Anti-Inflammatory Effects of Tegoprazan in Lipopolysaccharide-Stimulated Bone-Marrow-Derived Macrophages

The purpose of this study was to investigate the anti-inflammatory effect of tegoprazan (TEGO) in lipopolysaccharide (LPS)-stimulated bone-marrow-derived macrophages (BMMs). To this end, compared to methylprednisolone (MP; positive control), we evaluated whether TEGO effectively differentiates LPS-stimulated BMMs into M2-phenotype macrophages. Moreover, the expression of pro- and anti-inflammatory cytokines genes influenced by TEGO was measured using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. TEGO was found to reduce nitric oxide (NO) production in BMMs significantly. In addition, TEGO significantly decreased and increased the gene expression levels of pro-inflammatory and anti-inflammatory cytokines, respectively. In addition, we evaluated the phosphorylated values of the extracellular signal-regulatory kinase (ERK) and p38 in the mitogen-activated protein (MAP) kinase signaling pathway through Western blotting. TEGO significantly reduced the phosphorylated values of the ERK and p38. In other words, TEGO suppressed the various pro-inflammatory responses in LPS-induced BMMs. These results show that TEGO has the potential to be used as an anti-inflammatory agent.

The excessive secretion of inflammatory mediators is toxic to humans [2].Although steroidal medicines are used to decrease pro-inflammatory responses, steroids have several side effects, including reduced immunity, which can lead to severe infections [11].
Methylprednisolone (MP) has been utilized in the treatment of patients with inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, dermatomyositis, and acute spinal cord injuries [12].However, long-term/high-dose treatment with MP is associated with side effects including infections, pneumonia, and myopathy [13].Consequently, non-steroidal anti-inflammatory drugs (NSAIDs) are required to overcome the side effects of the steroid treatments in clinics [14].
Tegoprazan (TEGO) is a potassium-competitive acid blocker (P-CAB) used to treat acidrelated gastroenteritis [15,16] and gastro-esophageal reflux disease (GERD) [17][18][19].It has been approved as a treatment for GERD, Helicobacter pylori infections, and gastric ulcers in South Korea [20].It was also approved for erosive esophagitis in China [20].Although there are several reports that long-term use potentially increases the risk of hip fractures, it is reported that the potassium channel on macrophages is involved in the activation and the polarization [21].Based on previous studies, while TEGO has the potential to be used as an NSAID due to its role as a potassium-competitive inhibitor, to the best of our knowledge, there have been no investigations of the effects of TEGO on macrophages.
Hence, we aimed to investigate the anti-inflammatory effects of TEGO in lipopolysaccharide (LPS)-stimulated bone-marrow-derived macrophages (BMMs).
sitis, and acute spinal cord injuries [12].However, long-term/high-dose treatment with MP is associated with side effects including infections, pneumonia, and myopathy [13].Consequently, non-steroidal anti-inflammatory drugs (NSAIDs) are required to overcome the side effects of the steroid treatments in clinics [14].
Tegoprazan (TEGO) is a potassium-competitive acid blocker (P-CAB) used to treat acid-related gastroenteritis [15,16] and gastro-esophageal reflux disease (GERD) [17][18][19].It has been approved as a treatment for GERD, Helicobacter pylori infections, and gastric ulcers in South Korea [20].It was also approved for erosive esophagitis in China [20].Although there are several reports that long-term use potentially increases the risk of hip fractures, it is reported that the potassium channel on macrophages is involved in the activation and the polarization [21].Based on previous studies, while TEGO has the potential to be used as an NSAID due to its role as a potassium-competitive inhibitor, to the best of our knowledge, there have been no investigations of the effects of TEGO on macrophages.
Hence, we aimed to investigate the anti-inflammatory effects of TEGO in lipopolysaccharide (LPS)-stimulated bone-marrow-derived macrophages (BMMs).
In other words, the gene expression levels of pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α were most notably decreased in the LPS+TEGO group (Figure 4A-C), while anti-inflammatory cytokines including IL-4, IL-10, and TGF-β were significantly increased in the LPS+TEGO group (Figure 4D-F).

Discussion
This study determined the anti-inflammatory effects of TEGO in comparison to MP on LPS-stimulated BMMs.First, to avoid a cytotoxic concentration of TEGO, we utilized several concentrations (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 1, 10, 100, and 1000 µg/mL) of TEGO to BMMs (Figure 2A).Cell viability in excess of 80% was considered as a non-toxic density level [22].TEGO demonstrated no cytotoxicity up to 100 µg/mL.Based on this finding, we decided to use 0.5, 1, 10, and 100 µg/mL concentrations of TEGO in the subsequent experiments.
Macrophage colony-stimulating factor (M-CSF)-stimulated BMMs (M0-phenotype macrophages) can exhibit two distinct phenotypes: the M1 phenotypes (classically activated) and M2 phenotypes (alternatively activated).The M1-phenotype macrophages produce pro-inflammatory cytokines, whereas M2-phenotype macrophages produce anti-inflammatory cytokines [23].In the various inflammatory environments, it is reported that most macrophages polarize toward the M1 phenotype, while the M2-phenotype macrophage is displayed in only a small number of cells [24].Moreover, NO plays a role as a crucial inflammatory mediator to dilate the blood vessels, which induces infiltration of macrophages [25].Pacher et al. reported that accumulated M1 macrophages exacerbate the pro-inflammatory response [26].
Here, we observed that NO production in LPS-stimulated BMMs was significantly inhibited by 100 µg/mL of TEGO (Figure 2B).Specifically, TEGO showed the ability to differentiate LPS-stimulated macrophages into M2-phenotype macrophages (Figure 3).These results indicated that TEGO can effectively suppress the production of NO in inflammatory cells and induce differentiation from M0-phenotype macrophages to M2-phenotype macrophages.
M1-phenotype macrophages are classically polarized by LPS and produce the typical pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6.On the other hand, M2phenotype macrophages produce anti-inflammatory cytokines, including IL-4, IL-10, and TGF-β.We observed that TEGO downregulates the gene expression levels of pro- The results are expressed as the mean ± SD (n = 4 per group); not significant (ns); *** p < 0.001, a significant difference as compared to the control group and to each group.

Discussion
This study determined the anti-inflammatory effects of TEGO in comparison to MP on LPS-stimulated BMMs.First, to avoid a cytotoxic concentration of TEGO, we utilized several concentrations (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 1, 10, 100, and 1000 µg/mL) of TEGO to BMMs (Figure 2A).Cell viability in excess of 80% was considered as a non-toxic density level [22].TEGO demonstrated no cytotoxicity up to 100 µg/mL.Based on this finding, we decided to use 0.5, 1, 10, and 100 µg/mL concentrations of TEGO in the subsequent experiments.
Macrophage colony-stimulating factor (M-CSF)-stimulated BMMs (M0-phenotype macrophages) can exhibit two distinct phenotypes: the M1 phenotypes (classically activated) and M2 phenotypes (alternatively activated).The M1-phenotype macrophages produce pro-inflammatory cytokines, whereas M2-phenotype macrophages produce antiinflammatory cytokines [23].In the various inflammatory environments, it is reported that most macrophages polarize toward the M1 phenotype, while the M2-phenotype macrophage is displayed in only a small number of cells [24].Moreover, NO plays a role as a crucial inflammatory mediator to dilate the blood vessels, which induces infiltration of macrophages [25].Pacher et al. reported that accumulated M1 macrophages exacerbate the pro-inflammatory response [26].
Here, we observed that NO production in LPS-stimulated BMMs was significantly inhibited by 100 µg/mL of TEGO (Figure 2B).Specifically, TEGO showed the ability to differentiate LPS-stimulated macrophages into M2-phenotype macrophages (Figure 3).These results indicated that TEGO can effectively suppress the production of NO in inflammatory cells and induce differentiation from M0-phenotype macrophages to M2-phenotype macrophages.
M1-phenotype macrophages are classically polarized by LPS and produce the typical pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6.On the other hand, M2-phenotype macrophages produce anti-inflammatory cytokines, including IL-4, IL-10, and TGF-β.We observed that TEGO downregulates the gene expression levels of proinflammatory cytokines and upregulates the gene expression levels of anti-inflammatory cytokines in LPS-stimulated BMMs.Sone et al. reported that the mRNA expression levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were suppressed by treatment with TEGO in dinitrobenzene sulfonic acid-induced mouse models [27].Collectively, our findings were similar to those of other recent studies, showing the potential anti-inflammatory effects of TEGO in the inflammatory response.
TEGO is a novel development as a P-CAB that inhibits gastric H + /K + -ATPase for the treatment of gastric-acid-related diseases, including gastroenteritis and GERD.Recently, it was reported that P-CABs show an anti-inflammatory effect by suppressing the MAPK signaling pathways [28].Furthermore, Yeo et al. reported significant anti-inflammatory effects of PPIs by inactivating the Akt signaling, the NF-κB signaling pathway, and inflammatory cytokines in Helicobacter pylori infections that induce chronic gastric inflammation [29].Collectively, our results and recent studies by others suggest that TEGO, already approved by the Korean FDA, can potentially be an NSAID for clinical use.
MAPK signaling is implicated in cellular processes including apoptosis, cell survival, stress response, and inflammation [30].Among the MAPK signaling pathways, phosphorylation of ERK and p38 have an impact on the inflammatory response [31].In this study, we conducted a further investigation of the signal pathways to investigate the antiinflammatory effects of TEGO (Figure 5), showing that p/t volumes of ERK and p38 in the TEGO-treated BMMs were significantly decreased compared to those in the LPS-stimulated BMMs.Although the p/t volumes of ERK and p38 in the MP-treated group were the lowest among the groups, TEGO has the advantage of being suitable for long-term high-dose treatment [32].Here, while we did not conduct in vivo studies, to the best of our knowledge, our study is the first to demonstrate that TEGO inhibits pro-inflammatory genes and induces anti-inflammatory genes when used on LPS-stimulated BMMs in vitro.

Preparation of TEGO and LPS
TEGO was purchased from Med Chem Express (Monmouth Junction, NJ, USA) and solubilized in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific Inc., Waltham, MA, USA).For the in vitro method, the various concentrations of TEGO were diluted in Dulbecco's modified Eagle's medium (DMEM, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) including 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (P/S, Gibco).LPS was obtained from Sigma Aldrich (Sigma, St. Louis, MO, USA).It was dissolved in distilled water and diluted with DMEM 0.1 µg/mL.

Isolation of BMMs and Differentiation into Macrophages
All rat experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University (protocol code 220010, January 2022) and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).The isolation of the monocytes followed a previous method [8,33,34].Briefly, the monocytes were extracted from the tibia/femurs of Sprague Dawley rats at four weeks.Afterwards, the monocytes were placed in a 100 × 20 mm petri dish (Corning Inc., Corning, NY, USA).The monocytes were cultured in DMEM including 10% FBS, 1% P/S, and 10 ng/mL of macrophage colony-stimulating factor (M-CSF, Peprotech, NJ, USA) in a 5% CO 2 incubator at 37 • C.After 2 days of preculture, non-adherent cells in the supernatant were removed.Afterwards, adherent cells were considered macrophages.A detailed description of the isolation and differentiation of BMMs is provided in the Supporting Information.

Cell Toxicity Test
The cell viability kits used were obtained from EZ-Cytox (Daeil Lab Service, Seoul, Republic of Korea).BMMs (5 × 10 5 cells/well, n = 4 per group) were seeded into a 96-well cell culture plate (Falcon Becton Dickinson, NJ, USA) and cultured with various concentrations of TEGO (0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 10, 100, and 1000 µg/mL) for 24 h.After 24 h, BMMs were washed with Dulbecco's Phosphate Buffered Saline (DPBS, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).Subsequently, the cells were incubated with free media containing cell counting kit-8 (CCK-8) for two hours.The intensity was determined with a microplate absorbance reader at a wavelength of 450 nm.Each observation of groups was normalized relative to the control group.

NO Production
The NO production measurements were performed as described previously [8,33,35].Briefly, BMMs (1 × 10 6 cells/well, n = 4 per group) were seeded into 48-well culture plates (Falcon) to prepare the seven experimental groups (control (only BMMs), 0.1 µg/mL of LPS, 1 µg/mL of MP, and 0.5, 1, 10, and 100 µg/mL of TEGO).After 24 h, NO products accumulated in the supernatant were detected using a Griess reagent system (Promega, Madison, WI, USA).Briefly, the supernatant and sulfanilamide were mixed in equal amounts.Each group was measured at a wavelength of 548 nm using a microplate absorbance reader (Bio-Rad, Hercules, CA, USA).The NO concentration in the supernatant fluid was measured with a standard curve generated with sodium nitrite.

ICC Staining
The ICC stainings were performed as described previously [36].BMMs were seeded and cultured for one day in DMEM with MP (1 µg/mL) and TEGO (100 µg/mL) containing LPS (0.1 µg/mL).After one day, cells were fixed using 4% paraformaldehyde.Fixed cells were incubated with anti-CD86 and anti-CD206 antibodies at 4 • C overnight.Afterwards, Alexa 488 and Alexa 647 secondary antibodies were stained for two hours at room temperature.The nuclei were stained with 4 ,6 -diamidino-2-phenylindole dihydrochloride (DAPI).The fluorescent intensity was detected using a Zeiss LSM 880 confocal microscope.A detailed description of the ICC staining methods is provided in the Supporting Information.

Western Blotting
BMMs (1 × 10 6 cells/well, n = 4 per group) were seeded into six-well culture plates (Falcon) and treated with each concentration of MP (1 µg/mL) and TEGO (100 µg/mL) containing LPS (0.1 µg/mL) for 24 h.After 24 h, cells were lysed by adding radio immune precipitation assay lysis buffer (RIPA lysis buffer, Sigma) with protease (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Sigma) for 30 min at 4 • C. The p38 progressed 30 min after drug treatment.Subsequently, the protein was extracted by centrifugation at 13,000 rpm for 10 min.The concentration of the extracted protein was measured using a microplate absorbance reader (Bio-Rad) at a wavelength of

Figure 2 .
Figure 2. (A) Effect of TEGO on the cell viability of bone-marrow-derived macrophages (BMMs).(B) Effect on nitric oxide (NO) production in BMMs treated with lipopolysaccharide (LPS), 1 µg/mL methylprednisolone (MP), and TEGO.The results are expressed as the mean ± standard deviation (SD, n = 4 per group); *** p < 0.001, a significant difference as compared to the control group and to each group.

Figure 2 .
Figure 2. (A) Effect of TEGO on the cell viability of bone-marrow-derived macrophages (BMMs).(B) Effect on nitric oxide (NO) production in BMMs treated with lipopolysaccharide (LPS), 1 µg/mL methylprednisolone (MP), and TEGO.The results are expressed as the mean ± standard deviation (SD, n = 4 per group); *** p < 0.001, a significant difference as compared to the control group and to each group.

Figure 3 .
Figure 3.Immunocytochemistry (ICC) staining and fluorescence-activated cell sorting (FACS) of BMMs treated by TEGO.(A) ICC staining of cluster of differentiation (CD) 86 and CD206 in groups treated with LPS, LPS+MP, and LPS+TEGO (scale bar = 10 µm).(B) Quantitative analysis of the CD86 fluorescence intensity levels.(C) Quantitative analysis of the CD206 fluorescence intensity levels.(D) CD86-positive and CD206-positive (green) BMM populations selected for analysis among CD68-positive (red).Quantitative analysis of the (E) CD86 and (F) CD206 fluorescence intensity levels using FACS.The results are expressed as the mean ± SD (n = 4 per group); not significant (ns), ** p < 0.01 and *** p < 0.001, a significant difference as compared to the control group and to each group.

Figure 5 .
Figure 5. (A) Effect of TEGO on the phosphorylation values of extracellular regulatory kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and nuclear factor kappa B (NF-κB) in BMMs stimulated by LPS.Quantitative analysis of the p/t forms of (B) ERK, (C) JNK, (D) p38, (E) NF-κB, and (F) β-actin.The results are expressed as the mean ± SD (n = 4 per group); not significant (ns); *** p < 0.001, a significant difference as compared to the control group and to each group.

Figure 5 .
Figure 5. (A) Effect of TEGO on the phosphorylation values of extracellular regulatory kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and nuclear factor kappa B (NF-κB) in BMMs stimulated by LPS.Quantitative analysis of the p/t forms of (B) ERK, (C) JNK, (D) p38, (E) NF-κB, and (F) β-actin.The results are expressed as the mean ± SD (n = 4 per group); not significant (ns); *** p < 0.001, a significant difference as compared to the control group and to each group.