Male C57BL6 mice (8 weeks old, 18-20 g, n=50) were purchased from the Central Laboratory of Kunming medical university. Mice were fed on normal rodent feedstuff (cholesterol <0.02%) or a lithogenic diet (1.25% cholesterol plus 0.5% cholic acid and 15.8% buffer) for 4 weeks and divided into five groups (10 mice/group): i) ND (mice fed with normal diet), ii) ND + nicotine (H) (mice fed with normal diet and treated with 6.6 mg/kg/2 days nicotine), ii) LD (mice fed with lithogenic diet), iii) LD + nicotine (L) (mice fed with lithogenic diet and treated with 1.1 mg/kg/2 days nicotine), and v) LD + nicotine (H) (mice fed with lithogenic diet and treated with 6.6 mg/kg/2 days nicotine). Mice of all groups (except group ND) were treated with nicotine (1.1 mg and 6.6 mg) for 10 weeks after being weighed every two days (20-23). All mice in groups received free access to water and were kept under the controlled condition at room temperature (22±3˚C), with a relative humidity of 60-70% and a 12-h light/dark cycle. The mice were anesthetized with 4% chloral hydrate (300 mg/kg) by intraperitoneal injection. Mice were sacrificed with 150 mg/kg pentobarbital sodium by intraperitoneal (i.p.) injection. The animal experiments were approved by the Institutional Animal Care and Use Committee of Kunming medical university (approval no. kmmu2021058).

At week 10 of the diet feeding, non-fasted animals were weighed and anesthetized with an i.p. injection of 35 mg/kg pentobarbital. After cholecystectomy, gallbladder volume was measured by weighing the whole gallbladder and equating gallbladder weight (including stones) with gallbladder volume. Gallbladders were then opened and 5 µl of fresh gallbladder bile was examined for solid and liquid crystals and gallstones. The pooled gallbladder biles were centrifuged at 100,000 x g for 30 min at 37˚C and filtered through a preheated (37˚C) Swinnex-GS filter (0.22 µm) assembly (MilliporeSigma), samples were frozen and stored at -20˚C for further lipid analyses. Mouse blood was collected from the vena cava and separated serum samples were stored in a -80˚C biofreezer. The liver and gallbladder were isolated and frozen in liquid N₂ until required for analysis.

The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, HDL-cholesterol, phospholipids and triglycerides were determined using an automated Hitachi Clinical Analyzer (model 7020; Hitachi Ltd.).

Levels of bile cholesterol, phospholipid, bile salts as well as cholesterol and triglyceride in bile were determined respectively following the manufacturer's instructions. Bile from 10 mins' drainage was sampled. Bile cholesterol and phospholipid concentrations were determined by Cholesterol E-Test Wako kit (cat. no. 999-02601; FUJIFILM Wako Pure Chemical Corporation) and Phospholipid C-Test Wako kit (cat. no. 433-36201; FUJIFILM Wako Pure Chemical Corporation), respectively. The bile salts concentrations were measured by a Nexera X2 Ultra High-Performance Liquid Chromatography system (Shimadzu, Kyoto, Japan) according to the method of Paulusma et al (24). Briefly, 5 µl of diluted bile (1:100) were spiked with 20 µl of internal standard solution in methanol:water (1:1 v/v). Protein precipitation was performed with 30 µl of methanol, followed by 10,000 x g centrifugation for 10 min at 4˚C. The supernatant was used for LC-MS/MS analysis.

Paraffin-embedded gallbladder and liver sections (5 µm in thickness) were dewaxed twice in xylene solutions for 10 min each and rehydrated in descending alcohol series. Then paraffin sections were stained with hematoxylin for 5 min at room temperature and differentiated with 0.1% hydrochloric acid ethanol for 1 min, followed with eosin for 5 min at room temperature, dehydrated in ascending alcohol series, followed by being cleared twice in xylene solutions for 10 min each. They were observed at x100 magnification under a light microscope.

To confirm the expression of megalin and FXR, 10% formalin (48 h at 20-22˚C)-fixed tissues were embedded in paraffin. The 5 µm paraffin sections were subjected to anti-FXR (1:500, Novus Biologicals) or anti-megalin antibody (1:200, Abcam) overnight at 4˚C and stained with a DAB (3,3'Diaminobenzidine) kit (MXB Biotechnologies) for 15 min at 20-22˚C. The positive staining was taken photograph under a light microscope (magnification, x40).

All samples were blindly inspected by two independent pathologists. Positive immunostaining was visualized as brown granules contained in the cytoplasm. The immunostaining was scored by evaluating the intensity and percentage of positively stained cells. The intensity of FXR or megalin staining was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong. The percentage scores were assigned, as follows: 1, ≤25; 2, 26-50; 3, 51-75; and 4, >75%. These scores were multiplied to arrive at a final score ranging between 0 and 12.

The human gallbladder epithelial cell line GBEC were purchased from iCell Bioscience Inc. and were cultured in low-glucose DMEM supplemented with 2 mM glutamine, 1% MEM non-essential amino acids solution, 1% MEM vitamin solution, 7.5% FBS and P/S (all from Gibco; Thermo Fisher Scientific, Inc.). The cells were seeded at ~70-80% confluence in complete media for 24 h and maintained at 37˚C in 5% CO₂. For CDCA and nicotine treatments, GBECs were plated at 3x10⁴ cells/cm² for 48 h and subsequently cultured overnight at 37˚C in the low-serum medium. 10 µM CDCA was added medium for 6 h, then 2 µM nicotine was added. Cells were cultured at 37˚C in 5% CO₂ for another 18 h for reverse transcription-quantitative (RT-q) PCR and 48 h for western blot analysis.

Total RNA was isolated from 1x10⁵ cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions and the concentration of RNA was calculated by spectrophotometry. cDNA was prepared using the PrimeScript RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). Reverse transcription PCR was performed u sing SYBR Green Premix Ex Taq (Takara) according to the manufacturer's protocols Sequences for the primers used are in Table I. Primer sequences were synthesized by TsingKe Biological Technology.

For reverse transcription PCR, the PCR mixture was denatured at 95˚C for 10 sec, annealed at 60˚C for 20 sec and then extended at 72˚C for 30 sec. This process was repeated for a total of 40 cycles. The relation of NPC1 like intracellular cholesterol transporter 1 (NPC1L1) and sterol regulatory element-binding protein 2 (SREBP-2) mRNA expression with β-actin was calculated based on the threshold cycle (Ct) values. The relative mRNA expression of β-actin was calculated by the inverse log of ΔΔCq (25). All experiments were performed in triplicate.

Protein were lysed using RIPA lysis buffer (Beyotime Biotechnology, Inc.). Protein concentration was measured using the bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology, Inc.). Protein samples (20 µg) were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (MilliporeSigma). Non-specific binding to the membrane was blocked for 1 h at room temperature with 5% fat-free milk in TBST (Tris-buffered saline with 0.1% Tween 20 detergent) and then the membranes were incubated with 1:2,000 FXR primary antibody (cat. no. NB300-259; Novus Biologicals, LLC), 1:1,000 megalin primary antibody (cat. no. ab56014; Abcam) and 1:1,000 cubilin primary antibody (ab251050, Abcam) respectively at 4˚C overnight. Then, the membrane was washed four times with TBST and incubated with a 1:5,000 dilution of the HRP conjugated secondary antibody (cat. no. ab205718; Abcam) at room temperature for 45 mins. After the membrane was washed twice with TBST, membrane-bound antibody was visualized using an enhanced chemiluminescent kit (MilliporeSigma) according to manufacturer's instructions. The densities were quantified via photodensitometric scanning using Quantity One-4.2.3 software (Bio-Rad Laboratories, Inc.).

Values are given as the mean ± SD. Statistical differences between multiple groups were compared using Kruskal-Wallis analysis. When statistical significance was identified based on the nonparametric test, Dunn's test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The liver weight-to-body weight ratio was not significantly different among the different treatment groups. H&E staining of the liver in Fig. 1 did not reveal obvious histomorphological differences among different groups, except the high-dose nicotine treatment which caused liver injury (Fig. 1A).

Serum ALT and AST levels did not change after low-dose nicotine treatment, but increased in the high-dose nicotine treatment group (Fig. 1B and C). In addition, no significant differences were observed in the levels of inflammatory factors following nicotine treatment (Fig. 1D-F). This suggested that low-dose nicotine treatment showed no significant toxic side effects on liver function after 10 weeks.

Mice fed with a normal diet treated with high-dose nicotine showed no abnormal behavior and no gallbladder stone formation. Crystals were observed in the lithogenic diet groups. Gross analysis of the gallbladder showed that LD + nicotine groups did not exhibit significant lower stone formation compared to the LD group (Fig. 2A). The gallbladder volume of mice in the LD + nicotine (H) group was lower than that in the LD group, while low concentrations of nicotine did not change gallbladder volume (Fig. 2B; Table II).

Compared with the LD group, the cholesterol level in the gallbladder bile from the LD + nicotine (H) group was significantly reduced, (Fig. 3A). The cholesterol level did not significantly decrease in LD + nicotine (L) group compared with the LD group. Compared with the LD group, there were no significant differences in the phospholipids and bile salts in the bile of the experimental groups (Fig. 3B and C).

Serum lipid levels between different groups were also compared, including triglycerides (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) and total bile acid (TBA). The results showed that compared with the ND group, the serum levels of TG, TC and LDL-C in the LD group significantly increased, while HDL-C and TBA significantly decreased (Fig. 3D-H). After low-concentration nicotine treatment, the levels of these indicators are unchanged. Compared with the LD group, the serum TG, TC and LDL-C of the mice treated with the high-dose nicotine group decreased significantly, while the levels of HDL-C and TBA increased (Fig. 3D-H).

The present study examined major genes involved in bile acid metabolism in liver tissue from mice with or without nicotine treatment. As shown in Fig. 4, compared to the ND group, FXR, cholesterol 7α-hydroxylase (CYP7A1) and oxysterol 7α-hydroxylase (CYP7B1) and Na⁺-taurocholate cotransporting polypeptide (NTCP) mRNA levels increased, while CYP7A1 and sterol regulatory element-binding protein 2 (SREBP-2) decreased in the LD group. These effects were reversed by high-dose nicotine treatment (Fig. 4). Notably, no changes in some genes expression were observed following nicotine treatment, including 3-hydroxy-3-methylglutaryl-CoA reductase, sterol 12-α-hydroxylase (CYP8B1), organic solute transporter α (OSTα), bile salt export pump (BSEP), organic anion transporting polypeptide 1 (OATP1), multidrug resistance-associated protein (MRP)2, MRP3, MRP4. This suggests that nicotine may induce changes in cholesterol in other ways in addition to partially affecting bile acids.

The present study investigated the cholesterol metabolism in the gallbladder epithelial cells. It detected the mRNA levels of NPC1L1, ABCG5/G8, SR-BI and megalin/cubilin expression by RT-qPCR. The results showed that compared with the ND group, the expression level of NPC1L1 in the LD group significantly decreased, while megalin, ABCG5/G8 and SR-BI increased. The administration of low and high concentrations of nicotine could attenuate their expression levels (Fig. 5A).

Generally, the FXR-megalin/cubilin signaling pathway regulates cholesterol balance and participates in the formation of stones (17). Therefore, the present study also detected the levels of FXR-megalin/cubilin expression by western blotting and immunohistochemistry. Similar to mRNA expression, nicotine did not alter cubilin protein levels, but restored LD-downregulated FXR and megalin levels (Fig. 5B and C). These results suggest that nicotine can affect the formation process of gallstones by regulating cholesterol metabolism in the gallbladder.

As shown in Fig. 6A, the FXR agonists CDCA increased the expression of megalin in GBEC. CDCA clearly increased the expression of FXR and megalin in GBEC, while not changing the expression of cubilin. The nicotine inhibited CDCA-induced expression of FXR and megalin in GBEC. Immunofluorescence analyses of megalin expression in GBEC showed that megalin is expressed in perinuclear and vesicular, decreased by nicotine (Fig. 6B). It suggested that nicotine could regulate FXR and megalin, which might be involved in the regulation of the expression of the genes related to bile acid metabolism.