Omeprazole (IO0030) was purchased from (Solarbio, Beijing, China). TRIzol (15596) was from Invitrogen (Carlsbad, CA, USA). First-Strand cDNA Synthesis Kit (AORT-0020) and All-in-One qPCR Mix (QP002) were from GeneCopoeia (Rockville, MD, USA). Dipyridamole (DIP; S1895) was obtained from Selleck (Shanghai, China). MHY1485 (HY-B0795) was purchased from (MedChemExpress, NJ, USA). Other reagents were obtained from Biosharp (Hefei, China). The antibodies used in this study were mTOR (ab134903, abcam, Cambridge, UK, 1:2000 for western blotting, WB), phosphodiesterase-4 (PDE4; ab14628, Abcam Cambridge, UK, 1:2000 for WB); PDE4B/C/D phospho (Ser133/119/190) (YP0668, Immunoway, SuZhou, China, 1:2000 for WB, 1:400 for immunofluorescence, IF); mTOR (phosphor s2448)(ab109268, Abcam, Cambridge, UK, 1:2000 for WB, 1:400 for IF), caspase-1 (ab179515, Abcam, Cambridge, UK, 1:1000 for WB,1:300 for IF); NLRP3 (ab270449, Abcam, Cambridge, UK, 1:2000 for WB); IL-18 (A1115, Abclonal, Wuhan, China, 1:2000 for WB); IL-1β (A19635, Abclonal, Wuhan, China, 1:1000 for WB); GSDMD (A17308, Abclonal, Wuhan, China, 1:2000 for WB); gasdermin E (GSDME; 13075-1-AP, Proteintech, Chicago, IL, 1:1000 for WB), α-tubulin (AC007,1:4000 for WB); β-actin (AC026, 1:5000 for WB). IL-1β enzyme-linked immunosorbent assay (ELISA) kit (ab229384) and IL-18 ELISA kit (ab215539) as well as lactate dehydrogenase (LDH) assay kit (ab102526) were obtained from Abcam (Cambridge, UK).

GES-1 and BGC823 cells were cultured in Dulbecco’s Modified Eagle Medium (Kalamazoo, MI, USA) supplied with 10% fetal bovine serum. For treatment, cells were incubated with omeprazole at a final concertation of 300 mM for 48 h. DIP (DIP) was used at a final concentration of 4 µM, for transfection, mRFP-GFP-LC3 plasmid was purchased from (YouBio, Changsha, China) and transfected into cells with lipofectamine 3000 (Invitrogen, catalog no. L3000001).

As described in an earlier study (Wang et al., 2021), after stimulation with or without omeprazole in a 6-well plate, 1 mL TRIzol was added to replace with cell medium to lyse cell. The whole RNA was extracted to convert into cDNA and amplified by qPCR assay using a cDNA Synthesis Kit combined with an All-in-One qPCR Mix, respectively. Primer used as followed: IL-1β: Forward: 5′-ATGATGGCTTATTACAGTGGCAA-3′, Reverse: 5′-GTCGGAGATTCGTAGCTGGA-3′; IL-18: Forward: 5′-TCTTCATTGACCAAGGAAATCGG-3′, Reverse: 5′-TCCGGGGTGCATTATCTCTAC-3′; GSDMD: Forward: 5′-GTGTGTCAACCTGTCTATCAAGG-3′, Reverse: 5′-CATGGCATCGTAGAAGTGGAAG-3′; NLRP3: Forward: 5′-GATCTTCGCTGCGATCAACAG-3′, Reverse: 5′-CGTGCATTATCTGAACCCCAC-3′; ASC: Forward: 5′-TGGATGCTCTGTACGGGAAG-3′, Reverse: 5′-CCAGGCTGGTGTGAAACTGAA-3′; Caspase-1: Forward: 5′-CCTTAATATGCAAGACTCTCAAGGA-3′, Reverse: 5′-TAAGCTGGGTTGTCCTGCACT-3′; UBC: Forward: 5′-ATTTGGGTCGCGGTTCTTG-3′ and Reverse: 5′-TGCCTTGACATTCTCGATGGT-3′.

After treatment, the supernatant was harvested to centrifuge to remove debris, and the concentration of IL-1β and IL-18 in the content were measured and quantitated by the corresponding ELISA according to the kit protocol, respectively.

LDH (lactate dehydrogenase) is released due to the cell membrane is destroyed caused by pyroptosis, and the severity of pyroptosis can be reflected by the LDH release level. After treatment with or without omeprazole, the supernatants from BGC823 cells were collected to detect using an LDH assay kit. Relative cell death was calculated as described in the study by Xie et al. (2021).

This was performed as described earlier (Xu et al., 2017). Briefly, the lysate was harvested and the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After incubation with Tris-buffered saline Tween-20 with 5% milk, the membrane was incubated with appropriate antibodies at 4°C for 24 h, followed by washing with PBST for three times for 5 min. The membrane was further incubated for another 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody. The protein was detected using an enhanced chemiluminescence kit (Perkin Elmer, catalog no. NEL105001EA).

Immunofluorescent staining was performed as described in our previous study (Xu et al., 2017). Fixed and permeabilized cells were treated overnight at 4°C with primary antibodies, followed by incubation for 1 h with secondary antibodies conjugated with Alexa-488 or 594 at room temperature. Coverslips were mounted onto the glass slides with the prolong gold antifade reagent. The stained cells were imaged with a Leica TCS SP8 inverted fluorescence microscope (Leica Microsystems).

Data was displayed as mean ± SD and performed by GraphPad Prism V software (La Jolla, CA, USA). A p-value < 0.05 was considered statistically significant.

In our previous study, we found that omeprazole could decrease de novo lipogenesis in gastric epithelial cells (Chen et al., 2020), and saturated fatty acid was reported to trigger caspase-4/5 in human monocytes to induce IL-1β and IL-18 release (Pillon et al., 2016). These findings focused us to explore the possible role of omeprazole in cell pyroptosis. The result from the LDH release assay revealed that omeprazole treatment alone led to a decrease in the enhanced LDH release caused by LPS in BGC823 and GES-1 cells (Fig. 1A). Further results from qPCR and WB analyses showed that omeprazole treatment could inhibit IL-18 and IL-1β expression at mRNA and protein level (Figs. 1B and 1C). Consequently, the content of IL-18/1β in supernatant from BGC823 cells was drastically reduced in response to omeprazole stimulation (Fig. 1D). Most importantly, a pivotal executioner of cell pyroptosis (Poh et al., 2019; Zhang et al., 2019) GSDME, not GSDMD, was significantly inhibited in gastric epithelial cells treated with omeprazole (Fig. 1E). These finding suggested that omeprazole has a potential function in suppressing cell pyroptosis in gastric epithelial cells, which could alleviate inflammation.

NLRP3 inflammasome activation is an important event for GSDME and IL-18/1β maturation. Thus, NLRP3 inflammasome attracted our attention in assessing the effect of omeprazole on NLRP3 inflammasome (Liu et al., 2021; Li et al., 2021). As expected, the results from quantitative PCR displayed that omeprazole stimulation resulted in a strong inhibition of NLRP3 and Caspase 1 activation, while no remarkable change was observed in the expression of apoptosis-associated speck-like protein containing a CARD (ASC) mRNA in BGC823 and GES-1 cells (Fig. 2A). In line with this, we observed that the expression of NLRP3 and caspase-1, not ASC, was inhibited in BGC823 and GES-1 cells after omeprazole treatment (300 µM) (Fig. 2B). Moreover, omeprazole stimulation significantly reversed NLRP3 inflammasomes activation in the presence of LPS (500 ng/mL) in BGC823 cells (Fig. 2C). These findings suggest that omeprazole suppressed cell pyroptosis in gastric epithelial cells to ameliorate inflammation by reducing caspase-1-mediated NLRP3 inflammasome activation.

Autophagy has been well demonstrated to block pyroptosis (Guo et al., 2021; Yu et al., 2019; Lin et al., 2021). To investigate whether autophagy is involved in omeprazole-mediated pyroptosis, we examined the expression of several autophagy-related genes in response to omeprazole treatment. As presented in Fig. 3A, real-time PCR and WB analysis of BGC823 and GES-1 cells treated with omeprazole for 48 h, respectively, showed significant induction of autophagy characterized by enhanced LC3II and ULK1 expression as well as decreased p62 expression, which was attributed to suppression of mTOR phosphorylation caused by omeprazole treatment (Figs. 3A and 3B). To further monitor the effect of omeprazole on autophagy flux, the mRFP-GFP-LC3 plasmid was transfected into BGC823 cells. As expected, the number of autophagosomes (indicated as yellow puncta) was increased caused by omeprazole stimulation (Fig. 3C).

Initiation of autophagosome formation is regulated by mTOR inhibition. Insulin receptor withdrawal, amino acid starvation, and low ATP, result in mTOR inhibition, resulting in ULK1 activation and initiation of autophagy (Zhang, 2015). Next, MHY1485, an activator of mTOR, was used to further confirm omeprazole-regulated autophagy in an mTOR-dependent manner. As expected, MHY1485 treatment in BGC823 cells led to a reversal of omeprazole-mediated autophagy and also counterbalanced reduced caspase-1 expression caused by omeprazole (Fig. 3D), which further restored IL-18 level (Fig. 3E). This suggested that omeprazole might trigger mTOR-mediated autophagy to attenuate cell pyroptosis in BGC823 cells.

AMP-activated protein kinase (AMPK)/mTOR act as a cellular energy sensor, which is controlled in response to increased AMP:ATP ratio (Szrejder et al., 2020; Jayarajan et al., 2019). Intracellular cAMP degradation is mediated by PDE4 (Schick and Schlegel, 2022); this incited us to ask whether PDE4 is involved in omeprazole-induced autophagy. As shown in Fig. 4A, mTOR phosphorylation and PDE4 activity were significantly attenuated in BGC823 cells treated with omeprazole and DIP, an inhibitor of PDE4, respectively, which could induce autophagy. In addition, the levels of cleaved caspase-1 and GSDME were significantly reduced, while that of LC3II was enhanced in BGC823 cells treated with DIP along with omeprazole compared with omeprazole treatment alone (Fig. 4B). The level of IL-1β and IL-18 was also markedly decreased in response to DIP combined with omeprazole (Fig. 4C), Thus, the inhibition of PDE4 by DIP enhanced omeprazole-mediated autophagy and pyroptosis through mTOR.

Our above-mentioned experiments showed that omeprazole could enhance mTOR-mediated autophagy, which further suppressed cell pyroptosis. Based on this, we examined the relationship between mTOR phosphorylation and caspase-1 in clinical samples. A total of 38 gastric mucosa samples, including 24 (63.2%) boys and 14 (36.8%) girls in ages between 3 to 12 years (median age = 7.5), were enrolled from Guangzhou Women and Children’s Medical Center after clinical ethical review. Detailed clinical information of subjects could be obtained based on reasonable request.

Next, we examined the possible changes in patients who received omeprazole treatment and found that phosphorylation of mTOR and caspase1 expression as well as PDE4 phosphorylation were significantly reduced in gastric mucosa with H. pylori infection after omeprazole treatment (Figs. 5A–5C). These findings suggest impaired autophagy and enhanced cell pyroptosis in the patients with H. pylori-infected gastric mucosa, and omeprazole not only inhibited acid secretion, but was also able to suppress inflammation by suppressing pyroptosis via PDE4/mTOR-mediated autophagy.