Development of a standardized murine model for investigating cholesterol crystallization and deposition in the gallbladder wall: effects of dietary cholesterol and pharmacological modulation

Abstract

Objective

The aim of this study is to establish a standardized murine model for cholesterol crystallization and deposition in the gallbladder wall and evaluate the effects of differential dietary cholesterol intake and pharmacological interventions on this pathophysiological process.

Methods

A total of 45 eight-week-old C57BL/6 mice were randomly assigned to nine experimental groups (n = 5 per group) receiving different dietary and pharmacological interventions designed to modulate mucin expression and reverse cholesterol transport over a 12-week period (details in Methods ). Blood and gallbladder tissues were collected under anesthesia for serum lipid profiling and histopathological evaluation. Oil Red O staining was used to detect lipid droplet accumulation in the submucosal layer of the gallbladder wall.

Results

No lipid droplets were observed in the control group or the group receiving prostaglandin. Minimal droplet formation was noted in the high-cholesterol (HC) and HC combined with bile acid groups (p > 0.05 vs. control). A significant increase in submucosal lipid accumulation was observed in the HC + celecoxib and HC + bile acid + celecoxib groups (p < 0.05). The addition of methionine to these regimens (HC + celecoxib + methionine and HC + bile acid + methionine) further enhanced lipid droplet formation (p < 0.05), with no statistically significant difference between the two. The most substantial lipid aggregation was detected in the HC + bile acid + celecoxib + methionine group (p < 0.05 vs. all other groups).

Conclusion

Impaired mucosal cholesterol reverse transport, particularly under combined inhibition of both mucin expression and RCT, was associated with the most pronounced submucosal lipid deposition and the highest incidence of gallstone formation. To our knowledge, this study is the first to establish an animal model of GCPs integrating mucoprotein and RCT regulation, providing a platform for mechanistic studies and potential therapeutic exploration.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-026-03934-8.

Introduction

Cholesterol, a lipid essential for maintaining physiological metabolic functions, is predominantly synthesized in the liver and obtained through dietary intake. However, excessive cholesterol levels or dysregulated metabolism can lead to pathological accumulation, contributing to various disorders. A key manifestation is the deposition of cholesterol within vascular walls, which promotes atherosclerotic plaques formation and accelerates atherosclerosis progression [1]. Additionally, cholesterol is recognized as a key lipotoxic agent that accumulates in hepatocytes, contributing to the progression from simple steatosis to steatohepatitis, and subsequently to liver fibrosis, cirrhosis, and hepatocellular carcinoma [2, 3]. Therapeutic strategies targeting cholesterol metabolism have been actively examined in oncological research and clinical practice [4].

In the gallbladder, excessive cholesterol deposition within the gallbladder wall promotes the formation of cholesterol-rich foam cells and mucosal protrusions, known as gallbladder cholesterol polyps (GCPs)—the most prevalent type of gallbladder polyp [5]. Although GCPs are generally considered benign with low malignant potential, distinguishing them from adenomas or malignant lesions remains clinically challenging. Polyps exceeding 10 mm in diameter typically necessitate surgical intervention, whereas those smaller than 10 mm are managed conservatively through observation. Currently, no pharmacological treatment has proven effective for GCPs [6].

Cholesterol accumulation in the gallbladder wall has therefore been implicated in the pathogenesis of various gallbladder disorders Table 1. Although animal models of cholesterol gallstone formation are well established, existing models have predominantly focused on lithogenesis and have provided limited insight into the metabolic mechanisms within the gallbladder mucosa. High-cholesterol diet-induced models of cholesterol gallstone (GCS) formation have facilitated research into lithogenesis and therapeutic development [7]. The lithogenic model using C57BL/6 mice, as reported by Fujihira et al. remains a well-established experimental system for investigating GCS pathophysiology [8].

Table 1.

Control the feed formula in groups

Ingredients of feed	GroupA	GroupB	GroupC	GroupD	GroupE	GroupF	GroupG	GroupH	GroupI
Basic feed	+	−	−	+	−	−	−	−	−
High-fat diet	−	+	+	−	+	+	+	+	+
ursodeoxycholic	−	−	+	−	−	−	+	+	+
Prostaglandin	−	−	−	+	−	−	−	−	−
Celecoxib	−	−	−	−	+	+	+	−	+
Methionine	−	−	−	−	−	+	−	+	+

" + " indicates the ingredient is added

"−" indicates the ingredient is not added

Preliminary studies suggest that GCPs and cholesterol stones develop due to impaired mucin secretion and dysregulated reverse cholesterol transport in the gallbladder mucosa, particularly under hypercholesterolemic conditions. Downregulation of mucins and cholesterol transporters contributes to polyp pathogenesis. Experimental findings indicated that mucins may inhibit excessive cholesterol infiltration into the submucosal layer, while ATP-binding cassette (ABC) family transporters facilitate the removal of excess cholesterol from this compartment. In patients with GCPs, reduced expression of Mucin-1 (MUC1)—a mucin with protective functions—and ATP binding cassette transporter G1 (ABCG1)—a cholesterol efflux transporter—has been observed [9].

Despite these clinical insights, a significant research gap exists due to the lack of an animal model that accurately recapitulates GCP pathogenesis. The widely used high-cholesterol diet-induced GCS models are ill-suited for GCP research for three primary reasons: (1) They exhibit a pathological mechanism disparity, as they primarily focus on stone formation rather than the submucosal cholesterol deposition and foam cell formation characteristic of GCPs; (2) They lack key regulatory factors, failing to incorporate the synergistic downregulation of mucoprotein (MUC) secretion and reverse cholesterol transport (RCT) observed in patients; and (3) They provide inadequate simulation of the diseased microenvironment, particularly the imbalance in mucosal protective mechanisms. Consequently, they provide limited simulation of the specific diseased microenvironment involved in mucosal cholesterol handling and submucosal lipid accumulation. Our model was therefore designed to specifically target mucin regulation and mucosal reverse cholesterol transport, which remain poorly characterized in existing animal models.

To address these specific shortcomings, this study developed a novel model within the C57BL/6 lithogenic mouse system. By co-administering celecoxib (to inhibit COX-2/MUC1) and methionine (to inhibit ABCG1-mediated RCT), we successfully achieved a synergistic disruption of the mucosal barrier and cholesterol clearance, mirroring the key aspects of human GCPs. This model could serve as a valuable tool to investigate the pathogenic mechanisms of cholesterol polyp formation and assess potential therapeutic strategies.

Materials and methods

Eight-week-old C57BL/6 inbred mice, body weight 25–27 g/each, procured from commonly utilized as a standard model for gallstone formation, were used in the present study. When subjected to a high-cholesterol diet, a gallstone formation rate of approximately 50% has been reported after 8 weeks [7]. The experimental protocol was approved by the Animal Experiment Ethics Committee of Xuanwu Hospital, Capital Medical University (Approval No.: 20220104-1). The experiment was conducted in compliance with The ARRIVE Guidelines 2.0 and local and national regulations [10].

The 8-week-old male C57BL/6 mice used in the experiment were purchased from the Animal Laboratory Center of Xuanwu Hospital, Capital Medical University. The mice were housed in a pathogen-free (SPF) environment maintained at 22 °C with a 12-h light–dark cycle, and were kept in groups of five per cage. Prior to the start of the experiment, the mice underwent a week-long acclimatization period.

Feed preparation

Basic diet

Prepared according to the AIN-93 standard formula recommended by the American Institute of Nutrition for rodents [10].

High-cholesterol diet

Comprised of 10% sucrose, 15% lard, 2% cholesterol, 0.2% sodium cholate, and 72.8% basic diet. The preparation process involved dissolving sucrose in warm water, followed by dissolution of lard in the resulting solution. This mixture was thoroughly combined with the basic diet and remaining components, molded into 3 cm diameter cylinders, and subsequently dried for storage and use.

Cholic acid enrichment

The proportion of bile cholic acid was increased, and ursodeoxycholic acid (UDCA, 5 g/kg) was incorporated into the diet [11].

Mucin secretion promotion

Prostaglandin (PG, 500 mg/kg) was added to the feed to stimulate gallbladder mucin production.

Mucin secretion inhibition

Celecoxib (200 mg/kg) was selected based on established experimental precedent in rodent models to effectively inhibit mucin secretion in the gallbladder mucosa, rather than to reflect clinically relevant exposure [12].

Inhibition of cholesterol reverse transport

Methionine (Met, 30 g/kg) was included to inhibit RCT mechanisms [13, 14].

Grouping and feeding protocol

Based on the widely used mouse lithogenesis model, the experimental feeding scheme was designed [14]. The 8-week-old mice were randomly allocated into 9 experimental groups, with 5 animals in each group $(\text{total}\text{n}=45,\text{n}=9\times {\left(Z_{\alpha /2}+{\text{Z}}_{\mathrm{\beta }}\right)}^2\times {\sigma }^2/{\delta }^2$ with a significance level α = 0.05 (two-tailed) and a power of 1-β = 0.8. Combined with preliminary experimental data showing that the intra-group coefficient of variation in Oil Red O staining area is < 15%). Each group was fed a distinct diet containing specific pharmaceutical additives for a duration of 12 weeks (as detailed in Table 1).

Group A Normal diet: basal feed.

Group B High-cholesterol diet: high-cholesterol feed.

Group C High-cholesterol + high-bile acid diet: high-cholesterol feed + UDCA.

Group D Normal diet + MUC promotion: normal feed + PG.

Group E High-cholesterol diet + MUC inhibition: high-cholesterol feed + celecoxib.

Group F High-cholesterol diet + MUC inhibition + RCT inhibition: high-cholesterol feed + celecoxib + Met.

Group G High-cholesterol + high-bile acid diet + MUC inhibition: high-cholesterol feed + UDCA + celecoxib.

Group H High-cholesterol + bile acid diet + RCT inhibition: high-cholesterol feed + UDCA + Met.

Group I High-cholesterol + bile acid diet + MUC inhibition + RCT inhibition: high-cholesterol feed + UDCA + celecoxib + Met.

General observations

Survival rates were recorded throughout the experimental period. After administering 10% chloral hydrate overdose (0.3ml) intraperitoneally to euthanized experimental mice, researchers measured body weight and systematically recorded the weight data of all groups before skin preparation and tissue collection [15].

Histological processing and staining

Gallbladder specimens were cryosectioned at a thickness of 4–8 μm. Sections were air-dried at room temperature for 15–20 min and fixed in 100% isopropanol for 5 min. Subsequently, sections were stained with 0.5% Oil Red O solution for 7–8 min, rinsed in 85% isopropanol for 3 min, and subjected to double dehydration. Hematoxylin counterstaining was performed for 1–1.5 min, followed by an additional dehydration step. Submucosal lipid droplet aggregation was evaluated microscopically.

Immunohistochemical analysis

To assess the regulatory effects on mucin secretion and RCT, MUC1 and ABCG1 protein expression levels were examined. After paraffin embedding of the gallbladder tissue, routine sections were taken. MUC1 (#A19081, Abclonal, diluted 1:400, RRID:AB_2862573) and ABCG1 (#A4328, Abclonal, diluted 1:100,RRID:AB_2765627) were stained using an automated immunohistochemistry platform (Ventana BenchMark ULTRA, Roche Diagnostics) according to the manufacturer’s operating procedures. Protein localization and expression profiles were subsequently evaluated under a light microscope.

Analysis of Oil Red O staining

Stained tissue sections were imaged using an optical microscope (Nikon Eclipse E100, Nikon, Tokyo, Japan) at 100× magnification. Image analysis and interpretation were conducted using CaseViewer software (version 2.4.0.119028) [16].

Immunohistochemical results for MUC and RCT protein staining were independently evaluated by two senior pathologists. The assessors performed the analysis without knowledge of the sample grouping, pathological grading or clinical data. Interpretation was based on both the proportion of positively stained cells and the intensity of staining, as previously described [14]. Negative staining was defined as the presence of positively stained cells in fewer than 10% of the visual field and/or complete absence of staining (scored as 0). Staining involving ≥ 10% but < 50% of cells was classified as positive (1 point), while ≥ 50% positive cells were considered strongly positive (2 points).

Staining intensity was graded according to the following criteria:

• (1) Negative (0 points): absence of positive staining.

• (2) Weak positive (1 point): greater intensity than negative but less than moderate.

• (3) Moderate positive (2 points): greater than weak but less than strong;

• (4) Strong positive (3 points): greater than moderate positive.

An average staining score was calculated for each group to facilitate comparative analysis. Representative images illustrating the immunohistochemical scoring criteria are provided in the Supplementary Materials Fig. S1.

Statistical analyses were conducted using SPSS software version 26.0. Measurement data conforming to a normal distribution were expressed as means ± standard deviation $\left(\overline{\chi }\pm s\right)$, and intergroup comparisons were conducted using one-way analysis of variance. Categorical data were presented as frequencies and percentages (%), and comparisons between groups were performed using a chi-squared (χ²) test. The Duncan test was chosen as the post hoc comparison method. Post hoc comparisons were performed using Duncan’s multiple range test, as this method provides greater sensitivity for detecting differences among multiple experimental groups with expected biological variability, while maintaining acceptable control of type I error. A p-value < 0.05 indicates statistical significance, and the confidence interval is 95%.

Results

All mice in both control and experimental groups survived throughout the 12-week feeding period, resulting in a 100% survival rate. No statistically significant differences in body weight were observed among the groups (p > 0.05; see Fig. 1). Immunohistochemical staining for MUC1 in the gallbladder mucosa indicated expression scores of 3.40 ± 0.70 in the high-cholesterol diet + cholic acid + MUC inhibition + RCT inhibition group, and 4.80 ± 0.42 in the high-cholesterol diet + cholic acid + RCT inhibition group. This difference was statistically significant (p < 0.001), indicating that celecoxib effectively inhibited mucin expression (Fig. 2). The expression scores of ABCG1 in the gallbladder mucosa were 1.50 ± 0.53 in the high-cholesterol diet + cholic acid + MUC inhibition + RCT inhibition group, and 3.40 ± 0.52 in the high-cholesterol diet + cholic acid + MUC inhibition group. The difference between these groups was statistically significant (p < 0.001), suggesting that methionine suppressed RCT (Fig. 3). Quantification of Oil Red O-stained areas indicated the following mean area ratios (mean ± SD): Normal diet group: 0.00 ± 0.00% (95% CI 0.00%–0.00%), High-cholesterol diet group: 0.20 ± 0.10% (95% CI 0.76%–0.33%), High-cholesterol diet + hypercholic cholic acid group: 0.74 ± 0.19% (95% CI 0.50%–0.98%), Normal diet + MUC promotion group: 0.00 ± 0.00% (95% CI 0.00%–0.00%), High-cholesterol diet + MUC inhibition group: 1.40 ± 0.31% (95% CI 1.02%–1.78%), High-cholesterol diet + MUC inhibition + RCT inhibition group: 4.38 ± 0.48% (95% CI 3.78%–4.98%), High-cholesterol diet + hypercholic cholic acid + MUC inhibition group: 2.08 ± 0.73% (95% CI 1.17%–2.99%), High-cholesterol diet + hypercholic cholic acid + RCT inhibition group: 4.80 ± 1.15% (95% CI 3.37%−6.23%). High-cholesterol diet + bile acid-enriched diet + MUC inhibition + RCT group inhibition 8.30 ± 1.49% (95% CI 6.45%–10.15%, p < 0.05, Fig. 4).

Fig. 1.

The weight of mice. Group A normal diet group, Group B high cholesterol diet group, Group C high cholesterol diet + bile acid-enriched diet group, Group D normal diet + promote MUC group, Group E high cholesterol diet + inhibit MUC group, Group F high cholesterol diet + inhibit MUC + inhibit RCT group, Group G high cholesterol diet + bile acid-enriched diet + inhibit MUC group, Group H high cholesterol diet + bile acid-enriched diet + inhibit RCT group, Group I high cholesterol diet + high cholic acid + inhibit MUC + inhibit RCT group

Fig. 2.

MUC 1 staining in the mouse gallbladder (×200). A High cholesterol diet + cholic acid + inhibition of MUC + inhibition of RCT Group, B High cholesterol diet + cholic acid + inhibition of RCT Group

Fig. 3.

ABCG 1 staining in the mouse gallbladder (×200). A High cholesterol diet + cholic acid + inhibition of MUC + inhibition of RCT Group, B High cholesterol diet + cholic acid + inhibition of MUC Group

Fig. 4.

The oil red O staining area ratio (%), n = 5

When compared with the normal diet group, no significant differences in Oil Red O staining area were observed in the high-cholesterol diet group, high-cholesterol diet + hypercholic cholic acid group, or normal diet + MUC promotion group (p > 0.05). Similarly, no significant difference was detected between the high-cholesterol diet group and the high-cholesterol diet + hypercholic cholic acid group (p > 0.05).

A significant increase in lipid deposition was observed in the high-cholesterol diet + MUC inhibition group and the high-cholesterol diet + cholic acid + MUC inhibition group compared with the high-cholesterol diet group (p < 0.05). However, no significant difference was detected between the MUC inhibition group and the cholic acid + MUC inhibition group (p > 0.05).

In contrast, the high-cholesterol diet + cholic acid + MUC inhibition + RCT inhibition group exhibited a significantly greater Oil Red O staining area than the cholic acid + MUC inhibition group alone (p < 0.05). This group demonstrated the highest degree of lipid accumulation when compared with all other groups (p < 0.05; Fig. 5).

Fig.5.

Oil red O staining in frozen sections of the mouse gallbladder (×200). A Normal diet group, B high cholesterol diet group, C high cholesterol diet + bile acid-enriched diet group, D normal diet + promote MUC group, E high cholesterol diet + inhibit MUC group, F high cholesterol diet + inhibit MUC + inhibit RCT group, G high cholesterol diet + bile acid-enriched diet + inhibit MUC group, H high cholesterol diet + bile acid-enriched diet + inhibit RCT group, I high cholesterol diet + bile acid-enriched diet + inhibit MUC + inhibit RCT group. Arrows indicate representative foam cells and lipid droplet clusters

Discussion

Cholesterol polyps in the gallbladder are histologically characterized by cholesterol crystal aggregates adherent to the mucosal surface via slender pedicles, accompanied by microvascular branching, villous protrusions, and densely distributed foamy macrophages. Ultrastructural analysis conducted by Satoh et al. using electron microscopy indicated that cholesterol within gallbladder epithelial cells undergoes esterification to form lipid droplets in the endoplasmic reticulum [15]. These lipid droplets are subsequently secreted into the intercellular space and phagocytosed by macrophages. Macrophages that internalize substantial quantities of lipids exhibit cytoplasmic vacuolization, forming what are referred to as “foam cells”.

Due to their increased cellular volume, foam cells exhibit impaired mobility through the restricted interendothelial spaces and narrow lumina of small lymphatic vessels. This impaired transit promotes cellular accumulation and consequent compromise of local lymphatic drainage. This resultant stasis induces villous edema with subsequent protrusion of the gallbladder mucosa into the lumen, ultimately manifesting as macroscopically discernible cholesterol polyps.

The underlying mechanism of cholesterol accumulation within the gallbladder remains incompletely understood, with multiple contributing factors hypothesized. Findings from prior research on GCPs indicate that cholesterol deposition in the gallbladder wall may be associated with both injurious factors related to bile composition and protective mechanisms within the gallbladder mucosa.

The primary injurious factor involves the infiltration of cholesterol-rich bile into the gallbladder mucosa. Dietary enrichment with cholic acid induces bile cholesterol supersaturation, thereby increasing the likelihood of cholesterol penetrating the submucosal layer. In contrast, protective mechanisms are primarily mediated by mucins (MUC) and the process of RCT, both of which are secreted or regulated by the gallbladder mucosa. However, our data indicate that cholic acid-induced bile cholesterol supersaturation alone is insufficient to trigger pathological lipid deposition, and its pathogenic effect becomes evident only when mucin-mediated protection or reverse cholesterol transport is compromised.

Mucins serve a protective function analogous to the mucus layer lining the gastrointestinal tract. Specific MUC subtypes form a physical barrier that protects epithelial cells from bile-induced injury, while others may participate in the regulation of bile composition. RCT facilitates the efflux of intracellular cholesterol to the extracellular space, enabling its transport back to the liver for metabolic processing and excretion. This process plays a key role in modulating cholesterol concentration within the bile.

Under physiological conditions, a dynamic balance is maintained between these injurious and protective factors. However, when the protective mechanisms are compromised—either through reduced mucin secretion or impaired cholesterol efflux—cholesterol tends to accumulate in the submucosal layer, potentially contributing to the formation of GCPs (see Fig. 6).

Fig. 6.

Schematic representation of the submucosal aggregation of cholesterol

The high-cholesterol diet-induced gallstone mouse model and the high-cholesterol diet along with cholic acid stone-dissolution mouse model are among the most commonly used animal models for investigating gallstone pathogenesis. These models are widely used in preclinical research. Celecoxib, a selective cyclooxygenase-2 inhibitor known for its mucin-suppressive properties, has been extensively applied in atherosclerosis research [17]. Methionine, which promotes foam cell formation in atherosclerotic models, has not yet been widely adopted in studies of gallbladder polyps. In this experimental design, celecoxib was used as a MUC inhibitor, while methionine was used to suppress RCT in animal models. In addition, factors such as cholesterol saturation and bile acid ratios were considered to assess their potential contribution to submucosal cholesterol accumulation and foam cell formation.

Building on prior studies comparing gallbladder wall characteristics in patients with cholesterol polyps and gallbladder cholesterol stones, it has been hypothesized that elevated cholesterol concentrations in the gallbladder mucosa, reduced secretion of protective mucins, particularly MUC1, and impaired RCT are key risk factors associated with the development of GCP.

Under conditions of elevated bile cholesterol levels, bile may become supersaturated. In the presence of an adequate concentration of bile acids, emulsified cholesterol is less likely to precipitate or aggregate. However, insufficient production of conjugated mucins by the gallbladder epithelium facilitates the entry of emulsified cholesterol into the mucosal and submucosal layers. When RCT activity is diminished, cholesterol clearance from submucosal tissues becomes inefficient, resulting in lipid accumulation. Macrophages subsequently internalize excess cholesterol, leading to foam cell formation—an event believed to contribute to the formation of GCP [9].

Bile acids, which are metabolic derivatives of cholesterol, constitute the primary component of bile and play a key role in facilitating the digestion and absorption of dietary lipids within the intestinal tract. Most of the cholic acid is reabsorbed via the enterohepatic circulation, with only a limited amount synthesized de novo on a daily basis. Prior studies have reported elevated cholic acid concentrations in the bile of patients with GCP compared to those with gallbladder adenomas or patients without gallbladder disease [18].

A comparison of dietary and pharmacological interventions demonstrated distinct effects on mucosal and submucosal cholesterol accumulation, as detailed in the Results section. These findings indicate that elevated cholic acid alone may not constitute an independent risk factor for submucosal cholesterol deposition. However, when mucosal protective mechanisms—particularly mucin secretion and RCT—are compromised, increased cholic acid concentrations promote cholesterol emulsification. The resulting emulsified cholesterol exhibits enhanced permeability across mucosal barriers, facilitating its uptake by mucosal cells and subsequent accumulation within submucosal tissues. This process ultimately contributes to cholesterol sequestration in the gallbladder wall.

Mucin is a high-molecular-weight glycoprotein expressed in the gallbladder mucosa that contributes to the formation of the protective mucus layer covering the mucosal surface [19]. Major mucin types expressed in the gallbladder include MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6. From their distribution and function, mucins are categorized into membrane-bound and secretory types [20]. The primary role of mucus secreted by gallbladder epithelial cells is to form a protective barrier that limits the penetration of high-concentration bile constituents into the submucosal tissue. This function is analogous to the role of gastric mucus in shielding the gastric epithelium from acid-induced injury. Mucins may be further classified as either membrane-bound, which remain attached to epithelial cells, or secreted, which are oriented toward the bile-facing surface [21].

Prior studies demonstrated that membrane-bound MUC1, located in proximity to epithelial cells within the mucus layer, is closely associated with inhibiting cholesterol entry into the mucosa. Findings from the present study indicated a reduction in MUC1 expression in the gallbladder mucosa following celecoxib administration. In groups receiving celecoxib, the Oil Red O staining area ratio in the gallbladder mucosa was significantly increased compared with corresponding groups that did not receive celecoxib, despite being on the same dietary regimen (p < 0.05).

These results indicate that celecoxib may attenuate the mucin-mediated barrier function, thereby increasing the likelihood of contact between supersaturated bile cholesterol and the gallbladder mucosa. Under conditions of mucin deficiency, the mucosal epithelium exhibits increased vulnerability to cholesterol-saturated bile. This pathophysiological state promotes cholesterol permeation into the submucosal layer, creating a microenvironment conductive to cholesterol aggregation and tissue deposition.

Excess cholesterol within the gallbladder wall is eliminated primarily through RCT. The translocation of cholesterol into the gallbladder wall depends on a concentration gradient facilitated by scavenger receptor class B type 1. Conversely, RCT mediated by ABC family transporters—predominantly expressed on the serosal surface of the gallbladder—facilitates the efflux of intracellular cholesterol into systemic circulation for subsequent hepatic metabolism and excretion [22].

Experimental findings demonstrated that the Oil Red O staining area ratio in the gallbladder mucosa was significantly higher in the methionine-treated group, in which RCT was inhibited, compared with the group receiving the same basal diet without methionine supplementation (p < 0.05). These observations indicate a key role for RCT in the prevention of submucosal cholesterol accumulation and indicate its involvement in the pathogenesis of GCP. Prior studies have reported notable expression of ABC family transporters in the gallbladder mucosa of patients with GCP, with ABCG1 identified as a major mediator of cholesterol efflux [23].

GCP formation appears to be influenced by the balance between the quantity of cholesterol entering the gallbladder wall and the efficiency of RCT-mediated clearance. In the absence of adequate mucin protection, cholesterol conjugated with cholic acid is more likely to penetrate into the submucosal layer, a mechanism that resembles the role of cholic acid in enhancing lipid absorption in the intestine. In this study, ABCG1 expression was significantly reduced in mice administered methionine compared to those not receiving methionine, resulting in impaired cholesterol efflux capacity. Correspondingly, the Oil Red O staining area in the gallbladder mucosa was significantly greater in the methionine-treated group (p < 0.05), supporting the hypothesis that impaired RCT leads to submucosal cholesterol accumulation.

Collectively, these findings support a two-hit model for gallbladder cholesterol polyp (GCP) formation. In this framework, pathological lipid accumulation does not arise from cholesterol-rich bile exposure alone, but instead requires the simultaneous failure of mucosal barrier protection and cholesterol clearance mechanisms. Inhibition of mucin secretion compromises the first line of defense against bile-derived cholesterol, while impaired reverse cholesterol transport limits the removal of cholesterol that has entered the gallbladder wall. The convergence of these two defects results in maximal submucosal cholesterol deposition and foam cell formation, as observed in Group I.

Application of the Model: This standardized murine model advances research into non-invasive diagnostic markers, targeted pharmacological interventions, and preventive therapies for GCPs. Beyond mechanistic insights, it bridges preclinical discovery with clinical application. For diagnostics, it enables longitudinal evaluation of serum/urine biomarkers (e.g., MUC1-related glycoproteins, ABCG1-mediated metabolites) correlating with submucosal lipid accumulation. In pharmacological testing, it supports assessing MUC1 agonists (strengthening mucosal barrier) and ABCG1 activators (enhancing reverse cholesterol transport) to prevent early GCP formation. Its well-defined protocols facilitate preclinical testing of preventive strategies (dietary interventions, repurposed drugs) to inhibit GCP initiation. By quantifying lipid deposition and pathway activation, this model accelerates translation into clinical trials, including dose optimization and safety profiling of preventive agents for at-risk populations. Furthermore, the model's ability to recapitulate the early, pre-polyp stages of GCP pathogenesis makes it uniquely suited for screening and validating primary preventive strategies. This shifts the therapeutic paradigm from post-diagnosis intervention to pre-emptive risk mitigation, thereby highlighting its significant translational potential beyond mere mechanistic elucidation.

While this model does not fully reproduce advanced polypoid lesions observed in clinical settings, it captures key early pathological events implicated in human gallbladder cholesterol polyp development, including mucosal cholesterol infiltration and submucosal foam cell formation.

This study also has some limitations. We did not include groups receiving methionine or celecoxib alone, thus precluding explicit evaluation of their independent effects on gallbladder wall lipid deposition; future studies should supplement single-agent experiments to validate individual factor mechanisms. This limitation reflects the study’s primary focus on synergistic mechanisms rather than isolated pharmacological effects. Additionally, the immunohistochemical analysis of MUC1 and ABCG1 was performed primarily to validate the foundational model and was not extended to all experimental groups, which limits the mechanistic interpretation of the pharmacological interventions. This study, for the first time, achieved synergistic simulation of mucosal barrier disruption and cholesterol clearance impairment by combining celecoxib (COX-2/MUC1 inhibition) and methionine (ABCG1-mediated RCT inhibition).

This model may serve as a preclinical framework for exploring therapeutic approaches aimed at preserving mucosal barrier function and enhancing cholesterol clearance in the gallbladder.

In summary, this study employed a C57BL/6 inbred mouse model of high-cholesterol diet-induced lithogenesis to investigate the pathogenesis of cholesterol polyp development. By systematically modulating critical factors—including gallbladder mucosal mucin expression, RCT, and bile acid intake—we demonstrated that cholesterol esterification leads to characteristic lipid droplet accumulation within the submucosal layer. These results establish the essential regulatory roles of mucin barrier function and RCT efficiency in the cholesterol polyps pathogenesis. The established experimental model offers a valuable platform for future research on the mechanisms and potential preventive strategies targeting cholesterol accumulation and polyp formation in the gallbladder.

Abbreviation

ABC

ATP-binding cassette transporter

MUC

Mucin

RCT

Reverse cholesterol transport

GCP

Gallbladder cholesterol polyps

GCS

Gallbladder cholesterol stones

PG

Prostaglandin

URSO

Ursodeoxycholic acid

SR-B1

Scavenger receptor class B type 1

CCK

Cholecystokinin

ANOVA

Analysis of variance

SPSS

Statistical package for the social sciences

Author contributions

Conception and design of the research: Yamin Zheng. Acquisition of data:Shuang Liu, Chen Xu Analysis and interpretation of the data: Xiang Gao, Yamin Zheng Statistical analysis: Wei Gao, Chen Xu Writing of the manuscript: Chen Xu, Xiang Gao Critical revision of the manuscript for intellectual content: Wei Gao, Shuang Liu All authors read and approved the final draft.

Funding

No external funding received to conduct this study.

Data availability

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

All experiments were evaluated and approved by the Animal Ethical Committee of Xuanwu Hospital, Capital Medical University (XW-20220104-01) and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.