2.2.1 Preparation of S. chirayita extract

To extract the active compounds, 250 g of S. chirayita was subjected to two rounds of extraction with 2000 mL of 80 % ethanol under reflux for 90 min each. The resulting extracts were then combined, concentrated, and freeze-dried to yield the final S. chirayita extract.

2.2.2 Analysis of serum pharmacochemistry of S. chirayita

Sprague-Dawley rats (SCXK (Xiang) 2019–0004) were obtained from Hunan Slaughterhouse Laboratory Animal Co., Ltd (Changsha, China) and were housed at the Level III Laboratory of Pharmacology Research in Chinese Medicine (TCM-09–315) at Chengdu University of Traditional Chinese Medicine. The facilities were managed by the State Administration of Traditional Chinese Medicine and maintained a 12-hour day/night cycle with a consistent temperature of 24 ± 2 °C. The rats were fed standard chow (provided by Hunan Laboratory Animal Co., Ltd., Hunan, China) throughout the study.

Based on the clinical dosage of S. chirayita in humans (6–9 g/70 kg) (Swati et al., 2023), we calculated a dosage of 156 mg/kg for rats considering the acquisition rate of 80 % ethanolic extract from S. chirayita (23.07 %). To enhance the presence of chemical components, we increased the dosage to 1.56 g/kg for the test. The experimental procedure was as follows: after a seven-day adjustment period, the rats were randomly divided into two groups: a control group (treated with saline, 10 mL/kg) and a S. chirayita test group (administered an 80 % ethanolic extract of S. chirayita, 1.56 g/kg). The treatment was continued for seven more days through gavage. Prior to experimental blood sampling, the rats underwent a standard fasting period of 12 h with access to water. Blood samples were collected at specific time intervals after feeding: 5, 10, 15, 30, 45, 60, 90, 120, 240, and 480 min. These samples were then left at room temperature for one hour and centrifuged at 3500 r/min for 15 min at 4 °C. The clear top layer was stored at −80 °C. Following the completion of the treatment and blood sampling process, all Sprague-Dawley rats were humanely euthanized in accordance with the guidelines provided by the American Veterinary Medical Association (AVMA) for the Euthanasia of Animals. The euthanasia protocol was part of our experimental design approved by the Animal Care and Use Committee at Chengdu University of Traditional Chinese Medicine, with the ethics approval number 2018–15, demonstrating our commitment to the ethics of animal research and the principles of 3Rs (Replacement, Reduction, and Refinement).

2.2.3 Preparation of UPLC-Q-Exactive Orbitrap MS analysis

First, the extract samples of S. chirayita was prepared for the study of chemical composition of it based on the method of UPLC-Q-Exactive Orbitrap MS. Briefly, to obtain a preparative concentration of 20 mg/mL of solubles, 20 mg of S. chirayita extract was added to a chromatographic grade methanol solution. Next, the serum samples (blank and S. chirayita groups) of SD rat were explored based on the means of serum pharmaceutical chemistry. Specifically, the blank serum or drug-containing serum sample were added to a 1:5 (v/v) acetonitrile solution for chromatography and obtained the supernatant through centrifugation. Next, 300 μL of chromatographic grade methanol was added to dissolve the serum sample. Finally, all samples were be filtered through a 0.22 μm membrane. Finally, the samples were analyzed using UPLC on a Vanquish model coupled to a Q-Exactive Orbitrap MS (Thermofisher, Waltham, MA, USA).

2.2.4 Chromatographic conditions

Sample separation was performed using a Waters XB-C18 column (100 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.3 mL/min. The column temperature was maintained at 40 °C, and an injection volume of 2 μL was used. The mobile phase consisted of a 0.1 % aqueous formic acid solution (solvent A) and acetonitrile (solvent B). The elution gradient was programmed as follows: the proportion of solvent B was increased from 2 % to 10 % over 0–5 min, held at 10 % B from 5 to 10 min, gradually increased from 10 % to 65 % B between 10 and 30 min, further increased from 65 % to 95 % B during 30–45 min, and finally decreased from 95 % to 2 % B between 45 and 55 min.

2.2.5 Mass spectrometry conditions

A Q-Exactive Orbitrap mass spectrometer (MS) equipped with an electrospray ionization (ESI) feature was used to operate in both positive and negative ion modes. The specific parameters for data acquisition were set as follows: the spray voltage was maintained at 3500 V for both positive and negative ion modes; the ion transfer tube temperature was set at 320 °C, and the extra air heating temperature was set at 350 °C. The full MS resolution was set to 35,000, while the secondary mass spectral resolution was set to 17,500. The scan mass ranged from 100 to 1500 Da, and the collision energy gradient was set at 20, 40, and 60 Ev.

2.3.1 Potential targets prediction

Using network pharmacological analysis, this study aimed to investigate the interaction between S. chirayita and ALI. Initially, the SwissTargetPrediction database (http:/www.swisstargetprediction.ch) was utilized to identify potential targets of the prototype blood components. Subsequently, ALI-related targets were gathered from the GeneCards (https://www.genecards.org/) and OMIM (https://omim.org/) databases. These targets were then standardized using the UniProt database (https: liww.uniprot.org/). Finally, Venn analysis (http://bioinfogp.cnb.csis.es/tools/venny/index.html) was performed to identify potential S. chirayita targets for ALI intervention.

2.3.2 Enrichment analysis

The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analytical tools were utilized to identify potential pathways associated with these common targets. Additionally, a protein–protein interaction (PPI) network was created using the STRING database (http://string-db.org/) by inputting the relevant target genes.

2.5.1 Animals and experimental design

Kunming mice were obtained from Beijing SPIFU Laboratory Animal Co., Ltd. (Beijing, China) and housed at the Level III Laboratory of Pharmacology Research in Chinese Medicine (TCM-09-315) at Chengdu University of Traditional Chinese Medicine, managed by the State Administration of Traditional Chinese Medicine. The mice were kept under a 12 h day/night cycle with a constant temperature of 24 ± 2 °C. After acclimating for 7 days, 54 mice were divided into six groups. The dosage for each group was determined based on references (Tian et al., 2014, Zhang et al., 2015) as follows: the Control group (received saline, 10 mL/kg daily), the Model group (also given saline, 10 mL/kg daily), the Silymarin group (received Silymarin, 0.2 g/kg daily), and low-, medium-, and high-dose STW groups (STW-L, 0.07 g/kg daily, STW-M, 0.14 g/kg daily and STW-H group, 0.28 g/kg daily respectively). These dosages were administered for 14 consecutive days. Two hours after the final dose, the Control group received an intraperitoneal injection of a peanut oil solution (10 mL/kg), while the rest of the groups were given intraperitoneal injections of 0.5 % CCl₄ mixed with the peanut oil solution (10 mL/kg). After 20 h, the blood samples were collected through the ophthalmic venous plexus. Additionally, the liver of each mouse was divided into two parts and stored in 4 % paraformaldehyde and −80 °C, respectively. The euthanasia protocol for mice followed the same procedure as described in section 2.2.2. The Chengdu University of Traditional Chinese Medicine Animal Use Committee approved all procedures, which were conducted in accordance with their guidelines.

2.5.2 Liver index analysis

The liver index is a commonly used metric in the field of toxicology, which measures the weight of an animal's liver relative to its total body weight. It is used to assess the severity of liver enlargement. The liver index is calculated using the formula: Liver index (%) = [Liver weight (g) / Mouse weight (g)] × 100. A higher liver index indicates a more severe case of hepatomegaly.

2.5.3 Biochemical analysis of the serum

The mice blood samples were collected into centrifuge tubes without heparin and sodium and allowed to coagulate at room temperature for an hour. Afterward, the samples were centrifuged at 3500 r/min for 15 min at 4 °C. The resulting serum samples were utilized to evaluate the concentrations of AST, ALT, TNF-α, and IL-6, following established protocols.

2.5.4 Biochemical analysis of the liver

Liver tissues from mice weighing 200 mg were homogenized with 1 mL of PBS buffer in a 1.5 mL centrifuge tube using a homogenizer set at 60 Hz for 90 s at 4 °C. This homogenization procedure was repeated four times. The homogenates were then centrifuged at 3500 r/min for 15 min at 4 °C. The supernatant was collected and used as the liver tissue homogenate sample. Subsequently, the levels of AST, ALT, MDA, and SOD in the liver tissue homogenates were measured following the manufacturer's instructions.

2.5.5 Histological analysis of the liver

For histopathological analysis, liver tissues were fixed in 4 % neutral formaldehyde and then stained with hematoxylin and eosin (H&E). The tissue samples were subjected to a series of steps, including gradient dehydration with ethanol, clarification, and embedding, before being sectioned. Microscopic images of the sections were captured using an Olympus microscope (Tokyo, Japan) after H&E staining.

2.5.6 Analysis of qRT-PCR

For the analysis of gene expression, total RNA was extracted from approximately 60 mg of liver tissue using Trizol reagent. The concentration and purity of the total RNA were measured with the Nanodrop Micro Nucleic Acid Analyzer (Thermo Fisher Scientific, USA). Subsequently, cDNA was synthesized from 1000 ng of total RNA, and a qRT-PCR reaction was performed on the Quantitative Real-Time Fluorescence PCR Detection System (Rocgene, Beijing, China). GAPDH was used as the reference gene to normalize RNA content. The primer sequences used for this process are provided in Table 1. The qPCR analysis parameters were set as follows: initial denaturation at 95 °C for 3 min, followed by 45 amplification cycles involving denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 60 s. The relative expression levels of the target genes were then calculated using the 2^−Δ(ΔCt) method.

2.5.7 Analysis of molecular dynamics

To further identify the key targets of STW for intervention in ALI, Molecular dynamics simulations including RMSD, RMSF, SARA, etc. of STW in complex with critical was performed using Gromacs2022 software. Specifically, Amber14sb was selected as the protein force field and Gaff2 was selected as the ligand force field.

2.5.8 Analysis of immunohistochemistry

Liver tissue sections were dewaxed and hydrated. Antigen repair was then performed in heated sodium citrate buffer. Closure was performed after removal of endogenous peroxidase. Finally, primary and secondary antibodies were incubated.

3.1.1 Mangiferin

The compound under investigation, known as Mangiferin, exhibited a retention time of 12.71 min. Its molecular formula is C₁₉H₁₈O₁₁, and the mass spectral signal for its quasi-molecular ion peaks [M−H]^- was detected at m/z 421.0782, with a negligible error of 1.42 ppm. The compound's secondary mass spectra revealed characteristic fragment ions at m/z 331.0463, m/z 301.0357, and m/z 271.0251. Notably, the ions observed at m/z 331.0471 and m/z 301.0375 correspond to [M−H−C₃H₆O₃]^- and [M−H−C₄H₈O₄]^- mass spectrometric fragments, respectively, resulting from internal sugar breakage. These findings, along with a reference to previous work (Xu et al., 2008), confirm the identification of the compound as Mangiferin. The cleavage pattern of Mangiferin is depicted in Fig. 3A.

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Fig. 3

Diagram of cleavage pattern of chemical composition of 80% ethanolic extract of S. chirayita.

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3.1.2 1-Hydroxy-2,3,4,5-tetramethoxyxanthone

The compound of interest exhibited a retention time of 17.9 min. Its molecular formula was determined as C₁₇H₁₆O₇ and the mass spectral signal of its quasi-molecular ion peaks [M+H]⁺ was recorded at m/z 333.0970, with a deviation of 0.30 ppm. The secondary mass spectra displayed characteristic fragment ions at m/z 318.0736, m/z 303.0501, m/z 318.0736, m/z 285.0390, and m/z 275.0550. The ion m/z 318.0739 represents the fragment [M+H-CH₃], which is formed by the removal of one methyl molecule from the precursor ion [M+H]⁺. Meanwhile, m/z 303.0515 corresponds to the fragment [M+H]⁺ formed by the simultaneous loss of two methyl group molecules [M+H-2CH₃]⁺. Further, fragments at m/z 285.0385 and 275.0589 are likely the ions [M+H-2CH₃-H₂O]⁺ and [M+H-2CH₃-CO]⁺, respectively, resulting from neutral losses of H₂O (18 Da) and CO (28 Da) at m/z 303.0515. Hence, based on this analysis, the compound has been identified as 1-Hydroxy-2,3,4,5-tetramethoxyxanthone (Mou et al., 2020). The cleavage pattern of 1-Hydroxy-2,3,4,5-tetramethoxyxanthone is presented in Fig. 3B.

3.1.3 Caffeic acid

The compound under analysis had a retention time of 16.63 min. Its molecular formula was determined as C₉H₈O₄ and the mass spectral signal of its quasi-molecular ion peaks [M−H]⁻ was recorded at m/z 179.0358, with a deviation of 4.47 ppm. The secondary mass spectra exhibited characteristic fragment ions at m/z 135.0445, m/z 107.0495, and m/z 89.0235. Based on these findings and referencing to previous work (Xiao et al., 2022), the compound was identified as caffeic acid. The cleavage pattern of caffeic acid is illustrated in Fig. 3C.

3.1.4 Isoorientin

The compound of interest exhibited a retention time of 14.58 min and was identified as C₂₁H₂₀O₁₁ based on the inferred molecular formula. Mass spectral analysis showed a quasi-molecular ion peak [M−H]^- at m/z 447.0937, with a minimal error of 1.12 ppm. The secondary mass spectra of the compound displayed characteristic fragment ions at m/z 429.0831, m/z 411.0738, m/z 357.0702, m/z 327.0515, m/z 299.0563, and m/z 285.0411. Notably, the ions observed at m/z 357.0617 and m/z 327.0514 could indicate neutral losses of 90 Da and 120 Da, respectively, which are common features in C-glycosides. These findings, along with support from existing literature (Liu et al., 2019), confirm the identification of the compound as isoorientin. The fragmentation pathway of isoorientin is depicted in Fig. 3D.

3.1.5 Ursolic acid

The compound exhibited a retention time of 37.73 min. Its molecular formula was determined to be C₃₀H₄₈O, and the mass spectral signal showed quasi-molecular ion peaks [M+H]⁺ at m/z 457.3697, with a minimal error of 4.59 ppm. Secondary mass spectra exhibited characteristic fragment ions at m/z 439.3596, m/z 411.3626, m/z 249.1849, and m/z 191.1795. The losses of neutral molecules H₂O (18 Da) and HCOOH (46 Da), as well as the retro-Diels-Alder (RDA) fragmentation of the C ring, were identified based on previous literature (Wang et al., 2015). Therefore, this compound can be identified as ursolic acid. The fragmentation pattern is illustrated in Fig. 3E.

3.3.1 Potential targets prediction

To clarify the potential targets of S. chirayita against ALI, we used the SwissTargetPrediction database to predict the potential targets of 10 blood-entry prototype components of S. chirayita. And 310 relevant targets were obtained. Additionally, 1,803 ALI-associated targets we identified based on GeneCards and OMIM databases. By using the Venny online tool, 148 potential common targets were obtained (Fig. 6A). These 148 targets are considered as potential targets for S. chirayita to exert its anti-ALI effects.

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Fig. 6

Hub genes of the chemical components of 80 % ethanolic extract of S. chirayita absorbed in blood and acute liver injury. (A) Venn diagram and candidate targets of the chemical components of 80 % ethanolic extract of S. chirayita absorbed in blood and acute liver injury, (B) Protein-protein interaction (PPI) network, (C) KEGG enrichment diagram, (D) GO including CC, MF, and BP analysis diagram, (E) Molecular docking heat map of 10 core targets with 10 critical compounds. The color represents the binding ability. The greener the color, the lower the binding ability and the higher the affinity between the receptor and the ligand.

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3.3.2 PPI network analysis and enrichment analysis

To investigate the potential function and mechanism of action of S. chirayita on ALI, we conducted PPI network analysis and enrichment analysis on the 148 cross-targets mentioned earlier. Firstly, the STRING database was utilized to construct a PPI network comprising these 148 common targets (Fig. 6B). This network was crucial in understanding how S. chirayita intervenes in ALI, as all of these genes were included. Subsequently, the cytoHubba plug-in in Cytoscape 3.7.1 were employed to filter the top targets based on their degree value, resulting in the identification of the top 10 targets, which included EGFR, CASP3, CTNNB1, STAT3, SRC, ESR1, PTGS2, PPARG, TLR4 and MMP9 (Fig. 6C). Taken together, these 10 targets were deemed as the core targets responsible for S. chirayita's anti-ALI effects. GO analysis revealed that these ten typical blood components could potentially regulate ALI through biological processes such as drug response and positive regulation of apoptosis (Fig. 6D). Furthermore, KEGG analysis suggested that S. chirayita might govern ALI through various signaling pathways, including the PI3K/Akt pathway, HIF-1 pathway, MAPK pathway, VEGF pathway, AMPK signaling pathway, FoxO signaling pathway, JAK/STAT signaling pathway, NF-κB signaling pathway, among others (Fig. 6E and Table 4).

3.6.1 Analysis of liver index

An evaluation of the liver index following STW administration offers insights into the therapeutic impact of S. chirayita on liver pathology. In mouse models subjected to CCl₄-induced hepatic injury, treatment with STW significantly decreased the liver index, serving as an indicator of reduced organ swelling and damage. Compared to the blank group, those in the model group exhibited a statistically significant increase in liver index (p < 0.01). In contrast, administration of STW at varying doses, as well as silymarin, distinctly attenuated the liver index, suggesting a dose-responsive protective effect against the hepatic injury (p < 0.05) (Fig. 8A). These findings underscore the potential of STW as a mitigating agent in CCl₄-induced liver damage.

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Fig. 8

(A) Effect of the liver index, the biochemistry of serum and liver tissue in CCl₄-induced acute liver injury model. (I) liver index, (II) concentration of ALT in serum, (III) concentration of AST in serum, (IV) concentration of TNF-α in serum, (V) concentration of IL-6 in serum, (VI) concentration of ALT in liver, (VII) concentration of AST in liver, (VIII) concentration of MDA in liver, (IX) concentration of SOD in liver. (B) STW ameliorates induced histopathological changes in the liver tissues of CCl₄-induced acute liver injury model. ^#p < 0.05, ^##p < 0.01, vs. the control group; *p < 0.05, **p < 0.01, vs. the model group.

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3.6.2 Biochemical analysis of the serum

When exposed to high quantities of CCl₄, the liver is attacked, resulting in an excessive production of free radicals. This, in turn, increases the permeability of cell membranes, leading to the infiltration of inflammatory neutrophils and a significant influx of ALT, AST, TNF-α, and IL-6 into the bloodstream. These factors contribute to the elevated serum levels (Masuda 2006, Niu et al., 2017). Our findings demonstrate a statistically significant reduction in ALT levels among subjects treated with silymarin or STW-H groups compared to the model group, indicating a protective effect on the liver (p < 0.05). Similarly, AST, TNF-α, and IL-6 levels were significantly lower in all STW dosed groups (low, medium, and high), as well as the silymarin-treated group, in comparison to the model group. This highlights the potential of the treatment to alleviate the inflammatory response associated with hepatic injury (Fig. 8A). These differences emphasize the therapeutic potential of STW in mitigating CCl₄-induced hepatic damage.

3.6.3 Biochemical analysis of the liver

The therapeutic efficacy of STW in acute CCl₄-induced hepatic injury was evaluated by examining liver biochemical parameters. Since the liver is the primary site of acute CCl₄-induced ALI (Dong et al., 2013), it was important to assess the effects of STW. Compared to the control group, all STW treatment protocols (low, medium, and high doses) as well as silymarin administration showed significant improvements in hepatic ALT, AST, MDA, and SOD levels (p < 0.01). These findings not only support the serum biochemical results but also provide additional evidence that STW has a hepatoprotective effect against CCl₄-induced damage. This reinforces the potential use of STW for intervention in CCl₄-induced ALI (Fig. 8A).

3.6.4 Histological analysis of the liver

Histopathological examinations were conducted using H&E staining to provide a detailed understanding of the hepatoprotective impact of STW. In the control group, liver sections exhibited well-preserved lobular architecture, orderly arranged hepatocytes, and intact liver capsule structures without any swelling or inflammatory infiltration. In contrast, liver samples from the CCl₄-induced model group showed extensive inflammatory infiltrate surrounding vascular structures, along with significant hepatocellular degeneration and necrosis. Treatment with different dosages of STW (low, medium, and high) and silymarin resulted in significant improvements in these pathological changes. The extent of improvement in inflammatory infiltration, edema, degeneration, and necrosis of hepatocytes varied depending on the treatment concentration, but was observed in all treated groups (Fig. 8B). Notably, the group receiving the highest dosage of STW (STW-H) exhibited the most substantial amelioration of CCl₄-induced liver damage, highlighting the potential of STW in counteracting hepatic insult.

3.6.5 Analysis of qRT-PCR

To substantiate the interactions proposed by our network pharmacology framework, we used qRT-PCR to evaluate the gene expression of pivotal targets. The results showed that STW administration significantly downregulated the mRNA levels of EGFR (Fig. 9A), CASP3 (Fig. 9B), CTNNB1 (Fig. 9C), STAT3 (Fig. 9D), ESR1 (Fig. 9F), PTGS2 (Fig. 9G), PPARG (Fig. 9H), TLR4 (Fig. 9I) and MMP9 (Fig. 9J). Interestingly, SRC expression was found to be upregulated upon STW treatment (Fig. 9E). These expression patterns not only confirmed the network pharmacology predictions but also validated the target-related pathways involved in the therapeutic action of STW. The alignment of these qRT-PCR findings with the in-silico data provides strong evidence for the regulatory role of STW on these key genetic targets, strengthening the credibility of the computational strategies used to understand the molecular basis of S. chirayita's pharmacological effects.

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Fig. 9

Potential targets for STW intervention in a CCl₄-induced acute liver injury model. (A) mRNA level of EGFR, (B) mRNA level of CASP3, (C) mRNA level of CTNNB1, (D) mRNA level of STAT3, (E) mRNA level of SRC, (F) mRNA level of ESR1, (G) mRNA level of PTGS2, (H) mRNA level of PPARG, (I) mRNA level of TLR4, (J) mRNA level of MMP9. (K) RMSD analysis of ESR1 and MMP9 with STW, (L) RMSF analysis of ESR1 and MMP9 with STW, (M) Rg analysis of ESR1 and MMP9 with STW, (N) SARA analysis of ESR1 and MMP9 with STW, (O) Free energy landscape map analysis of ESR1 and MMP9 with STW, (P) H-Bounds number analysis of ESR1 and MMP9 with STW, (Q) IHC analysis of ESR1 and MMP9 in liver. ^#p < 0.05, ^##p < 0.01, vs. the control group; *p < 0.05, **p < 0.01, vs. the model group.

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3.6.6 Analysis of molecular dynamics and immunohistochemistry

To enhance the validation of molecular docking individual qRT-PCR results, we combined both expression levels and selected the target with an expression level greater than 15 and a molecular docking score lower than −6.5 kcal·mol⁻¹ for further validation. The molecular dynamics findings indicated that STW effectively bound to both ESR1 and MMP9. Specifically, the RSMD analysis revealed that the RMSD curves of ESR1 and MMP9 proteins in complex with STW stabilized after initial fluctuations, with fluctuations remaining within 0.5 nm, indicating a stable binding formation between them and STW (Fig. 9K). The RMSF curves for ESR1 and MMP9 proteins in complex with STW did not fluctuate beyond 1 nm, with no amino acid residues showing significant fluctuations, suggesting that STW did not affect the overall structural stability of the proteins (Fig. 9L). Rg (Fig. 9M) and SASA (Fig. 9N) results confirmed that the addition of STW did not notably impact the overall structural stability of the protein. The free energy landscape map displayed a single minimum energy cluster for both ESR1 and MMP9 in association with STW, indicating a high stability of the complex formed by them with STW (Fig. 9O). The results from the analysis of hydrogen bond quantity revealed that ESR1, MMP9, and STW all formed stable hydrogen bonding interactions (Fig. 9P). Additionally, to further elucidate the protein levels of ESR1 and MMP9, an immunohistochemistry assay was conducted. The findings demonstrated that STW notably enhanced the protein expression levels of ESR1 and MMP9 (Fig. 9Q), providing further evidence that these targets could be crucial for the intervention of swertiamarin in acute liver injury. The consistency between the results of immunohistochemistry, molecular dynamics, and PCR underscored the promising potential of bioinformatics analysis in drug research.