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

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.

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.

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.

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.

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.

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.