Novel Potassium-Competitive Acid Blocker, Tegoprazan, Protects Against Colitis by Improving Gut Barrier Function

Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder characterized by prolonged inflammation of the gastrointestinal tract. IBD can result from gut barrier dysfunction, altered gut microbiota, and abnormal intestinal immunity induced by environmental factors in genetically susceptible individuals. Proton pump inhibitors (PPIs) such as rabeprazole are frequently employed for gastric acid inhibition. However, long-term PPI administration can alter the intestinal microbiome composition, possibly worsening IBD severity. The present study revealed that tegoprazan, a potassium-competitive acid blocker, significantly improved colitis in mice and enhanced the intestinal epithelial barrier function. Tegoprazan alleviated gut microbiota dysbiosis and enhanced the growth of Bacteroides vulgatus. In turn, B. vulgatus alleviated intestinal inflammation by inhibiting epithelial adhesion of pathogenic bacteria. Unlike rabeprazole, tegoprazan did not induce gut dysbiosis. Our findings provide novel insights into the potential role of tegoprazan as an intestinal protectant for IBD and as a therapeutic agent for gastric acid-related diseases.


Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded colon sections were deparaffinized in xylene and ethanol and rehydrated in water. Antigen retrieval was performed in a heated sodium citrate buffer (pH 6.0) for 10 min. Sections were washed with distilled water, quenched in 0.3% hydrogen peroxide to block endogenous peroxidase activity, and blocked in 3% bovine serum albumin (BSA) diluted in Tris-buffered saline plus 0.1% Tween-20 (TBS-T) for 30 min at room temperature. Then, sections were incubated with anti-Zo-1 antibody (1:500, ab59720, Thermo Fisher Scientific) diluted in blocking buffer overnight at 4°C, following which biotinylated anti-rabbit secondary antibody (1:500, BP-9100-50, Vector Laboratories, Burlingame, CA, USA) was applied for 30 min at room temperature. VECTASTAIN ® Elite ABC-HRP Kit (PK-6100, Vector Laboratories) was employed, and staining was visualized using the DAB Substrate Kit (SK-4100, Vector Laboratories). Slides were counterstained with hematoxylin, dehydrated, mounted, and observed under a microscope. qRT-PCR, western blotting, and flow cytometric analysis are described in the Supporting Information.

Metagenomic analysis of the microbiome
Bacterial genomic DNA was obtained from fecal and tissue samples using SPIN for Soil Kit (116560200, MP Biomedicals), according to the manufacturer's instructions. Extracted DNA was amplified using bar-coded primers flanking the V3-V4 region of the 16S rRNA gene by ChunLab Inc. (Seoul, Korea). For bacterial amplification, fusion primers of 341F (5′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3′; underlining sequence indicates the target region primer) and 805R (5′-CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3′). The Fusion primers are constructed in the following order which is P5 (P7) graft binding, i5 (i7) index, Nextera consensus, Sequencing adaptor, and Target region sequence. The amplifications were carried out under the following conditions: initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72 °C for 30 s, with a final elongation at 72°C for 5 min. The PCR product was analyzed by using 1% agarose gel electrophoresis and visualized under a Gel Doc system (BioRad, Hercules, CA, USA). The amplified products were purified with the CleanPCR (CleanNA). Equal concentrations of purified products were pooled together and short fragments (non-target products) were removed with CleanPCR (CleanNA, Waddinxveen, The Netherlands). The quality and product size were assessed on a Bioanalyzer 2100 system (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled and the sequencing was carried out at Chunlab, Inc., with Illumina MiSeq Sequencing system (Illumina, CA, USA) according to the manufacturer's instructions.
Reads obtained from different samples were sorted by unique barcodes of each PCR product. Processing raw reads start with quality check and filtering of low quality (<Q25) reads by Trimmomatic 0.32 (52). After QC pass, paired-end sequence data are merged using PANDAseq (53). Primers are then trimmed with ChunLab's in-house program at a similarity cutoff of 0.8. Non-specific amplicons that do not encode 16S rRNA are detected by HMMER's hmmsearch program (54) with 16S rRNA profiles. Sequences are denoised using DUDE-Seq (55) and non-redundant reads are extracted by . The EzBioCloud database is used for taxonomic assignment using USEARCH (8.1.1861_i86linux32) (56) followed by more precise pairwise alignment (57). UCHIME (58) and the non-chimeric 16S rRNA database from EzBioCloud are used to detect chimera on reads with <97% similarity. Reads that are not identified to the species level (with <97% similarity) in the EzBioCloud database are compiled and UCLUST (56) and CDHIT (59) are used to perform de-novo clustering to generate additional OTUs. Finally, OTUs with single reads (singletons) are omitted from further analysis. The alpha diversity indices (60) are estimated by in-house code. Microbiome data were analyzed with the 16S-based microbial taxonomic profiling (MTP) platform of EzBio-Cloud (ChunLab Inc.). All 16S rRNA sequences were deposited in the ChunLab EzBioCloud Microbiome Database (63). Herein, we utilized the eztaxon-e taxonomic structure (64) as the backbone phylogenetic tree. Both UniFrac and Fast Unifrac algorithms were developed by Knight and colleagues (65). We analyzed operational taxonomic units and beta diversity using principal coordinate analysis (PCoA) and alpha diversity analysis using diversity indices (Chao1, Ace, Shannon, and Simpson indices). Permutational multivariate analysis of variance (PERMANOVA) was used to analyze significant differences in alpha diversity. Bacterial community abundance was determined using CLcommunity (ChunLab Inc.).

Immunofluorescence analysis
Caco-2 cells were fixed with 10% neutral buffered formalin for 30 min at room temperature and washed with phosphate-buffered saline (PBS). Next, cells were blocked with 5% BSA for 1 h at room temperature and incubated with rabbit anti-E-cadherin antibody (1:200, Cell Signaling Technology, Inc., Beverly, MA, USA) diluted in blocking buffer overnight at 4°C. Cells were then washed with PBS, incubated with the secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG H&L (1:200, A-11008, Invitrogen, Cambridge, UK) for 1 h at room temperature, and nuclear stained with diamidino-2-phenylindole (DAPI, 300 nM, D1306, Thermo Fisher Scientific) for 3 min. Confocal images were obtained using a Zeiss LSM700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Western blotting
Caco-2 cells were lysed with Pierce RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was measured with Pierce ™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Proteins were separated according to their molecular weight by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes.

Bacterial adhesion assay
HT-29 cells were plated in 12-well plates in fresh medium supplemented with 10% FBS. The cells were treated with B. vulgatus and S. typhimurium for 3 h at a ratio of 50:1. Cells were then washed and removed from plates and exposed to 100 μL of 0.1% Triton-X 100 for 10 min. Next, 900 μL of nutrient broth (Difco, Becton & Dickinson, Sparks, MD, USA) was added to the plates, and the cell lysates were resuspended. Finally, cell lysates were serially diluted and plated on nutrient agar plates with 100 µg/ml ampicillin to enumerate the CFUs of the adhered bacteria. Figure S1. Tegoprazan action is beyond the effect of filgotinib. Colitis was induced in 8-week-old male mice by administering 2.5% DSS in distilled drinking water for 5 days, followed by normal drinking water for 4 days. Tegoprazan and filgotinib (30 mg/kg) were administered orally daily throughout the experimental period q.d. and b.d., respectively. All drugs were dissolved in 0.5% (w/v) methylcellulose. a, Survival rate from survival model. CON (n = 7), DSS+Veh, DSS+TEGO, and DSS+FILGO (each n = 13). Survival analyses were performed using Kaplan-Meier plots for overall survival and differences were compared using a log-rank test. We observed that Tregs (CD3 + CD4 + CD25 + Foxp3 + cells) were significantly induced in the DSS+TEGO group when compared with the DSS+Veh group, indicating that tegoprazan facilitates Treg induction. Data represent the mean ± standard error of the mean (S.E.M).