Fos-related antigen 1 is required for Helicobacter pylori-induced inflammatory response in gastric epithelial cells through NF-κB pathway

Introduction

Chronic gastritis affects more than half of the global population and represents a major long-term health burden. Despite the fact it is a key step in the development of gastric cancer, gastritis is often underestimated in clinical practice (1). Helicobacter pylori (H. pylori) infection is the leading cause of gastritis (2,3) with approximately 60% of the Chinese population infected (4). This gram-negative bacterium colonizes the gastric mucosa and induces inflammatory effect (5). Long-term colonization of H. pylori in vivo can cause direct damage to gastric epithelial cells, in addition to the activation of immune cells, triggering inflammation and immune responses (6,7). As a result pro-inflammatory mediators such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)α are markedly upregulated in H. pylori-infected gastric tissues (8-11).

Fos-related antigen 1 (FRA-1), encoded by the Fos-like antigen 1 (FOSL1) gene, is a transcription factor implicated in both stomach inflammation and malignancy (12,13). Elevated FRA-1 expression has been observed in multiple types of human cancer (14,15), and emerging evidence suggests its potential involvement in gastric inflammation (13,16). However, the functional role of FRA-1 in H. pylori-induced gastritis remains poorly understood, prompting us to investigate its effects in this context.

The nuclear factor-κB (NF-κB) signaling pathway is crucial in gastritis caused by H. pylori infection (17). As an important central nuclear factor, NF-κB plays an important role in cell proliferation, immune reactions, inflammation, and other physiological and pathological processes by regulating the transcription of multiple genes (18,19). Consequently, H. pylori infection frequently activates the NF-κB pathway (8), leading to increased expression of inflammatory cytokines in gastric epithelial cells (20). Additionally, research has indicated that H. pylori infection induces the recruitment of c-Jun and c-Fos to the promoter of IL-6 and IL-8, further modulating their expression (21,22). Moreover, it has been observed that FRA-1 expression exhibits a time- and dose-dependent increase in response to the infection of H. pylori, although the underlying mechanism remains unclear (23). The precise role of FRA-1 in H. pylori-driven gastritis and the mechanisms through which signaling events such as NF-κB activation affect FRA-1 expression are not yet well understood.

In this study, H. pylori-infected gastric epithelial cells are used for investigating the function of FRA-1 in inflammatory responses based on the conditional knockdown of FRA-1. We provide evidence of the regulatory role of FRA-1 on the expression of inflammatory cytokines and explore potential mechanisms involved in FRA-1-mediated inflammation. We present this article in accordance with the MDAR reporting checklist (available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-188/rc).

Methods

Patients and samples

A total of 32 FFPE tissues were acquired from the pathology department of Taizhou People’s Hospital between October 2020 and December 2020. These tissues were obtained from patients with histology-confirmed gastritis who were consecutively subjected to immunohistochemistry (IHC), 16 of which were positive for H. pylori infection. All tissues were randomly selected from the patients diagnosed with gastritis, and there were no significant differences in the age and sex distribution between these groups. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of the Jiangsu Taizhou People’s Hospital (Taizhou, China; No. KY 202011801). Written informed consent was waived due to the retrospective nature of the study and the absence of any interaction with patients or access to their identifiable private information.

Cell culture

The immortalized gastric epithelial mucosa GES-1 cell line was sourced from the Beijing Institute for Cancer Research. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and incubated at 37 ℃ in a 5% CO₂ humidified atmosphere. For experiments, cells were seeded in culture plates and allowed to reach confluence prior to infection.

Cell transfection

In this experiment, si-FRA-1 (5’-GCCAAGCAUCAACACCAUGTT-3’ sense and 5’-CAUGGUGUUGAUGCUUGGCTT-3’ antisense) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). For targeted knockdown of human FRA-1, transfection was performed in GES-1 cells (plated in 6-well plates at a density of 3.5×10⁵ cells/mL) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc., Waltham, USA), following the manufacturer’s instructions. After 48 h post-transfection, RNA and protein were extracted and then used for real-time quantitative polymerase chain reaction (RT-qPCR) and western blot to analyze.

Bacteria and infection experiments

The H. pylori cytotoxin-associated gene A (CagA)-positive type strain 11637 was maintained in the laboratory. Bacteria were routinely cultured on Columbia agar plates supplemented with 10% sheep blood and incubated at 37 ℃ under microaerophilic conditions. For infection experiments, bacteria were harvested, suspended in medium. Host cells were then infected at a range of multiplicity of infection (MOIs): 0, 10, 50, and 100.

RNA extraction and RT-qPCR analysis

Total RNA from cells was obtained from cells with TRIzol Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions. Then, cDNA was synthesized from 1,000 ng of total RNA using HiScript QRT SuperMix (Vazyme Biotech Co., Ltd., Nanjing, China). RT-qPCR was conducted using TransStart Top Green qPCR Super Mix (TransGen Biotech Co., Ltd., Beijing, China) on an ABI Step One Plus Real Time PCR System (Thermo Fisher Scientific, Inc.). The sequences of the RT-qPCR primers were synthesized by Shanghai Sunny Biotech Co., Ltd. (Shanghai, China). Gene expression was analyzed by the 2^−DDCt quantitative method and normalized to GAPDH. The sequences of primers are listed in Table 1.

Western blot analysis

Western blot was performed as described previously with minor modifications (24). Cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) with phenylmethanesulfonyl fluoride and phosphatase inhibitors, mixed on ice for 30 min. Then centrifuged at 12,000 ×g for 30 min, and protein concentrations were measured by a bicinchoninic acid assay protein assay kit (Thermo Fisher Scientific, Inc.). Next, equal amounts of protein were resolved on 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were then incubated with 5% non-fat dry milk powder in tris-buffered saline (TBS) containing 0.1% tween-20 for 2 h at room temperature. The membranes were incubated with primary antibodies at 4 ℃ overnight and then incubated with secondary antibody (dilution, 1:2,000; catalog number sc-2004, sc-2005; Santa Cruz) at room temperature for 1 h before visualized using an enhanced chemiluminescence system (Image Quant LAS 4000 mini). The levels of target proteins were analyzed by densitometry, normalized to GAPDH, and the relative fold change compared to the control group was calculated. Following were primary antibodies: Anti-FRA-1 (dilution, 1:1,500; catalog number ab124722; Abcam), GAPDH (dilution, 1:1,000; catalog number sc-32233; Santa Cruz), anti-NF-κB P65 (catalog number 8242), anti-P-NF-κB P65 (catalog number 76778), anti-IL-1β (catalog number 12242), anti-IL-6 (catalog number 12912), anti-IL-8 (catalog number 94407), anti-TNFα (catalog number 8184), anti-A20 (catalog number 5630), anti-NF-κB P105 (catalog number 4717), anti-IκB Kinase (IKB)α (catalog number 61294), anti-IKKβ (catalog number 2370), anti-IKBα (catalog number 15595), anti-P-IKBα (catalog number 5209), anti-NF-κB P50 (catalog number 13586, anti-glycogen synthase kinase 3 beta (GSK3β) (catalog number 9315) and anti-P-GSK3β (catalog number 5558) (dilution, 1:500; Cell Signaling Technology, Inc., Danvers, USA).

Immunohistochemistry

Immunohistochemical analysis was performed on gastric tissue samples. The tissue was fixed in 10% buffered formalin, paraffin-embedded and cut into serial sections of 4-µm thickness. Endogenous peroxidase activity was quenched by treating the sections with 3% hydrogen peroxide for 10 min. Non-specific binding sites were blocked using 5% bovine serum albumin (BSA; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Subsequently, the sections were incubated with a primary antibody against targeting (dilution, 1:150) overnight at 4 ℃ in a humidified chamber. Finally, antibody binding was visualized by applying 3,3'-diaminobenzidine (DAB, SA1020; Wuhan Boster Biological Technology, Ltd.) as the chromogen and counterstained with hematoxylin for microscopic examination. The expression levels of FRA-1 protein were scored as the percentage distribution of positive-stained cells. Image J software was used in the quantitative analysis (25).

Immunofluorescence

For immunofluorescence analysis, cells grown on glass coverslips were fixed with 4% formaldehyde for 30 min, permeabilized with 0.1% triton-X 100 for 20 min, and blocked with 1% bovine serum albumin (BSA) for 1 h. The cells were then incubated overnight at 4 ℃ with a primary antibody against NF-κB P65 (Cell Signaling Technology, Inc.). After washing with PBS, the samples were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Inc.) for 30 min at room temperature Nuclei were counterstained with DAPI for 5 min. Coverslips were mounted and imaged using an inverted wide-filed fluorescence microscope (DeltaVision Elite, GE Healthcare Life Sciences). Representative images from three independent experiments are shown.

Cytokine measurements

Cytokine levels in cell culture supernatants were quantified using enzyme-linked immunosorbent assay (ELISA). Prior to analysis, samples were clarified by centrifugation at 3,000 ×g for 10 min to remove cellular debris. The processed supernatants were either assayed immediately, or aliquoted and stored at ≤−20 ℃ for subsequent use. Concentrations of the bioactive cytokines IL-6, IL-8 and TNFa were determined according to the manufacturer’s protocol using commercial ELISA kits (Thermo Fisher Scientific, Inc.).

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 8.0.2). Data from three independent experiments are presented as the mean ± SD or SE. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post-hoc test for pairwise comparisons. Differences between two groups were evaluated using randomized controlled t-tests. A P value less than 0.05 was considered statistically significant.

Results

FRA-1 is increased in patients with gastritis

To determine the relationship between FRA-1 expression and H. pylori infection in gastritis, immunohistochemical staining of FRA-1 was performed in gastric biopsy specimens from both H. pylori-infected and uninfected patients. Scoring of FRA-1 protein expression was performed by the percentage distribution of positive-stained cells. As shown in Figure 1, the accumulation of FRA-1 was significantly higher in H. pylori-infected gastric biopsies than uninfected ones (P<0.05) as determined by immunohistochemistry.

Figure 1 FRA-1 is up-regulated in human gastric mucosa of patients infected with H. pylori strains. FRA-1 protein expression and localization in the gastric mucosa of patients without H. pylori infection and those infected with H. pylori was determined by IHC. The percentage distribution of positive-stained cells is shown in the right graph. Magnification, ×200; scale bar =20 µm. *, P<0.05. FRA-1, Fos-related antigen 1; H. pylori, Helicobacter pylori; IHC, immunohistochemistry.

H. pylori infection upregulates FRA-1 expression in GES-1 cells

GES-1 cells were inoculated with H. pylori for varying durations (0, 4, 6, and 8 hours) and different MOIs (0, 10, 50, 100). Optical microscopy observations revealed that as the infection time increased, cells progressively became deformed, shrinking, and swelling. There was a decrease in adherent cells and an increase in suspended/dead cells, membrane blurring, and the appearance of a “hummingbird” phenotype (Figure 2A). H. pylori infection induced an increase in FRA-1 mRNA and protein expression compared to uninfected controls, peaking at 8 hours post-infection using an MOI of 50. The expression level of FRA-1 was upregulated by 3.01-fold following H. pylori infection (Figure 2B,2C).

Figure 2 Active H. pylori and cell interaction upregulates FRA-1 and transfection of siRNA specific for FRA-1 into GES-1 cells. (A) White light images were observed by phase-contrast microscopy in GES-1 cells following infection with H. pylori for 0, 4, 6 and 8 h. Images in (i), (ii), (iii) and (iv) are at a magnification of ×40, whereas those in (v), (vi), (vii) and (viii) were taken at a magnification of ×200. Time points are as follows: (i, v) 0 h; (ii, vi) 4 h; (iii, vii) 6 h; (iv, viii) 8 h post-infection. Scale bar =100 µm (for ×200 images). (B,C) GES-1 cells were infected with H. pylori for 8 h at an MOI of 0, 10, 50 and 100 before FRA-1 mRNA and protein expression analysis. (D,E) mRNA and protein collected from FRA-1 siRNA GES-1 cells with or without infection of H. pylori (MOI =50:1). FRA-1 in response to H. pylori infection and siRNA was assessed by western blot analysis and RT-qPCR. Experiments were performed in triplicate with similar results. *, P<0.05; **, P<0.01; ***, P<0.001. FRA-1, Fos-related antigen 1; H. pylori, Helicobacter pylori; MOI, multiplicity of infection; RT-qPCR, real-time quantitative polymerase chain reaction.

To explore the contribution of FRA-1 to H. pylori-induced inflammation, GES-1 cells were transfected with small interfering RNA (siRNA) targeting FRA-1 and subsequently infected with H. pylori (MOI 50 for 8 hours). As expected, infection resulted in a 1.57-fold increase in FRA-1 mRNA expression compared to uninfected controls. Besides, uninfected controls were reduced by 10.15-fold following FRA-1 knockdown. Furthermore, FRA-1 knockdown by siRNA reduced this infection-induced expression by 2.83-fold relative to the Control group (Figure 2D). The protein expression of FRA-1 showed similar trends: it was significantly induced by H. pylori but decreased in si-FRA-1 cells (Figure 2E). This expression pattern was further confirmed by immunofluorescence (Figure S1). Based on these findings, an in vitro cell model of H. pylori infection with conditional FRA-1 knockdown was established.

FRA-1 increases H. pylori-induced inflammatory cytokine expression

To examine the effects of FRA-1 on cytokine expression in GES-1 cells during H. pylori infection, we measured the expression levels of proinflammatory genes using RT-qPCR. Our results showed that H. pylori increased the expression of IL-1β, IL-6, IL-8, and TNFα (by 2.74, 1.86, 4.08, and 4.71-fold, respectively) in vitro. However, in si-FRA-1 cells, the expression of these cytokines was reduced following H. pylori infection compared to control cells (Figure 3A). Protein expression was assessed by Western blot analysis, which revealed that H. pylori strongly stimulated the production of IL-1β, IL-6, IL-8, and TNFα in both wild-type and si-FRA-1 cells, but the levels were notably lower in si-FRA-1 cells compared to wild-type cells (Figure 3B). Additionally, ELISA measurements of cytokine concentrations in cell culture supernatants showed that H. pylori-induced expression of IL-1β, IL-6, IL-8, and TNFα was suppressed in si-FRA-1 cells compared to control cells (Figure 3C). These findings support our previous observations and identify FRA-1 as a key regulator of H. pylori-driven inflammatory responses by modulating proinflammatory cytokine gene expression.

Figure 3 FRA-1 knockdown alters H. pylori-induced expression of inflammatory cytokines in GES-1 cells in vitro. mRNA and protein were collected from FRA-1 siRNA-treated GES-1 cells with or without H. pylori infection (MOI =50:1) for 8 h. (A) RT-qPCR of the expression of inflammatory cytokines. (B) Western blot analysis of inflammatory cytokines. (C) Quantitation of cytokine levels in the supernatants of FRA-1 siRNA-treated GES-1 cells with or without H. pylori infection (MOI =50:1) for 8 h. Experiments were performed in triplicate with similar results. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. FRA-1, Fos-related antigen 1; H. pylori, Helicobacter pylori; MOI, multiplicity of infection; RT-qPCR, real-time quantitative polymerase chain reaction.

FRA-1 augments H. pylori-induced inflammation during gastritis by promoting the activation of NF-κB

The NF-κB pathway plays a crucial role in the inflammation associated with H. pylori infection, controlling the expression of multiple inflammatory genes. To determine the regulatory role of FRA-1 on NF-κB activity under the experimental conditions, we assessed the levels of total P65 and phosphorylated P65 in H. pylori-treated and FRA-1-siRNA cells using Western blot analysis. Treatment of GES-1 cells with H. pylori led to a significant activation of the P65 pathway, with an increase in P65 phosphorylation normalized to total P65. However, the knockdown of FRA-1 resulted in reduced levels of phosphorylated P65 (Figure 4A). This suggests that the P65 signaling pathway may be primarily responsible for the H. pylori-induced increase in FRA-1 expression in GES-1 cells.

Figure 4 Effect of FRA-1 deficiency on the NF-κB pathway in GES-1 cells in vitro. Protein was collected from FRA-1 siRNA-treated GES-1 cells with or without H. pylori infection (MOI =50:1) for 8 h. (A) NF-κB P65 and phosphorylation of P65 in response to H. pylori infection were assessed by western blot analysis. (B) The expression of major proteins in the NF-κB pathway in si-FRA-1-transfected cells before and after H. pylori infection was studied by western blot analysis. (C) The effects of FRA-1 on the phosphorylation of IKBα stimulated by H. pylori were observed by western blot analysis. Experiments were performed in triplicate with similar results. *, P<0.05; **, P<0.01; ***, P<0.001. FRA-1, Fos-related antigen 1; H. pylori, Helicobacter pylori; IKBα, IκB kinase α; MOI, multiplicity of infection; NF-κB, nuclear factor kappaB.

We then examined key molecules in the NF-κB pathway, finding that the expression levels of A20, NF-κB P105, NF-κB P50, IKKα, IKKβ, and P-GSK3β/GSK3β were significantly elevated by H. pylori and decreased in si-FRA-1 cells (Figure 4B). Further analysis of the effects of FRA-1 on IKBα and P-IKBα expression revealed that, following H. pylori stimulation, the expression of P-IKBα, normalized to total IKBα, increased; however, this effect was diminished in si-FRA-1 cells (Figure 4C). These data indicate that H. pylori-mediated upregulation of FRA-1 accelerates NF-κB activation, leading to increased transcription of inflammatory factors.

Immunofluorescence analysis showed that, following H. pylori infection, P65 translocated from the cytoplasm to the nucleus, resulting in increased nuclear P65 levels. Moreover, si-FRA-1 inhibited this translocation (Figure 5A). These findings indicate that the infection of H. pylori increases the expression of FRA-1, which promotes the phosphorylation of P65 and its translocation into the nucleus through NF-κB pathway activation, thereby regulating the expression of downstream inflammatory factors and enhancing the inflammatory response (Figure 5B).

Figure 5 FRA-1 knockdown in GES-1 cells inhibits H. pylori-induced nuclear translocation of P65 in vitro. (A) Immunofluorescence staining was performed to determine the location of NF-κB P65. Magnification, ×600; scale bar =10 µm. Experiments were performed in triplicate with similar results. (B) Proposed model for the role of FRA-1 in promoting H. pylori-induced transcription of proinflammatory cytokines through the acceleration of NF-κB translocation. FRA-1, Fos-related antigen 1; H. pylori, Helicobacter pylori; NF-κB, nuclear factor kappaB.

Discussion

Chronic gastritis is a persistent inflammatory condition affecting the gastric mucosa (26); long-term H. pylori infection can lead to gastric mucosal atrophy, which is a primary cause of chronic gastritis (27). Additionally, H. pylori-induced atrophic gastritis is epidemiologically linked to the development of gastric cancer (28). Although some evidence supports this association, the underlying mechanisms remain not fully understood (29-31). A Swedish cohort study indicated that within 20 years, the risk of developing gastric cancer is 1 in 85 for patients with chronic gastritis, 1 in 50 for those with chronic atrophic gastritis, 1 in 39 for individuals with intestinal-type gastritis, and 1 in 19 for those with gastric mucosal hyperplasia (32). Therefore, identifying molecular abnormalities associated with these conditions may enhance our understanding of gastritis and facilitate early disease detection.

FRA-1 participates in the regulation of inflammation, cellular proliferation and other biological activities (23). Our research group’s previous studies identified several proteins with differential expression in response to H. pylori infection and gastritis. Notably, FRA-1 expression was elevated following H. pylori infection, suggesting that gastritis may be associated with H. pylori-induced dysregulation of FRA-1. In this study, we demonstrated that FRA-1 mediates the production of proinflammatory cytokines induced by H. pylori. As shown in Figure 1A, FRA-1 levels were significantly higher in gastric mucosal tissues infected with H. pylori compared to uninfected tissues, suggesting that FRA-1 is involved in the pathogenesis of H. pylori- associated gastritis. In vitro research in vitro further confirmed that H. pylori infection upregulates d FRA-1 expression in GES-1 cells, consistent with the findings reported by Ding et al. (23). Proinflammatory cytokines are crucial in the pathogenesis and progression of H. pylori-associated diseases (5), as well as cause persistent inflammation, which in turn leads to chronic inflammation (33,34). Under normal conditions, GES-1 cells secreted minimal amounts of cytokines. However, exposure to H. pylori significantly increased the levels of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, and TNFα. In contrast, FRA-1-deficient cells exhibited reduced levels of these cytokines, both in the presence and absence of H. pylori infection. We therefore propose a pathogenic pathway in which FRA-1 promotes H. pylori-induced gastritis via its role in promoting inflammatory cytokine expression.

FRA-1 can activate the NF-κB signaling pathway, which is a crucial response mechanism in gastric mucosal epithelial cells exposed to H. pylori (8). Ferrero et al. (35) demonstrated that H. pylori infection maintains NF-κB signaling in an active state and is vital for initiating the transcription of IL-8, TNFα, and IL-1β. Vaz et al. found that the FRA-1 transcription factor complex exacerbates lipopolysaccharide-induced lung injury and mortality of mice by modulating proinflammatory cytokine expression through c-Jun and NF-κB signaling (36). In this study, key molecules of NF-κB pathway increased after H. pylori infection but were downregulated in the FRA-1 knockdown group These findings suggest that H. pylori infection upregulates FRA-1, which in turn activates the NF-κB pathway, thereby promoting inflammatory responses. Specifically, siRNA against FRA-1 was found to inhibit the phosphorylation of H. pylori-stimulated IKBα and reduce P65 nuclear localization. Based on the results, the associated mechanism might be associated with the FRA-mediated upregulation of IKKα and IKKβ, thus promoting the phosphorylation of IKBα stimulated by H. pylori. Besides, we observed that H. pylori infection led to increased expression of A20, p105, and phosphorylated GSK3β (Ser9) (Figure 4B). While these molecules are generally recognized as negative regulators of the NF-κB pathway, their upregulation upon infection likely reflects the activation of compensatory negative feedback mechanisms in response to sustained pro-inflammatory signaling. A20 is a classical NF-κB target gene that functions to terminate NF-κB activation through ubiquitin editing, thereby preventing excessive inflammation (37). Similarly, p105 serves both as a precursor for p50 and as an IκB-like molecule that retains NF-κB dimers in the cytoplasm, and its increased expression may represent a regulatory attempt to modulate NF-κB activity (38). Phosphorylation of GSK3β at Ser9 inhibits its kinase activity, which has been reported to suppress TLR-induced inflammatory responses (39). Thus, the concurrent upregulation of these molecules likely reflects a dynamic balance between H. pylori-induced inflammation and the induction of regulatory mechanisms aimed at maintaining immune homeostasis.

Conclusions

This study highlights FRA-1 as a key regulatory factor in the enhanced inflammatory response to H. pylori infection. Our findings suggest that FRA-1 mediates H. pylori-induced inflammation by regulating the production of pro-inflammatory cytokine. Thus, FRA-1 may serve as a potential molecular target for the treatment of H. pylori infection and associated gastritis.

Acknowledgments

The authors would like to acknowledge the generous support of the Clinical Laboratory Center of the Jiangsu Taizhou People’s Hospital. The authors would also like to thank Spandidos Publications (https://www.spandidospublications.com/languageediting) for the English language editing.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-188/rc

Data Sharing Statement: Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-188/dss

Peer Review File: Available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-188/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://cco.amegroups.com/article/view/10.21037/cco-2025-1-188/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Ethics Committee of the Jiangsu Taizhou People’s Hospital (Taizhou, China; No. KY 202011801). Written informed consent was waived due to the retrospective nature of the study and the absence of any interaction with patients or access to their identifiable private information.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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