Hypoxia exacerbates the malignant transformation of gastric epithelial cells induced by long-term H. pylori infection

ABSTRACT Helicobacter pylori is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that H. pylori infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, H. pylori infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the in vivo situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to H. pylori under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and H. pylori for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained H. pylori stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the in vitro results, we conducted an experiment involving Mongolian gerbils infected with H. pylori for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in H. pylori-related apoptosis and autophagy. In summary, continuous stimulation by H. pylori affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process. IMPORTANCE Deciphering the collaborative effects of Helicobacter pylori infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term H. pylori stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium’s nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine in vivo infection scenario by repeatedly exposing gastric epithelial cells to H. pylori under hypoxic conditions for up to 30 generations. The results show that chronic exposure to H. pylori in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of H. pylori infection. By accurately replicating the in vivo gastric environment, this study enhances our comprehension of H. pylori’s pathogenic mechanisms in gastric cancer.

G astric cancer is a significant global health concern, ranking as the fifth most common cancer and the second leading cause of cancer-related deaths worldwide.In 2020, there were over 1 million new cases of gastric cancer and approximately 770,000 related deaths, posing a serious threat to the well-being of individuals across the globe (1).The development of gastric cancer is influenced by various factors, and one notable and preventable cause is the infection of Helicobacter pylori.H. pylori is a Gram-negative bacterium that thrives in the stomach's microaerophilic environment.It is the most prevalent bacterial infection worldwide and is closely associated with gastritis, gastric ulcer, and even gastric cancer.In fact, H. pylori has been classified as a class I carcinogen for human gastric cancer (2)(3)(4)(5).
During the colonization in the mucosa, H. pylori consistently releases a diverse range of bacterial toxins, including cytotoxin-associated gene product A (CagA), vacuolat ing cytotoxin A (VacA), lipopolysaccharide, and peptidoglycan (6)(7)(8)(9)(10)(11). Additionally, H. pylori secretes outer membrane vesicles (OMV) containing various bacterial toxins and antigens (11).These components actively participate in modulating crucial functions of host cells, such as proliferation, autophagy, apoptosis, migration, and invasion.As a result, they promote cellular malignant transformation and contribute to gastric carcinogenesis (11)(12)(13).Our previous investigation unveiled the ability of H. pylori to deliver CagA toxin protein to gastric epithelial cells via OMV in the lysate, thereby inducing cellular transformation toward cancer through the Nod1 pathway.However, the precise underlying mechanism by which H. pylori triggers gastric cancer remains to be fully elucidated.
Despite numerous studies on H. pylori's pathogenicity, previous research often used short-term stimulation to mimic in vivo infection.However, H. pylori infection is a chronic process, and short-term cellular experiments don't replicate the true in vivo scenario.Additionally, the oxygenation characteristics of human gastrointestinal tract are highly distinctive (14).Oxygen levels vary, with oxygen partial pressure (pO 2 ) ranging from 13.33 to 14.66 kPa in lung alveoli, 7.73 kPa in the stomach, and an anaerobic environment in the gastrointestinal lumen (14)(15)(16)(17).Previous studies predominantly operated under normal oxygen partial pressure, which was higher than the stomach environment, thus failing to replicate the true impact of H. pylori infection on gastric epithelial cell function under realistic conditions.
In this study, we artificially created a hypoxic environment to simulate the authentic gastric conditions during continuous H. pylori infection.GES-1 cells were co-incubated with H. pylori under intermittent hypoxia conditions for 30 generations.Simultaneously, a control cell line was established through intermittent hypoxia culture of GES-1 cells for 30 generations.We further investigated the effects of long-term H. pylori stimulation under hypoxic conditions on cell functions and the Nod1 signaling pathway.Our findings demonstrate that the hypoxic gastric environment exacerbates the malignant transfor mation of gastric epithelial cells induced by sustained H. pylori infection.

Cell treatment
In the experimental group (Hp 30 -GES-1), GES-1 cells were co-cultured with H. pylori for 30 consecutive generations under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ).H. pylori strain ATCC43504 (6 × 10^8 CFU) was added to GES-1 cells at a bacteria-to-cell ratio of 1,000:1.After a 24-hour co-culture with H. pylori under hypoxic conditions, the cells underwent a PBS wash to remove the bacteria.Subsequently, the cells were cultured for another 24 hours under aerobic conditions (20% O 2 , 5% CO 2 , and 75% N 2 ) in a sterile environment to restore their vitality, marking one generation in continuous culture.To assess the impact of continuous hypoxia culture on cells, a hypoxia control group (Hy 30 -GES-1) was established.The Hy 30 -GES-1 cells were cultured for 30 generations under microoxygen conditions of 5% O 2 , 10% CO 2 , and 85% N 2 .The culture method of the Hy 30 -GES-1 cell line mirrors that of Hp 30 -GES-1, except it does not involve the introduction of H. pylori throughout the entire process.To serve as a control, untreated wild-type cells (B-GES-1) were cultured for 30 consecutive generations, where each generation represented a 48hour culture period.

Detection of H. pylori infection activity
GES-1 cells were co-cultured with H. pylori for 24 hours, and the cell supernatant was collected.After centrifugation, the supernatant was discarded, and the precipitated bacteria was resuspended in PBS.A 200 µL bacterial suspension was inoculated on Campylobacter Agar Base (Karmali) plates and incubated for 48-72 hours in a micro aerobic environment at 37°C.Subsequently, bacterial identification was conducted through Gram staining microscopy, catalase testing, urease testing, and oxidase testing to confirm H. pylori.

Colony formation assay
B-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells (500 cells/well) were plated in new 6-well plates and cultured in DMEM medium containing 10% FBS under 5% CO 2 at 37°C.After 14 days, the cells were fixed and stained with 0.1% crystal violet.Visible colonies were counted using ImageJ software, with each well assessed in triplicate.

CCK-8 assay
B-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells were counted and plated in 96-well plates at a density of 3,000 cells per well.After cell attachment, 10 µL of CCK-8 reagent (Vazyme, China, A311-01) was added to each well at 0 hours, 12 hours, 24 hours, and 48 hours and incubated at 37°C for 2 hours.The absorbance at a wavelength of 450 nm was measured using a microplate reader, and these values were used to generate cell proliferation curves.Each treatment was performed in quintuplicate

Wound healing assay
C-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells were seeded in 6-well plates and cultured in DMEM medium containing 10% FBS.When the cells reached 90% confluence, a wound was created by discarding the medium, followed by washing the cells twice with PBS to remove any floating cells.

Transwell migration and invasion assay
For the migration assay, B-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells (3 × 10 4 cells in 200 µL) were placed in the upper chamber of a Transwell migration chamber with serum-free medium.Medium containing 10% FBS was added to the lower chamber.After 24 hours of incubation, the cells were fixed, stained with 0.1% crystal violet, and then counted.The assays were performed in triplicate.
For the invasion assay, 3 × 10 4 cells were seeded into the upper chamber of a Transwell invasion chamber with serum-free media, while medium containing 10% FBS was added to the lower chamber.After a 24-hour incubation, the cells were fixed, stained with 0.1% crystal violet, and counted.The assays were performed in triplicate.

mCherry-EGFP-LC3 transfection
B-GES-1, Hy30-GES-1, and Hp30-GES-1 cell suspensions with a density of 2.5 × 10 4 cells/mL were prepared in complete medium and added to a six-well plate.After cell attachment, the old medium was discarded, and the cells were washed with PBS.Medium containing a 50-mL titer of 10 8 TU/mL mCherry-EGFP-LC3 lentivirus (Hanbio Biotechnology Co. Ltd., Shanghai, China) and 8 mg/mL Polybrene was added to the culture.After 48 hours of infection, the fluorescence expression of cells was observed using fluorescence microscopy.When the cell infection efficiency reached approximately 80%, the cells were cultured in medium containing H. pylori for an additional 24 hours.The images of mCherry-EGFP-LC3-transfected cells were captured using laser scanning confocal microscopy.

Apoptosis assay
Flow cytometry was employed to detect the effects of H. pylori on the apoptosis of B-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme, China, A213-01) according to the manufacturer's instructions.The rate of apoptosis was analyzed using the LSR Fortessa Flow Cytometer at 488 nm.

Reverse transcriptase polymerase chain reaction
Total RNA was isolated from B-GES-1, Hy 30 -GES-1, and Hp 30 -GES-1 cells using TRIzol Reagent (Vazyme, China, R401-01).Single-stranded DNA was synthesized from 1 mg total RNA using reverse transcriptase-bound oligonucleotide (DT) primers.Each cDNA sample (2 mL) was subjected to reverse transcriptase polymerase chain reaction amplification using specific primers as detailed in Table S1.The data were collected and analyzed.The values were compared with the experimental controls after normalization to those of GAPDH.

H. pylori-infected Mongolian gerbil model
Five Mongolian gerbils weighing 60-80 g were used to establish the in vivo H. pylori infection model, with five H. pylori-negative gerbils as a control group.Gerbils were obtained from the Capital Medical University and housed in secondary biosafety laboratories at the Chinese Center for Disease Control and Prevention.They were kept in standard plastic cages with a 12-hour light/dark cycle and provided with free access to food and water throughout the experiments.Gerbils aged 6-8 weeks were orally infected with H. pylori ATCC 43504 strain solution using a 0.5 mL dose of 2 × 10 9 CFU/mL.Prior to challenge, the gerbils were fasted for 12 hours.Oral gavage was performed five times at 48-hour intervals.Before euthanasia, the colonization of H. pylori in gerbils was confirmed through 13 C urea breath test and PCR detection.After 90 weeks of infection, the gerbils were euthanized, and gastric mucosa samples were collected for H&E staining and immunohistochemistry (IHC).

Immunohistochemistry
Stomach sections were incubated overnight at 4°C with rabbit monoclonal Nod1 (CST, USA, 3545S, diluted 1:1,000 for IHC) and RIP2 (Abcam, UK, ab8428, diluted 1:1,000 for IHC).Subsequently, the sections were incubated with the corresponding biotinyla ted secondary antibody.Cell nuclei were counterstained with hematoxylin, and the samples were dehydrated in a gradient series, vitrified with dimethylbenzene, and finally mounted with neutral balsam.

Statistical analysis
All statistical analyses were performed using SPSS v19.0 software.Data were presented as mean ± standard deviation.Independent sample t-test and one-way analysis of variance (ANOVA) were employed to determine the significance of differences between the results.A P value of less than 0.05 was considered statistically significant.

Establishment of the GES-1 cell line with H. pylori continuous infection under hypoxic conditions
During the establishment of gastric epithelial cell lines continuously exposed to H. pylori (Hp 30 -GES-1) or not (Hy 30 -GES-1) under hypoxic conditions, we employed two methods to verify H. pylori infection.First, microscopic imaging systems were used to observe cell morphology.GES-1 cells exhibited a typical hummingbird phenotype after co-culturing with H. pylori for 24 hours under hypoxic conditions, while cells cultured solely under hypoxia and the control group did not display this phenotype (Fig. 1A).Second, after co-culturing cells with H. pylori under hypoxia for 24 hours, the culture supernatant was collected for bacterial culture, and H. pylori strains were successfully cultivated.The existence of live bacteria was confirmed by morphology strain, Gram staining micro scopy, catalase test, urease test, and oxidase test (Fig. 1B and C).All the results confirm that active H. pylori continuously attack GES-1 cells during the establishment of the cell lines.

Sustained exposure to H. pylori under hypoxic conditions promotes cell proliferation
To investigate the regulation of H. pylori on cell growth under hypoxic conditions, proliferation assays were performed.The colony formation assay revealed that Hp 30 -GES-1 cells exhibited higher activity than B-GES-1 and Hy 30 -GES-1 cells, while Hy 30 -GES-1 cells showed greater activity than B-GES-1 cells (Fig. 2A).This finding was further supported by the CCK-8 assay (Fig. 2B).These findings demonstrate that prolonged exposure to hypoxic conditions significantly amplifies the functional capacity of GES-1 cells.Additionally, H. pylori infection has the potential to synergistically augment the cell proliferation stimulated by hypoxia.Furthermore, the cells maintain robust activity even after long-term exposure to H. pylori and hypoxia.

Sustained exposure to H. pylori under hypoxic conditions promotes cell migration and invasion
To investigate the regulation of gastric epithelial cell migration and invasion by H. pylori, wound healing and transwell assays were conducted.The wound healing assay revealed that Hy 30 -GES-1 cells had significantly inhibited migratory ability compared with B-GES-1 cells, while Hp 30 -GES-1 cells showed significantly enhanced migratory ability (Fig. 3A).The transwell migration assay yielded consistent results (Fig. 3B), indicating that long-term exposure to H. pylori under hypoxic conditions significantly promoted cell migration.
Regarding the cell invasion assay, long-term exposure to hypoxic conditions did not significantly affect the invasive ability of the cells.However, the invasive ability of Hy 30 -GES-1 cells was significantly enhanced compared with B-GES-1 and Hp 30 -GES-1 cells (Fig. 3C).The observed phenomenon may be attributed to the intrinsic robust invasive capacity and comparable week migratory propensity of the GES-1 cell line Hp30-GES-1: GES-1 cells co-cultured with H. pylori for 30 consecutive generations under microaerophilic conditions.(18).Extended hypoxia conditions and subsequent recovery do not alter the invasive potential of GES-1 cells but rather exert an additional suppressive effect on their migratory capabilities.These results indicated that sustained exposure to H. pylori under hypoxic conditions promotes cell invasion, while hypoxia treatment alone does not affect the invasive ability.

Continuous treatment with H. pylori under hypoxic conditions inhibits autophagy
Hp 30 -GES-1, Hy 30 -GES-1, and B-GES-1 cells were collected for western blot assays to detect the expression of LC3-I and LC3B-II.The magnitude of the LC3-II/LC3-I ratio can estimate the level of autophagy.The results indicated that the level of LC3-II/LC3-I in Hp 30 -GES-1 cells was significantly lower than that in both B-GES-1 and Hy 30 -GES-1 cells (Fig. 4A).Conversely, the LC3-II/LC3-I ratio in Hy 30 -GES-1 cells was upregulated compared with B-GES-1 cells (Fig. 4A).These findings demonstrated that long-term exposure to H. pylori under hypoxia inhibited autophagy in GES-1 cells, while hypoxic treatment without H. pylori promoted autophagy in gastric epithelial cells.
We also evaluated the expression of p62, which is regulated by the production and removal of autophagosomes along with LC3B-II.Western blot assays showed that the expression of p62 was increased in Hp 30 -GES-1 cells, while it remained unchanged in Hy 30 -GES-1 cells compared with B-GES-1 cells (Fig. 4B).Moreover, B-GES-1, Hy30-GES-1, and Hp30-GES-1 cells were transfected with the mCherry-EGFP-LC3 lentiviral vector  to detect autophagy flux.The EGFP signal in the mCherry-EGFP-LC3 fusion protein is quenched under acidic pH in autophagolysosomes, making autophagolysosomes (GFP negative/RFP positive; red dots) and autophagosomes (GFP positive/RFP positive; yellow dots) easier to detect (Fig. 4C).In Hp 30 -GES-1 cells, the yellow puncta were more numerous than the red puncta, which contrasted with B-GES-1 and Hy30-GES-1 cells (Fig. 4C).These observations suggest that long-term treatment of H. pylori with hypoxia inhibits autophagy flux in GES-1 cells.

Constant treatment of H. pylori under hypoxic conditions inhibits cell apoptosis
We then investigated whether H. pylori regulates the apoptosis of GES-1 cells under hypoxic condition.The flow cytometry analysis was conducted, and the results showed that apoptosis of Hp 30 -GES-1 cells was significantly inhibited compared with B-GES-1 and Hy 30 -GES-1 cells, while apoptosis of Hy 30 -GES-1 cells decreased compared with that inB-GES-1 cells (Fig. 4D).We also detected the activity of caspase-3, and the results further supported the aforementioned findings (Fig. 4E).These results indicate that long-term treatment of H. pylori under hypoxia inhibits the apoptosis of GES-1 cells.

Sustained exposure to H. pylori under hypoxic conditions induces epithelial mesenchymal transition
Previous studies have demonstrated that epithelial mesenchymal transition (EMT)induced carcinogenesis is a prevalent cause of various malignancies (19,20).Therefore, we hypothesized that long-term H. pylori infection under hypoxia conditions could promote malignant progression.To investigate this, we assessed the expression of mesenchymal marker N-cadherin, transcription factor Snail, epithelial markers E-cad herin, and tight junction protein zonula occludens 1 (ZO-1).Our findings revealed that the expression of N-cadherin and Snail in Hp 30 -GES-1 cells was significantly higher than in both B-GES-1 and Hy 30 -GES-1 cells (Fig. 5A and B).The level of ZO-1 in Hp 30 -GES-1 cells was higher than that in B-GES-1 cells, but lower than that in Hy 30 -GES-1 cells (Fig. 5C).E-Cadherin levels in Hp 30 -GES-1 and Hy 30 -GES-1 cells were downregulated compared with B-GES-1 cells (Fig. 5D).
Furthermore, we examined the mRNA expression levels of two gastric cancer biomarkers, chloride channel-3 (CLC-3) and actin-related protein 2/3 complex subunit 3 (ARPC3) (21)(22)(23).Our results showed that the mRNA levels of CLC-3 in Hy 30 -GES-1 cells increased significantly compared with those in B-GES-1 cells, while the CLC-3 mRNA levels in Hp 30 -GES-1 cells were even higher than those in in Hy 30 -GES-1 cells (Fig. 5E).Additionally, ARPC3 mRNA levels in Hp 30 -GES-1 cells decreased significantly compared with those in both control groups (Fig. 5E).These findings suggest that long-term stimulation by H. pylori under hypoxia induces EMT and contributes to the malignant transformation of gastric epithelial cells.
We evaluated the mRNA and protein expression levels of key genes involved in the Nod1-RIP2-MAPK/ERK-FOXO4 and Nod1-RIP2-NF-κB pathways.Our results demonstrated that the mRNA and protein expression levels of Nod1 and receptor-interacting protein 2 (RIP2) were significantly upregulated in Hp 30 -GES-1 cells compared with B-GES-1 and Hy 30 -GES-1 cells (Fig. 6A through C).Additionally, the phosphorylation level of IKKα and ERK increased in Hp 30 -GES-1 cells (Fig. 6D), while the mRNA level of FOXO4 was inhibited in Hp 30 -GES-1 cells without statistically significant differences (Fig. 6A).The protein expression of FOXO4 decreased significantly compared with B-GES-1 and Hy 30 -GES-1 cells (Fig. 6E).Moreover, the mRNA expression levels of TRAF1/2 and BCL-2 in Hp 30 -GES-1 cells were significantly higher than in Hy 30 -GES-1 and B-GES-1 cells (Fig. 6A).These findings indicate that long-term stimulation of H. pylori under hypoxic conditions regulates the function of GES-1 cells through the Nod1 receptor pathway.

Long-term infection of H. pylori under hypoxia inhibits autophagy and apoptosis through the Nod1 receptor pathway in vivo
We have previously confirmed that long-term infection of H. pylori inhibits autophagy and apoptosis of gastric epithelial cells in vivo (24).In this study, we investigated the influence of H. pylori infection on autophagy and apoptosis in vivo.We infected gerbils with the H. pylori ATCC 43504 strain for 85 weeks and collected gastric tissues.Immuno histochemistry was performed to detect the expression of Nod1 and RIP2 in gastric epithelial cells.The results showed a remarkable increase in the expression levels of Nod1 and RIP2 in gerbils continuously exposed to H. pylori infection compared with the control group (Fig. 7A through D).Furthermore, pathological analysis revealed that H. pylori infection induced dysplasia in the gastric mucosa of gerbils (Fig. 7E).These findings provide additional evidence supporting the involvement of H. pylori in facilitating the malignant transformation of gastric epithelial cells.All the data verify the key roles of the Nod1 receptor pathway in H. pylori-induced inhibition of autophagy and apoptosis in vivo.

DISCUSSION
H. pylori infects about half of the global population and is the most important cause of cancer infection, accounting for approximately 90% of new cases of gastric can cer worldwide (25)(26)(27)(28).Currently, numerous studies have elucidated the pathogenic mechanisms of H. pylori both in vivo and in vitro.However, it is noteworthy that most in vitro experiments are conducted under normal pO 2 conditions (20.265 kPa).This approach falls short of accurately replicating the growth environment of gastric epithelial cells in vivo, primarily due to the considerably lower oxygen levels present in the human stomach (pO 2 = 8 kPa) (16,17,29).Furthermore, as a microaerophile, H. pylori is greatly influenced by oxygen concentration, and this, in turn, affects its form and pathogenicity (14,30).H. pylori cultured under a 7% O 2 concentration exhibit the bacillary form, whereas the cells cultured under aerobic conditions adopt the coccoid form (14,30).The conversion from the bacillary to the coccoid form leads to a significant reduction of bacterial toxicity (31).Certain characteristics displayed by H. pylori cultivated under microaerobic conditions more closely resemble the physiology of organisms grown in the human gastric mucosa (32).Therefore, in this study, we established a cell line infected with H. pylori under hypoxic conditions, simulated the gastric environment, and explored the pathogenic mechanism of persistent H. pylori infection under hypoxic conditions.
Cell migration and invasion play crucial roles in tumor development.,H. pylori infection has been demonstrated to enhance the migratory and invasive capabilities of gastric cells in a CagA-dependent manner (33).Cells infected with CagA + H. pylori, as compared with CagA-H.pylori, exhibit upregulation of miR-543, a potent inducer of cell migration and invasion (34).Additionally, studies have shown that CagA protein activates the NLRP3 inflammasome, promoting migration and invasion of gastric cancer cells (35).Moreover, previous research by Guo et al. has indicated that hypoxia contributes to the migration and invasion of MGC-823 cells (36).Notably, AGS cells demonstrated height ened invasive capabilities under a hypoxic microenvironment (37).In our research, we observed that prolonged hypoxic stimulation of H. pylori significantly enhanced the migration and invasion abilities of GES-1 cells, consistent with previous reports (33)(34)(35).However, interestingly, prolonged hypoxic stimulation did not enhance the migration and invasion of GES-1 cells compared with the control group.This lack of enhancement could be attributed to unique characteristics of different cell lines and variations in their origins.In general, our results support the notion that prolonged H. pylori stimulation contributes to cell migration and invasion under a hypoxic condition more similar to the in vivo environment.Autophagy plays a pivotal role in the survival and carcinogenesis of H. pylori.Prior investigations have documented that H. pylori infiltrates gastric epithelial cells, disrupts autophagic lysosomal acidification, and evades them to bolster survival and colonization (38)(39)(40).Suppression of H. pylori-induced autophagy fosters the progression of gastric cancer by impairing DNA damage repair and inducing genome instability (41).In our study, we validated the inhibition of cellular autophagic flux during both hypoxia and prolonged H. pylori stimulation.This inhibition potentially contributes to the prolonged survival, proliferation, and immune evasion of bacteria.
The balance between cell proliferation and apoptosis is intricately regulated, and disruption of this balance can lead to the malignant transformation of cells.Various components produced by H. pylori have regulatory effects on the proliferation of gastric epithelial cells (42).H. pylori toxins, such as UreA and CagA, have been confirmed to promote the proliferation of gastric epithelial cells and increase the risk of cancer, as demonstrated in a guinea pig model infected with H. pylori (43).Additionally, H. pylori infection has been shown to inhibit apoptosis in gastric epithelial cells (44,45).Our research revealed that prolonged exposure to H. pylori under a hypoxic environment stimulates the proliferation of gastric epithelial cells, hinders cell apoptosis, and induces epithelial-to-mesenchymal transition.These results once again confirmed the promotive role of H. pylori infection in carcinogenesis.
Our findings suggest that prolonged exposure to H. pylori in a hypoxic environment not only inhibits autophagy and apoptosis but also significantly enhances the migration and invasion capabilities of GES-1 cells.Meanwhile, hypoxia alone does not exert the same effects on cell migration and invasion as those observed in H. pylori-infected cells.It is important to note that H. pylori thrives in the microaerobic environment of the human gastric mucosa (2).Notably, under hypoxic conditions, H. pylori assumes a bacillary form, which is more virulent and better equipped to colonize the gastric mucosa (14).Therefore, our study underlines the synergistic effects of hypoxic conditions and H. pylori infection, providing a more accurate simulation of the in vivo gastric environment.
The Nod1 receptor pathway plays a crucial role in the regulation of host cell functions in response to H. pylori (46,47).Nod1 receptors recognize H. pylori's peptidoglycan, thereby modulating the autophagy and apoptosis of host cells (48).In our previous studies, we have identified the involvement of the Nod1 pathway in regulating gastric epithelial cell autophagy and apoptosis during prolonged stimulation with H. pylori lysate (24).Therefore, in this study, we investigate the role of the Nod1 pathway in regulating host cell functions under long-term hypoxic stimulation by H. pylori, and the results were consistent with our previous findings.Furthermore, existing literature reports suggest that CagA regulates autophagy and apoptosis of gastric epithelial cells through the NF-κB and MAPK/ERK pathways (45,49).Based on this, we hypothesize that during prolonged H. pylori stimulation, CagA is internalized into host cells via the T4SS and then binds to SHP-2, thereby regulating downstream NF-κB and MAPK/ERK pathways.Other bacterial toxins such as VacA and peptidoglycan are delivered into host cells through OMV.These toxins modulate the downstream NF-κB and MAPK/ERK pathways through Nod1 recognition.Collectively, all these pathways work together to promote tumorigenesis (Fig. 8).
In summary, our study provides insights into the synergistic effects of H. pylori infection and hypoxic conditions on the function of gastric epithelial cells.Prolonged exposure to H. pylori under hypoxia significantly enhanced cell migration, invasion, and epithelial mesenchymal transition, while inhibiting autophagy and apoptosis.These findings underscore the importance of the Nod1 signaling pathway in mediating the inhibitory effects of H. pylori on autophagy and apoptosis.The establishment of an H. pylori-infected cell line under hypoxic conditions provides a more accurate cell model for studying the pathogenic mechanisms of H. pylori in gastric carcinogenesis.

FIG 1
FIG 1 Establishment and detection of GES-1 cell line with sustained exposure to H. pylori under hypoxic condition.(A) Microscopic imaging systems were used to observe cell morphology of Hp 30 -GES-1 and Hy 30 -GES-1 cells.(B and C) The culture supernatant of GES-1 cells co-cultured with H. pylori under hypoxia for 24 hours was collected for bacterial culture, and H. pylori strains were successfully cultivated.Catalase test, urease test, and oxidase test (B) and Gram staining microscopy (C) were performed to confirm the activity of H. pylori.Hy30-GES-1 cells: GES-1 cells cultured for 30 generations under microoxygen conditions.

FIG 4 (
FIG 4 (Continued) ratio of red spots to yellow spots.The yellow spots indicate autophagosomes, while the red spots indicate autolysosomes.If the phagosome and lysosome fuses normally, then the red fluorescence is greater than the yellow fluorescence.If downstream autophagy is blocked, the phagosome and lysosome cannot fuse normally, and then, yellow fluorescence is the main color visualized.(D) Apoptosis of Hp 30 -GES-1, Hy 30 -GES-1, and B-GES-1 cells was detected by flow cytometry.

FIG 8 A
FIG 8 A model of the Nod signal pathway.