2.1 Animal Experiments

C57BL/6J male mice (6–8 weeks old) were provided by SPF (Beijing) Biotechnology Co. Ltd. All mice were raised in a specific pathogen-free (SPF) facility of the Animal Experiment Center of Peking University People's Hospital with a temperature of 21°C ± 2°C, relative humidity of 40%–60%, and normal access to food and water. A 12-h light/dark cycle was maintained. Following a 6-h fasting period, the mice were euthanized via cervical dislocation. This study was approved by the Institutional Medical Ethics Review Board of Peking University People's Hospital (document no. 2021PHE023).

After a 1-week acclimation period, all mice were fed either a normal diet (ND) or a high-fat diet (HFD) for 4 or 8 weeks, based on the specific research requirements. The HFD composition contained 60% fat, 20% carbohydrate, and 20% protein by energy content, with a caloric density of 5.24 kcal/g (formulated by Research Diets Inc., New Brunswick, NJ, USA). Following the dietary intervention period, mice were randomly assigned to two groups: the sham-operated group (Sham) and the post cholecystectomy group (PC). Following an 8-h fast, mice underwent either cholecystectomy or sham surgery. Three months later, the mice were euthanized, and tissues including the liver and ileum were dissected and preserved. Detailed research procedures and group schematic are illustrated in Figures 1a and 2a.

2.2 Haematoxylin and Eosin (H&E) Staining

After euthanizing the mice, liver tissue specimens were fixed in 4% paraformaldehyde solution for at least 24 h, subjected to gradient ethanol dehydration, and then embedded in paraffin to produce 4 μm sections. The liver tissue sections were stained with H&E and examined under a microscope. NAFLD activity scores (NAS) for each group of mice were assessed by two observers using an optical microscope [28], with the scoring criteria detailed in Table S1.

2.3 Liver Triglyceride and Total Cholesterol Measurement and Bradford Assay for Determining Liver Protein Concentration

Liver samples (50 mg) were homogenised in 1 mL lysis buffer using a tissue homogeniser (Bio-Gen Pro200). The homogenate was incubated at room temperature for 10 min, and the supernatant was transferred to a fresh tube. The extract was heated at 70°C for 10 min, followed by centrifugation at 2000 rpm for 5 min (room temperature). The resulting supernatant was collected for analysis according to the instructions provided with the respective kits: The High Fatty Sample Triglyceride (TG) Content Assay Kit (Applygen Technologies Co. Ltd., Beijing, China), The High Fatty Sample Cholesterol Total (CHOL) Content Assay Kit (Applygen Technologies Co. Ltd., Beijing, China), and the Bradford Protein Concentration Assay Kit (Beyotime Biotechnology Co. Ltd., Shanghai, China) to measure liver triglyceride, total cholesterol, and tissue protein content.

2.4 Determination of Serum Triglycerides (TG), Total Cholesterol (TC), Alanine Aminotransferase (ALT), and Aspartate Aminotransferase (AST) Levels

Serum or cell supernatant levels of TG, TC, ALT, and AST were determined according to the instructions provided with the following kits: The Liquid Sample TG Content Assay Kit (Applygen Technologies Co. Ltd., Beijing, China), The Liquid Sample TC assay kit (Applygen Technologies Co. Ltd., Beijing, China), The Liquid Sample ALT Activity Assay Kit (Applygen Technologies Co. Ltd., Beijing, China), and The Liquid Sample AST Activity Assay Kit (Applygen Technologies Co. Ltd., Beijing, China).

2.5 RNA Extraction and Real-Time Quantitative PCR

Total RNA was extracted from liver tissue or HepG2 cells using TRIzol reagent (Thermo Fisher Scientific). Subsequently, mRNA from liver tissue was reverse transcribed into cDNA using the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). qRT-PCR was performed on an Applied Biosystems StepOne Plus Real-Time PCR System, employing SYBR Green Master Mix (Thermo Fisher Scientific). Primer sequences are provided in Tables S2 and S3.

2.6 BAs Detection

All BAs standards were obtained from Steraloids and TRC Chemicals Inc., or synthesised in the Metabolomics Laboratory (China). Ten stable isotope-labelled standards were obtained from C/D/N Isotopes (Canada) and Steraloids (USA) Internal standard (IS) concentrations were maintained consistently across all calibration points. BAs were quantitatively analysed using an ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC Xevo TQ-S, Waters), with assistance from Shanghai Metware Biotechnology Co. Ltd. Raw data files from UPLC-MS/MS bile acid assays were processed using MassLynx software (v4.1, Waters, USA) for peak integration, calibration, and quantification of each metabolite. Data analysis was performed using SIMCA 14.1 (32-bit) (Umetrics).

2.7 Flow Cytometry Analysis

Mouse liver tissue was isolated and mechanically homogenised in 15 mL of precooled PBS. The homogenate was filtered through a 70 μm cell strainer, washed with PBS, and centrifuged at 500 g for 5 min. Cells were resuspended in PBS and aliquoted into tubes, with 100 μL added to each tube and stained with flow cytometry antibodies as per the manufacturer's instructions (Anti-mouse CD16/CD32, BioLegend, San Diego, CA, USA). Staining was performed at 4°C in the dark for 30 min, followed by the addition of 1 mL of PBS and centrifugation at 700 g for 5 min. The cell pellets were resuspended in 200 μL of PBS, and the proportion of B and T lymphocytes was analysed by flow cytometry. All data were acquired on a Gallios flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and analysed using FlowJo 10.8.1 (BD Biosciences, San Jose, CA, USA) or Kaluza Analysis Software 2.1 (Beckman Coulter Inc., Brea, CA, USA).

2.8 Cell Isolation and Culture

HepG2 cells, a human liver cancer cell line, were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Cell revival, culture, and subculturing were performed according to the ATCC cell culture guidelines. Hepatocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (10,000 U/mL penicillin, 10 mg/mL streptomycin). The culture medium was replaced every 48 h, and cells were passaged at a 1:3 ratio every 3–4 days. All cultures were maintained in a humidified incubator at 37°C with 5% CO. Human neutrophils used in this study were isolated from the peripheral blood of healthy volunteers. The isolation process, based on the referenced literature [29], is summarised as follows: 5 mL of Polymorphprep lymphocyte separation medium was added to a 15 mL sterile centrifuge tube. 5 mL of peripheral blood was carefully layered on top of the separation medium and centrifuged. The white cell layer (1–1.5 mL) was aspirated into a new centrifuge tube, resuspended in RPMI 1640 medium, centrifuged again, and the supernatant was discarded while the pellet was retained. A 10 μL aliquot of the cell suspension was diluted 10-fold, and cell counts were performed using a haemocytometer under a microscope. Based on the cell count, the cell concentration was adjusted to 2 × 10⁶ cells/mL for subsequent experiments.

2.9 Oleic Acid (OA) and Palmitic Acid (PA) Treatment of Hepatocytes to Simulate a High-Fat State

To determine the optimal ratio of OA to PA, HepG2 liver cells were divided into six groups and treated with varying concentrations (0–1200 μM) of OA and PA for 24 h. The cell supernatants were collected to measure ALT levels. Cells were also collected for TG content analysis and Oil Red O staining.

2.10 Bile Acid Exposure Experiment

The HepG2 liver cells were divided into 8 groups (Figure 6a). For the bile acid groups, cells were treated with deoxycholic acid (DCA) or taurodeoxycholic acid (TDCA). The OA and PA treatment groups received 1 mM OA and PA solution for 24 h. Subsequently, in the bile acid exposure groups, cells were treated with different concentrations of individual BAs for 24, 48, and 72 h. The control and non-bile acid groups were treated with complete culture medium under the same conditions. Cell supernatants were collected, and CXCL1 and CXCL2 levels were measured according to the instructions of the respective ELISA kits (Human CXCL1/KC ELISA Kit, MultiSciences Biotech Co. Ltd., Hangzhou, China; Human CXCL2/GRO-β ELISA Kit, MultiSciences Biotech Co. Ltd., Hangzhou, China). Following cell collection, chemokine and Farnesoid X receptor (FXR) expression levels were detected using qRT-PCR as described above, with primers listed in Table S3.

2.11 Neutrophil Chemotaxis Assay and Co-Culture Experiments With Hepatocytes and Neutrophils

Neutrophil chemotaxis assays and co-culture experiments with hepatocytes and neutrophils were performed using Transwell plates (24-well, 3.0 μm, Corning Incorporated, America). Detailed experimental procedures and group assignments are outlined in Figure 7a. For the OA and PA treatment groups, cells were exposed to 1 mM OA and PA solution for 24 h. In the bile acid exposure groups, cells were stimulated with 100 μM deoxycholic acid for 48 h and 200 μM TDCA for 72 h. Control and non-bile acid groups were treated with a complete culture medium under the same conditions. Cell supernatants were collected for subsequent neutrophil chemotaxis assays, and hepatocytes were used for co-culture experiments with neutrophils.

In the neutrophil chemotaxis assay, cell supernatants were added to the lower chambers of 24-well Transwell plates, while the upper chambers received 200 μL of neutrophil suspension (2 × 10⁶ cells/mL). The plates were incubated in a cell culture incubator (37°C, 5% CO₂) for 2 h. The Transwell chambers were then carefully removed, fixed with 4% paraformaldehyde, and cells on the membrane surface were gently removed with a cotton swab. The chambers were stained with 0.1% crystal violet, and images were captured under a microscope. Cell counting was performed using ImageJ software.

For the hepatocyte and neutrophil co-culture experiments, HepG2 cells were plated in 24-well plates, and Transwell inserts with 3 μm pore size were placed in the 24-well plates. The upper chambers were filled with 200 μL of neutrophil suspension (2.5 × 10⁶ cells/mL) and incubated in a cell culture incubator (37°C, 5% CO₂) for 20 h. Afterward, the lower chamber supernatants were aspirated, and the wells were washed twice with 1× PBS. Oil Red O staining and qRT-PCR experiments were then conducted to assess lipid accumulation in hepatocytes and the expression levels of lipid metabolism-related genes. Primers are listed in Table S3.

2.12 Statistical Analysis and Data Visualisation

All data were represented as mean ± Standard error. GraphPad Prism 9.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis. Independent sample t-tests were used to compare means between two groups, one-way ANOVA was employed for comparing means across multiple groups, and pairwise comparisons were conducted using the LSD method. Non-parametric tests were used for data that did not meet normality assumptions. In correlation analyses, quantitative variables were tested for normality. Pearson correlation was used for normally distributed data, while Spearman correlation was applied for non-normally distributed data. GraphPad Prism 9.0 software was utilised for plotting, calculating correlation coefficients (r values), and determining p values, with p < 0.05 considered statistically significant.

3.1 Cholecystectomy Does Not Cause Abnormalities in Hepatic Lipid Metabolism Under Normal Dietary Conditions

To investigate the impact of cholecystectomy on hepatic lipid accumulation and inflammation, a PC mouse model was established. The mice were kept on a ND for the entire duration of the study (Figure 1a). There were no significant differences between the two groups in absolute body weight or weight gain compared to the week of surgery at 3 months post-cholecystectomy (Figure 1b–d). Hepatic lipid accumulation was assessed by comparing liver-to-body ratio between the two groups, with no significant differences observed (Figure 1e). H&E staining was performed to analyse hepatic tissue, specifically for hepatic steatosis, ballooning degeneration, and inflammation. The NAFLD activity scores (NAS scores), depicted in Figure 1f,g, revealed no variations among the groups. The levels of hepatic TG and TC were measured to assess hepatic lipid accumulation. Interestingly, there were no significant differences observed at 1, 2, or 3 months post-cholecystectomy (Figure 1h,i). These results suggest that cholecystectomy under normal dietary conditions does not affect hepatic lipid content or induce liver inflammation.

To further confirm the impact of cholecystectomy on hepatic lipid metabolism, including lipid synthesis, degradation, and transport, the expression of relevant genes was measured using real-time quantitative PCR (RT-qPCR). The majority of genes involved in triglyceride synthesis were expressed without significant differences between the groups, except for an increase of Dgat1 in the PC group at 3 months after modelling (Figure 1j). No significant differences in Cpt1 and Pparα expression were observed between the PC and Sham groups under ND conditions, indicating similar liver fatty acid β-oxidation levels (Figure 1k). Moreover, genes related to lipid transportation, except for Apob at 3 months, were similarly expressed between the two groups (Figure 1l). These results suggest that under normal dietary conditions, cholecystectomy has no significant effect on hepatic lipid metabolism or inflammation.

3.2 Cholecystectomy Exacerbates Hepatic Lipid Metabolism Disorders Under a High-Fat Diet

To determine the timing when mice exhibit systemic glucose and lipid metabolic disorders without hepatic lipid deposition, mice were initially fed with either a ND or a HFD, with dynamic monitoring of glucose and lipid metabolism as well as hepatic lipid accumulation. After 1 week on the HFD, mice in this group showed significant increases in blood TG, TC, and fasting blood glucose (FBG) compared to the ND group. Among these, TC levels continued to rise with prolonged high-fat feeding, resulting in a stable and significant difference between the two groups by week 3 (Figure 2a). In contrast, differences in blood TG and FBG between the groups emerged after 1 week on the high-fat diet but receded by week 2; significant differences reappeared by week 3 and remained stable and significant by week 4 (Figure 2b,c). To further clarify the metabolic state at this time, glucose tolerance tests were conducted to assess insulin resistance, revealing significant insulin resistance and impaired glucose tolerance in the HFD group after 4 weeks compared to the ND group (Figure 2d). Concurrently, dynamic monitoring of serum ALT levels and comprehensive liver pathology examination showed no significant difference in ALT levels and no signs of hepatic steatosis or liver damage under microscopy in the HFD group compared to the ND group after 4 weeks (Figure 2e,f), indicating the absence of hepatic lipid deposition and hepatocyte damage at this stage. Based on these findings, we decided to perform cholecystectomy on the HFD group after 4 weeks of high-fat diet feeding, and then to sacrifice the mice and collect blood and liver tissues after 3 months (Figure 2g).

To investigate the impact of cholecystectomy on hepatic lipid metabolism and inflammation under conditions of pre-existing insulin resistance and systemic glucose and lipid metabolic disturbances, the PC mouse model was established after 4 weeks on a HFD, with continuous high-fat diet administration throughout the study. Under HFD conditions, cholecystectomy resulted in a faster rate of weight gain in the PC group compared to the Sham group (Figure 3a). The absolute body weight of the PC group was higher than that of the sham-operated group at 3 months post-surgery (Figure 3b). The body weight gain, and the liver/body ratio of the PC group at 3 months post-surgery was significantly greater than that of the Sham group (Figure 3c,d). Both groups exhibited marked hepatic steatosis and ballooning degeneration; however, the PC group also displayed scattered inflammation within liver lobules. NAS scores were elevated in the PC group compared to the Sham group (Figure 3e,f). Circulating and hepatic TG and TC content were used to evaluate systemic metabolic disturbances and liver lipid accumulation, respectively; and circulating ALT and AST levels were assessed to evaluate liver injury. All of these parameters were significantly higher in the PC group relative to the Sham group (Figure 3g,h), suggesting that cholecystectomy under a HFD contributes to increased hepatic lipid accumulation and inflammation and may also impact systemic metabolic disorders.

The expression of key genes involved in hepatic triglyceride synthesis was significantly elevated in the PC group (Figure 3i). Additionally, the expression levels of Ppara and Cpt1a were measured to evaluate the rate of fatty acid β-oxidation in the liver. Results indicated that Cpt1a expression was significantly reduced in the PC group compared to the Sham group (Figure 3j). Additionally, there were no significant differences in the expression of lipid transport genes between the PC and Sham groups (Figure 3k); while Tnfα, the inflammatory cytokine, was significantly higher in the HFD-fed PC mice (Figure 3l). These results suggested a disbiotic situation for HFD-dieted PC animals in hepatic triglyceride synthesis, metabolism, and liver inflammation.

3.3 Impact of Cholecystectomy on the Gut-Liver-Bile Acid Axis and Transport Systems in Mice

Orthogonal partial least squares discriminant analysis (OPLS-DA) was initially employed to observe the overall structural differences in BAs between the Sham group and the PC group in mouse ileal contents and liver tissues. The results show that bile acid distributions in the ileal contents and liver tissues of the two groups are markedly different (Figure 4a,f) indicating distinct characteristics in the bile acid profiles of the gut and liver between the groups.

Quantitative analysis of bile acid concentrations in the ileal contents revealed a significant increase in total bile acid levels in the HFD-fed PC mice. Additionally, the ratio of secondary to primary BAs was significantly elevated in the PC group, attributable to the increased absolute concentration of secondary BAs, while no significant difference was observed in the absolute concentration of primary BAs between the two groups. Both 12α-hydroxylated (12-OH) and non-12-OH BAs were significantly increased in both groups under the high-fat diet, although the ratio of these two types of BAs did not differ significantly between the groups (Figure 4b). As to the bile acid contents in the liver tissues, no significant difference exists in total bile acid levels between the Sham and PC groups under a high-fat diet. Although the absolute concentrations of primary and secondary BAs were not significantly different between the groups, the ratio of secondary to primary BAs was significantly higher in the PC group compared to the Sham group. Additionally, the ratio of conjugated to unconjugated BAs was markedly elevated in the PC group due to a significant decrease in unconjugated BAs. Furthermore, the concentration of non-12-OH BAs was significantly lower in the PC group, resulting in a significantly higher ratio of 12-OH to non-12-OH BAs compared to the Sham group (Figure 4g).

Subsequently, we categorised the BAs in the distal ileum contents of the two groups of mice according to their types and assessed the changes in the concentrations of each bile acid. The results showed that, under a high-fat diet, compared to the Sham group, the proportions of DCA, lithocholic acid (LCA, and hyodeoxycholic acid) HDCA were significantly increased, while the proportion of chenodeoxycholic acid (CDCA) was significantly decreased in the PC group (Figure 4c). Categorization of BAs in liver tissues showed no significant differences in the proportions of Muricholic Acid (MCA), CA, CDCA, UDCA, and LCA between the PC and Sham groups, except for DCA and HDCA (Figure 4h).

Differential BAs were identified based on their concentrations. Using the VIP values from the first principal component of the OPLS-DA model, we identified differential gut BAs as DCA, LCA, TDCA, muroCA, alloCA, and HDCA, all of which were upregulated in the HFD-dieted PC mice (Figure 4d,e). For liver BAs, TDCA, DCA, CDCA, UDCA, βCA, βMCA and alloCA were revealed as differential liver BAs. Among these, TDCA and DCA were upregulated, while CDCA, UDCA, βCA, βMCA and alloCA were downregulated (Figure 4i,j).

The findings suggest that a high-fat diet following cholecystectomy induces substantial changes in bile acid profiles in both the intestine and liver, potentially affecting receptors and transporters associated with the enterohepatic circulation of BAs. Therefore, an evaluation was conducted on the expression of primary bile acid receptors and transporters involved in the enterohepatic circulation.

Intestinal bile acid transporters, such as the apical sodium-dependent bile acid transporter (ASBT), cooperating with the ileal bile acid binding protein (IBABP) and the organic solute transporter alpha/beta (OST α/β), mediated BAs transportation from the intestinal lumen to the portal vein. We found the mRNA levels of ASBT and OST β are significantly elevated in the PC group, whereas IBABP levels do not show a significant difference (Figure 4k). Hepatic bile acid transporters, including the sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporting polypeptides (OATP), and the bile salt export pump (BSEP), are responsible for transporting BAs from the portal vein into hepatocytes and bile ducts. Data showed that, compared to the Sham group, mRNA levels of NTCP and BSEP are significantly increased in the PC group, whereas OATP levels are significantly reduced (Figure 4l).

Other genes involved in bile acid metabolism were also evaluated, including bile acid receptors, signalling molecules, and synthesising enzymes, such as the intestinal Farnesoid X receptor (iFxr), fibroblast growth factor 15 (Fgf15), fibroblast growth factor receptor 4 (Fgfr4), liver FXR (lFxr), cholesterol 7α-hydroxylase (Cyp7a1), and cholesterol 27α-hydroxylase (Cyp27a1). Analysis of the mRNA expression levels of these genes revealed that, following cholecystectomy under a high-fat diet, the gene expression levels of the intestinal Fxr-Fgf15-Fgfr4 signalling axis were significantly downregulated. Additionally, the gene expression level of the key bile acid synthesis enzyme Cyp27a1 did not show significant variation, whereas the expression of Cyp7a1 was notably upregulated (Figure 4m).

These findings indicate that cholecystectomy under a high-fat diet markedly influences the bile acid profiles in both the intestine and liver, as well as the enterohepatic circulation of BAs. Given the essential role of BAs in energy metabolism and inflammatory responses, these findings suggest that changes in bile acid levels are likely involved in the development of hepatic lipid metabolic disorders and inflammation following cholecystectomy on a high-fat diet.

3.4 Impact of Cholecystectomy on Hepatic Immune Cells in Mice

The flow cytometry was performed to elucidate the underlying mechanisms of hepatic lipid metabolism disorders and exacerbated inflammation following cholecystectomy under high-fat diet conditions. The results demonstrated that, 3 months after cholecystectomy under ND, there was a significant increase in the proportion of B220⁺ B cells in the liver of the PC compared to the Sham group (Figure 5a). No significant differences were observed in the proportions of other immune cell types between the groups, including CD11b⁺F4/80⁺ macrophages (Figure S2a), CD11b⁺Ly6G⁺ neutrophils (Figure S2b), CD11c⁺ dendritic cells (Figure S2c), CD4⁺ helper T cells (Figure S2d), and CD8⁺ cytotoxic T cells (Figure S2e). These findings indicated that, under normal dietary conditions, cholecystectomy results in a notable increase in hepatic B cell proportions 3 months after modelling, while the proportions of other immune cell types do not exhibit significant changes.

In HFD-fed PC mice, there was a significant increase in the proportions of CD11b⁺Ly6G⁺ neutrophils and B220⁺ B cells in the liver (Figure 5b,c). Conversely, CD4⁺ helper T cells were significantly reduced (Figure 5d). No significant changes were observed in the proportions of CD11c⁺ dendritic cells (Figure S2f), CD11b⁺F4/80⁺ macrophages (Figure S2g), and CD8⁺ cytotoxic T cells (Figure S2h) between the PC and Sham groups. These findings indicate that, under high-fat dietary conditions, cholecystectomy results in increased hepatic proportions of B cells and neutrophils, and a decrease in CD4⁺ helper T cells after 3 months.

3.5 Correlation Between Differential BAs and Immune Cell Changes

Based on the aforementioned results, since cholecystectomy exacerbated liver lipid metabolism disorders exclusively under high-fat diet conditions, this suggests that immune cells, specifically neutrophils and CD4⁺ T cells, may be involved in the progression of liver lipid metabolism disorders and inflammation. Therefore, correlations between differential BAs, that is, DCA and TDCA, and the proportion of neutrophils and CD4⁺ T cells in the liver were analysed in both PC and Sham groups. The results indicated that differential BAs exhibited strong correlations with changes in liver neutrophil proportions (Figure 5e,f), rather than CD4⁺ T cells (Figure 5g,h). This suggests that neutrophils likely play a significant role in the exacerbation of liver lipid metabolism disorders and inflammation following bile acid changes post-cholecystectomy.

3.6 Alterations in Neutrophil Markers and Chemokine Levels in Liver Tissue

To further validate changes in neutrophil numbers and functions in liver tissue following cholecystectomy under a high-fat diet, the gene expression levels of neutrophil-associated markers myeloperoxidase (Mpo) and the chemokines CXC motif ligand 1 (Cxcl1) and CXC motif ligand 2 (Cxcl2) were assessed. The results indicated that, under a ND, the mRNA expression levels of Mpo, Cxcl1, and Cxcl2 were similar between the PC and Sham groups. In contrast, under a high-fat diet, the PC group exhibited significantly elevated mRNA expression levels of Mpo and Cxcl1 in liver tissue (Figure 5i–k). These findings revealed that in mice on a high-fat diet, undergoing cholecystectomy resulted in increased expression of markers and chemotactic factors that attract neutrophils to the liver, indicating changes in both the quantity and function of neutrophils.

3.7 1 mM OA: PA (2:1) Solution Simulates Hepatocytes Under High-Fat Conditions

When HepG2 hepatocytes were exposed to mixtures of OA and PA at various concentrations (ranging up to 1000 μM), there was a clear dose-dependent increase in both intracellular TG levels and the size of lipid droplets, as observed through Oil Red O staining (refer to Figure S1a,b). In addition, it should be noted that there was no significant rise in ALT levels in the cell supernatant at concentrations up to 1000 μM. Nonetheless, a notable increase was detected at 1200 μM (Figure S1c). The results indicate that treatment of hepatocytes with an OA: PA (2:1) solution leads to lipid deposition within the cells without causing cellular damage. With adherence to the animal experiments, our aim was to simulate a state of lipid metabolism disorders that does not involve cellular injury and investigate the consequences of bile acid alterations on hepatocytes after cholecystectomy. For this purpose, we chose a specific concentration (1 mM) of an OA: PA (2:1) solution that leads to visible lipid deposition without causing any harm to the cells for the following experiments.

3.8 Enhanced Chemokine Release and Decreased FXR Expression From Liver Cells Following Stimulation With DCA or TDCA

The liver cells were divided into two groups: one treated with OA and PA solutions to simulate a high-fat state, and the other left untreated to represent a normal state. The cells were subjected to various concentrations of DCA and TDCA (0, 50, 100, and 200 μM) for different time periods (0, 24, 48, 72 h). The expression level of key chemokines, CXCL1 and CXCL2, was measured at both the mRNA and the protein levels (Figure 6a).

Results showed that liver cells treated with 1 mM OA and PA solutions exhibit significantly increased mRNA expression levels of the chemokines Cxcl1 and Cxcl2 when exposed to 100 μM DCA. With a further increase in DCA concentration to 200 μM, the expression levels of Cxcl1 and Cxcl2 showed a more pronounced increase. Compared to untreated liver cells exposed to the same concentration of DCA, liver cells treated with OA and PA exhibited a significantly higher level of Cxcl2 expression, while Cxcl1 expression was significantly elevated at DCA concentrations of 100 and 200 μM (Figure 6b). In contrast, for liver cells treated with 1 mM OA and PA, although there was a trend toward increased Cxcl1 expression with rising TDCA concentrations, no significant difference was observed. However, at a TDCA concentration of 200 μM, Cxcl2 expression was significantly increased. Compared to untreated liver cells exposed to the same TDCA concentration, Cxcl1 expression was significantly higher at TDCA concentrations of 50 μM and 100 μM, and both Cxcl1 and Cxcl2 expressions were markedly elevated at 200 μM TDCA (Figure 6c). Collectively, these results suggest that liver cells treated with OA and PA show a notable increase in Cxcl1 and Cxcl2 expression when exposed to 100 μM DCA or 200 μM TDCA.

To assess the concentrations of the chemokines CXCL1 and CXCL2 released into the culture supernatant, liver cells were stimulated with either 100 μM DCA or 200 μM TDCA for different durations. Stimulation with 100 μM DCA led to a time-dependent increase in CXCL1 and CXCL2 concentrations in the supernatant of liver cells treated with OA and PA, with significant differences observed. The supernatant of OA and PA-treated liver cells showed a significant increase in the levels of CXCL1 and CXCL2 after 48 h of DCA stimulation, in contrast to untreated liver cells (Figure 6d). Similarly, stimulation with 200 μM TDCA resulted in a significant increase in CXCL1 and CXCL2 concentrations in the supernatant of OA and PA-treated liver cells after 72 h, with markedly higher levels compared to untreated liver cells (Figure 6e). These results indicate that exposure of liver cells to 100 μM DCA for 48 h or 200 μM TDCA for 72 h significantly enhances the release of CXCL1 and CXCL2.

To further investigate the regulatory effects of DCA and TDCA stimulation on FXR in hepatocytes, we systematically analysed FXR expression under different experimental conditions. The results revealed that exposure to 100 μM DCA for 48 h significantly decreased FXR expression in OA/PA-treated groups compared with unstimulated controls (including normal controls and OA/PA-treated groups without DCA) (p < 0.05). Notably, while DCA stimulation reduced FXR expression in non-OA/PA-treated hepatocytes, this reduction did not reach statistical significance (p > 0.05). Similarly, 200 μM TDCA intervention for 72 h induced a significant FXR expression decrease in OA/PA-treated hepatocytes relative to unstimulated controls (p < 0.05). However, the same TDCA dosage in normal hepatocytes only exhibited a downward trend in FXR expression (p > 0.05). These findings collectively suggest that both DCA and TDCA treatments suppress FXR expression in hepatocytes, and hyperlipidemic conditions may enhance hepatocyte sensitivity to bile acids (Figure 6f,g).

3.9 Bile Acid Enhances Neutrophil Chemotaxis and Disrupts Lipid Metabolism in Liver Cells

In liver cells under high-fat conditions, the stimulation of bile acid has been found to notably enhance the release of CXCL1 and CXCL2. In order to evaluate the impact of chemokines on neutrophils, they were isolated from human peripheral blood and then tested in chemotaxis assays. The results revealed that DCA stimulation not only enhances neutrophil chemotaxis in untreated liver cells, but also significantly increases it in liver cells treated with OA and PA. Neutrophils from untreated liver cells did not show a significant chemotactic response to TDCA stimulation. However, when liver cells were treated with OA and PA, TDCA greatly enhanced their chemotaxis (Figure 7b–d). The findings of this research demonstrated that the introduction of DCA or TDCA to liver cells in a high-fat state leads to a significant amplification of neutrophil chemotaxis.

In order to delve deeper into the effects of bile acid stimulation and neutrophil chemotaxis on liver cells, we conducted co-culture experiments involving both liver cells and neutrophils. The accumulation of lipids in liver cells was evaluated, along with the expression levels of genes related to lipid metabolism. The results showed that when liver cells were treated with OA and PA and then stimulated with DCA or TDCA, they had a higher number of lipid droplets compared to cells that were not stimulated with BAs, indicating a more pronounced lipid accumulation. Some liver cells stimulated with DCA even showed morphological changes (Figure 7e). Furthermore, mRNA expression levels of lipid metabolism genes Fasn, Acaca, and Cpt1a were analysed. While Fasn and Acaca showed no significant differences in high-fat state liver cells with or without bile acid stimulation, Cpt1a expression was significantly reduced in bile acid-stimulated high-fat state liver cells compared to those without stimulation (Figure 7f). The findings indicate that, after cholecystectomy, the varying stimulation of BAs in high-fat state liver cells and the resulting neutrophil chemotaxis may worsen lipid accumulation in liver cells, potentially caused by decreased fatty acid β-oxidation.