Reagents

G. japonica total alkaloid extract (TA) was acquired from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). To confirm the adequate extraction of active components, the PA content within the TA was evaluated using a previously described method [8]. First, the TA was solubilized in 5% HCl, and the pH of the solution was meticulously adjusted to a neutral range of 6–7 by the addition of 5 mM NaOH. Finally, to achieve the desired working concentration, saline was incorporated into the TA mixture. The Cga purified for this investigation was obtained from Shanghai Standard Technology Co. Ltd. (Shanghai, China).

Animal experiments

C57BL/6J male mice aged eight weeks were procured from the Laboratory Animal Center at the Shanghai University of Traditional Chinese Medicine (SHUTCM). The experiment was conducted with mice housed under conditions free from specific pathogens. These conditions included a stable temperature maintained at 23 ± 2 ℃ and a consistent relative humidity of 55 ± 5%. The ventilation in the room was adjusted to cycles between 12 and 18 times per hour, complemented by a light/dark cycle maintained for 12 h each. The mice were maintained on a standardized diet provided in the laboratory, with free access to tap water. Oversight by the SHUTCM Experimental Animal Ethical Committee ensured that humane care was uniformly administered to all the subjects.

According to our research group’s previous studies, a mouse model of acute liver injury was established using TA [24]. Initially, the study involved dividing thirty mice into five distinct groups, each consisting of six individuals. These groups included vehicle, TA, and different concentrations of Cga (20 mg/kg, 40 mg/kg, and 80 mg/kg). Treatment commenced with a single dose of 100 mg/kg TA or the corresponding vehicle, which was administered intragastrically. Subsequent doses of Cga were administered at 6 h and again at 30 h following the initial TA administration. At 48 h post-TA administration, the animals were euthanized for immediate collection of samples from the blood, liver, ileum, and feces.

In a subsequent phase of the experiments, wild-type (WT) mice and whole-body Fxr knockout (Fxr-KO) mice were allocated into six distinct clusters, each containing six mice. These groups were designated WT-vehicle, WT-TA, WT-TA + Cga, Fxr-KO-vehicle, Fxr-KO-TA, and Fxr-KO-TA + Cga. A single dose of 100 mg/kg TA or the corresponding vehicle was administered intragastrically, followed by two subsequent doses of Cga (40 mg/kg each), which were administered at 6 h and 30 h after the initial TA dose. After 48 h of TA administration, all the mice were sacrificed, and both blood and liver samples were subsequently collected for subsequent analysis.

In the third experimental series, a total of 30 mice were distributed into five separate groups, with each group comprising six animals: (1) vehicle, (2) TA, (3) TA + Cga, (4) TA + Cga + EX 527, and (5) EX 527 (HY-15452, MedChemExpress, Shanghai, China) groups. A single oral administration of 100 mg/kg TA or its corresponding vehicle was given. Two doses of Cga (40 mg/kg) were orally administered at 6 and 30 h following TA treatment. EX 527 was administered via intraperitoneal injection at a dose of 20 mg/kg one hour before the initial Cga treatment commenced. Two days after the TA was applied, the mice were sacrificed to facilitate the immediate collection of both liver and blood samples.

Serum biochemistry analysis

To determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bile acid (TBA) in the serum, fresh blood samples were processed to isolate the serum. An Enzymatic Assay Kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) was subsequently used to measure the concentrations of ALT, AST, and TBA in strict accordance with the instructions provided by the manufacturer.

Histopathological analysis

Freshly obtained liver samples were preserved in 4% paraformaldehyde before being encapsulated in paraffin. Staining of the paraffin sections was performed using hematoxylin and eosin (H&E), which enabled detailed observation through a light microscope (Olympus Corp., Japan).

Quantitative real-time PCR (qRT-PCR) analysis

Hepatic samples were processed to extract RNA via an EZB-RN001-plus kit supplied by EZBioscience in the USA. The subsequent synthesis of cDNA was facilitated by the addition of Evo M-MLV RT Premix (AG11706, Accurate Biology, Hunan, China), which adheres to the protocols recommended by the manufacturer. The quantitative analysis of mRNA was conducted with a SYBR® Green qPCR Kit (AG11718, Accurate Biology, Hunan, China) with a real-time PCR detector. The expression levels of specific genes were normalized to those of GAPDH via the 2−ΔΔCt method and are reported as a ratio to the gene expression level of the vehicle group. The primer sequences are provided in Table 1.

Table 1 Primer sequences for qRT-PCRRNA-sequencing analysis

RNA was extracted from freshly obtained liver samples using TRIzol reagent according to the manufacturer’s instructions. The total amount and integrity of the extracted RNA were determined using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent Technologies, USA). This RNA served as the input material for subsequent sample preparation steps. The mRNA purification procedure included several steps: the use of poly-T oligo-attached magnetic beads, followed by fragmentation, reverse transcription, end repair, the addition of a single ‘A’ base, adaptor ligation, purification, and enrichment via PCR. The resulting library fragments were then purified using the AMPure XP system (Beckman Coulter, USA), and the final library quality was assessed using an Agilent 2100 Bioanalyzer. High-quality libraries were sequenced on an Illumina NovaSeq 6000 platform.

Western blotting (WB)

Liver tissue samples were homogenized using RIPA buffer (89900, Thermo Fisher Scientific, USA) supplemented with a protease inhibitor cocktail (539134, Merck Millipore, Germany), following methodologies outlined in previous studies [25]. Protein concentrations were quantitatively assessed through densitometric analysis via ImageJ software. The relative fold differences in expression levels were standardized to the GAPDH expression levels. The primary antibodies utilized included anti-FXR (72105), anti-SHP (3759), anti-SIRT1 (9475), and anti-GAPDH (5174) from Cell Signaling Technology, USA and anti-BSEP (ab155421), anti-MRP2 (ab203397), anti-CYP7A1 (ab65596), and anti-CYP8B1 (ab191910) from Abcam, USA. The secondary antibodies used, specifically the HRP-conjugated AffiniPure goat anti-rabbit or mouse IgG (H + L) (SA00001-2 or SA00001-1), were sourced from Proteintech Group, USA.

Immunoprecipitation (IP) assay

Liver tissue extraction was conducted via IP lysis buffer (87787, Thermo Fisher Scientific, USA) containing a protease inhibitor cocktail (539134, Merck Millipore, Germany). IP assays utilized antibodies against FXR (72105) and Acetylated-Lysine (9441, Cell Signaling Technology, USA), along with Dynabeads ™ Protein G Immunoprecipitation Kit (10007D, Thermo Fisher Scientific, USA), which strictly followed the protocols provided by the manufacturers.

Analysis of bile acid pools

The levels of bile acids in the serum, liver, ileum and feces were determined according to a previously established method [26]. The specific methods used were as follows: cold methanol was added to the serum, liver, ileum and feces (tissue samples were first homogenized) for protein precipitation, the mixture was vortexed, the mixture was centrifuged, the entire supernatant was took out, and then vacuum concentration was conducted. Then, a solution of 50% methanol containing the internal standard (CA-d4) was added for dissolution, the mixture was vortexed, and the mixture was centrifuged, after which the supernatant was aspirated for detection. The bile acid concentrations within the samples were quantified via ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (AB SCIEX 6500 Series HPLC–MS/MS, USA).

Luciferase analysis

HEK-293 T cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and Dulbecco’s Modified Eagle’s Medium (DMEM) enriched with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, California, USA). After 24 h, the cells were transfected with FXR plasmids, RXR-ɑ plasmids, ECRE plasmids or Renilla plasmids in DMEM for 6–8 h. The medium was then replaced with medium containing GW4046 or Cga (10, 25, 50, or 100 μM). Finally, the cell lysates were collected after 24 h and subjected to analysis with a Dual-Lumi™ Luciferase Reporter Gene Assay Kit (RG088S, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol.

Statistical analysis

Data analysis was performed via the GraphPad Prism 8 software developed by GraphPad Software, USA. The experimental results are presented as the mean ± SEM. WB results were obtained from 3 independent samples per group, while the results of serum biochemistry, bile acid pool analysis, and qRT-PCR were obtained from six independent samples per group. To evaluate the statistical significance of differences between groups, parametric data were analyzed using either Student’s t test (for two groups) or one-way analysis of variance (ANOVA) (for multiple groups). The P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) for all statistical tests.