagus. This figure details how mesenchymal stem cells affect smooth muscle contraction-related proteins in gastroesophageal reflux disease rat esophagus. A: Immunohistochemical staining and western blot images for alpha-smooth muscle actin; B: Immunohistochemical staining and representative western blot images for myosin light chain kinase, phosphorylated-myosin light chain kinase, myosin phosphatase target subunit 1, and phosphorylated-myosin phosphatase target subunit 1. Error bars represent SEM. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 for gastroesophageal reflux disease group vs normal group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 for mesenchymal stem cell group vs gastroesophageal reflux disease group. NS: Not significant; Nor: Normal; GERD: Gastroesophageal reflux disease; MSC: Mesenchymal stem cell; α-SMA: Alpha-smooth muscle actin; MYLK: Myosin light chain kinase; p-MYLK: Phosphorylated-myosin light chain kinase; MYPT1: Myosin phosphatase target subunit 1; p-MYPT1: Phosphorylated-myosin phosphatase target subunit 1.

The expression of ANO1 and CACNA1C, important for calcium flux, was assessed. CACNA1C protein levels were significantly decreased in the esophageal tissue of GERD rats compared to Normal controls (P < 0.001) (Figure 4A and B). MSC treatment significantly upregulated the expression of both ANO1 and CACNA1C compared to the GERD group (P < 0.001) (Figure 4A and B).

Figure 4 Mesenchymal stem cells modulate on channels in gastroesophageal reflux disease rat esophagus. This figure demonstrates the modulation of ion channels by mesenchymal stem cells in gastroesophageal reflux disease rat esophagus. A: Western blot images for anoctamin 1 and calcium voltage-gated channel subunit alpha1 C; B: Immunofluorescence staining images for anoctamin 1 and calcium voltage-gated channel subunit alpha1 C. Error bars represent SEM. ^cP < 0.001 for gastroesophageal reflux disease group vs normal group; ^fP < 0.001 for mesenchymal stem cell group vs gastroesophageal reflux disease group. NS: Not significant; Nor: Normal; GERD: Gastroesophageal reflux disease; MSC: Mesenchymal stem cell; ANO1: Anoctamin 1; CACNA1C: Calcium voltage-gated channel subunit alpha1 C.

MSCs reverse TNF-α-accelerated cellular senescence and protein dysregulation in HESMCs in vitro

To further elucidate the cellular mechanisms, HESMCs were used for in vitro studies. Cellular senescence was assessed using β-gal staining in HESMCs at different passages and under varying conditions (Figure 5A). P22 HESMCs, used as a model of replicative senescence, exhibited a higher percentage of β-gal positive cells compared to P6 HESMCs. To mimic the GERD-associated cellular aging and inflammatory phenotypes, P22 HESMCs were further stimulated with TNF-α (1 ng/mL for 96 hours). This treatment significantly exacerbated senescence, leading to an increase in β-gal positive cells compared to untreated P22 HESMCs, thereby establishing an in vitro GERD-like senescent model (GM group). Crucially, co-culture of TNF-α-stimulated P22 HESMCs with MSCs (MSC group) for 48 hours effectively reversed this induced senescence, significantly reducing the percentage of β-gal positive cells compared to the GM group (and P22 HESMCs). These in vitro findings robustly demonstrate that MSCs can directly mitigate both intrinsic and inflammation-accelerated cellular senescence in esophageal smooth muscle cells. For muscle contraction-related proteins, the GM group exhibited significant downregulation of key proteins including MYLK, p-MYLK, α-SMA, ANO1, and CACNA1C, compared to P6 HESMCs as control (all P < 0.001) (Figure 5B). Interestingly, the GM group also exhibited an increased expression of MYPT1 and p-MYPT1, and an elevated p-MYPT1/MYPT1 ratio in HESMCs (all P < 0.001) (Figure 5B). MSC co-culture effectively counteracted these changes in the GM group. Specifically, MSC co-culture significantly upregulated the protein levels of MYLK, p-MYLK, α-SMA, ANO1, and CACNA1C compared to the GM group (all P < 0.001) (Figure 5B). Moreover, MSC co-culture also led to a further increase in MYPT1, p-MYPT1, and the p-MYPT1/MYPT1 ratio beyond the levels seen in the GM group (P < 0.001) (Figure 5B). These findings suggest that MSCs can directly modulate HESMC senescence and the expression of contraction-related proteins, mirroring the in vivo observations.

Figure 5 Mesenchymal stem cells ameliorate tumor necrosis factor-α-accelerated cellular senescence, protein dysregulation, and calcium signaling in human esophageal smooth muscle cells. This figure investigates the effects of mesenchymal stem cells on human esophageal smooth muscle cells under tumor necrosis factor-α exposure. A: Β-galactosidase staining images indicating cellular senescence; B: Western blot images and quantification for myosin light chain kinase, phosphorylated-myosin light chain kinase, alpha-smooth muscle actin, myosin phosphatase target subunit 1, phosphorylated-myosin phosphatase target subunit 1, anoctamin 1 and calcium voltage-gated channel subunit alpha1 C protein levels; C: Fluorescence images of Fluo-3, AM staining and quantification of intracellular Ca²⁺ intensity. Error bars represent SEM. ^aP < 0.05 for gastroesophageal reflux disease model group vs control group; ^cP < 0.001 for mesenchymal stem cell group vs gastroesophageal reflux disease model group; ^dP < 0.05 for mesenchymal stem cell group vs gastroesophageal reflux disease model group; ^fP < 0.001 for mesenchymal stem cell group vs gastroesophageal reflux disease model group; ^gP < 0.001 for si-ANO1/(+)Cacna1C group vs mesenchymal stem cell group. TNF: Tumor necrosis factor; NS: Not significant; Ctr: Control; GM: Gastroesophageal reflux disease model; MSC: Mesenchymal stem cell; α-SMA: Alpha-smooth muscle actin; MYLK: Myosin light chain kinase; p-MYLK: Phosphorylated-myosin light chain kinase; MYPT1: Myosin phosphatase target subunit 1; p-MYPT1: Phosphorylated-myosin phosphatase target subunit 1; ANO1: Anoctamin 1; CACNA1C: Calcium voltage-gated channel subunit alpha1 C; si-NC: Negative control small interfering RNA.

MSCs enhance intracellular calcium concentration in senescent HESMCs via ANO1 and CACNA1C

Given the significant changes observed in ANO1 and CACNA1C expression following TNF-α treatment and MSC co-culture, we further investigated their mechanistic role in MSC-mediated regulation of intracellular Ca²⁺ levels in HESMCs under GERD-like conditions. To control for potential non-specific effects of the transfection procedure, we included HESMCs transfected with a non-targeting si-NC in both the GERD model (GM + si-NC) and MSC co-culture (MSC + si-NC) settings. Using Fluo-3 AM staining, we observed that the MSC group significantly increased intracellular Ca²⁺ concentration compared to the GM group (P < 0.001) (Figure 5C), indicating that MSC treatment effectively restores Ca²⁺ homeostasis. Notably, Ca²⁺ levels in the GM + si-NC group showed no significant difference from the untransfected GM group, and levels in the MSC + si-NC group were comparable to the untransfected MSC group, confirming that the transfection process itself did not alter the baseline phenotype or the therapeutic effect of MSCs. To determine the specific contribution of ANO1 and CACNA1C to this MSC-induced Ca²⁺ upregulation, we performed gene knockdown experiments within the MSC co-culture system. The MSC-induced increase in intracellular Ca²⁺ (observed in the MSC + si-NC group) was significantly attenuated when ANO1 was knocked down (in MSC + siANO1 group), leading to a reduction in Ca²⁺ levels compared to the MSC + si-NC group (P = 0.043) (Figure 5C). Similarly, knockdown of CACNA1C (in MSC + siCACNA1C group) also significantly reduced the MSC-mediated Ca²⁺ elevation compared to the MSC + si-NC group (P < 0.001) (Figure 5C). Furthermore, simultaneous knockdown of both ANO1 and CACNA1C (in MSC + siCACNA1C + siANO1 group) resulted in an even greater reduction of MSC-induced intracellular Ca²⁺ levels compared to either single knockdown group (MSC + siANO1, P = 0.001), suggesting a cooperative and additive role of these channels in mediating MSC effects on Ca²⁺ dynamics (Figure 5C).

MSCs modulate muscle contraction-related proteins through DNMT3a and HIF1α pathways in senescent HESMCs

Further mechanistic studies explored the involvement of DNMT3a and HIF1α in MSC-mediated effects. In the MSC group, the mRNA levels of both DNMT3a and HIF1α showed a progressive upregulation over time, with significant increases observed at 24 hours and 48 hours compared to 12 hours (P < 0.001) (Figure 6A). Western blot analysis demonstrated that DNMT3a and HIF1α protein levels were significantly downregulated in the GM group compared to the control group (P < 0.001) (Figure 6B and C), an effect that was reversed by MSC co-culture (P < 0.001) (Figure 6B and C). Notably, pharmacological inhibition of DNMT3a with SGI-1027 led to a significant downregulation of HIF1α protein expression (P < 0.001) (Figure 6C). Overexpression of HIF1α in HESMCs significantly upregulated the protein levels of MYLK, p-MYLK, the p-MYLK/MYLK ratio, MYPT1, p-MYPT1, the MYPT1/p-MYPT1 ratio, and α-SMA compared to control cells (P < 0.001) (Figure 6D). Conversely, inhibition of DNMT3a using SGI-1027 resulted in a significant downregulation of MYLK, MYPT1, and α-SMA protein levels (P < 0.001) (Figure 6E). These findings suggest that MSCs exert their effects on muscle contraction proteins, at least in part, by modulating DNMT3a and HIF1α.

Figure 6 Mesenchymal stem cells modulate contraction-related proteins through DNA methyltransferase 3a and hypoxia-inducible factor 1 alpha pathway. This figure explores the molecular pathways by which mesenchymal stem cells modulate contraction-related proteins. A: Time-dependent mRNA expression levels of DNA methyltransferase 3a (DNMT3a) and hypoxia-inducible factor 1 alpha (HIF1α), likely as line graphs; B and C: Western blot images and quantification for DNMT3a and HIF1α protein levels in human esophageal smooth muscle cells (HESMCs), respectively; D: Western blot images and quantification for alpha-smooth muscle actin, anoctamin 1, calcium voltage-gated channel subunit alpha1 C, myosin light chain kinase, phosphorylated-myosin light chain kinase, ratio analysis, myosin phosphatase target subunit 1, phosphorylated-myosin phosphatase target subunit 1, ratio analysis in HESMCs with HIF1α overexpression; E: Western blot images and quantification for myosin light chain kinase, myosin phosphatase target subunit 1, and alpha-smooth muscle actin in HESMCs treated with a DNMT3a inhibitor. Error bars represent SEM. ^cP < 0.001 vs control; ^gP < 0.001 vs gastroesophageal reflux disease model group. TNF: Tumor necrosis factor; MSC: Mesenchymal stem cell; Ctr: Control; GM: Gastroesophageal reflux disease model; α-SMA: Alpha-smooth muscle actin; MYLK: Myosin light chain kinase; p-MYLK: Phosphorylated-myosin light chain kinase; MYPT1: Myosin phosphatase target subunit 1; p-MYPT1: Phosphorylated-myosin phosphatase target subunit 1; ANO1: Anoctamin 1; CACNA1C: Calcium voltage-gated channel subunit alpha1 C; DNMT3a: DNA methyltransferase 3a; HIF1α: Hypoxia-inducible factor 1 alpha.

Transcriptomic analysis reveals global gene expression changes and pathway modulation by MSCs in GERD esophagus

Transcriptome sequencing was performed on esophageal tissues to provide a comprehensive view of gene expression changes. Comparison between GERD and normal groups identified 13425 significantly upregulated (up_DGNG) and 1160 significantly downregulated (down_DGNG) genes (Figure 7A). Following MSC treatment, comparison between MSC and GERD groups identified 11910 upregulated (up_DGMG) and 1671 downregulated (down_DGMG) genes (Figure 7A). KEGG enrichment analysis of the DGNG (differential genes in GERD rats compared to normal rats) revealed significant enrichment in pathways related to endocytosis, various cancer pathways, and critical signaling pathways, including phosphatidylinositol-3-kinase (PI3K), Rap1, mitogen-activated protein kinase, nuclear factor kappa B (NF-κB), chemokine, and tumor necrosis factor signaling (Figure 7B). For DGMG (differential genes in MSC rats compared to GERD rats), KEGG analysis mainly highlighted pathways involved in cancer and infection (Figure 7C). A Venn diagram analysis revealed 11215 common genes (CGS) shared between DGNG and DGMG (Figure 7D). KEGG enrichment analysis of these CGS showed enrichment in pathways involved in infection, cancer, Rap1 signaling, NF-κB signaling, and PI3K signaling (Figure 7E). Further trend analysis within the CGS identified 2504 genes that were downregulated in GERD rats compared to normal, and subsequently upregulated after MSC treatment (Figure 7F). GO enrichment analysis of the CGS, especially focusing on muscle-related clusters, demonstrated that esophageal muscle function and structure were significantly affected in GERD rats and importantly regulated by MSC treatment, highlighting biological processes such as “muscle contraction”, “smooth muscle cell proliferation”, and “regulation of cell adhesion” (Figure 7G). Additionally, common DEGs revealed significant involvement in several key biological processes, including signal transduction and inflammatory response (e.g., JUN kinase activity, leukocyte degranulation), cellular structure and motility (e.g., lamellipodium organization, establishment of cell polarity), and intracellular transport (e.g., protein import into nucleus, endosome transport) (Figure 7H).

Figure 7 Transcriptomic analysis reveals mesenchymal stem cell-mediated pathway modulation. This figure presents a series of bioinformatics analyses comparing gene expression. A: Bar chart illustrating the number of significantly differentially expressed genes; B: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes differentially expressed between the gastroesophageal reflux disease and normal groups (DGNG); C: KEGG pathway enrichment analysis of genes differentially expressed between the gastroesophageal reflux disease and mesenchymal stem cell groups (DGMG); D: Venn diagram illustrating the overlap between differentially expressed genes in DGNG and DGMG; E: KEGG pathway enrichment analysis of common genes; F: Trend analysis of common genes; G: Gene Ontology (GO) enrichment analysis specifically focusing on muscle-related clusters within common genes; H: GO enrichment analysis of common genes. The bubble plot shows enriched pathways, with bubble size indicating the number of genes and color representing the adjusted P-value in KEGG and GO enrichment analysis. DEGs: Differentially expressed genes; KEGG: Kyoto Encyclopedia of Genes and Genomes; CGS: Common genes; GERD: Gastroesophageal reflux disease; MSC: Mesenchymal stem cell; GO: Gene Ontology.

DISCUSSION

The current study provides compelling evidence that MSC treatment effectively restores esophageal contractility and ameliorates ED in GERD both in vivo and in vitro models. This therapeutic effect is mediated by the modulation of multifaceted cellular and molecular pathways, offering a promising strategy for a condition where conventional acid suppression often fails.

Our in vivo findings demonstrate significant esophageal contractile dysfunction in GERD rats, characterized by a robust 25.65 mmHg reduction in ex vivo esophageal contraction force (Figure 1). Crucially, MSC treatment reversed this functional deficit, yielding a 16.53 mmHg increase in contraction force, underscoring the direct restorative potential of MSCs. Intriguingly, histological examination revealed smooth muscle cell hyperplasia and hypertrophy in GERD (Figure 2A), yet this increased muscle mass paradoxically coincided with impaired contractility. This striking structural-functional dissociation suggests a maladaptive hypertrophic response, where the enlarged smooth muscle tissue is functionally ineffective or minimally contributes to force generation. The observed functional impairment, despite the presence of structural hypertrophy, likely results from a disruption in excitation-contraction coupling - potentially along with changes in cytoskeletal organization or the development of interstitial fibrosis, which can increase passive stiffness and compromise effective force transmission. Our data supported the core mechanism of excitation-contraction uncoupling, as molecular analyses showed a significant downregulation of key contractile proteins and their activated forms, including α-SMA, MYLK, and p-MYLK, alongside a decreased p-MYLK/MYLK ratio in GERD rats (Figure 3A and B). Following MSC treatment, we observed a robust upregulation of α-SMA, MYLK, and p-MYLK, and an increased p-MYLK/MYLK ratio (Figure 3A and B), indicating a shift towards a more mature and functional smooth muscle phenotype. Simultaneously, MSCs notably augmented ANO1 and CACNA1C expression (Figure 4), vital for smooth muscle excitation-contraction coupling through their roles in calcium influx[13]. These in vivo observations were strongly corroborated by our in vitro HESMC experiments, where MSC co-culture effectively counteracted the TNF-α-accelerated senescence-induced downregulation of these essential contractile proteins and ion channels (Figure 5B), highlighting a direct cellular mechanism for MSC-mediated enhancement of smooth muscle function.

Further mechanistic insight emerged from the dynamic balance in the myosin light chain phosphorylation pathway. GERD rats exhibited decreased MYLK and p-MYLK, signifying impaired contractile initiation (Figure 3B). Concurrently, we observed a more modest, yet significant, elevation in MYPT1, p-MYPT1, and the p-MYPT1/MYPT1 ratio in GERD (Figure 3B). Given that p-MYPT1 inhibits myosin light chain phosphatase to promote contraction[14], this concomitant change may represent a compensatory attempt to counteract the severe loss of contractile drive from the diminished MYLK/p-MYLK “on” signal. However, the magnitude of this change suggests it is insufficient to restore function.

MSC administration not only normalized MYLK/p-MYLK but also induced a further, coordinated increase in MYPT1, p-MYPT1, and their ratio (Figure 3B). This pattern suggests that MSCs orchestrate a rebalancing of the phosphorylation-dephosphorylation switch. While the principal therapeutic effect is the rescue of the deficient “on” signal, the concomitant rise in p-MYPT1 serves to fine-tune the contractile apparatus. By inhibiting myosin light chain phosphatase, p-MYPT1 lowers the threshold for contraction and increases calcium sensitivity, thereby enabling the muscle to generate greater force for a given level of stimulatory input. Thus, MSC therapy facilitates optimal contractile force through a dual mechanism: The primary restoration of the contractile trigger, complemented by a strategic sensitization of the contractile machinery[14,15].

A novel and significant finding is the identification of the DNMT3a-HIF1α axis as a key signaling pathway mediating MSCs’ effects on muscle contraction-related proteins (Figure 6). We demonstrated that DNMT3a and HIF1α were significantly downregulated in senescent HESMCs (GM group), and MSC co-culture consistently reversed this (Figure 6B and C). We established a regulatory link where pharmacological DNMT3a inhibition suppressed HIF1α expression, and conversely, HIF1α overexpression robustly upregulated key contractile proteins (MYLK, p-MYLK, MYPT1, p-MYPT1, α-SMA) (Figure 6C and D). The direct impact of DNMT3a on contractile protein expression was also confirmed by its inhibition (Figure 6E). This elucidates a sophisticated regulatory cascade: MSCs modulate DNMT3a, which epigenetically influences HIF1α[16], a master transcription factor[17], thereby governing the expression of crucial contractile machinery components. This adds a profound layer of understanding to MSCs’ therapeutic actions.

Our comprehensive transcriptome analysis (Figure 7) further corroborated these mechanistic insights by providing a global perspective on gene expression changes. The extensive DEGs in GERD and their significant modulation by MSCs underscore the broad impact of both pathology and intervention. KEGG enrichment analysis highlighted that GERD primarily involves pathways related to inflammation, cellular stress, and even potential precancerous changes, such as PI3K, Rap1, mitogen-activated protein kinase, NF-κB, chemokine, and TNF signaling[18,19] (Figure 7B). Critically, MSC treatment significantly modulated many of these shared pathways, with enriched CGS showing involvement in infection, cancer, Rap1, NF-κB, and PI3K signaling (Figure 7E). The identification of 2504 genes with a “downregulated in GERD, upregulated by MSCs” trend (Figure 7F) is particularly valuable for pinpointing future therapeutic targets. Furthermore, GO enrichment analysis, specifically focusing on muscle-related clusters within shared genes, directly linked these genomic shifts to the restoration of esophageal muscle function and structure, via processes like “muscle contraction”, “smooth muscle cell proliferation”, and “regulation of cell adhesion” (Figure 7G). The transcriptomic enrichment of pathways like NF-κB and PI3K-Akt signaling in GERD tissues aligns with the observed inflammatory milieu (elevated TNF-α) and cellular senescence. The modulation of these pathways by MSC treatment, as seen in the sequencing data, provides a genomic-level corroboration for the functional rescue of senescence and contractility, suggesting that MSCs may act, in part, by normalizing these dysregulated signaling networks.

Despite these findings, our study has certain limitations that warrant future investigation. While our GERD rat model is well-established, it may not fully capture the chronicity, multifactorial etiology, or complete reflux spectrum of human GERD. The relatively short-term follow-up means long-term efficacy and persistence of MSC effects remain to be determined. Although paracrine effects were inferred in our Transwell co-culture, the specific soluble factors secreted by MSCs that mediate senescence amelioration and DNMT3a/HIF1α pathway modulation were not identified. Future research should prioritize longer-term follow-up, fate-mapping studies of MSCs to assess their integration and differentiation, and targeted investigations into specific MSC-secreted factors that activate the identified molecular pathways in HESMCs. While the restoration of ex vivo contractile force provides a fundamental measure of intrinsic smooth muscle health, future studies employing in vivo manometry or bolus transit assays will be essential to fully evaluate the restoration of coordinated peristaltic function and its translational relevance following MSC therapy. Furthermore, while the pharmacological inhibition and overexpression data support the involvement of the DNMT3a-HIF1α axis, future studies employing techniques such as chromatin immunoprecipitation to directly examine DNMT3a binding to the HIF1α promoter would help to further establish the precise transcriptional regulatory relationship. Additionally, the translation of MSC therapy into clinical practice for GERD would need to address key translational challenges, including optimal delivery routes, dosing regimens, long-term safety, and efficacy in human patients.

CONCLUSION

In conclusion, this study establishes MSC treatment as a potent therapeutic strategy for ED in GERD. MSCs exert their effects through multifaceted mechanisms, including direct restoration of esophageal contractility, alleviation of cellular senescence, improvement of calcium handling (via ANO1 and CACNA1C), and sophisticated regulation of muscle contraction-related proteins through a novel DNMT3a-HIF1α pathway. These comprehensive insights, integrating functional, histological, molecular, and transcriptomic data, provide a strong foundation for the potential clinical translation of MSC-based therapies to address the underlying dysmotility and improve outcomes for GERD patients.

ACKNOWLEDGEMENTS

We extend our sincere gratitude to Beijing RM Bio-tech Co., Ltd. (Beijing, China) for their generous donation of human umbilical cord mesenchymal stem cells, which were indispensable for the successful completion of this study. Their support significantly facilitated our research efforts.