Fexuprazan safeguards the esophagus from hydrochloric acid-induced damage by suppressing NLRP1/Caspase-1/GSDMD pyroptotic pathway

Abstract

Introduction: Proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) are widely used to manage gastric acid-related disorders by inhibiting hydrochloric acid (HCl) secretion from parietal cells in the stomach. Although PPIs are known to have anti-inflammatory properties beyond their role in inhibiting gastric acid secretion, research on P-CABs is lacking. In this study, we aimed to investigate whether all available P-CABs exhibit anti-inflammatory effects in gastroesophageal reflux-induced esophagitis and to elucidate the underlying mechanisms.

Methods: Het-1A cells, normal esophageal epithelial cells, were treated with HCl (pH 4) for 30 min. Esomeprazole, a representative PPI, and three currently marketed P-CABs (vonoprazan, tegoprazan, and fexuprazan) were used for pretreatment. Total RNA sequencing was performed using Het-1A cells pretreated with 1% DMSO or fexuprazan, followed by exposure to HCl. Pyroptosis was measured using lactate dehydrogenase (LDH) release and Annexin V-FITC/PI staining. Western blotting, qRT-PCR, and ELISA were used to determine the expression of the related genes.

Results: Pretreatment with esomeprazole, vonoprazan, tegoprazan, and fexuprazan significantly inhibited the HCl-induced pro-inflammatory cytokines, including IL-6, IL-8, IL-1β, and TNF-α. Fexuprazan and vonoprazan significantly attenuated the HCl-induced pyroptosis rate, as assessed by elevated LDH release and Annexin V-FITC/PI staining, whereas esomeprazole and tegoprazan did not. RNA sequencing revealed that NOD-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) was significantly reduced in Het-1A cells pretreated with fexuprazan compared to those treated with DMSO. Fexuprazan and vonoprazan markedly reduced the HCl-induced transcriptional and translational expression of genes involved in the pyroptosis pathway, including NLRP1, Caspase-1, gasdermin D, and IL-1β. Notably, fexuprazan reduced the HCl-induced increase in pyroptosis and IL-1β using siRNA, even in the presence of NLRP1 knockdown. Fexuprazan, tested on inflammatory THP-1 macrophage cells, significantly reduced NLRP1 expression and inhibited lipopolysaccharide-induced pyroptosis.

Conclusion: Our findings reveal that all p-CABs exhibit anti-inflammatory properties, while fexuprazan inhibits inflammation and pyroptosis of esophageal cells caused by the gastric acid. Therefore, it is presumed to have additional benefits in gastroesophageal reflux disease in addition to suppressing gastric acid secretion.

Keywords: NLRP1; P-CABs; esophagus; fexuprazan; hcl; pyroptosis.

Figure 1

Fexuprazan reduced inflammatory responses caused by HCl. (A) Het-1A cells were treated with HCl (pH 4) as indicated in various time points (min: minutes). The indicated genes were analyzed by real-time PCR assay. (B) The LDH release (%) was measured at various concentrations of fexuprazan (FXZ) before and after treatment under basal condition s (left) and HCl treatment (right, at pH 4). (C) Het-1A cells were treated with fexuprazan (FXZ, 30 µM) as indicated in various time points (hours). The indicated genes were analyzed by real-time PCR assay. (D) Het-1A cells were pretreated with fexuprazan (FXZ, 30 µM), vonoprazan (VNZ, 30 µM), tegoprazan (TGZ, 30 µM), esomeprazole (ESO, 30 µM) for 6 h followed by HCl (pH 4) for 30 min. The indicated genes were analyzed by real-time PCR assay. Data represented the mean ± s.e.m. n = 3, t test, ns: not significant; *p < 0.05, **p < 0.01, ***P < 0.001 versus control.

Figure 2

Differentially expressed genes (DEG) detected by RNA-seq. (A) Total RNA-sequencing on Het-1A cells pre-treated with 1% DMSO (control) or fexuprazan (test, FXZ, 30 µM) followed by HCl treatment. (B) Top ten functional categories of DEGs. (C) Volcano plot of cluster analysis for DEGs between immune response and inflammatory response of RNA-seq. (D) Heatmap of pyroptosis-related DEGs on Het-1A cells pre-treated with 1% DMSO (DMSO+HCl_1, DMSO+HCl_2) or fexuprazan (FXZ+HCl_1, FXZ +HCl_2).

Figure 3

Fexuprazan reduced the expression of pyroptosis related genes upregulated by HCl. (A, B) mRNA and protein expression of pyroptosis related genes were detected with Het-1A cells pretreated with DMSO (control) or drugs followed by exposure to HCl. (C) Het-1A cells were treated as indicated condition and supernatant of IL-1β was determined by ELISA assay. (D) Het-1A cells were assessed for pyroptosis using flow cytometry based on Annexin V-FITC/PI staining. Pyroptosis was defined as Annexin V+/PI+. All of the data are from three independent experiments. Data represented the mean ± s.e.m. n = 3, ns: not significant; t test, *p < 0.05, **p < 0.01, ***P < 0.001 versus con group. DMSO (control, 1%) or fexuprazan (FXZ, 30 µM), vonoprazan (VNZ, 30 µM), tegoprazan (TGZ, 30 µM), esomeprazole (ESO, 30 µM) followed by exposure to HCl.

Figure 4

Fexuprazan attenuated LPS-induced pyroptosis in THP-1 macrophages. (A) THP-1 cells were treated with LPS as indicated in various time and dose points. The indicated genes were analyzed by real-time PCR assay. (B) mRNA expression of NLRP1 was detected with THP-1 cells pretreated with DMSO or fexuprazan followed by treatment to LPS. (C) The LDH assay was performed to detect pyroptosis in response to fexuprazan and LPS treatment. (D) NLRP1, GSDMD and GSDMD-NT, Pro caspase-1 and cleaved caspase-1, and Mature IL-1β protein expression were assessed by immunoblotting. (E) THP-1 cells were assessed for pyroptosis using flow cytometry based on Annexin V-FITC/PI staining. Pyroptosis was defined as Annexin V+/PI+. (F) Schematic showing how fexuprazan safeguards the esophagus from hydrochloric acid-induced damage. All of the data are from three independent experiments. Data represented the mean ± s.e.m. n = 3, ns: not significant; t test, *p < 0.05, **p < 0.01, ***P < 0.001 versus con group.

Figure 5

Fexuprazan demonstrated a pyroptosis inhibitory effect, similar to the administration of NLRP1 siRNA. (A) mRNA expression of NLR gene family was detected with Het-1A cells pretreated with DMSO or fexuprazan followed by exposure to HCl. (B) After transfection with NLRP1 siRNA (siNLRP1), treatment with fexuprazan, and simultaneous co-treatment with siNLRP1 and fexuprazan, the mRNA (left) and protein (right) expression of NLRP1 were compared. (C) After transfection with NLRP1 siRNA (siNLRP1), treatment with fexuprazan, and simultaneous co-treatment with siNLRP1 and fexuprazan, the impact on the HCl-induced increase in NLRP1 mRNA and protein was observed. (D) The LDH assay was performed to detect pyroptosis in response to NLRP1 siRNA transfection, treatment of fexuprazan, and siNLRP1 and fexuprazan co-treatment followed by exposure to HCl. (E) Het-1A cells were treated as indicated condition and supernatant of IL-1β was determined by ELISA assay. All of the data are from three independent experiments. Data represented the mean ± s.e.m. n = 3, ns: not significant; t test, *p < 0.05, **p < 0.01, ***P < 0.001 versus control or siCT.