IntroductionDuctal reaction (DR) is a common injury repair response in many hepatobiliary diseases and is histologically characterized by reactive bile duct hyperplasia [1]. The dominant feature of DR is hyperplasia of reactive-appearing ductlike cells, including hepatic progenitor cells (HPCs), cholangiocytes and hepatocytes, the proliferation of which contributes to the development of DR [2, 3]. Abnormal DR is a characteristic pathological change in hepatobiliary diseases, such as sclerosing cholangitis and hepatic fibrosis [4, 5]. Without early intervention, abnormal DR may deteriorate and eventually lead to cirrhosis and even hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) [6].Epidemiological studies have identified toxic exposure and poor lifestyle choices as the main causes of chronic hepatobiliary diseases, with environmental toxic exposure being one of the most representative risk factors [7, 8]. Benzo(a)pyrene (B[a]P) is a common environmental toxin classified by the International Agency for Research on Cancer (IARC) as a Group I carcinogen and is formed by the incomplete combustion of organic materials in many industrial and food-cooking processes [9, 10]. After it enters the body, the absorbed B[a]P is mainly distributed in the liver, which contains several enzymes essential for the bioactivation of B[a]P. Therefore, compared with other organs, B[a]P is more toxic to the liver [11, 12]. Our previous research revealed that B[a]P promotes multidrug resistance in HCC [13]. Current research has focused mainly on the toxic effects of B[a]P on hepatocytes; however, the relationship between B[a]P and DR remains inadequately explored.Glucose-regulated protein 75 (GRP75) is a unique member of the HSP70 family located predominantly in the mitochondrial matrix that regulates a variety of cellular processes, including cell survival, growth and metabolism [14]. In addition, GRP75 is thought to bridge mitochondria and the endoplasmic reticulum (ER) through the assembly of the inositol triphosphate receptor (IP3R)-GRP75-voltage-dependent anion channel (VDAC) complex and play a crucial role in regulating the distance and interaction between mitochondria and the ER [15]. GRP75, an important mitochondrial chaperone, is closely associated with the development of various diseases, such as CCA, hepatic lipogenesis and colon cancer [16,17,18]. Our latest study revealed that GRP75 plays a key role in the B[a]P-promoted progression of HCC [19]. However, the effects of GRP75 on B[a]P-induced abnormal DR and the underlying molecular mechanisms remain unclear. This study aimed to explore the molecular mechanisms of GRP75 in B[a]P exposure-induced abnormal DR in cellular and animal models through a variety of molecular biology techniques and to investigate novel potential targeted intervention approaches.Materials and methodsReagents and drugsB[a]P (C20H12, >96.0% purity) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, C14H21NO4, 99.0% purity) were purchased from Sigma-Aldrich China Co (Shanghai, China). Corn oil and luteolin (C15H10O6, >99.5%purity) were purchased from MedChemExpress (Shanghai, China). All other reagents were of analytical grade or the highest grade available.Mice and in vivo treatmentThis study was approved by the Nanjing Medical University Institutional Animal Care and Use Committee (the permit No. IACUC-2209058), and the animals were treated humanely to alleviate suffering. Male C57BL/6 J mice (8 weeks old, 25 ± 2 g) were purchased from the Animal Laboratory Center of Nanjing Medical University. All mice were kept under 12 h:12 h dark/light cycle at a consistent temperature (25 °C) and provided with standard chow with access to sterile water freedom. The NC group was administered 0.1 mL of corn oil for 4 weeks. In the B[a]P treatment group, mice were administered B[a]P via oral gavage at a dose of 12.5 mg/kg (dissolved in corn oil) daily for 4 weeks [20]. In the DDC (served as a positive control) treatment group, mice were fed with containing 0.1% DDC for 4 weeks. For the prevention model, mice were treated with luteolin by oral gavage at a dose of 10 mg/kg, daily for 4 weeks [21]. After treatment, all mice were euthanized simultaneously, and the liver tissue and serum were subjected to further investigation.Cells and cell cultureFor the isolation of primary bile duct cells (BECs), the entire procedure was conducted on a sterile operating table. C57BL/6 J mice were euthanized under anesthesia with pentobarbital sodium. An abdominal midline incision was made to expose the liver, which was then perfused with 1× HEPES-buffered saline containing 0.02% (wt/vol) egtazic acid (Sigma-Aldrich) until pale, indicating effective blood clearance. Perfusion continued using 0.02% (wt/vol) collagenase (Sigma-Aldrich). Subsequently, the liver was removed and placed in a sterile 10 cm dish containing ice-cold DMEM/F12 medium (Gibco, Grand Island, NY). After removing the liver peritoneum, it was placed in collagenase IV (MedChemExpress, Shanghai, China) digestive solution and digested at 37 °C on a shaker for 10 min. The liver parenchymal cells were brushed away to extract the biliary tree. The bile duct was cut into pieces and digested with prewarmed 0.5 g/L collagenase I and 4.2 g/L dispase (Gibco) solution in DMEM/F12 medium at 37 °C for 15 min. The suspension was filtered through a 100 μm cell strainer (Corning, NY, USA), and the residual tissue fragments were re-digested. The collected cell suspension was washed by centrifugation, and the primary BECs were aggregated as precipitates [22]. Primary BECs precipitate was resuspended in DMEM/F12 medium to a cell density of 1 × 105/mL with careful pipetting. The cells were inoculated into 25 mL plastic culture flasks coated with type I rat tail collagen (Sigma-Aldrich) and cultured in a 37 °C, 5% CO2 incubator [23].The cholangiocarcinoma cell line (SG231) was obtained from and short tandem repeat identified by Shanghai Honsun Biological Co. Ltd (Shanghai, China); while the human hepatic stellate cell (HSC) line (LX-2) was obtained from and short tandem repeat identified by KeyGen Co. Ltd (Nanjing, China). SG231 cells were cultured in RPMI-1640 medium (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Beyotime Co. Ltd., Shanghai, China), and 100 μg/mL streptomycin (Beyotime). While LX2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were routinely cultured at 37°C in a humidified incubator containing 5% CO2.Molecular biology experimentsThe experimental procedure for cell transfection, DNA methylation analysis, Fluorescence mitochondrial calcium and ER calcium imaging, transmission electron microscopy (TEM), reactive oxygen species (ROS) staining and quantification, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining (IF), histological analyses, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and data mining and bioinformatics analysis please see the detailed information listed item by item in the supplementary materials and methods section.Drug screening and functional analysisThe Coremine (https://coremine.com/), high-throughput experiment and reference-based database (HERB, http://herb.ac.cn/) and symptom mapping (SymMap, http://www.symmap.org/) databases were used to identify traditional Chinese medicines (TCMs) with therapeutic effects on cholangitis; the traditional Chinese medicine integrated pharmacology (TCMIP, http://www.tcmip.cn/TCMIP/index.php/) database was used to reveal potentially effective key monomeric chemicals in TCMs; and the PyMOL software was used to simulate molecular docking of chemicals with the GRP75 protein. For the combination of recombinant GRP75 protein and luteolin, as we described previously [24], the protein-ligand interactions were measured by biolayer interferometry (BLI) assay.Statistical analysisStatistical analysis was performed by GraphPad Prism (version 9.0.0 for Windows; San Diego, California, USA; www.graphpad.com). The statistical significance was determined using a two-tailed Student’s t-test, or a one-way analysis of variance (ANOVA) followed by Tukey’s t-test. The data were presented as mean ± standard deviation (SD). A p-value