Ambroxol mitigates cyclophosphamide-induced liver injury by suppressing TLR-4/NF-κB signaling and oxidative stress and upregulating cytoglobin, TXNRD1 and HMGB1

Abstract

BACKGROUND

Cyclophosphamide (CP) is a potent chemotherapeutic and immunosuppressant agent, but its hepatotoxicity remains a significant concern. Ambroxol (ABX) is a mucolytic agent with emerging beneficial effects against oxidative stress and inflammation.

AIM

To investigate the hepatoprotective effects of ABX against CP-induced liver injury, focusing on oxidative stress, inflammation, and the possible role of cytoglobin, thioredoxin reductase 1 (TXNRD1) and high-mobility group box 1 (HMGB1).

METHODS

ABX (20 mg/kg) was orally administered for 7 days, and the rats received a single injection of CP (100 mg/kg) on day 5. Blood and liver samples were collected for analyses, and the affinity of ABX towards cytoglobin, TXNRD1, and HMGB1 was evaluated using molecular docking.

RESULTS

CP administration significantly elevated alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, reduced albumin, and caused multiple histopathological alterations in the liver. ABX effectively restored liver function biomarkers and attenuated histopathological alterations. CP-induced oxidative stress was evidenced by increased malondialdehyde and decreased glutathione and antioxidant enzyme activities, all of which were ameliorated by ABX. CP upregulated toll-like receptor 4 (TLR-4), nuclear factor-kappaB (NF-κB) p65 and pro-inflammatory cytokines, while downregulating cytoglobin, TXNRD1 and HMGB1. ABX suppressed TLR-4/NF-κB signaling and pro-inflammatory cytokines, and upregulated cytoglobin, TXNRD1 and HMGB1. In silico molecular docking revealed the affinity of ABX to bind with cytoglobin, TXNRD1, and HMGB1.

CONCLUSION

ABX protects against CP hepatotoxicity by mitigating oxidative stress, suppressing TLR-4/NF-κB signaling, and upregulating cytoglobin, TXNRD1 and HMGB1. ABX showed binding affinity towards cytoglobin, TXNRD1 and HMGB1. These findings suggest that ABX has therapeutic potential in alleviating hepatotoxicity associated with CP treatment.

Key Words: Chemotherapy; Ambroxol; Oxidative stress; Inflammation; Hepatotoxicity; Cyclophosphamide; Acute liver injury

Core Tip: Cyclophosphamide (CP) is a cornerstone chemotherapeutic and immunosuppressive agent, but its hepatotoxicity limits its clinical utility. While oxidative stress and inflammation are known contributors to CP-induced liver injury, effective therapeutic strategies to counteract these effects remain limited. This study is the first to demonstrate that ambroxol exerts potent hepatoprotective effects against CP hepatotoxicity. Our findings not only elucidate the molecular mechanisms underlying the hepatoprotective effects of ambroxol but also position it as a promising adjunct therapy to mitigate CP hepatotoxicity. Given the lack of effective hepatoprotective agents against CP toxicity, this study has significant translational implications.

INTRODUCTION

Drug-induced liver injury (DILI) is a significant clinical and public health concern, accounting for approximately 10% of all reported cases of acute hepatitis and being the leading cause of acute liver failure in many developed countries[1-3]. DILI is responsible for large number of drug withdrawals and black box warnings issued by regulatory agencies, underscoring its impact on drug safety and patient health[3]. Among the drugs implicated in hepatotoxicity, chemotherapeutic agents are responsible for a large number of cases of liver injury due to their toxic nature and widespread use in cancer treatment[4]. Cyclophosphamide (CP), a potent alkylating agent, is a cornerstone in the management of solid tumors, leukemias, and lymphomas[5-7]. Its therapeutic efficacy stems from its ability to crosslink DNA strands, thereby inhibiting DNA replication and transcription, ultimately leading to cancer cell death[5-7]. However, its clinical use is often limited by its severe side effects, particularly hepatotoxicity[4-7]. The detrimental effects of CP are provoked by its metabolites, phosphoramide mustard and acrolein, which are generated via cytochrome P-450-mediated metabolism[8]. Among these metabolites, acrolein, characterized by its high reactivity and short biological half-life, plays a significant role in inducing oxidative stress by stimulating the generation of reactive oxygen species (ROS)[9].

CP hepatotoxicity is characterized by inflammation, oxidative stress, and cellular damage, which can compromise liver function and exacerbate patient morbidity[5,10,11]. The central role of oxidative stress and inflammation in the pathogenesis of CP-induced liver injury is associated with ROS generation and acrolein, a highly reactive aldehyde, which deplete cellular antioxidants such as reduced glutathione (GSH) and induce lipid peroxidation (LPO), and oxidative protein and DNA damage[12,13]. These oxidative insults provoke inflammation responses by activating nuclear factor-kappaB (NF-κB)[14,15]. Excess ROS possess the capacity to activate a cascade of signaling molecules, including toll-like receptor 4 (TLR-4), which subsequently triggers the activation of NF-κB. This activation leads to the transcriptional upregulation and release of numerous pro-inflammatory mediators, thereby amplifying the inflammatory response[14,15]. This vicious cycle of oxidative stress and inflammation exacerbates hepatocellular damage, highlighting the need for therapeutic interventions that simultaneously target these interconnected pathways. Accordingly, activation of key cellular proteins with diverse functions in oxidative stress, inflammation, and tissue repair could be a valuable strategy to mitigate CP hepatotoxicity. Cytoglobin (Cygb), a member of the globin family, is ubiquitously expressed in various tissues, including the liver, where it prevents oxidative stress by scavenging ROS and maintaining cellular redox homeostasis[16]. Thioredoxin reductase 1 (TXNRD1) is a selenoprotein which regulates cellular redox balance by reducing oxidized proteins and scavenging ROS. TXNR1 also modulates NF-κB signaling, thereby influencing inflammatory responses[17]. High-mobility group box 1 (HMGB1), a non-histone nuclear protein, has dual roles depending on its cellular localization. Intracellularly, HMGB1 regulates DNA repair, transcription, and autophagy, while extracellularly, it acts as a damage-associated molecular pattern, promoting inflammation and tissue repair by activating TLR4 and other immune receptors[18,19]. Owing to their cytoprotective roles, upregulation of Cygb, TXNRD1, and HMGB1 can mitigate CP-induced liver injury.

The mucolytic ambroxol (ABX) has recently shown other pharmacological properties, including beneficial effects in pneumonia, coronavirus disease 2019, Parkinson disease, Gaucher disease, and others[20-23]. Clinically, ABX is employed to manage chronic obstructive pulmonary disease, asthma, and other respiratory conditions characterized by excessive mucus production. Beyond its mucolytic action, ABX has been shown to protect against neurotoxicity[24], ischaemic stroke[25], and renal and hepatic ischemia-reperfusion (I/R) injury[26-28]. The protective efficacies of ABX have been associated with suppression of inflammation[26], suggesting its potential utility in protecting against CP-induced liver damage. However, nothing has yet been reported on the efficacy of ABX against CP hepatotoxicity. The aim of this study is to explore the effects of ABX on CP-induced hepatotoxicity in rats, emphasizing its modulatory role on oxidative stress, inflammation, and the expression of Cygb, TXNRD1, and HMGB1.

MATERIALS AND METHODS

Animals and treatments

Male Wistar rats weighing 180 ± 10 g were housed in a controlled environment with a temperature of 23 °C ± 2 °C, relative humidity of 50%-60%, and a 12-hour light/dark cycle. The animals were provided free access to a standard rodent diet and water. A total of 24 rats were randomly divided into four experimental groups (n = 6) to evaluate the hepatoprotective effects of ABX against CP hepatotoxicity. All experimental protocols were approved by the Institutional Animal Ethics Committee of Al Azhar University, Egypt (Approval No. AZ-AS/PH-REC/3/25), and conducted in accordance with their guidelines.

ABX and CP were prepared as suspensions in 0.5% carboxymethyl cellulose (CMC; Sigma, United States) and physiological saline, respectively. The experimental groups were designed as follows:

Group I (Control): Received 0.5% CMC.

Group II (ABX): Received 20 mg/kg ABX (GNP, Egypt)[27].

Group III (CP): Treated with 100 mg/kg CP (Endoxan; Baxter Oncology, Germany)[29].

Group IV (CP + ABX): Co-administered 100 mg/kg CP and 20 mg/kg ABX.

ABX was administered via oral gavage for seven days and a single intraperitoneal dose of CP was administered on day 5. Following treatments, blood samples were collected under ketamine/xylazine-induced anesthesia. Serum was separated for biochemical analysis. The liver was promptly excised following euthanasia. Portions of the liver tissue were fixed in 10% neutral buffered formalin (NBF), while others were homogenized in Tris-HCl buffer (pH 7.4), and the homogenates were centrifuged to obtain supernatants for biochemical assays.

Biochemical assays

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and albumin were determined using commercially available kits (Biodiagnistic, Egypt). ELISA kits supplied by Elabscience (China) were used to quantify tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the liver homogenate. Malondialdehyde (MDA), superoxide dismutase (SOD), GSH, and catalase were assessed in the liver homogenate using Biodiagnostic (Egypt) kits.

Histopathological and immunohistochemical evaluations

Liver tissues fixed in 10% NBF were processed for paraffin embedding. Sections (5 μm thick) were stained with hematoxylin & eosin (H&E) to examine tissue architecture. Prussian blue, periodic acid-Schiff (PAS), and Sirius red were employed to stain other sections. To assess changes in TLR-4, NF-κB p65, Cygb, TXNRD1, and HMGB1, immunohistochemical staining was employed. Briefly, paraffin-embedded sections were subjected to antigen retrieval using citrate buffer (50 mmol/L, pH 6.8). The sections were incubated in 0.3% hydrogen peroxide (H₂O₂) to block peroxidase, and a protein-blocking solution was then added before incubation with the primary antibodies (Biospes, China) overnight at 4 °C. After washing and incubation with secondary antibodies (Biospes, China), DAB was used for color development and hematoxylin was employed for counterstaining. To measure staining intensity, 6 randomly selected fields per rat, at 400 × magnification, were evaluated using ImageJ software (NIH, United States).

Molecular docking

The binding affinity of ABX to Cygb (PDB ID: IUMO), TXNRD1 (PDB: 2ZZ0), and HMGB1 (PDB ID: 1HME) was investigated through molecular docking studies. The 3D structures of the target proteins were downloaded from the RCSB Protein Data Bank and prepared using AutoDock Tools (v1.5.6) by removing water molecules, adding polar hydrogens, and assigning Kollman charges. The ABX ligand structure was retrieved from the PubChem database, converted to PDB format, and energy-minimized using the Universal Force Field in PyRx (v0.8). Docking was performed using AutoDock Vina integrated in PyRx (v0.8)[30], where the grid boxes were centered on the active or binding site of each protein, as identified from literature or based on co-crystallized ligand positions when available. The docking results were ranked by binding affinity scores. PyMOL (v2.3.2) and LigPlot+ (v2.2.8)[31] were employed to visualize interactions and binding modes.

Statistical analysis

All results are presented as mean ± standard deviation (SD). Intergroup comparisons were carried out using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism (version 8). A P value < 0.05 was considered significant.

RESULTS

ABX mitigates CP-induced liver damage

The hepatoprotective effects of ABX against toxicity induced by CP were evaluated through biochemical (Figure 1) and histopathological examinations (Figure 2). CP significantly increased circulating ALT (67.83 ± 9.77 U/L; Figure 1A), AST (154.4 ± 21.48 U/L; Figure 1B), and ALP (445.6 ± 63.78 U/L; Figure 1C), along with a notable reduction in albumin levels (1.06 ± 0.18 g/dL; Figure 1D) compared to the control group [P < 0.001; ALT (17.30 ± 2.09 U/L), AST (41.60 ± 3.32 U/L), ALP (89.75 ± 12.98 U/L) and albumin (2.39 ± 0.22 g/dL)]. ABX significantly mitigated these changes in hepatic biomarkers [P < 0.001; ALT (38.79 ± 7.22 U/L), AST (82.72 ± 11.10 U/L), ALP (239.1 ± 36.67 U/L) and albumin (1.89 ± 0.26 g/dL)].

Figure 1 Ambroxol ameliorated liver function markers in cyclophosphamide-administered rats. A: Serum alanine aminotransferase; B: Aspartate aminotransferase; C: Alkaline phosphatase; D: Albumin. Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group. ^fP < 0.001 vs cyclophosphamide. ABX: Ambroxol; CP: Cyclophosphamide; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase.

Figure 2 Ambroxol prevented liver tissue injury in cyclophosphamide-administered rats. A-D: Photomicrographs of hematoxylin & eosin-stained sections from the control (A and B) and ABX-supplemented rats (C and D) showing normal liver architecture, including hepatocytes (arrowheads), hepatic lamina (L), central veins (arrows), and sinusoids (S); E-H: Cyclophosphamide-treated rats showing congested vessels (arrows), hepatocyte vacuolation (black arrowheads), enlarged hepatocytes with enlarged nuclei (white arrowheads), inflammatory cells (IC) infiltration, dilated sinusoids (S), and disorganized hepatic laminae (L); and I and J: ABX treatment prevented tissue injury and hepatic tissues relatively appear as control group showing normal non-congested vessels (arrows), hepatocytes (arrowheads), hepatic laminae (L), and sinusoids (S). A, C, E, and I: 100 × and scale bar = 200 µm; B, D, F, G, H and J: 400 × and scale bar = 50 µm. ABX: Ambroxol; CP: Cyclophosphamide; S: Sinusoids; L: Laminae; IC: Inflammatory cells.

Histopathological examination of liver tissues of control (Figure 2A and B) and ABX-supplemented rats (Figure 2C and D) revealed normal hepatic architecture, including intact hepatocytes, sinusoids, and central veins. CP administration caused severe hepatic injury, characterized by congested vessels, hepatocyte vacuolation, enlarged hepatocytes with enlarged nuclei, inflammatory cells infiltration, dilated sinusoids, and disorganized hepatic laminae (Figure 2E-H). ABX noticeably alleviated these pathological alterations (Figure 2I and J). In addition, the liver of control and ABX-supplemented rats revealed normal collagen and mucopolysaccharides (MPS), and no iron deposition (Figure 3). In contrast, accumulation of collagen, MPS, and iron were observed in CP-administered rats; effects that were reversed by ABX.

Figure 3 Ambroxol prevented cyclophosphamide-induced collagen, mucopolysaccharides, and iron deposition in rat liver. Photomicrographs of Sirius red-, periodic acid-Schiff (PAS)- and Prussian blue-stained liver sections. Sirius red staining shows few collagen fibers (arrows) in hepatic parenchyma around the central vein in control and ambroxol (ABX)-treated rats, increased collagen fibers (arrows) in cyclophosphamide (CP)-administered rats and normal collagen fiber (arrows) content in CP-administered rats treated with ABX. The liver of control and ABX-treated rats shows normal PAS stain intensity and distribution (arrows), whereas CP-administered rats show an increase in the PAS stain intensity (arrows). ABX treatment decreased PAS staining in CP-administered rats. Control and ABX-supplemented rats show negative Prussian blue staining affinity, CP-administered rats show hemosiderin deposits (arrows), and CP-administered rats treated with ABX show few hemosiderin deposits (arrows). (400 × and scale bar = 50 µm). ABX: Ambroxol; CP: Cyclophosphamide; PAS: Periodic acid-Schiff.

ABX attenuates CP-mediated hepatic oxidative stress

CP remarkably elevated liver MDA (59.11 ± 9.76 nmol/100 mg; Figure 4A) and led to a significant depletion of GSH (9.68 ± 1.98 nmol/100 mg), SOD (4.48 ± 0.79 U/g), and catalase (5.14 ± 0.51 U/g) in the liver (Figure 4B-D) as compared to the control group [MDA (20.65 ± 3.72 nmol/100 mg), GSH (19.01 ± 3.24 nmol/100 mg), SOD (10.52 ± 1.26 U/g), and catalase (12.71 ± 1.55 U/g)]. Treatment with ABX significantly reduced MDA (31.68 ± 5.75 nmol/100 mg) while restoring GSH (18.71 ± 4.39 nmol/100 mg) and SOD (9.41 ± 1.15 U/g) and catalase (10.54 ± 1.31 U/g) activities (P < 0.001).

Figure 4 Ambroxol attenuated cyclophosphamide-induced oxidative stress. A-D: Ambroxol decreased hepatic malondialdehyde (A), and increased reduced glutathione (B), superoxide dismutase (C), and catalase (D) in cyclophosphamide (CP)-administered rats. Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP. MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Reduced glutathione; ABX: Ambroxol; CP: Cyclophosphamide.

ABX downregulates TLR-4/NF-κB signaling and suppresses inflammation in CP-treated rats

CP administration significantly upregulated TLR-4 and NF-κB p65 expression in the liver (Figure 5A-C), and increased hepatic levels of TNF-α (Figure 5D), IL-1β (Figure 5E). ABX supplementation effectively suppressed hepatic TLR-4, NF-κB p65, TNF-α, IL-1β levels in CP-administered rats.

Figure 5 Ambroxol suppressed inflammation in cyclophosphamide-treated rats. A-C: Ambroxol decreased liver toll-like receptor-4 (A and B), nuclear factor-kappaB (NF-κB) p65 (A and C); D: Tumor necrosis factor-α; E: Interleukin-1β. Data are expressed as mean ± SD (n = 6). ^bP < 0.01 and ^cP < 0.001 vs control group, ^eP < 0.01 and ^fP < 0.001 vs cyclophosphamide. ABX: Ambroxol; CP: Cyclophosphamide; TLR: Toll-like receptor; NF-κB: Nuclear factor-kappa B; TNF: Tumor necrosis factor; IL: Interleukin.

Modulation of Cygb, TXNRD1, and HMGB1 in CP-administered rats by ABX

CP administration significantly downregulated Cygb (Figure 6A and B), TXNRD1 (Figure 7A and B) and HMGB1 (Figure 8A and B) protein levels in the liver of rats. ABX enhanced hepatic Cygb, TXNRD1, and HMGB1 in CP-intoxicated rats (P < 0.001). In silico results showed the binding of ABX with Cygb (Figure 6C), TXNRD1 (Figure 7C) and HMGB1 (Figure 8C) with energies of -5.1, -6.6 and -4.9 kcal/mol, respectively (Table 1). The analysis revealed 4, 7, and 4 residues mediating the hydrophobic interaction of ABX with Cygb, TXNRD1, and HMGB1, respectively. Polar bonding of ABX with TXNRD1 and HMGB1 involved 2 and 3 residues, respectively (Table 1).

Figure 6 Ambroxol upregulated cytoglobin in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) enhanced the expression of cyctoglobin in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^cP < 0.001 vs control group and ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and cytoglobin. ABX: Ambroxol; CP: Cyclophosphamide.

Figure 7 Ambroxol increased thioredoxin reductase 1 in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) upregulated the expression of thioredoxin reductase 1 (TXNRD1) in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^aP < 0.05 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and TXNRD1. ABX: Ambroxol; CP: Cyclophosphamide; TXNRD1: Thioredoxin reductase 1.

Figure 8 Ambroxol upregulated high-mobility group box 1 in the liver of cyclophosphamide-administered rats. A and B: Ambroxol (ABX) increased the expression of high-mobility group box 1 (HMGB1) in the liver of rats that received cyclophosphamide (CP). Data are expressed as mean ± SD (n = 6). ^bP < 0.01 and ^cP < 0.001 vs control group, ^fP < 0.001 vs CP; C: Molecular docking showing the interaction between ABX and HMGB1. ABX: Ambroxol; CP: Cyclophosphamide; HMGB1: High-mobility group box 1.

Table 1 Binding affinities and interaction of ambroxol with different protein targets.

	Lowest binding energy (kcal/mol)	Polar bonds	Hydrophobic interactions
Cytoglobin	-5.1		Val47, Val51, Arg48, Pro69
TXNRD1	-6.6	Glu477, Thr480	Val474, Cys475, Ile347, Pro344, Thr481, Val478, Phe406
HMGB1	-4.9	Ser12, Ala13, Asn46	Lys39, Gly42, Ala38, Phe14

TXNRD1: Thioredoxin reductase 1; HMGB1: High-mobility group box 1.

DISCUSSION

The use of CP in chemotherapy is associated with significant hepatotoxicity, which limits its clinical application and poses a serious health risk to patients. Oxidative stress and inflammatory responses have been implicated in CP hepatotoxicity[5,10,11], representing a potential therapeutic target. Despite extensive research efforts dedicated to understanding CP-induced hepatotoxicity, the available preventive measures remain insufficient, highlighting the urgent necessity for innovative strategies to mitigate liver damage. This study investigated the effects of ABX against CP hepatotoxicity, pinpointing oxidative stress, inflammation, and the expression of Cygb, TXNRD1, and HMGB1.

CP-induced hepatotoxicity was evident from the significant elevation in liver function markers, ALT, AST, and ALP, alongside a reduction in albumin levels. These findings are consistent with our previous studies showing elevated serum transaminases and declined albumin in CP-treated rats[5,10,11]. These biomarkers are indicative of hepatocellular damage and impaired liver synthetic function. ALT and AST are released into the bloodstream upon hepatocyte membrane disruption, while ALP elevation reflects bile duct injury[32]. The reduction in albumin, a protein synthesized by the liver, further underscores the impairment of hepatic synthetic function. Histopathological examinations corroborated these findings, revealing congested blood vessels, hepatocyte vacuolation, enlarged hepatocytes with enlarged nuclei, inflammatory cells infiltration, dilated sinusoids, and disorganized hepatic laminae. Additionally, Sirius red staining revealed remarkable accumulation of collagen fibers and increased PAS staining revealed an increase in MPS following CP administration. An increase in PAS-positive material in the liver may indicate the accumulation of these substances, which can be associated with pathological conditions, such as altered metabolic processes or cellular stress responses, potentially leading to or indicating liver damage[33]. Excessive accumulation of MPS may disrupt normal liver function, contributing to hepatocyte injury, inflammation, or fibrosis[33]. Similarly, the increased Prussian blue staining points to increased iron deposition, which may reflect disrupted iron homeostasis or oxidative damage in the liver[34]. These findings collectively indicate that CP leads to pronounced metabolic disruptions and structural damage in the liver. ABX substantially countered these effects, alleviating liver function indicators and enhancing hepatic tissue integrity. The observed decreases in collagen, iron, and MPS suggest that ABX not only mitigates acute hepatocellular injury but also prevents chronic liver damage. The hepatoprotective efficacy of ABX has been highlighted in studies on a rat model of I/R-liver injury authored by Jiang et al[27] and Gultekin et al[28]. In both studies, ABX effectively restored serum transaminases and suppressed inflammation in rats exposed to hepatic I/R[27,28]. In rats challenged with cisplatin, ABX conferred hepatoprotective effect evidenced by ameliorated serum transaminases and pro-inflammatory mediators[35]. Our findings support the hepatoprotective efficacy of ABX and highlight its potential as a therapeutic agent to counteract CP-induced hepatotoxicity, preserving liver function and structure.

ABX has been shown to effectively reduce hepatic oxidative and inflammatory responses in preclinical liver injury models, including those induced by I/R and cisplatin[27,28,35]. Given the role of oxidative and inflammatory responses in CP hepatotoxicity[5,10,11], the hepatoprotective effect of ABX could be attributed to attenuation of these processes. Here, CP increased MDA, TLR-4, NF-κB, TNF-α, and IL-1β, and suppressed SOD, catalase and GSH, demonstrating redox imbalance and inflammation in the liver of rats. Oxidative stress is a central mechanism underlying CP-induced hepatotoxicity. CP metabolism generates ROS and acrolein, leading to the depletion of antioxidants and triggering LPO[9], as evidenced by elevated MDA levels. The reduction of GSH, a critical intracellular antioxidant, disrupts redox homeostasis, rendering hepatocytes vulnerable to oxidative damage[36]. The reduction in antioxidant enzymes, including SOD and catalase, further exacerbates oxidative stress[37]. SOD converts superoxide radicals into H₂O₂ and catalase decomposes it into water and oxygen. The decline in these enzymes impairs the cellular defense system[37], leading to the accumulation of ROS and subsequent cellular damage. Excess ROS induced by CP can inflict significant cellular damage through mechanisms such as LPO, protein oxidation, depletion of endogenous antioxidants, and DNA impairment. LPO compromises membrane integrity by altering permeability and fluidity and deactivating proteins, ultimately resulting in cell damage[38]. ROS can induce conformational changes in structural proteins and modify the catalytic sites of protective enzymes, thereby disrupting cellular homeostasis and exacerbating oxidative stress-induced damage[38]. Furthermore, excess ROS can activate inflammatory signaling molecules, resulting in an inflammatory response. Accordingly, ROS can activate TLR-4/NF-κB signaling, resulting in the transcriptional upregulation and release of pro-inflammatory mediators, thereby amplifying the inflammatory response[14,15], effects that were reported in the current study. Activation of the TLR-4/NF-κB signaling pathway is a key contributor to several liver disorders, including NAFLD[39]. The released cytokines (TNF-α and IL-1β) upon activation of this signaling pathway perpetuate hepatic inflammation and damage by attracting immune cells, enhancing ROS generation, and driving fibrogenesis[40]. Elevated levels of ROS and pro-inflammatory mediators contribute to mitochondrial dysfunction and the induction of apoptotic pathways. Excessive ROS destabilize the mitochondrial membrane potential, enhancing its permeability and triggering the release of cytochrome c which then associates with Apaf-1 and caspase-9 to assemble the apoptosome. Subsequently, caspase-3 is activated to trigger the execution stage of apoptosis[41].

ABX treatment significantly reduced MDA levels and restored GSH, SOD, and catalase, indicating its potent antioxidant properties. ABX alleviates oxidative stress by limiting LPO and boosting antioxidants, thereby protecting cells from damage. Furthermore, ABX downregulated TLR-4 and NF-κB, resulting in decreased production of pro-inflammatory cytokines. This anti-inflammatory response appears to be associated with the suppression of ROS formation. By suppressing the TLR-4/NF-κB signaling cascade, ABX attenuates hepatic inflammation and prevents further tissue damage. The reduction in inflammatory cell infiltration and fibrogenesis observed in histopathological examinations further supports the anti-inflammatory effects of ABX. The suppressive efficacy of ABX on oxidative and inflammatory responses is supported by investigations on experimental models of I/R-liver injury[27,28] and cisplatin hepatotoxicity[35]. In these models, ABX conferred protective effects associated with suppression of LPO and pro-inflammatory cytokines. To further investigate the role of ABX in mitigating CP-induced inflammation and oxidative stress, we evaluated changes in key proteins involved in the regulation of redox homeostasis and inflammation. The results showed upregulation of Cygb, TXNRD1, and HMGB1 in the liver of CP-intoxicated rats following treatment with ABX. Cygb is ubiquitously expressed in various tissues, including the liver, where it serves a critical protective function against oxidative stress[16]. As a potent scavenger of ROS, Cygb helps mitigate the damaging effects of free radicals, which are often elevated during DILI. By neutralizing ROS, Cygb contributes to the preservation of cellular redox homeostasis, preventing oxidative damage to lipids, proteins, and DNA[16]. This protective mechanism is particularly important in the liver, as it is a primary site for drug metabolism and is highly susceptible to oxidative stress-induced damage. Furthermore, emerging evidence suggests that Cygb may also play a role in regulating cellular signaling pathways associated with inflammation and apoptosis, further enhancing its hepatoprotective effects[16]. TXNRD1 is a critical selenoprotein that plays a central role in maintaining cellular redox homeostasis[17,42,43]. It functions by catalyzing the reduction of oxidized proteins and scavenging ROS, thereby protecting cells from oxidative damage. Beyond its antioxidant properties, TXNRD1 is also involved in the regulation of key signaling pathways, including the modulation of NF-κB activity[17,42,43]. By influencing NF-κB signaling, TXNRD1 exerts control over inflammatory responses, which are often implicated in tissue injury and disease progression. This dual role in redox regulation and inflammation underscores the importance of TXNRD1 in cellular defense mechanisms, particularly in contexts such as DILI, where oxidative stress and inflammation are major contributors to tissue damage. HMGB1 is a multifunctional non-histone nuclear protein that exhibits distinct roles based on its cellular localization[18,19]. Within the cell, HMGB1 is primarily located in the nucleus, where it plays a critical role in maintaining genomic stability by facilitating DNA repair processes, regulating transcription, and modulating autophagy[18,19]. These intracellular functions are essential for cellular homeostasis and survival under stress conditions. ABX upregulated the expression of Cygb, TXNRD1, and HMGB1, enhancing cellular antioxidant defenses and reducing oxidative stress and inflammation. These findings pinpoint the role of Cygb, TXNRD1, and HMGB1 in hepatoprotective effects of ABX. To further support these findings, we explored the in silico binding affinity of ABX towards Cygb, TXNRD1, and HMGB1. The results revealed the binding affinities between ABX and Cygb, TXNRD1, and HMGB1, mediated through hydrogen bonding and hydrophobic interactions. ABX formed stable complexes with these proteins, suggesting direct interactions that may enhance their functional activities. The binding of ABX to Cygb and TXNRD1 likely enhances their antioxidant capacities, while its interaction with HMGB1 may prevent its extracellular release and subsequent activation of TLR-4. These findings provide mechanistic insights into the hepatoprotective effects of ABX and highlight its potential as a therapeutic agent for mitigating CP-induced liver injury. Although this study introduces novel information on the hepatoprotective efficacy of ABX, its preclinical nature, employment of a single ABX dose, and the absence of long-term outcome results might be considered as limitations. Mechanistic validation using gene knockout or overexpression models, which would help to confirm the specific roles of Cygb, TXNRD1, and HMGB1 in mediating the hepatoprotective effects of ABX could be a valuable direction of future studies.

CONCLUSION

This study demonstrates that the protective efficacy of ABX against CP hepatotoxicity is mediated by ameliorating oxidative stress, inflammation, and histopathological alterations. ABX upregulates Cygb, TXNRD1, and HMGB1, enhances antioxidant defenses, and suppresses the TLR-4/NF-κB signaling cascade and reducing pro-inflammatory cytokine production. Molecular docking studies further support these findings, revealing the binding affinities between ABX and Cygb, TXNRD1, and HMGB1 through hydrogen bonding and hydrophobic interactions, suggesting a potential direct modulation of these targets. These results suggest that ABX has the potential to be employed as an adjunctive therapy to mitigate CP-induced hepatotoxicity, thereby improving the outcomes of cancer chemotherapy. Further studies are required to validate the hepatoprotective role of ABX in clinical settings and investigate its full therapeutic potential in DILI.

ACKNOWLEDGEMENTS

Princess Nourah bint Abdulrahman University Researchers Supporting Project Number (PNURSP2025R381), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.