Coptidis Rhizoma Alkaloids Alleviate Acetaminophen-Induced Liver Injury by Regulating GSH Metabolism and the TNF Signaling Pathway

TL;DR

Coptidis Rhizoma alkaloids protect the liver from acetaminophen overdose by boosting glutathione and reducing inflammation.

Contribution

This study reveals how CRA protects against APAP-induced liver injury via GSH metabolism and TNF signaling.

Findings

CRA increased hepatic cysteine and GSH levels by 2.2-fold and 1.8-fold, reducing oxidative stress.

CRA suppressed ERK and NF-κB phosphorylation by 39.2% and 38.0%, respectively, reducing inflammation.

CRA showed dose-dependent protection against APAP-induced liver injury in vitro and in vivo.

Abstract

Acetaminophen (APAP) overdose is a major global cause of drug-induced liver injury (DILI), and the rising incidence of APAP-induced hepatotoxicity has raised substantial concern in the medical community, highlighting an urgent need for effective therapeutic approaches. Coptidis Rhizoma alkaloids (CRAs) have shown hepatoprotective effects in multiple hepatic disease models. This study aimed to investigate the therapeutic efficacy and the underlying mechanisms of CRA in acetaminophen (APAP)-induced acute liver injury. After identifying 18 alkaloid components in CRA, we employed an integrated strategy of untargeted metabolomics and network pharmacological analysis to investigate the underlying mechanisms. The potential mechanisms were subsequently validated through histopathological examination and molecular biology assays. Our results showed that CRA exerted dose-dependent protection against APAP-induced liver injury in vitro and in vivo. This protective effect was mediated by enhanced hepatic glutathione (GSH) biosynthesis via increased intracellular cysteine (Cys) availability. In the mouse model, hepatic Cys and GSH levels were increased by 2.2-fold and 1.8-fold, respectively, relative to the model group, which consequently attenuated oxidative stress damage. Furthermore, CRA suppressed APAP-induced activation of ERK and NF-κB, reducing the phosphorylation levels by 39.2% and 38.0%, respectively. Accordingly, it also downregulated the subsequent expression of inflammatory mediators in the TNF signaling pathway. These findings provide crucial mechanistic insights into the hepatoprotective role of CRA against APAP-induced toxicity, establishing a valuable foundation for developing novel therapeutic or preventive strategies for APAP-induced liver injury.

Figures

CRA ameliorates APAP-induced liver injury by enhancing GSH biosynthesis in vivo: (A) Schematic diagram of the hepatoprotective effects of CRA in a mouse model of APAP-induced DILI. (B) Representative histology images of H&E-stained liver tissue (magnification: ×100 or ×200; scale bars: 100 μm and 50 μm). Serum levels of (C) ALT and (D) AST in mice. Hepatic levels of (E) L-Cys, (F) GSH, (G) ROS, and (H) MDA in mice. Serum levels of (I) L-Cys, (J) GSH, and (K) MDA in mice. Data are presented as mean ± SD, n = 6. ### p < 0.001 compared with the CON group; * p < 0.001 compared with the MOD group.

Full text

1. Introduction

The metabolism of drugs relies on drug-metabolizing enzymes. As the liver contains a wide variety and abundant quantity of these enzymes, it serves as the primary organ for drug metabolism. However, this function also makes it a major target for drug-induced toxicity [1]. Among the various drugs associated with drug-induced liver injury (DILI), acetaminophen (APAP) hepatotoxicity represents one of the leading causes of DILI worldwide. APAP, also known as paracetamol, is widely used in clinical practice as an antipyretic and analgesic agent [2]. It is also a primary active component in numerous over-the-counter cold remedies around the world, increasing the risk of unintentional overdose. Overdose of APAP can cause severe DILI, characterized by hepatocyte death and centrilobular necrosis, which may progress to acute liver failure [3]. The global annual incidence of APAP poisoning averages 7.4 cases per 100,000 people [4]. With the increasing occurrence of APAP-induced hepatotoxicity, there is growing concern and research interest within the medical community, underscoring the urgency of addressing DILI.

At recommended doses, approximately 10% of APAP is oxidized by cytochrome (CYP) enzymes to form N-acetyl-p-benzoquinone imine (NAPQI) [5,6]. NAPQI binds with glutathione (GSH) in the liver, and the conjugate is excreted through bile without causing any toxicity [7]. However, the overdose of APAP leads to excessive NAPQI formation, which depletes hepatic GSH stores and binds to cellular biomolecules, thereby causing oxidative stress and subsequent hepatocyte apoptosis and necrosis [8,9]. Necrotic hepatocytes release damage-associated molecular patterns (DAMPs) that are recognized by Kupffer cells, which in turn activate inflammatory pathways, release pro-inflammatory cytokines, and exacerbate liver injury [10]. Therefore, promoting GSH biosynthesis and alleviating inflammatory damage are potential therapeutic strategies for APAP-induced DILI.

N-acetylcysteine (NAC) is the only antidote approved by the FDA for the treatment of APAP-induced DILI [11]. As a precursor to cysteine (Cys), NAC acts as the rate-limiting step in GSH biosynthesis, effectively replenishing hepatic GSH reserves [12]. The mechanism confers the ability to alleviate APAP-induced hepatotoxicity via antioxidant ability. However, the efficacy of NAC depends on patients’ intrinsic capacity for GSH biosynthesis, which may lead to insufficient detoxification and treatment failure [13]. Consequently, the development of new therapeutic regimens remains a critical topic in research on APAP-induced hepatotoxicity. With pharmacological research on natural medicines, the therapeutic application of herbal medicines has demonstrated multiple clinical advantages in APAP-induced DILI [14,15,16].

Coptidis Rhizoma (CR), commonly known as “Huanglian”, is derived from the dried rhizomes of Coptis chinensis Franch. It is widely recognized that alkaloids serve as the primary bioactive constituents, with other components including organic acids, coumarins, and quinones [17,18]. In traditional Chinese medicine (TCM) theory, CR is recognized for its properties of heat-clearing, drying dampness, purging fire, and detoxification [19]. Given that DILI frequently manifests as dampness–heat syndrome, it is often recommended to treat it by clearing heat and eliminating dampness, an approach consistent with the indications for CR [20]. As the principal active fraction, CR alkaloids (CRAs) account for the majority of CR’s pharmacological activities [21]. Both CR and CRAs exert significant hepatoprotective effects, as well as antioxidant and anti-inflammatory properties, which are closely relevant to the key pathological processes of APAP-induced liver injury [9,22,23]. In rat models of carbon tetrachloride-induced (CCl_4_) acute and chronic hepatotoxicity, the aqueous extract of CR effectively ameliorates liver damage [23,24]. Berberine, a representative constituent of CRA, has demonstrated a potent hepatoprotective effect and alleviated CCl_4_-induced hepatic pathological abnormalities in vivo by regulating oxidative responses [25,26]. However, the hepatoprotective effects of CRA in APAP-induced liver injury have not yet been systematically evaluated, and the underlying mechanisms remain to be elucidated.

Our study aimed to investigate the therapeutic potential of CRA and its underlying mechanisms in APAP overdose-induced DILI. The results demonstrate that CRA effectively attenuates APAP-induced DILI by modulating GSH metabolism and the TNF signaling pathway. These findings enhance the mechanistic understanding and support the potential clinical applicability of CRA in the treatment of APAP-induced DILI.

2. Materials and Methods

2.1. Drugs and Reagents

CR was purchased from Nanjing Tongrentang (Nanjing, China) and identified at Dalian University by Professor Xuan Lu. APAP was acquired from Macklin (Shanghai, China). RIPA lysis buffer, malondialdehyde (MDA) assay kit, and BCA protein assay kit were purchased from Solarbio (Beijing, China). Assay kits for lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), L-Cys, glutamate (Glu), GSH, and reactive oxygen species (ROS) were supplied by Nanjing Jiancheng (Nanjing, China). NAC was procured from Aladdin (Shanghai, China). Tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β) ELISA kits were sourced from Shanghai Enzyme-linked (Shanghai, China). Primary antibodies against NF-κB (D221030), P-NF-κB (D155072), ERK (D260317), P-ERK (D290879), and GAPDH (D110016) were purchased from Sangon (Shanghai, China). The fluorescently labeled secondary antibody (SA00013-4) was purchased from Proteintech Group, Inc. (Wuhan, China).

2.2. Preparation of CRA

100 g of dried CR powder was mixed with 30 mL of 0.1% HCl solution and transferred into a percolation apparatus. An additional 1.5 L of 0.1% HCl solution was added to initiate percolation, and the percolate was collected. The percolate was loaded onto a cation-exchange resin for ion-exchange chromatography. Post-exchange, the resin was collected, dried, and mixed with concentrated ammonia solution, and then transferred to a Soxhlet extractor. Dichloromethane was added for water bath reflux extraction. Finally, the dichloromethane extract was concentrated via rotary evaporation to obtain the CRA sample.

2.3. Cell Culture and Treatment

HepG2 cells were obtained from Procell (Wuhan, China) and cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C under a 5% CO_2_ humidified atmosphere.

HepG2 cells were seeded into 96-well plates. Upon reaching approximately 60% confluency, they were treated with 10 mM APAP in the presence or absence of 10 μM NAC, 1 μg/mL CRA, or 10 μg/mL CRA for 24 h. Cell viability was assessed by LDH leakage assay, and the intracellular levels of L-Cys, Glu, GSH, MDA, and ROS were quantified using kits according to the manufacturers’ protocols.

2.4. Untargeted Metabolomics Analysis

Untargeted metabolomics analysis of HepG2 cell lysates was performed through UHPLC-MS/MS with metabolite identification services provided by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China), including technical consultation and analytical validation. Significantly altered metabolites were defined as those exhibiting a variable importance in projection (VIP) > 1 and p < 0.05. Subsequent KEGG pathway enrichment analysis identified biologically relevant affected metabolic pathways.

2.5. UHPLC-MS/MS Analysis of CRA

UHPLC (UltiMate 3000 UHPLC, Thermo Fisher Scientific, Waltham, MA, USA) and mass spectrometry (Q-Exactive MS, Thermo Fisher Scientific) operated in positive ion mode were used to separate and identify the small-molecular compounds in the CRA sample. The instrument analysis platform used was LC-MS, with a C18 column (Thermo Hypersil Gold C18, 1.9 μm, 2.1 mm × 100 mm). The chromatographic separation conditions included a flow rate of 0.3 mL/min, a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and an injection volume of 2 μL. The gradient elution program was as follows: 10% B (0–1 min), 100% B (15–17 min), and 10% B (19–20 min). Potential chemical constituents were identified via automated database searching against multiple repositories (V 3.2, Thermo Fisher Scientific, Carlsbad, CA, USA). Only components with high match scores or relatively high concentrations were retained for subsequent analysis. The sample testing and result analysis were conducted by Guangzhou Huaxi Testing Technology Co., Ltd. (Guangzhou, China).

2.6. Network Pharmacological Analysis

The active ingredients of CRAs and their associated targets were retrieved using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; https://www.tcmsp-e.com). The SMILES sequences of the active ingredients were input into the Swiss Target Prediction (STP; http://swisstargetprediction.ch/), with a probability threshold greater than 0 to predict the most probable protein targets. Additionally, protein targets associated with DILI were acquired from 3 disease databases: GeneCards (GC; https://www.genecards.org), the Open Targets Platform (OTP; https://platform.opentargets.org/), and the Comparative Toxicogenomics Database (CTD; http://ctdbase.org/). All collected targets underwent gene name standardization using the UniProt database (http://www.uniprot.org) to convert them into official Homo sapiens nomenclature. Potential therapeutic targets were identified by intersecting the component-action targets with DILI-related disease targets, followed by the construction of a Venn diagram. Gene Ontology (GO) functional annotation analysis, including encompassing biological processes (BP), cellular components (CC), and molecular functions (MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the screened potential targets using the Database for Annotation, Visualization and Integrated Discovery (DAVID; https://davidbioinformatics.nih.gov). All annotated entries were sorted by p-value, and the top 5 BP, CC, and MF terms, along with the top 10 KEGG pathways, were visualized using an online plotting tool (http://www.bioinformatics.com.cn/).

2.7. Immunofluorescence Staining

This experiment was performed following a previously described methodology [27]. Briefly, upon reaching approximately 60% confluence, HepG2 cells were treated with 10 mM APAP in the presence or absence of 10 μM NAC, 1 μg/mL CRA, or 10 μg/mL CRA for 24 h. The cells were incubated with P-NF-κB antibody at 4 °C for 12 h, washed three times with PBS, and then incubated with the fluorescent secondary antibody for 1 h. Following staining procedures, cellular images were acquired using a laser scanning confocal microscope (Olympus Fluoview FV3000, Tokyo, Japan).

2.8. Animals and Treatments

Male C57BL/6J mice (18–22 g) were purchased from Changsheng Biotechnology Co., Ltd. (Shenyang, China) and acclimatized for 5 days under standard housing conditions (22 ± 1 °C, 55 ± 5% humidity, 12 h light/dark cycle) with ad libitum access to food and water. The use and care of mice for the study described herein was approved (DW2023-033-1).

The mice were randomly divided into the following 5 groups (n = 6): the control group (CON), APAP-induced model group (MOD), NAC group (APAP + 100 mg/kg NAC), low-dose CRA group (APAP + 40 mg/kg CRA), and high-dose CRA group (APAP + 80 mg/kg CRA). The CON and MOD groups were administered the same volume of physiological saline as that given to the drug-treated group; both the NAC and CRA treatment groups received their respective administrations for five consecutive days. The detailed experimental procedure is illustrated in corresponding Results section. Following a 12 h fasting period, all groups except CON received 300 mg/kg APAP intraperitoneally. At 24 h post-APAP treatment, mice were sacrificed to collect liver tissues, and blood samples were obtained for serum separation. Serum levels of L-Cys, GSH, and MDA and hepatic levels of L-Cys, GSH, MDA, and ROS in mice were quantified using commercial assay kits according to the manufacturer’s protocols. TNF-α, IL-6, and IL-1β contents in mouse serum were determined by ELISA kits according to standardized instructions.

2.9. Histopathological Analysis of Liver Injury

Liver tissues were fixed in 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. Sections were baked, deparaffinized, and subjected to standard hematoxylin and eosin (H&E) staining. Histopathological assessment of hepatic damage was performed by light microscopy.

2.10. Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA was isolated from cells or liver tissues using RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Beijing, China). Following the manufacturer’s instructions, RNA was reverse-transcribed into cDNA. Subsequently, qRT-PCR amplification was performed using the synthesized cDNA. The 2^−ΔΔCT^ method was used to calculate the relative mRNA expression levels of target genes. The primers were synthesized by Sangon (Shanghai, China). The primer sequences are listed in Table 1.

2.11. Western Blotting Analysis

Cells and liver tissues were lysed using RIPA buffer, and the lysates were then centrifuged and quantified to obtain protein samples. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes, and incubated with primary antibodies and HRP-conjugated secondary antibodies. Immunoreactive signals were detected by the chemiluminescence imaging system GE Amersham Imager 600 and quantified using the ImageJ (version 1.53t; https://fiji.sc) analysis system.

2.12. Statistical Analysis

All data are shown as the mean ± standard deviation (SD), and statistical analysis was conducted using GraphPad Prism software 8.0, with intergroup comparisons performed using one-way ANOVA. A threshold of p < 0.05 indicated a statistical difference.

3. Results

3.1. Metabolomics Analysis Indicated That CRA Influenced the GSH Metabolism Pathway

Based on the detection method described in the literature, compositional analysis revealed that the extract of CR contained 97.6% alkaloids [28]. To investigate the hepatoprotective effects of CRA on APAP-induced hepatocyte damage in vitro, cell viability was evaluated using the LDH leakage assay. As shown in Figure 1A, compared to the MOD group, CRA treatments significantly increased cell viability in a dose-dependent manner, which suggested that CRA alleviated APAP-induced cytotoxicity in HepG2 cells. Untargeted metabolomics techniques were adopted to analyze the metabolites and associated pathways affected by CRA in HepG2 cells. Differential analysis identified 682 significantly altered metabolites, comprising 428 upregulated and 254 downregulated species, as visualized in the volcano plot (Figure 1B). Additionally, cluster analysis was conducted using a KEGG Sankey bubble chart (Figure 1C). The results showed that the crucial GSH metabolism pathway, associated with APAP-induced liver injury, was included among the top 10 signaling pathways influenced by CRA. Then, we analyzed the specific metabolites within the GSH metabolism pathway, as shown in Figure 1D–G. CRA markedly enhanced the levels of 5-oxoproline, γ-L-glutamyl-L-cysteine (γ-Glu-Cys), L-Cys, and GSH in the pathway. Studies have demonstrated that elevated GSH levels effectively detoxify NAPQI, the harmful metabolite of APAP, thereby mitigating hepatocyte injury [29]. The metabolomics analysis results suggested that the upregulation of GSH in HepG2 cells is a potential mechanism for the hepatoprotective effect of CRA.

3.2. Identification of Phytochemical Compounds in CRA

The phytochemical constituents present in CRA were identified by UHPLC-MS/MS (Figure 2A). Through compound analysis of CRA and cross-referencing the MS database, we identified 18 distinct chemical compounds (Table 2): magnoflorine, 8-oxocoptisine, demethyleneberberine, D-tetrahydropalmatine, protopine, 13-methylberberine, coptisine, epiberberine, jatrorrhizine, 8-oxyepiberberine, berberrubine, canadine, berberine, palmatine, dihydropalmatine, berberastine, dehydrocorydaline, and 8-oxyberberine (Figure 2B).

3.3. Network Pharmacological Analysis Revealed That CRA Modulated the TNF Signaling Pathway

For candidate targets, we obtained 174 targets from TCMSP, 412 from STP, 2603 from GC, 94 from OTP, and 6788 from CTD (Figure 3A). A total of 14 common targets were identified across all databases: GSH reductase (GSR), GSH synthetase (GSS), GSH peroxidase 1 (GPX1), GSH S-transferase Mu 1 (GSTM1), TNF, IL-1β, serine/threonine-protein kinase (AKT1), prostaglandin synthase 1 (PTGS1), prostaglandin synthase 2 (PTGS2), peroxisome proliferator-activated receptor gamma (PPARγ), superoxide dismutase [Cu-Zn] (SOD1), catalase (CAT), peroxiredoxin-1 (PRDX1), and bifunctional epoxide hydrolase 2 (EPHX2). GO and KEGG enrichment analyses were performed on these target proteins. BP terms primarily encompassed response to selenium ion, reactive oxygen species biosynthetic process, GSH metabolic process, response to lipopolysaccharide, and multicellular organismal-level homeostasis. CC analysis revealed associations with microbody, peroxisome, neuronal cell body, mitochondrial matrix, and cell body. MF terms included antioxidant activity, peroxidase activity, oxidoreductase activity, and protein homodimerization activity (Figure 3B). KEGG pathway analysis showed that the GSH metabolism pathway (involving GSS, GSR, GPX1, and GSTM1) had the lowest p-value. The TNF signaling pathway, composed of TNF, IL-1β, PTGS2, and AKT1, was also significantly enriched (Figure 3C). In APAP-induced DILI, the activation of the TNF signaling pathway promotes inflammatory responses and hepatocyte death, which exacerbates liver injury [16]. Hence, both GSH metabolism and the TNF signaling pathway play important roles in the progression of APAP-induced liver injury [17,30]. Integrated analysis of untargeted metabolomics and network pharmacology suggested that the underlying mechanism by which CRA alleviates liver injury involves modulating GSH metabolism and the TNF signaling pathway.

3.4. CRA Mitigated APAP-Induced Oxidative Stress by Promoting GSH Synthesis in Cells

Based on the results of untargeted metabolomics and network pharmacological analysis, we detected the levels of L-Cys, Glu, and GSH to explore the effect of CRA on the GSH metabolic process during APAP-induced DILI. As shown in Figure 4A–C, compared with the MOD group, treatment with NAC or CRA increased the levels of L-Cys and GSH. However, the levels of Glu were not significantly affected by NAC or CRA. The results indicated that CRA promoted GSH synthesis by increasing the content of the key substrate, L-Cys, in APAP-induced cells.

As the key endogenous antioxidant, GSH is essential for reducing oxidative stress [31]. To further evaluate the antioxidant effect of CRA, we analyzed the levels of MDA and ROS in cells. As shown in Figure 4D–F, APAP induced elevated levels of MDA and ROS in the MOD group. Meanwhile, the levels of MDA and ROS in the CRA treatment groups were both significantly decreased in a dose-dependent manner. The above results suggest that CRA alleviates APAP-induced oxidative stress in HepG2 cells by promoting GSH synthesis.

3.5. CRA Ameliorated APAP-Induced Inflammatory Responses by Regulating the TNF Signaling Pathway in Cells

To evaluate the TNF signaling pathway activity, we assessed the activation of downstream inflammatory proteins and the transcription of inflammatory genes in cells. Western blot analysis revealed that APAP treatment increased the phosphorylation levels of ERK and NF-κB, compared to the CON group. In contrast, CRA administration markedly suppressed the phosphorylation of ERK and NF-κB in a dose-dependent manner (Figure 5A–C). Furthermore, we evaluated the inhibitory effect of CRA on APAP-induced NF-κB nuclear translocation. As shown in Figure 5D, APAP stimulation promoted the translocation of NF-κB p65 from the cytoplasm to the nucleus, whereas NAC and CRA treatments significantly inhibited the process of nuclear translocation. During the progression of APAP-induced hepatotoxicity, inflammatory mediators upregulate the expression of pro-inflammatory cytokines [32]. We next examined the transcriptional levels of downstream inflammatory genes in the TNF signaling pathway. As shown in Figure 5E–G, the mRNA expression levels of TNF-α, IL-6, and IL-1β were significantly upregulated in the MOD group. After treatment with CRA, the expression of these inflammatory cytokines markedly reduced in a dose-dependent manner. In comparison with NAC, CRA exerted weaker antioxidant capacity but more potent anti-inflammatory activity in APAP-induced DILI cells, which suggested that the regulation of the TNF signaling pathway by CRA played a critical role in alleviating the inflammatory response associated with APAP-induced hepatotoxicity.

3.6. CRA Alleviated APAP-Induced Liver Injury In Vivo

We established an animal model to evaluate the protective role of CRA against APAP-induced DILI in vivo (Figure 6A). Histological analysis revealed liver structural damage to hepatocytes, hepatic membrane stenosis, nuclear atrophy, extensive infiltration of inflammatory cells, and significant hepatocyte necrosis in the APAP-induced MOD group. However, these pathological alterations were significantly attenuated by CRA treatment, particularly in the high-dose group (Figure 6B). The serum levels of ALT and AST, markers of liver injury, were measured to evaluate the protective effect of CRA on APAP-induced hepatotoxicity. As shown in Figure 6C,D, the serum levels of ALT and AST were significantly elevated in the MOD group, and CRA treatment reversed the upregulation of ALT and AST in a dose-dependent manner. These results indicated that CRA alleviates APAP-induced DILI in vivo.

CRA ameliorates APAP-induced liver injury by enhancing GSH biosynthesis in vivo: (A) Schematic diagram of the hepatoprotective effects of CRA in a mouse model of APAP-induced DILI. (B) Representative histology images of H&E-stained liver tissue (magnification: ×100 or ×200; scale bars: 100 μm and 50 μm). Serum levels of (C) ALT and (D) AST in mice. Hepatic levels of (E) L-Cys, (F) GSH, (G) ROS, and (H) MDA in mice. Serum levels of (I) L-Cys, (J) GSH, and (K) MDA in mice. Data are presented as mean ± SD, n = 6. ### p < 0.001 compared with the CON group; *** p < 0.001 compared with the MOD group.

3.7. CRA Exerted Antioxidant Effects by Promoting GSH Synthesis In Vivo

To examine the effect of CRA on hepatic GSH biosynthesis and oxidative stress during APAP-induced liver injury in vivo, we measured the levels of L-Cys, GSH, ROS, and MDA in liver tissue. The results demonstrated that APAP administration markedly depleted hepatic GSH and L-Cys stores, resulting in significant reductions in both GSH and L-Cys levels while concomitantly causing an increase in ROS and MDA levels. Notably, CRA treatment significantly upregulated L-Cys levels and enhanced hepatic GSH biosynthesis in a dose-dependent manner, resulting in 2.2-fold and 1.8-fold increases in hepatic Cys and GSH levels, respectively, compared to the MOD group. This effectively inhibited the accumulation of ROS and MDA and alleviated oxidative stress injury (Figure 6E–H). As the primary site for GSH synthesis and storage, liver injury depletes GSH, resulting in a systemic elevation of oxidative stress [31]. We further quantified the levels of L-Cys, GSH, and MDA in serum. Consistent with the hepatic tissue observations, CRA treatment significantly promoted the levels of L-Cys and GSH while attenuating the APAP-induced increase in serum MDA levels (Figure 6I–K). These results suggested that CRA enhanced hepatic GSH biosynthesis and mitigated APAP-induced oxidative stress in vivo.

3.8. CRAs Exert Anti-Inflammatory Effects by Modulating the TNF Signaling Pathway In Vivo

We further examined the anti-inflammatory mechanism of CRA in the APAP-induced mouse model. Western blot analysis revealed phosphorylation levels of both ERK and NF-κB were elevated in the MOD group, while CRA inhibited APAP-induced activation of ERK and NF-κB, reducing their phosphorylation levels by 39.2% and 38.0%, respectively (Figure 7A–C). The transcriptional levels of downstream inflammatory genes in the TNF signaling pathway were analyzed by RT-qPCR. As shown in Figure 7D–F, APAP upregulated hepatic mRNA expression of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. Notably, CRA treatment markedly mitigated APAP-induced elevation in the transcripts of these inflammatory genes. Moreover, we assessed the expression levels of pro-inflammatory cytokines in serum. Consistent with the results of RT-qPCR, CRA significantly attenuated APAP-induced inflammatory responses and dose-dependently reduced the serum levels of TNF-α, IL-6, and IL-1β (Figure 7G–I). Compared to the NAC group, the high-dose CRA group demonstrated a more potent anti-inflammatory effect. The results indicated that modulation of the TNF signaling pathway is a pivotal mechanism underlying the protective effects of CRA against the inflammatory response of APAP-induced hepatic injury in vivo.

4. Discussion

With the growing diversity of pharmacological agents, the global incidence of DILI has been rising annually [33]. Among various etiological factors, DILI resulting from excessive APAP intake accounts for a substantial proportion of cases worldwide, posing a serious threat to public health and highlighting an urgent need for novel therapeutic agents [34,35,36]. As a traditional Chinese herbal medicine, CR is recognized for its heat-clearing and detoxifying properties and has exerted hepatoprotective effects in animal models [37,38]. In the present study, CRA attenuated APAP-induced hepatocyte necrosis by ameliorating oxidative stress and inflammatory responses in vitro. Consistently, CRA treatment significantly reduced the accumulation of oxidative products, decreased pro-inflammatory cytokine levels, and lowered serum ALT and AST activities in vivo in the mouse model, thereby exhibiting marked hepatoprotective effects in DILI. To our knowledge, this is the first study to comprehensively demonstrate the efficacy of CRA against APAP-induced liver damage.

Excessive APAP metabolism generates NAPQI, which rapidly depletes intracellular GSH in hepatocytes and induces the formation of ROS. The overproduction of ROS when overdosed can initiate lipid peroxidation of cell membranes, leading to structural and functional mitochondrial impairment. This damage further aggravates cellular oxidative stress, which plays a central role in the pathogenesis of APAP-induced hepatotoxicity [39,40]. Furthermore, the accumulated ROS adversely affects the function of other major organ systems [41,42,43]. As a well-established clinical therapy, acetylcysteine supplementation serves to alleviate oxidative stress and mitigate hepatocyte injury [44]. Our findings indicated that CRA enhanced GSH biosynthesis, thereby effectively mitigating APAP-induced GSH depletion both in vivo in the mouse model and in vitro in HepG2 cells. Moreover, CRA reduced the accumulation of oxidative stress markers, such as ROS and MDA, in hepatic tissue and serum, and decreased ALT and AST, markers of hepatocyte injury. These results demonstrate the pivotal role of enhanced GSH biosynthesis in the protective effects of CRA against APAP-induced DILI.

Accumulating evidence indicates that berberine effectively inhibits multiple CYP isoforms, including CYP2E1, CYP3A4, and CYP2C9, which are involved in the metabolism of APAP to NAPQI [45,46]. Notably, CRA contains several compounds structurally similar to berberine, suggesting that it may produce analogous inhibitory effects on CYP enzymes. Therefore, CRA has the potential to exert synergistic antioxidant and hepatoprotective effects in APAP-induced DILI by enhancing GSH biosynthesis and suppressing CYP450-mediated NAPQI formation. However, this proposed dual regulatory mechanism requires further experimental validation.

The de novo GSH synthesis is accomplished through the consecutive addition of Cys to Glu, followed by the addition of glycine. Among these precursor amino acids, the cellular availability of Cys represents the primary rate-limiting factor for GSH biosynthesis [47,48]. Owing to its low oral bioavailability, direct GSH supplementation has shown limited efficacy in humans. Instead, NAC, a cysteine prodrug, is commonly administered to elevate hepatic GSH levels, thereby promoting the elimination of reactive metabolites and ROS [11]. Metabolomic analysis revealed that CRA treatment enhanced the intracellular levels of L-Cys, γ-Glu-Cys, which is the first intermediate in GSH biosynthesis, and GSH, without affecting other precursor amino acids. In both APAP-induced cell and mouse models, CRA also effectively attenuated the decrease in L-Cys levels resulting from GSH depletion. These results suggest that the mechanisms by which CRA and NAC influence GSH biosynthesis are similar, both acting by increasing the intracellular level of the key substrate, L-Cys.

The liver possesses a remarkable capacity for GSH biosynthesis and contains the highest GSH levels among all organs [49]. Furthermore, it is central to maintaining systemic GSH homeostasis by exporting synthesized GSH into the serum and bile [50]. Indeed, adequate GSH availability is essential for maintaining the cellular antioxidant defense system to alleviate toxicity induced by multiple factors [31]. GSH depletion is not only observed in DILI but also in alcohol-induced liver injury, where patients commonly exhibit markedly reduced GSH levels in both hepatic tissue and serum. This reduction contributes to hepatocyte apoptosis and necrosis [51,52]. Moreover, endogenous GSH levels govern the susceptibility of the liver and other tissues to endotoxin-induced injury [53]. Our findings demonstrate that CRA effectively enhances the GSH synthesis capacity of hepatocytes and increases systemic serum GSH levels. This suggests its potential for treating liver injury induced by other factors associated with GSH deficiency.

Inflammation is considered a fundamental mechanism underlying APAP-induced liver injury, with inflammatory cytokines such as TNF-α, IL-6, and IL-1β playing a significant role [54,55,56]. Excessive APAP intake causes hepatocyte damage and the release of DAMPs, which initiate immune responses and activate the TNF signaling pathway [30,57,58]. Upon activation, this pathway triggers downstream cascade reactions that involve key transcriptional regulators, such as ERK and NF-κB. These regulators undergo post-translational modifications and nuclear translocation, leading to the expression of inflammatory mediators, including TNF-α, IL-6, and IL-1β. Subsequently, the release of these cytokines exacerbates hepatic injury; IL-1β and IL-6 promote inflammatory amplification, while TNF-α further stimulates the synthesis of additional inflammatory factors [16]. In this study, CRA effectively inhibited the phosphorylation of ERK and prevented the nuclear translocation of NF-κB, thereby blocking the initiation of inflammatory gene transcription. The APAP-induced upregulation of IL-6, IL-1β, and TNF-α gene expression in hepatocytes was significantly reduced by CRA, along with a decrease in the serum levels of these inflammatory cytokines in mice. Our findings indicate that CRA suppresses APAP-induced activation of the TNF signaling pathway in vitro and in vivo, thereby disrupting the inflammatory amplification cycle and ultimately mitigating the inflammation-associated exacerbation of liver injury.

DILI involves a complex pathological network comprising multiple molecular processes, such as oxidative stress, hepatocyte death, fibrosis, and inflammatory activation [14,59]. Recently, the multi-target mechanism of herbal medicine has been recognized for offering notable benefits for the treatment of DILI [60,61,62]. As a TCM, CR is extensively utilized in clinical practice owing to its well-established efficacy and favorable safety profile, which provides a clinical foundation for the application of its active components in DILI treatment and mitigates the risks associated with translational development [63]. This study demonstrates that CRA exerts therapeutic effects on DILI through multiple pathways, including GSH metabolism and the TNF signaling pathway. From a clinical perspective, these findings suggest that CRA has potential clinical application value in the prevention and treatment of APAP-induced DILI. Furthermore, its favorable safety profile also supports its long-term potential for preventive administration in high-risk populations.

Although the present study provides preliminary evidence for the hepatoprotective effects and underlying mechanisms of CRA in both in vitro and in vivo APAP-induced liver injury models, there are some limitations to this study. Firstly, HepG2 cells were employed for in vitro experiments instead of normal primary hepatocytes. Multiple validations in other types of liver cells are needed to enhance the representativeness and statistical power of our findings. Secondly, oral administration of CRA in animal models has yielded valuable insights into its role, but this approach does not fully mimic the clinical application and metabolic characteristics of CRA in humans. Finally, given that liver injury develops much faster in mice than in humans after APAP overdose [64], CRA was administered in advance of APAP exposure in our study. This pre-treatment protocol differs from the clinical scenario, in which antidotal therapy is usually initiated after the onset of overdose or injury. Therefore, further research is required to explore the applicability and dosage requirements of CRA in humans.

5. Conclusions

In summary, our study demonstrates that CRA enhances GSH synthesis, mitigates oxidative stress, and suppresses the inflammatory response by modulating GSH metabolism and the TNF signaling pathway in APAP-induced cellular and mouse models (Figure 8). These findings provide key mechanistic insights into the protective role of CRA against APAP-induced hepatotoxicity, offering a valuable foundation for developing novel preventive approaches for APAP-induced liver injury.