Potential targets of Sch B were predicted using the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/), followed by standardization via the UniProt database (https://www.uniprot.org/) to obtain relevant target information. Disease-related targets for CCA were retrieved from the GeneCards (https://www.genecards.org/) and DisGeNET databases (https://www.disgenet.org/) using the keyword ‘Cholangiocarcinoma’. After merging datasets, non-human genes and duplicate entries were removed to generate a standardized target list. An EVenn online tool (http://www.ehbio.com/test/venn) was employed to construct a Venn diagram illustrating overlapping targets between Sch B and CCA.

The shared targets were imported into the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network under the ‘Homo sapiens’ setting. Disconnected nodes were hidden, and a high confidence interaction score of ≥0.900 was applied. The resulting network was exported in Tab-Separated Values format and visualized using Cytoscape 3.8.0 (https://cytoscape.org/). Core targets are identified based on three topological parameters: Degree centrality (DC), betweenness centrality (BC) and closeness centrality (CC).

The drug-disease intersection targets were uploaded to the Metascape platform (http://metascape.org/) for gene ontology (GO) (https://geneontology.org/) enrichment analysis, including biological process (BP), cellular component and molecular function (MF) categories. Analysis parameters included a minimum overlap of 3, a P-value cut-off of 0.01 and an enrichment of ≥1.5. The top 10 enriched terms from each GO category were selected based on ascending log P-values. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using the ClueGO plugin in Cytoscape, with thresholds set to include pathways containing ≥16 genes and a κ score >0.6. A total of 11 key pathways were identified as significantly enriched.

Molecular docking was performed with the selected core targets. The structure of Sch B in .mol2 format was obtained from the TCMSP database (http://tcmsp-e.com/) and prepared using AutoDock Tools-1.5.6 by removing water molecules, adding hydrogen atoms and assigning atom types. Rotatable bonds were defined, and the file was saved in .pdbqt format, which can be opened and used for molecular docking with AutoDock Vina (version 1.1.2; The Scripps Research Institute).

Crystal structures of MAPK1, EGFR, ESR1, HSP90AA1, AKT1, GRB2, SRC, and HRAS in PDB format were retrieved from the PDB database (https://www.wwpdb.org/). Using PyMOL 3.7.1, non-essential small molecules were removed from the protein structures. The proteins were then processed in AutoDock Tools-1.5.6 to remove water molecules, add hydrogens and assign atom types, and these were saved as .pdbqt files.

The prepared Sch B ligand and the eight core protein receptors were subjected to molecular docking using AutoDock Vina with a batch processing script. The binding site and grid parameters were appropriately defined for each target. The resulting binding modes were visualized and analyzed using PyMOL 3.7.1.

Human CCA HuccT1 cell line was sourced from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin (HyClone™; Cytiva) and maintained in a CO₂ incubator at 37°C. Upon reaching 80–90% confluency as observed under a microscope, cells were digested with 1 ml of trypsin containing 0.25% EDTA at 37°C for 1–3 min. Digestion was monitored microscopically and terminated immediately when cells became rounded and detached from the culture surface.

Sch B was purchased from Merck KGaA and dissolved in DMSO (Merk KGaA) to create a 100 mmol/l stock solution. This stock solution was further diluted with culture media to obtain the desired concentrations. The control groups received treatment with equivalent volumes of DMSO. N-acetyl-L-cysteine (NAC) was acquired from BD Biosciences, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Merck KGaA and Bay 11–7082 was purchased from Merck KGaA. Detailed information for all primary and secondary antibodies used in this study, including antibody names, catalog numbers, dilutions and suppliers, is provided in Table I.

Total RNA was extracted from treated HuccT1 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 1 µg of RNA from each sample was used for complementary DNA synthesis using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio, Inc.), according to the manufacturer's protocol. For RT-qPCR, TB Green^® Premix Ex Taq™ (Takara Bio, Inc.) was used on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 45 cycles of denaturation at 95°C for 5 sec and combined annealing/extension at 58°C for 30 sec. All reactions were performed in triplicate. The relative expression levels of target genes were normalized to GAPDH and calculated using the 2^−ΔΔCq method (23). The primer sequences are listed in Table II.

HuccT1 cells were treated with various concentrations of Sch B (0, 40, 80 and 160 µM) for 48 h at 37°C. After treatment, cells were harvested and lysed on ice using RIPA buffer (Beyotime Biotechnology) supplemented with a protease inhibitor cocktail (Roche Applied Science) for 15 min. The lysates were centrifuged at 13,500 × g for 30 min at 4°C to collect the supernatant. Protein concentration was determined using the BCA assay. Subsequently, 60 µg of total protein per sample was separated by SDS-PAGE on 10% polyacrylamide gels and electrophoretically transferred onto PVDF membranes (MilliporeSigma). Following transfer, the membranes were blocked with 5% skim milk prepared in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature. The membranes were then incubated overnight at 4°C with the indicated primary antibodies (Table I). After incubation, the membranes were washed three times for 10 min each with TBST. A total of two distinct detection systems were employed: Horseradish Peroxidase (HRP)-chemiluminescence detection: For HRP-based detection, the membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST, protein bands were visualized using an enhanced chemiluminescence substrate and imaged with a Gel Doc 2000 system (Bio-Rad Laboratories, Inc.). Alkaline Phosphatase (AP)-colorimetric detection: For AP-based colorimetric detection, an AP western detection kit (Vigorous Biotechnology Beijing Co., Ltd.) was used. After primary antibody incubation and washing, the membranes were probed with AP-conjugated secondary antibodies for 30 to 120 min at room temperature. The membranes were then thoroughly washed 3–5 times with TBST to remove any phosphate residues. A color development solution was freshly prepared immediately before use by adding 0.1 ml of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 0.1 ml of nitroblue tetrazolium (NBT) per 10 ml of reaction buffer (Vigorous Biotechnology, Beijing Co., Ltd.). After removing the wash buffer, sufficient development solution was added to completely cover the membrane, followed by incubation at room temperature for 5 to 15 min in the dark, with close monitoring of band intensity. The reaction was stopped by rinsing the membrane with distilled water once the target bands were clearly visible with minimal background. The membrane was air-dried and imaged directly.

HuccT1 cells were prepared as single cell suspensions and then seeded in 6-well plates at a density of 1×10⁶ cells per well in 100 µl of culture medium. Subsequently, the cells were treated with various concentrations of Sch B (0, 40 and 160 µM) for 24 h at 37°C in a CO₂ incubator. DCFH-DA was diluted with serum-free medium at a ratio of 1:1,000 to reach a final concentration of 10 µmol/l. The CCA cells were incubated with DCFH-DA at 37°C for 20 min. Following three washes with PBS, the cells were stimulated using a positive control for ROS. Finally, the excitation and emission wavelengths for the fluorescence enzyme were adjusted to 488 and 525 nm, respectively.

An LDH release assay was performed according to the manufacturer's protocol (Beyotime Biotechnology). The amount of color formed is proportional to the number of lysed cells. Cells treated with DMSO or 160 µmol/l Sch B + different concentrations of NAC (0, 0.5, 1, 3, 6 µmol/l) were seeded in a 96-well plate. After 24 h, LDH levels were determined by analyzing the amount of LDH released into the cell culture supernatant. Absorbance signals at 490 nm were obtained using a microplate reader. To determine the percentage of LDH release, the experimental LDH release quantities were calculated relative to the control LDH release quantities, as stated in the provided instructions.

HuccT1 cells were treated with Sch B (0 and 160 µM) in the presence or absence of various concentrations of NAC (0, 0.5, 1, 3 and 6 µM) for 24 h at 37°C. After treatment, cells were incubated with 2 µM calcein-AM (CAS 148504-34-1; MedChemExpress) for 20 min at 37°C, followed by a 5 min co-incubation with 4 µM propidium iodide (PI; CAS 25535-16-4; MedChemExpress) at 37°C. Fluorescence images were captured using a Cytation 3 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc.) with excitation at 488 nm and emission recorded at 525 nm for calcein-AM.

HuccT1 cells were seeded in 24-well plates and cultured for 24 h at 37°C in a 5% CO₂ atmosphere prior to transfection. Cells were co-transfected with 100 ng of reporter plasmid (pGL4.2–3×AP-1-Luc or pGL4.2-NF-κB-Luc, both from Promega Corporation) and 1 ng of internal control plasmid (pRL-TK, Promega Corporation), corresponding to a mass ratio of 100:1 (reporter:internal control), using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Transfection was performed at 37°C, and the medium was replaced with fresh culture medium 4 h post-transfection. At 24 h post-transfection, cells were treated with various concentrations of Sch B (0, 10, 20, 40, 80 and 160 µM) for an additional 24 h at 37°C. After treatment, the culture medium was removed, and cells were lysed with Reporter Gene Cell Lysis Buffer (Beyotime Biotechnology). A 20 µl aliquot of cell lysate was used to measure firefly and Renilla luciferase activities sequentially using the Dual Luciferase Reporter Gene Assay Kit II (cat. no. RG029; Beyotime Biotechnology) on a luminometer. Relative luciferase activity was calculated as the ratio of firefly luminescence to Renilla luminescence.

After cell counting, the HuccT1 cells were diluted to 1×10⁵ cells/ml, then 1×10⁴ cells/ml was inoculated on a 6-well plate (100 µl/well). After inoculation, complete medium was added (2 ml/well). After 1 week of culture at 37°C in 5% CO₂, the medium was removed and cells were rinsed twice with PBS or normal saline for 10 sec each, fixed with 4% paraformaldehyde for 15 min at room temperature, and washed twice with PBS twice for 10 sec each. Subsequently, 200 µl of crystal violet staining solution was added to cover the bottom of each well and incubation for 20 min at room temperature. The 6-well plate was then rinsed under running water for 10 sec, air-dried, and the number of colonies (containing >50 cells) was counted manually.

HuccT1 cells were cultured on circular coverslips in 6-well plates, fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with 0.3% Triton X-100 (Merck KGaA) for 15 min at room temperature. Following permeabilization, cells were incubated in a solution containing 5% bovine serum albumin (Merck KGaA) for 40–60 min at room temperature. After washing three times with PBS, the cells were incubated overnight at 4°C with the corresponding primary antibodies (dilution 1:100; Table I). The next day, cells were washed three times with PBS and incubated with fluorescence-labeled secondary antibodies (dilution 1:200; Table I) for 1 h at room temperature in the dark. Subsequently, cells were stained with Hoechst 33342 (cat. no. HY-D0983; MedChemExpress) for 15 min at room temperature in the dark. The coverslips were then mounted onto glass slides, and fluorescence images were captured using a Nikon fluorescence microscope (Nikon Corporation).

The data are presented as mean ± SD from at least three independent experiments. Statistical analyses were performed using SPSS 23.0 (IBM Corp.). All datasets were confirmed to follow a normal distribution by the Shapiro-Wilk test. For comparisons among multiple experimental groups with a single control group, homogeneity of variances was assessed using Brown-Forsythe test. When the assumption of homogeneity of variances was met, ANOVA followed by Dunnett's post hoc test was applied to compare each experimental group with the control group. In cases where variances were heterogeneous, Welch's ANOVA followed by Dunnett T3 post hoc test was employed. The specific statistical test used for each dataset is indicated in the corresponding figure legend. A value of *P<0.05, **P<0.01 and ***P<0.001 was considered to indicate a statistically significant difference.

Potential targets of Sch B and CCA were retrieved from relevant databases. A Venn diagram revealed 120 overlapping targets between Sch B and CCA (Fig. 1A). A PPI network was constructed, comprising 68 nodes and 158 edges, with node size proportional to the degree value (Fig. 1B). Using a DC threshold >6, a subnetwork with 20 nodes and 60 edges was obtained. Further applying criteria of DC >10, BC >147 and CC >0.35, a core network of 8 nodes and 21 edges was identified. A total of eight core targets were ultimately selected: MAPK1, EGFR, ESR1, HSP90AA1, AKT1, GRB2, SRC and HRAS (Fig. 1C).

GO enrichment analysis of the 120 overlapping targets was performed, covering BP, cellular component and MF categories. The top 10 enriched terms from each category are displayed in a bubble chart (Fig. 1D), suggesting that Sch B may regulate the migration and motility of CCA cells.

KEGG pathway analysis was conducted using the ClueGO plugin in Cytoscape 3.8.0, with thresholds set to include pathways containing ≥16 genes and a κ score >0.6. A total of 11 significantly enriched pathways were identified, primarily associated with ‘Pathways in cancer’ and the ‘MAPK signaling pathway’ (Fig. 2A). Molecular docking was performed between Sch B and the top 8 core target proteins (MAPK1, EGFR, ESR1, HSP90AA1, AKT1, GRB2, SRC and HRAS) identified from the PPI network. The lowest binding energies for all ligand-receptor complexes were below-5 kcal/mol, indicating spontaneous binding and stable conformations (Table III). Among them, MAPK1 exhibited the strongest binding affinity with Sch B. The binding modes of Sch B with MAPK1, HRAS, EGFR and HSP90AA1 were visualized using PyMOL 3.7.1 (Fig. 2B and C).

Based on the KEGG pathway enrichment analysis, which indicated strong associations between the predicted MAPK signaling pathway and key BPs such as chemical carcinogenesis, and supported by established literature evidence highlighting the pivotal role of oxidative stress as a key mediator in these pathways, the present study was guided to investigate the involvement of ROS in the mechanism of Sch B. Guided by these predictions, the biological effects were subsequently validated through a series of in vitro experiments. CCA cells were exposed to various concentrations of Sch B, ranging from 0 to 160 µmol/l, to assess the mRNA expression levels of BIP, CHOP and XBP1s. The results showed an increasing trend, indicative of a dose-dependent effect (Fig. 3A-C). Western blot analysis further confirmed the elevated expression levels of BIP, CHOP and XBP1s, showing an upward trend (Fig. 3D).

To support these findings, a ROS probe, DCFH-DA and flow cytometry were used to measure ROS levels in CCA cells. The results demonstrated a dose-dependent increase in ROS levels (Fig. 3E and F). Collectively, these results suggest that Sch B induces a dose-dependent increase in ROS levels within CCA cells, offering valuable insights into its molecular impact on cellular responses.

Treatment with 160 µmol/l Sch B led to a notable increase in the expression levels of BIP, CHOP and XBP1s in CCA cells. The addition of NAC showed a dose-dependent reduction in the expression levels of BIP, CHOP and XBP1s in the experimental group (Fig. 4A and B).

It is well known that NAC, as an antioxidant, can reduce ROS levels and protect cell membrane integrity (24,25), thereby lowering LDH release from cells. With increasing concentrations of NAC, the activity of LDH in the culture medium decreases (Fig. 4C), A reduction in Bax expression was observed only at the highest concentration of NAC (6 µM). Western blotting results in Fig. 4E are representative images and were not subjected to densitometric quantification. In parallel, Calcein-AM/PI staining showed that Sch B-induced cytotoxicity was alleviated by NAC co-treatment, as evidenced by increased green fluorescence intensity (Fig. 4D). Consequently, these results show that NAC may counter the inhibitory effects of Sch B on CCA cells proliferation and facilitate apoptosis by mitigating ROS upregulation.

CCA cells treated with different concentrations of Sch B ranging from 0 to 160 µmol/l showed a dose-dependent decrease in AP-1 expression, a key regulatory component of the MAPK signaling pathway, as measured by a double luciferase reporter assay (Fig. 5A). Additionally, western blot analysis indicated a decreasing trend in p-p38 expression with higher Sch B concentrations, further demonstrating inhibition of the MAPK signaling pathway (Fig. 5C).

Treatment with 160 µmol/l Sch B led to significant inhibition of CCA cell activity (Fig. 5B). The subsequent addition of SB203580, a p38 MAPK inhibitor, at 0.2 and 0.5 µmol/l concentrations resulted in a notable decrease in CCA cell activity compared with the control group (Fig. 5B), reflecting the effects seen with Sch B.

Furthermore, the anti-proliferative effect of Sch B on CCA cells was evidenced by a trend of reduced colony formation at 160 µM, an effect qualitatively similar to that observed with 0.5 µM SB203580 (representative images shown in Fig. 5D). These findings together highlight the effectiveness of Sch B in impeding the MAPK signaling pathway and reducing CCA cell proliferation.

Sch B exhibited dose-dependent suppression of the NF-κB signaling pathway in CCA cell lines. Treatment with Sch B (0,40, 80, 160 µmol/l) led to a corresponding decrease in the expression levels of IL-6, IL-8 and TNF-α. Although the 40 µmol/l treatment group generally did not show statistically significant differences compared to the control group, a trend of reduction was observed with increasing concentrations (Fig. 6A-C). The suppressive effect on NF-κB expression was shown by a dose-dependent reduction in NF-κB levels, as shown by a double luciferase assay (Fig. 6D).

In the experimental group, 160 µmol/l Sch B notably reduced CCA cell activity. To directly and visually assess the effect of Sch B on p65 translocation, the present study performed immunofluorescence staining for p65. The results demonstrate nuclear accumulation of p65 in control cells (without Sch B treatment), whereas in Sch B-treated cells, p65 was predominantly retained in the cytoplasm, with correspondingly lower nuclear levels (Fig. 6E). Bay 11–7082 is a selective NF-κB inhibitor that suppresses IκBα phosphorylation (26), thereby preventing IκBα degradation and NF-κB nuclear translocation, which ultimately inhibits downstream NF-κB-dependent gene transcription. Accordingly, CCA cell activity was significantly reduced following the addition of Bay 11–7082 in a dose-dependent manner, with both 2 and 5 µM concentrations showing significant decreases compared with the control group (Fig. 6F). These results highlight the effectiveness of Sch B in inhibiting the NF-κB signaling pathway, resulting in the suppression of CCA cell activity.