Animals and Treatment

All animal experiments were conducted according to the ARRIVE guidelines and approved by the Ethics Committee of Animal Experiments of Guangzhou Jennio Biotech Co., Ltd (Approval No. JENNIO-IACUC-2023-A056). 24 Male C57BL/6 J mice (6 weeks old, 18–22 g) were obtained from Laboratory Animal Center of Southern Medical University and kept in temperature-controlled room (20–24 °C), with a light–dark cycle of 12:12 h and free access to water and food. They were randomly divided into four groups (n = 6): (1) Control group: mice were fed a regular diet (Xietong Biotech Co., Ltd, Jiangsu, China) for 6 weeks; (2) LD group: mice was fed a diet (Xietong Biotech Co., Ltd) consisting of 15% fat, 1.25% cholesterol, and 0.5% cholic acid for 6 weeks; (3) LD + NXT629 group: after finishing the LD feeding, mice were received daily intraperitoneal injection of 30 mg/kg of NXT629 (Abmole, Shanghai, China) for 4 weeks; (4) LD + NXT629 + Ad-glycerol-3-phosphate acyltransferase mitochondrial (GPAM) group: Adenovirus-mediated GPAM overexpression (6 × 108 PFU/mouse) was injected through the tail vein. After 10 weeks, the body weight of each mouse was measured. The gallbladder and liver were dissected and photographed to record the stones.

Hematoxylin and Eosin (H&E) Staining

Hepatic specimens were fixed with 4% paraformaldehyde and paraffin-embedded. Paraffin blocks were divided into 5 μm-thick slices, followed by dewaxing and hydrating. Slices were stained with hematoxylin and eosin (H&E) solution (Sigma Aldrich, St. Louis, MO, the USA) for 10 min. Five different fields of the sections were observed to determine pathological changes in the liver.

Oil Red O staining

Hepatic specimens were taken for frozen Sects. (6 μm) and then stained with Oil Red O solution (Solarbio Science & Technology Co., Ltd, Beijing, China) according to the manufacture’s instructions, and the percentage of steatotic hepatocytes was assessed in terms of relative lipid content and quantified by ImageJ.

Analysis of Mouse Gallbladder Bile and Serum

The levels of total cholesterol (TC), triglycerides (TG) in mouse bile and in serum were determined using kits purchased from Jiancheng Biotech Co., Ltd (Nanjing, China). The phospholipids (PL) and total bile acid (TBA) levels in bile were measured using kits obtained from Abcam. The levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in mouse serum were detected with kits from Jiancheng Biotech Co., Ltd based on the manufacturer’s instructions.

Cholesterol Saturation Index (CSI) Calculation

Calculation of gallbladder bile CSI was performed on the basis of the Carey table [20]. That is, actual molar percentage of TC in bile/highest concentration of soluble TC for a given molar concentration of bile.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

Total RNA from hepatic specimens was extracted using TRIzol (Invitrogen, CA, USA). The RNA was converted into complementary DNA using the Reverse Transcription Kit (Takara, Dalian, China), Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was executed using the SYBR Select Master Mix (Yeasen, Shanghai, China). All reactions were performed using ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, the USA). Fold changes were calculated using the cycle threshold (2−ΔΔCT) method. The primers used in this research were listed in Table 1.

Table 1 The sequences of primers and oligonucleotides used in this studyWestern Blotting

Total protein from hepatic specimens were extracted using radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). And total protein concentration was quantified with BCA Protein Assay Kit (Beyotime). Separation of protein samples was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transferring onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibody against GPAM (1:500, Kanglang Biotechnology Co., Ltd, Shanghai, China) and GAPDH (1:3000; Affinity Biosciences, Cincinnati, OH, USA) at 4 °C overnight. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:5000; Affinity Biosciences), the bands were detected with enhanced chemiluminescence detection reagent (Beyotime) and analyzed using ImageJ software (NIH, Bethesda, MD, USA).

Statistical Analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences software version 21.0 (SPSS Inc., Chicago, IL, the USA). Data were expressed as mean ± standard deviation (SD). Differences between two groups were determined by Student’s t-test, whereas differences among multiple groups were analyzed using one-way or two-way analysis of variance. Differences were considered significant at P < 0.05.