Fig 2: A graphical hypothesis of this study. Weak acid exposure at pH 4–5 induces PGE2 production via TRPV4/ERK/cPLA2. Under normal conditions, oesophageal epithelial cells produce only a small amount of PGE2 in response to weak acid stimulation, which may contribute to biological protection. Meanwhile, in cases of high expression of COX-2 due to stimulations, such as CDCA, weak acid exposure at pH 4–5 produces excess PGE2, which may trigger various symptoms including heartburn and oesophageal cancer development. HST is effective in treating various GERD symptoms due to excessive PGE2 production by suppressing COX-2-dependent production. PGE2 prostaglandin E2; TRPV4 transient receptor potential vanilloid 4; ERK extracellular signal-regulated kinase; cPLA2 cytosolic phospholipase A2; COX-2 cyclooxygenase-2; CDCA chenodeoxycholic acid; HST hangeshashinto.

Fig 3: (A) State 1 mitochondrial H2O2 generation from permeabilized fibers of the TA muscle in the presence of different enzyme inhibitors. Addition of each inhibitor (chlorgyline (monoamine oxidase A), parglyline (monoamine oxidase B), apocynin (NADPH oxidase), AACOCF3 (PLA2) resulted in significantly reduced H2O2 generation compared to untreated fiber bundles at 14 and 21 days post-denervation. Histograms represent the mean and standard error of the mean for each experimental group (n = 4). *p < 0.05 - **p < 0.01 - ***p < 0.001 compared with its state 1. (B) Quantification of western blots of NADPH oxidase 2 subunits (Nox2/gp91phox and p67phox) and NADPH oxidase 4 (mitochondrial isoform) and (C) of cPLA2 and monoamine oxidase A content in TA muscle from control mice (non-denervated) and mice at 1, 3, 7, 14 and 21 days post-denervation. Histograms represent the mean protein content and standard error of the mean for each experimental group (n = 4). *p < 0.05 - **p < 0.01 - ***p < 0.001 compared with the control group.

Fig 4: Different effects of pH 4.5 medium and CDCA on mitogen-activated protein kinase (MAPK) activation. (a) Treatment with the extracellular signal-regulated kinase (ERK) inhibitor FR180204 (FR; 0.2 and 1 μmol/L) inhibited PGE2 production induced by pH 4.5 medium but it had little or no effect on CDCA (400 μmol/L)-induced production in KYSE-270 cells. (b) ERK and cPLA2 phosphorylation were increased after pH 4.5 stimulation for 5 min. (c) pH 4.5-induced cPLA2 phosphorylation was inhibited by ERK inhibitor FR180204 (FR; 0.2 μmol/L) treatment. These shown blots were cropped to improve the conciseness and the full-length blots were presented in Supplementary Fig. S7. PGE2 prostaglandin E2; CDCA chenodeoxycholic acid; cPLA2 cytosolic phospholipase A2. Data are presented as means ± SD (n = 3). Statistical significance was determined by Tukey–Kramer test; *P < .001.

Fig 5: WWL70 treatment had no effect on cPLA2 phosphorylation in CCI mouse sciatic nerve. a Representative blot of two tissue lysates made from sciatic nerve removed from sham and CCI injured mice at 7 days after injury probed with antisera specifically recognizing p-cPLA2 or total cPLA2. b Quantification of western blot data showing the relative ratio of p-cPLA2/cPLA2 each normalized to β-actin (mean ± S.E.M., n = 4)