Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly prescribed medications for arthritis, inflammation, and cardiovascular protection, particularly among patients over 65 years old1. NSAIDs damage the mucosa of the gastrointestinal (GI) tract, leading to severe complications such as bleeding and perforation. Because of the increasing aging of the population and the rising rates of major comorbidities, the frequency and severity of NSAID-related GI complications are important concerns. Although previous research primarily focused on NSAID-induced gastropathy, recent epidemiological studies indicate a rising incidence of NSAID-induced enteropathy2,3. Furthermore, patients with NSAID-related lower GI complications have prolonged hospitalization and increased mortality compared to those with upper GI complications4.

Discontinuation of NSAIDs is recommended in cases of NSAID-induced enteropathy. Nevertheless, given the difficulty in discontinuing NSAIDs for some patients with chronic conditions, strategies for the prevention and treatment of complications are needed. Although the incidence of upper GI complications of NSAIDs has decreased with use of proton pump inhibitors (PPIs)5, there are no medications specific for NSAID-induced enteropathy. Additionally, PPIs have been reported to exacerbate the damaging effects of NSAIDs on the small bowel mucosa6,7,8.

A recently developed potassium-competitive acid blocker (P-CAB), tegoprazan, has prompt and longer-lasting acid inhibitory effects than PPIs9 and is prescribed for acid-related disorders. P-CAB also showed a beneficial effect in preventing ulcer recurrence in patients receiving NSAIDs or low-dose aspirin10,11. However, the effects of tegoprazan on NSAID-related small bowel injury are unclear. Here, we evaluated the effect of tegoprazan on NSAID-induced enteropathy and investigated the underlying mechanisms in small intestinal epithelial cells.

Materials and methods

Cell culture

The human intestinal epithelial cell line HIEC-6 was obtained from the American Type Culture Collection (Manassas, VA, USA). HIEC-6 cells were maintained in Dulbecco’s modified Eagle’s medium and Ham’s F-12 Nutrient Mixture 1:1 (DMEM/F12; WELGENE, Gyeongsangbuk-do, Korea) supplemented with 10% fetal bovine serum (FBS; WELGENE), 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 5 ng/mL epidermal growth factor (EGF; MERCK, Darmstadt, Germany), and 1% penicillin–streptomycin solution (WELGENE) in a 5% CO2 atmosphere at 37 °C. Human colorectal cancer cell lines (Caco-2 and HT-29) were purchased from the Korean Cell Line Bank (Seoul, Korea). Caco-2 cells were maintained in Eagle’s minimum essential medium (MEM; WELGENE), and HT-29 cells were maintained in RPMI-1640 medium (WELGENE) containing 10% FBS and 1% penicillin–streptomycin in a 5% CO2 atmosphere at 37 °C.

Cell viability assay

Indomethacin and tegoprazan were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO). To determine the optimal concentrations of indomethacin and tegoprazan in HIEC-6 and the two colorectal cancer cell lines, we analyzed cell viability by 3(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay according to the manufacturer’s recommendations. Briefly, cells were cultured in 96-well plates for 24 h and incubated with predetermined concentrations of indomethacin (1–1000 µM) and tegoprazan (1–1000 µM) for 72 h in a 5% CO2 incubator at 37 °C. At the indicated times, 0.5 mg/mL MTT solution was added to the wells. After incubation for 3 h, the supernatant was discarded, and DMSO was added to the plates. The color intensity at 570 nm was measured using microplate reader (SpectraMax Plus 384; Molecular Devices, USA). Cell viability values are presented as percentages of the viability of control culture (DMSO, considered 100% viability). Tests were performed on all samples at least three times in the same assay run.

Quantitative RT-PCR

Total RNA was isolated from cells using TRIZOL (Thermo Fisher Scientific Inc., Waltham, MA, USA). mRNA was eluted in 20 µL of diethyl pyrocarbonate (DEPC) water (Thermo Fisher Scientific) and quantified using a NanoDrop ND-100 instrument (Thermo Fisher Scientific). Synthesis of cDNA was performed in mixtures containing 2 µg of RNA, oligo-dT, and AMV-reverse transcriptase (Promega, Madison, WI, USA), following the manufacturer’s instructions. Primers were designed using Primer3 version 0.4.0 (https://primer3.ut.ee/) and are shown in Table 1. Quantitative RT-PCR was performed using TOPreal SYBR Green qPCR PreMIX (Enzynomics, Daejeon, Korea) and the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in the same reaction to serve as an internal control for normalization. Fold changes in gene expression were measured using the comparative threshold cycle method (ΔΔCt). Tests were performed in triplicate in the same assay, with all experiments involving at least three replicates.

Table 1 Primer sequences used for mRNA quantitation.
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Western blotting

To verify the efficacy of tegoprazan against indomethacin in HIEC-6 and Caco-2, cells were seeded in six-well culture plates (SPL LIFE SCIENCES, Pocheon, Korea) at 1 × 106 per well. After 24 h, HIEC-6 cells were treated with 300 µM indomethacin, and Caco-2 cells were treated with 200 µM indomethacin. Each well was treated with indomethacin alone or in combination with tegoprazan at 30 and 100 µM. The cells were harvested after 24 and 48 h, respectively. To extract total protein, cells were lysed in RIPA buffer containing Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, Baker, TX, USA) and stored at − 80 °C until use. Protein extract was diluted with sample buffer (LPS SOLUTION, Daejeon, Korea) and denatured at 95 °C for 5 min. Protein samples were subjected to electrophoresis in sodium dodecyl sulfate polyacrylamide gels (10% SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare Life Science, Pittsburgh, PA). The membranes were incubated overnight at 4 °C with primary anti-occludin (#91131s), anti-ZO-1 (#8193s), anti-IL-1β (#12703s), anti-p53 (#9282T), anti-cleaved caspase 3 (#9661), anti-GAPDH (#97166, Cell Signaling, Danvers, MA, USA), and anti-caspase 1(#ab207802, Abcam, Cambridge, UK) antibodies. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and -mouse; Cell Signaling) for 1 h at room temperature. Bound antibodies were visualized using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). GAPDH was used as the loading control.

Immunofluorescence staining

Cells were grown on 18-mm glass coverslips in 12-well culture plates and fixed with freshly prepared 4% paraformaldehyde (PFA) for 10 min. Next, cells were permeabilized in 0.1% Triton X-100 for 10 min and blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. Cells were stained overnight at 4 °C using 1:200 dilutions of anti-occludin and anti-ZO-1 primary antibodies. After washing, cells were stained with a fluorescent secondary antibody (Alexa Fluor 488-conjugated anti-rabbit IgG‘ Cell Signaling) for 1 h at room temperature. Finally, cells were mounted in mounting solution containing Hoechst 33342 (Thermo Fisher Scientific). Images were acquired using ZEISS LSM 900 microscope with Airyscan 2 (Jena, Germany).

Statistical analysis

Experiments were repeated at least three times. Statistical significance was determined by paired t-test with IBM SPSS Statistics version 21.0 (IBM Inc., Chicago, IL, USA) and all tests were two-sided. An ANOVA test was applied to evaluate the significance of multiple experimental conditions. A significance values of P < 0.05 were considered to indicate statistical significance.

Results

Tegoprazan mitigates small bowel injury induced by indomethacin

To assess the effect of indomethacin and tegoprazan on cell proliferation, we conducted an MTT assay to evaluate cell viability. Both indomethacin and tegoprazan exhibited a dose-dependent suppression of cell viability (Fig. 1A–F). For indomethacin, IC50 values of 300, 200, and 300 µM were the optimal concentrations for HIEC-6, Caco-2, and HT-29 cells, respectively. In combination with indomethacin, tegoprazan was used at a concentration of 100 µM or less, which inhibited cell viability by less than 20%. The indomethacin-induced reduction of viability was mitigated by tegoprazan in HIEC-6, Caco-2, and HT-29 cells (Fig. 2). In HIEC-6 cells, tegoprazan at 1–100 µM showed therapeutic efficacy (Fig. 2A). In Caco-2 cells, the inhibition of cell viability by indomethacin was significantly alleviated by tegoprazan at 10–100 µM; in HT-29 cells, 10 µM tegoprazan showed significant efficacy (Fig. 2B,C).

Fig. 1
figure 1

Cell viability of HIEC-6, Caco-2, and HT-29 cells treated with indomethacin and tegoprazan. (A) Cell viability of HIEC-6 cells was assessed using an MTT assay after treatment with indomethacin at concentrations ranging from 1 to 1000 µM for 3 days. (B) Cell viability of HIEC-6 cells was evaluated using an MTT assay after treatment with tegoprazan at concentrations ranging from 10 to 1000 µM for 3 days. (C) Cell viability of Caco-2 cells was assessed using an MTT assay after treatment with indomethacin at concentrations ranging from 1 to 1000 µM for 3 days. (D) Cell viability of Caco-2 cells was assessed using an MTT assay after treatment with tegoprazan at concentrations ranging from 10 to 1000 µM for 3 days. (E) Cell viability of HT-29 cells was assessed using an MTT assay after treatment with indomethacin at concentrations ranging from 1 to 1000 µM for 3 days. (F) Cell viability of HT-29 cells was assessed using an MTT assay after treatment with tegoprazan at concentrations ranging from 10 to 1000 µM for 3 days. Indo, indomethacin; Tego, tegoprazan. −, not treated.

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Fig. 2
figure 2

Effect of tegoprazan on the indomethacin-induced inhibition of cell viability of HIEC-6, Caco-2, and HT-29 cells. (A) Cell viability of HIEC-6 cells was evaluated by MTT assay after treatment with indomethacin alone at 300 µM or in combination with tegoprazan at 1–100 µM for 3 days. Bar graph shows MTT results on days 1–3 after treatment with indomethacin alone and in combination with 10 µM tegoprazan. (B) Cell viability of Caco-2 cells after treatment with 200 µM indomethacin alone and in combination with tegoprazan by MTT assay for 3 days. Bar graph shows MTT results on days 1–3 after treatment with indomethacin alone and in combination with 100 µM tegoprazan. (C) Cell viability of HT-29 cells after treatment with 300 µM indomethacin alone and in combination with tegoprazan by MTT assay for 3 days. Bar graph shows MTT results on days 13 after treatment with indomethacin alone and in combination with 10 µM tegoprazan. *, †P-values < 0.05 were considered indicative of statistical significance. Indo, indomethacin; Tego, tegoprazan; +, treated; −, not treated. NS not significant.

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Tegoprazan restores indomethacin-induced small bowel permeability

Disruption of tight junctions and increased intestinal permeability are important factors in NSAID-induced enteropathy12. Because tight junctions regulate intestinal permeability, we investigated the effects of indomethacin and tegoprazan on the expression of two tight junction-specific factors, occludin and ZO-1. Quantitative RT-PCR revealed a significant decrease in the mRNA levels of occludin and ZO-1 induced by indomethacin in HIEC-6 and Caco-2 cells. However, these levels were effectively restored by tegoprazan (Fig. 3A,B). Additionally, the protein levels of occludin and ZO-1, which were reduced by indomethacin, were markedly increased by tegoprazan in HIEC-6 and Caco-2 cells (Fig. 3C,D). Immunofluorescence staining confirmed that tegoprazan attenuated the indomethacin-induced suppression of occludin and ZO-1 (Fig. 3E,F).

Fig. 3
figure 3

Tegoprazan-induced expression of the tight junction molecules occludin and ZO-1 in HIEC-6 and Caco-2 cells damaged by indomethacin. (A) HIEC-6 cells were treated with 300 µM indomethacin alone and in combination with tegoprazan at 1–100 µM for 2 days. Relative mRNA levels of occludin and ZO-1 were determined by qRT-PCR. (B) Caco-2 cells were treated with 200 µM indomethacin alone and in combination with tegoprazan for 2 days. Relative mRNA levels of occludin and ZO-1 were determined by qRT-PCR. (C) HIEC-6 cells were treated with 300 µM indomethacin alone or in combination with tegoprazan at 30 and 100 µM for 2 days. The protein levels of occludin and ZO-1 were analyzed by Western blotting. (D) Caco-2 cells were treated with 200 µM indomethacin alone or in combination with tegoprazan at 30 and 100 µM for 2 days. The protein levels of occludin and ZO-1 were analyzed by western blotting. GAPDH was used as the loading control. HIEC-6 (E) and Caco-2 (F) cells were treated with indomethacin alone and in combination with tegoprazan for 2 days, and the distributions of occludin and ZO-1 were analyzed by immunofluorescence staining and confocal microscopy (×100). Nuclei were stained with Hoechst 33,342. *, †P-values < 0.05 were considered indicative of statistical significance. Indo, indomethacin; Tego, tegoprazan; Hoechst, Hoechst 33,342 +, treated; −, not treated.

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Tegoprazan suppresses proinflammatory cytokine expressions and indomethacin-induced cell apoptosis

To analyze the effects of indomethacin and tegoprazan on proinflammatory cytokines, the mRNA levels of IL-1β, IL-6, TNF-α, IL-17α, and Muc2 were measured after treatment with indomethacin, with or without tegoprazan. The mRNA levels of proinflammatory cytokines were significantly increased by indomethacin in HIEC-6 and Caco-2 cells. Conversely, tegoprazan significantly reduced the levels of these cytokines (Fig. 4A,B). Western blotting was performed to assess the inhibitory effect of tegoprazan on indomethacin-induced inflammation responses and apoptosis. Indomethacin induced the expression of inflammation-related caspase 1 and the inflammatory cytokine IL-1β in HIEC-6 and Caco-2 cells, and induced apoptosis by increasing the levels of p53 and caspase 3. These effects induced by indomethacin were mitigated by tegoprazan in both HIEC-6 and Caco-2 cells (Fig. 5A,B).

Fig. 4
figure 4

Effect of tegoprazan on the mRNA levels of proinflammatory cytokines in HIEC-6 and Caco-2 cells damaged by indomethacin. (A) HIEC-6 cells were treated with 300 µM indomethacin alone and in combination with tegoprazan at 1–100 µM for 2 days. Relative mRNA levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-17α) were determined by qRT-PCR. (B) Caco-2 cells were treated with 200 µM indomethacin alone and in combination with tegoprazan for 2 days. Relative mRNA levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and Muc2) were determined by qRT-PCR. GAPDH was used as the loading control. *, †P-values < 0.05 were considered indicative of statistical significance. Indo, indomethacin; Tego, tegoprazan; +, treated; −, not treated.

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Fig. 5
figure 5

Protein levels of inflammation-related caspase 1, proinflammatory cytokine, IL-1β, and cell apoptotic molecules, p53 and cleaved caspase 3 induced by tegoprazan in HIEC-6 and Caco-2 cells damaged by indomethacin. (A) HIEC-6 cells were treated with 300 µM indomethacin alone and in combination with tegoprazan at 30 and 100 µM for 2 days. The protein levels of IL-1β, p53, and cleaved caspase 3 were analyzed by western blotting. (B) Caco-2 cells were treated with 200 µM indomethacin alone and in combination with tegoprazan at 30 and 100 µM for 2 days. The protein levels of caspase 1, IL-1β, p53, and cleaved caspase 3 were analyzed by western blotting. GAPDH was used as the loading control. Indo, indomethacin; Tego, tegoprazan; +, +, treated; −, not treated.

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Discussion

We investigated the effect of tegoprazan on NSAID-induced enteropathy. Indomethacin suppressed cell proliferation, and tegoprazan reversed this effect. In indomethacin-induced small bowel injury, tegoprazan restored intestinal permeability by upregulating the tight junction proteins, occludin and ZO-1. Also, tegoprazan reduced the expression levels of proinflammatory cytokines and inhibited apoptosis.

With the progression of diagnostic techniques for the small bowel, including capsule endoscopy and balloon-assisted enteroscopy, evidence indicates that NSAIDs cause damage to the small bowel, with estimated prevalences of 68% in short-term endoscopy studies and 71% in long-term users7,13. Over the past decade, the incidences of NSAID-related lower GI complications have increased significantly, whereas upper GI complications show a clear decreasing trend in incidence4. NSAIDs induce upper GI mucosal damage by inhibiting cyclooxygenase, causing a deficiency of prostaglandin (PG)12,14. However, the pathophysiology of small bowel damage differs because the small intestine is independent of gastric acid. In addition, there are no effect drugs to prevent NSAID-induced lower GI complications, although preliminary data indicate that misoprostol, rebamipide, antibiotics, and probiotics could be effective1,15.

NSAID-induced enteropathy has a multifactorial pathogenesis, commencing with topical effects on the small bowel and subsequent compromise of cellular gut barrier function, resulting in increased permeability to bacterial toxins1. Mitochondrial damage, together with the direct NSAID–phospholipid interaction, further increases intestinal permeability. Additional mucosal aggressors, such as bile and intestinal bacteria, contribute to subsequent small intestinal inflammation12,16. NSAID-induced mucosal injury manifests as increased small intestinal permeability in 50–70% of long-term NSAID users2. Our findings, indicating indomethacin-induced suppression of occludin and ZO-1, and an increase in the level of proinflammatory cytokines in HIEC-6 cells, confirm the importance of increased permeability and sustained inflammation in NSAID-induced injury to the small bowel. However, we did not conduct in vivo studies, thus, further studies using animal models of NSAID-induced enteropathy are required to confirm this mechanism.

By contrast to its established role in preventing NSAID-induced gastropathy5, PPIs do not prevent injury to the small bowel. A nationwide study in Taiwan revealed that an NSAID, low-dose aspirin, and PPI markedly increased the risk of lower GI complications, including bleeding17,18. A capsule endoscopy study reported small bowel injuries in 55% of patients taking naproxen and omeprazole, as compared to 16% in those taking celecoxib only and 7% in controls8. Additionally, Wallace et al. showed that PPIs exacerbated naproxen- and celecoxib-induced small bowel ulceration and bleeding in an animal model6. Furthermore, significant shifts in the microbiome were observed, including notable reductions in the abundances of Actinobacteria and Bifidobacterium spp. Restoring the microbiome prevented small bowel injury, suggesting that PPIs exacerbate the damaging effects of NSAIDs on the small bowel mucosa by inducing dysbiosis. In NSAID users, risk factors of small bowel injuries include advanced ages, concomitant use of antiplatelet agents, and existing comorbidities19. In real-world clinical practice, discontinuing NSAIDs is often challenging, and these patients share risk factors for upper GI complications5. Given that PPIs are not recommended for preventing NSAID-induced enteropathy due to their association with dysbiosis or leaky gut syndrome20, our findings suggest that tegoprazan could be integrated into current treatment protocols for individuals at high risk of complications. Further clinical studies are needed to evaluate the efficacy and long-term effects of tegoprazan in preventing and treating NSAID-induced small bowel injuries.

In this study, we investigated the effect of tegoprazan on intestinal permeability using small intestinal epithelial cells. Tegoprazan, a novel active P-CAB, exerted a greater inhibitory effect on gastric acid secretion than traditional PPIs and suppressed gastric acid secretion by distinct mechanisms. P-CAB is prescribed for gastroesophageal reflux disease, gastroduodenal ulcers, and eradication of Helicobacter pylori21,22,23,24. Recently, growing evidence has highlighted the potential of P-CABs in modulating intestinal permeability and exerting anti-inflammatory effects. Han et al. demonstrated that revaprazan, a P-CAB, inactivated Rho-GTPase via ERK signaling pathway in indomethacin-induced intestinal damage, which subsequently decreased myosin light chain phosphorylation and preserved tight junction proteins25. In addition, tegoprazan was shown to inhibit proinflammatory genes, IL-6, IL-1β, and TNF-α, by suppressing the MAPK signaling pathways in lipopolysaccharide-stimulated bone-marrow-derived macrophages in vitro26. However, the effect of P-CABs on enteropathy is inconsistent. Natadi et al. reported that vonoprazan, another potent P-CAB, exacerbated NSAID-induced injury to the small intestine by reducing the abundance of beneficial bacteria27. By contrast, Son et al. showed that tegoprazan significantly improved colitis in mice by enhancing intestinal barrier function and modulating the composition of the microbiome with an increased abundance of Bacteroides vulgatus28. The tegoprazan-mediated prevention of colitis could be explained by the different pKa values of P-CABs: 6.0 (revaprazan), 9.3 (vonoprazan), and 5.2 (tegoprazan). Tegoprazan, which has a pKa of 5.2, is activated only in the stomach and has a non-significant effect on the small intestine, unlike vonoprazan, which has a pKa of 9.3 and can exert effects in the alkaline environment of the small bowel. Previous studies have reported that drugs such as misoprostol and rebamipide exert a therapeutic effect on NSAID-induced enteropathy by enhancing mucosal defense through the increase of PG1. However, ulceration still occurred even at normal mucosal PG levels29, supporting PG deficiency may not be the sole factor to cause small bowel damage. Furthermore, recent studies have indicated that PPIs lead to an myosin light chain kinase-dependent increase in colonic permeability which, in turn, exaggerates experimental colitis and fails to mitigate indomethacin-induced increases in intestinal permeability25,30. Taken together, our results suggest that tegoprazan reduced indomethacin toxicity by promoting the expression of tight junction proteins and restoring intestinal permeability.

On the other hand, changes in the microbiome are crucial in NSAID-induced enteropathy, with a gram-negative bacteria playing a major role in the development of small intestinal ulcer. Similar to PPI-induced dysbiosis and 20% reduction of bacterial taxa, a recent meta-analysis found a significant decrease in alpha diversity with P-CAB treatments31,32. Further studies of the effect of tegoprazan on the microbiome in an animal model of small bowel injury could provide insight into its role in NSAID-induced enteropathy.

In conclusion, tegoprazan has the potential to ameliorate NSAID-induced injury to small intestinal epithelial cells by enhancing the expression levels of tight junction proteins and inhibiting those of proinflammatory cytokines and apoptosis. Larger-scale studies, including clinical studies, are warranted to validate our findings and uncover therapeutic targets for NSAID-induced enteropathy.