Medicinal herbs of AR was purchased from Sichuan Tianzhi Co. The extraction method was performed according to the previous study of our research group (Fu et al., 2022). Briefly, AR.WE was prepared with distilled water (1:50, w/v), and the ARPs fractions were collected by water extraction and alcohol precipitation. The polysaccharide content was determined by the phenol-sulfuric acid method (sulfuric acid: phenol: polysaccharide = 5:1:2). Concentrated sulfuric acid (95%) was added and left in a water bath at 100°C for 10 min and the absorbance was measured at 490 nm. Finally, for further study, the precipitate was washed with distilled water and freeze-dried. The ARPs fractions were identified by High-performance liquid chromatography (HPLC, Agilent, Palo Alto, CA, United States) and separated on a Shiseido C18 column (4.6 mm × 250 mm, 5 μm, pore size). The molecular weights of the fractions were determined by gel permeation chromatography (GPC) on a Viscotek GPCmax VE2001 (Malvern Instruments, UK) chromatograph.

C57BL/6J male mice, 6–8 weeks old were purchased from HFK Biological Technology Co. Ltd, and all mice were housed in standard procedures in the Animal Experiment Center of Southwest Medical University. They were kept in the Animal Experiment Center of Southwest Medical University with 12 h of light and dark cycles, 20°C–25°C, 50%–70% humidity, and free access to food and water. For ALIL model construction, APAP was dissolved in lukewarm water at 50°C and used as it was prepared. In this study, the drug was administered by gavage. In the prevention experiment, a total of 18 C57BL/6J mice were gavaged with AR.WE (250 mg/kg) (n = 6) or ARPs (250 mg/kg) (n = 6) for three consecutive days, and on the last day, the mice were fasted for 16 h and then given 300 mg/kg APAP to induce acute liver injury. In the treatment experiments, mice were gavaged with 300 mg/kg APAP to induce acute liver injury after 16 h of fasting. One hour later, mice were gavaged with AR.WE. (250 mg/kg) (n = 6). Ten hours after administration, mice were anaesthetised with isoflurane and blood and liver were taken for testing. All experimental designs were approved by the Ethics Committee of the Animal Experiment Center of Southwest Medical University.

HepG2 cells were cultured using medium containing 10% FBS and 1% penicillin-streptomycin and were incubated at 37°C in an incubator containing 5% CO₂. After pretreatment with ARPs (0.5 μg/mL and 1 μg/mL) for 1 h, APAP (2.5 mM) was given for stimulation for 24 h before subsequent experiments.

Blood was taken from mice after anesthesia and execution, and whole blood samples were centrifuged at 4°C at 6,000 rpm for 10 min, the supernatant was taken in a new tube. Next, the supernatant was centrifuged again at 4°C at 6,000 rpm for 10 min to remove impurities, and then plasma was collected. Plasma was stored in a refrigerator at 80°C and assayed with ALT and AST kits (Zhongsheng Technologies, China). 200 μL of assay solution was added to 5 µL of serum/standard/PBS (blank control) and immediately subjected to enzyme kinetic assay at 37°C.

The surface of mouse liver was washed with PBS, then added with saline homogenate, centrifuged at 4°C, 4,000 rpm for 10 min, and the supernatant was taken for measurement. The GSH content in the supernatant was detected using a GSH kit (Nanjing Jiancheng, China).

The surface of mouse liver was washed with PBS, then added with saline homogenization, centrifuged at 4°C, 4000rpm for 10 min, and the supernatant was taken for measurement. The SOD content in the supernatant was detected by using SOD kit (Nanjing Jiancheng, China).

The same leaf of mouse liver was taken and immediately fixed in 4% paraformaldehyde, then the dehydrated liver tissue was embedded in paraffin, and the tissue wax block was cut into sections of about 4 μm thickness. Each section was dewaxed, rehydrated and stained with hematoxylin and eosin. Microscope equipped with a DFC350FX digital camera (Leica, Milano, Leica) was used to observe the tissue morphology.

Whole blood samples were centrifuged at 4°C, 6,000 rpm for 10 min, and the supernatant was taken in a new tube next, and the supernatant was centrifuged again at 4°C, 6,000 rpm for 10 min to remove impurities, and then plasma was collected. The plasma was stored in a refrigerator at 80°C, and the concentration levels of serum IL-6, TNF-α (ml002095, mlbio, China) were determined using ELISA kits according to the manufacturer’s instructions. Add 10 µL of biotin labelled antibody and 50 µL of enzyme reagent to 40 µL of serum/standard/PBS. Warm bath at 37°C for 30 min and then washed with washing solution. Finally the absorbance was measured after adding the colour developer.

Total RNA from liver and HepG2 cells were extracted using TRIzol reagent (Vazyme, Nanjing, China), and 1,000 ng of RNA was reverse transcribed to complementary DNA using a PrimeScript RT Reagent Kit (Takara RR037A). 1,000 ng of RNA was extracted using SYBR^® Green Realtime PCR Master Mix (Novizan, Nanjing, China) was used to perform real-time quantitative PCR on a Bio-Rad CFX ConnectTM Real-Time system (Bio-Rad, United States). The following procedure was used: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 20 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s, for a total of 39 cycles. The relative gene expression levels were assessed using the 2^−ΔΔCT method with 18s or GAPDH as control. Primer sequences are shown in Supplementary Table S1.

Besides the data from DIA-based quantitative proteomics, other data were statistically analyzed using Graph Pad Prism 9.5 software (San Diego, CA, USA). Student's t-test was used for comparisons between two groups. Comparisons between multiple groups were analyzed using one-way ANOVA, and post hoc test adjustments were made using Bonferroni correction. Post hoc tests were performed only when F reached p < 0.05 and there was no significant heteroscedasticity.

APAP-induced liver injury is mainly characterized by histopathological changes and hepatic necrosis (Chen et al., 2023). We assessed the preventive effects of AR.WE on acute liver injury induced by APAP using a dose of 250 mg/kg (Figure 1A). The hallmark of APAP-induced liver injury is centrilobular liver necrosis (Yan et al., 2018). H&E results showed that pretreatment of AR.WE significantly reduced pathological changes and necrotic area of mouse liver (Figures 1B,C). Furthermore, ALT and AST expression in serum AST and ALT are specific aminotransferases in the liver. When hepatocytes are damaged, ALT and AST are released in large amounts into the blood, and serum ALT and AST levels are increased (Lv et al., 2019). APAP-treated mice showed high ALT and AST levels in the blood test, the biochemical outcomes demonstrated that the AR.WE pretreatment considerably decreased the blood ALT and AST levels induced by APAP (Figure 1D).

Superoxide Dismutase (SOD) is an important antioxidant enzyme that clears harmful free radicals. APAP-induced liver injury often leads to reduced SOD levels, which triggers oxidative stress in the body (Li et al., 2023a). However, we found that AR.WE pretreatment significantly increased hepatic SOD levels, thereby reducing the occurrence of oxidative stress (Figure 2A). Meanwhile, Glutathione (GSH) depletion is a critical step in APAP-induced liver injury. We detected variations in liver GSH activity in mice. The results demonstrated that APAP significantly reduced the activity of hepatic GSH compared to the control group, whereas AR.WE preconditioning significantly increased the activity of hepatic GSH (Figure 2A). APAP-induced liver injury is often accompanied by a powerful inflammatory response (Xu L. et al., 2022). To assess the inflammatory state in the liver, enzyme activity and protein expression levels of a certain factor involved in the inflammatory pathway were detected. We examined the serum pro-inflammatory cytokine protein and mRNA levels such as TNF-α by using ELISA analysis (Figure 2B) and RT-PCR analysis (Figure 2C), respectively. APAP significantly increased serum IL-6 and TNF-α levels and expression of genes involved in inflammation compared to controls. As expected, AR.WE pretreatment prevent inflammatory response induced by APAP.

Our previously results have shown that pretreatment of AR.WE for 3 days significantly prevent against APAP-induced liver injury, whether this herb is effective against the liver damage that has been caused is still unclear. Thus, mice were gavaged with AR.WE at 1 h after APAP administration, and were sacrificed 10 hour later (Figure 3A). Similarly, AR.WE treatment after APAP administration significantly reduced pathological changes and necrotic area of mouse liver, as revealed by H&E analysis (Figures 3B, C). Meanwhile, the liver function markers ALT and AST were also improved by AR.WE treatment (Figure 3D). Moreover, we found that AR.WE treatment significantly increased hepatic SOD levels and decreased the activity of hepatic GSH compared to the control group, therefore play an important role in antioxidant defense and stress resistance (Figure 4A). We then examined serum pro-inflammatory cytokine expression levels and the mRNA expression of genes related to inflammatory response. Consistently, APAP-induced inflammatory response in liver and circulation was significantly abolished by AR.WE (Figures 4B, C). Thus, we may conclude that AR.WE treatment after APAP administration mitigated APAP-induced liver injury.

ARPs is the main water-soluble component of AR, have been proved to play a significant role in antitumor, anti-inflammatory, antivirus, antihyperglycemic, anti-aging and anticoagulant activities (Liu et al., 2020). We then performed experiment to determine whether ARPS was the main component of AR.WE that exerts hepatoprotective effects. Polysaccharides from AR were usually extracted by the method of water extract and alcohol precipitate, followed by protein removal using the Sevag method, as shown in our previous research (Figure 5A). Before APAP administration, Mice were pretreated with ARPs at a dose of 250 mg/kg for 3 days (Figure 5B). As revealed by H&E analysis, ARPs preconditioning for 3 days ameliorated APAP-induced liver injury, much less hepatocellular injury and necrosis was observed in ARPs-treated group, indicating the protection of ARPs against APAP-induced liver toxicity (Figures 5C, D). Meanwhile, ARPs treatment reduced both ALT and AST levels significantly (Figure 6A). In addition, ARPs preconditioning significantly improved hepatic SOD and GSH activities compared to the APAP group (Figure 6B). Then, RT-PCR and ELISA assays were conducted to detect the expression change of the angiogenic factor and inflammatory factors at the mRNA levels and protein levels. ARPs-treated mice also exhibited significantly decreased pro-inflammatory factor expression levels which were consistent with the previous experimental results (Figures 6C–E). The above findings indicated that ARPs pretreatment effectively prevented acute liver damage induced by APAP.

To further test the potential effect of ARPs on hepatocytes, we performed in vitro experiment. HepG2 cell lines were preconditioned with ARPs for 1 h and then stimulated with APAP for 24 h. As revealed by RT-PCR analysis, ARPs preconditioning significantly upregulated genes involved in inflammation such as TNF-α, F4/80, and il-1β in a dose-dependent manner (Figure 7). The NLRP3 inflammasome is a cytosolic complex for early inflammatory responses, increasing evidence shows that the NLRP3 inflammasome is activated in APAP-induced liver injury in mice. Consistently, APAP exposure significantly increased the expression of NLRP3 in HepG2 cell, while the treatment of ARPs effectively reversed this phenotype (Figure 7). Thus, these in vitro results further verified the hepatoprotective effect in APAP-induced liver injury.

To further explore the potential mechanisms by which ARPs ameliorates liver injury, we conducted a Data-independent acquisition (DIA)-based quantitative proteomics research. PCA analysis showed that the three patterns were distinct (Figure 8A). In total, 74,977 peptide fragments and 6,257 quantitative proteins were identified (Figure 8B). A total of 729 differentially expressed proteins (DEPs) (356 upregulated proteins and 373 downregulated proteins) were found between the APAP and ARPs groups (Figure 8C). The DEPs screening criteria was as follows: fold change (FC) ≥1.5 or ≤0.67 and P < 0.05. Volcano plot showing differential expressed genes between APAP and ARPs groups (Figure 8D).

We conducted subcellular localization of DEPs between the APAP group and the ARPs group and demonstrated the top 12 most enriched. In the first place was the cytoplasm, indicating that 27.75% of DEPs were located in the cytoplasm, followed by extracell (26.1%), nucleus (17.31%), and plasma membrane (10.16%), respectively (Figure 8E). Then Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed based on these DEPs. The GO annotation divides these DEPs into three sections: biological processes, cellular component, and molecular function. For biological processes, DEPs in the APAP and ARPs groups were mainly enriched in innate immune response (Figure 8F). For cell contents, DEPs were mainly enriched in extracellular components. In terms of molecular function, DEPs were mainly enriched in antigen binding (Figure 8F). KEGG enrichment revealed that these DEPs were most enriched in regulation of actin cytoskelenon, and phagosome pathway (Figure 8G). We further analysed the phagosome pathway based on animal model and research direction. KEGG pathway map demonstrated that vacuolar H⁺-ATPases (vATPases) played an important role in the phagosome pathway (Figure 9). We therefore speculated that downregulation of vATPase activity was a potential target of ARPs for the treatment of liver injury.

We next assess the protein levels involved in apoptosis, inflammation, necroptosis, and oxidative stress, all of which important pathogenesis of APAP-induced liver injury. As shown in heatmaps, APAP exposure significantly upregulated the protein levels involved in pro-apoptosis, pro-inflammation, pro-necroptosis, and oxidative stress, while these phenotypes were prevented by ARPs treatment (Figures 10A–D). Hepatic autophagy is an essential mechanism for liver regeneration, and impaired autophagy is found to be related to dysfunction of various processes of hepatic regeneration (Wang et al., 2024). Thus, improving autophagy is an important therapeutic method to alleviate hepatic injury. Our data showed that ARPs could promote autophagy by transcriptionally activating autophagy-related genes, which indicated that ARPs may ameliorate APAP-induced liver injury by restoring autophagy (Figure 10E). APAP can aggravate oxidative stress and induce ferroptosis, a mode of cell death mediated by lipid peroxidation and cellular free iron when protective mechanisms such as glutathione peroxidase activity have been compromised (Jaeschke et al., 2021). In our data, ferroptosis-related genes were significantly regulated by ARPs treatment (Figure 10F). Consequently, we could speculate ARPs protects against drug-induced liver injury by inhibiting hepatocyte ferroptosis. To validate this conjecture, we performed RT-PCR analysis and found that Liver P62 gene expression level was significantly higher in APAP group mice compared to control group (P = 0.01). After ARPs administration, P62 gene expression level decreased (P = 0.17). Whereas, APAP stimulation resulted in decreased hepatic LC3B expression level in mice (P = 0.03). After ARPs administration, LC3B gene expression level increased (P = 0.004) (Figures 11A, B). In addition, the gene expression levels of GPX4, FTH1, ATG5, and XCT were reduced in the livers of mice in the APAP group compared to the control group, and the administration of ARPs reversed these trends (Figures 11C–F). All these data suggest that ARPs may ameliorate liver injury by regulating autophagy and ferroptosis.