Introduction

The liver plays a crucial role in various physiological functions, such as metabolism, detoxification, and production of essential biomolecules, which are essential for maintaining homeostasis and overall health1,2. Due to its central role in processing endogenous and exogenous substances, it is particularly susceptible to damage from drugs, toxicants, and environmental agents. Drug-induced liver injury (DILI) is a leading cause of acute liver failure and contributes significantly to global morbidity and mortality. In the United States, medications are implicated in over half of the approximately 2,000 acute liver failure cases annually, with paracetamol (PCM) accounting for 39% of these cases3. Risk factors for DILI include age, sex, genetic predisposition, alcohol use, and comorbidities. Elderly individuals are at higher risk due to reduced hepatic clearance and drug interactions, while females are more susceptible to certain drugs4.

PCM is primarily metabolized in the liver through glucuronidation and sulfation, which render the drug water-soluble for renal excretion. However, a minor fraction—approximately 5%—is metabolized by the cytochrome P450 enzyme CYP2E1, resulting in the formation of a highly reactive intermediate, N-acetyl-p-benzoquinone imine (NAPQI). Under normal conditions, glutathione (GSH) conjugates and detoxifies NAPQI, rendering it harmless. In cases of paracetamol overdose, the glucuronidation and sulfation pathways become saturated, and excessive NAPQI is produced. The hepatic glutathione stores are rapidly depleted, leading to the accumulation of free NAPQI. This toxic metabolite binds covalently to essential cellular proteins, including mitochondrial proteins, initiating a cascade of oxidative stress and cellular damage. A critical consequence of this process is the uncoupling of oxidative phosphorylation within mitochondria, impairing ATP synthesis. This leads to energy failure, the development of lactic acidosis, and the pathological release of ionized calcium from mitochondrial stores. These disturbances collectively contribute to mitochondrial dysfunction, activation of cell death pathways, and ultimately hepatocellular apoptosis and necrosis5,6,7. In addition to direct cellular injury, PCM-induced hepatotoxicity triggers a sterile inflammatory response characterized by the activation of resident Kupffer cells and the recruitment of immune cells8. Damaged hepatocytes release danger-associated molecular patterns, which are recognized by pattern recognition receptors such as Toll-like receptor 4 (TLR4), leading to downstream activation of key inflammatory signaling pathways, notably the nuclear factor kappa B (NF-κB) pathway. Activation of NF-κB promotes the transcription of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, which amplify hepatic inflammation by promoting leukocyte infiltration, increasing oxidative stress, and exacerbating hepatocellular damage9. Additionally, interleukin-17 (IL-17), primarily produced by Th17 cells, contributes to neutrophil recruitment and the sustained inflammatory environment, further aggravating liver injury10. Thus, the coordinated action of TLR4-mediated signaling, NF-κB activation, and elevated cytokines including IL-17 plays a critical role in initiating and perpetuating the inflammatory cascade during PCM-induced liver damage11. Despite advances in drug discovery, the treatment of drug-induced liver injury remains a significant challenge, necessitating the development of novel hepatoprotective agents12.

Benzimidazole and pyrazole are two pharmacologically significant heterocycles that have garnered considerable attention due to their diverse and potent biological activities13. Benzimidazole, a fused bicyclic compound composed of benzene and imidazole rings14, forms a core structural motif in several clinically used drugs such as pantoprazole, omeprazole, candesartan, and mebendazole15. Its presence in biologically vital molecules like vitamin B12 and the amino acid histidine underscores its intrinsic medicinal relevance. Benzimidazole derivatives have been extensively reported to exhibit a wide range of activities including anti-inflammatory16, antihyperlipidemic17, antihypertensive18, antibacterial, antifungal19, antiviral, anti-hepatitis C virus (HCV)20, and anti-cancer effects21. Notably, benzimidazoles are also involved in modulating lipid biosynthesis and improving lipid profiles22, making them valuable in managing metabolic disorders and cardiovascular risk factors.

On the other hand, pyrazole moieties have demonstrated significant pharmacological potential, contributing to antimicrobial23, anti-ulcer24, anti-inflammatory, antitumor25, and antioxidant properties26. Their versatile biological profile has made them a focal point in medicinal chemistry, particularly when hybridized with benzimidazole structures. Benzimidazole- Pyrazole conjugates have emerged as potent agents capable of mitigating oxidative stress and inflammation, thereby showing promise in therapeutic applications ranging from cancer to gastrointestinal diseases27,28,29. Synthetic approaches such as 1,3-dipolar cycloaddition have facilitated the development of diverse pyrazole derivatives, further broadening their biomedical applications30.

In recent decades, the incorporation of fluorine into heterocyclic scaffolds has revolutionized drug design and development. Fluorine, the most electronegative element, imparts significant electronic effects due to its high polarization potential (μ C − F = 1.41 D), enhancing drug-target interactions and metabolic stability. Fluorinated compounds now constitute roughly one-quarter of all pharmaceuticals on the market31. The strategic addition of fluorine not only improves aqueous solubility—a critical parameter for drug absorption and bioavailability—but also increases pharmacokinetic and physicochemical properties such as lipophilicity, permeability, and resistance to metabolic degradation32. The rise of fluoroquinolones in the 1980s marked a turning point, firmly establishing fluorine as a cornerstone in medicinal chemistry33.

Together, the fusion of benzimidazole and pyrazole moieties, with the added benefit of fluorine substitution, creates a privileged scaffold with enhanced therapeutic potential. This hybrid framework offers a multi-targeted pharmacophore system with robust anti-inflammatory and antioxidant making it an attractive candidate for further investigation in the management of oxidative liver injuries and associated pathologies. The compound investigated in this study, 2-((1H-benzo[d]imidazol-2-yl) thio)-1-(3-(2-fluorophenyl)-5-phenyl-1H-pyrazol-1-yl) ethanone (M3F) as shown in Fig. 1, is a novel benzimidazole-pyrazole derivative with no reported pharmacological activity to date, and that a structurally similar compound has demonstrated anti-ulcer effect24. Both benzimidazole and pyrazole moieties are well documented in the literature for their antioxidant, anti-inflammatory, antifungal and anticancer activities13,21,29,34. Given this structural foundation and known pharmacophore relevance, this study intends to determine the hepatoprotective activity of M3F, using in silico and in vivo approaches while elucidating the underlying mechanisms via molecular analysis.

Fig. 1
figure 1

2D Structure of M3F (2-((1H-benzo[d]imidazol-2-yl) thio-1-(3-(2-fluorophenyl)-5-phenyl-1Hazol-1-yl) ethanone).

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Materials and methods

Chemicals

PCM, Silymarin (SIL) (Sigma-Aldrich), diaminobenzidine (DAB) (Sigma-Aldrich), sulfuric acid, paraformaldehyde, xylene, 1-Chloro-2,4-Dinitrobenzene (CDNB) and 5,5'-Dithio-bis-(2-Nitrobenzoic Acid) (DTNB) (Sigma-Aldrich, St. Louis, MO, USA). Other reagent used in this study adhered to high-purity specifications.

Animal housing

Thirty-six healthy BALB/cAnN male mice (6–8 weeks old, weighing 28–36 g), procured from the National Institute of Health, Islamabad, are housed under regulated conditions at the Department of Pharmacy, Quaid-i-Azam Universsity. The experiments were designed to minimize harm to the animals. Mice were housed in polypropylene cages under controlled conditions, including a 12-h light–dark cycle, 50–60% relative humidity and a temperature of 22 ± 3 °C, with unrestricted access to water and standard food. All methods and procedures were conducted in accordance with relevant guidelines and regulations. This study received approval from the Bioethics Committee of Quaid-i-Azam University (Reference Number # BEC-FBS-QAU2024-686).

Computational studies

ADME and toxicity predictions

Absorption, Distribution, Metabolism, and Excretion (ADME) evaluations were performed using Swiss ADME (http://www.swissadme.ch/) and ProTox software (https://tox.charite.de/protox3/index.php?site=compound_input)35. These software predicted pharmacokinetic properties and drug-likeness based on parameters like lipophilicity and molecular similarity to existing drugs, and the M3F’s drug-likeness was assessed according to Lipinski’s rule36. Toxicity classification ranged from highly toxic (LD₅₀ ≤ 50 mg/kg) to non-toxic (LD₅₀ > 5000 mg/kg)37.

Protein preparation

The 3D structures of target proteins (Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-17 (IL-17), Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB), Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), Heme Oxygenase-1 (HO-1), and Toll-Like Receptor 4 (TLR4) were retrieved from the Protein Data Bank (PDB), with corresponding PDB IDs: 1ALU, 9ILB, 2AZ5, 4HR9, 1VKX, 2FLU, 1IRM, 3VQ2, respectively. These protein structures were opened and prepared using BIOVIA Discovery Studio Visualizer 2020. Water molecules and non-essential ligands were removed, and the cleaned protein structures were saved in PDB format for docking38.

Ligand preparation

The ligand M3F, was drawn using ChemDraw and converted into 3D format. The 3D ligand file was imported into PyRx 0.8 for energy minimization and converted into the .pdbqt format for docking.

Molecular docking protocol

Molecular docking was performed using AutoDock Vina integrated with PyRx 0.8. The grid center was placed in the active site pocket center. Protein and ligands were converted to pdb.qt files. Default docking algorithms were set in conformation with the standard docking protocol. Independent docking runs were carried out for each ligand–protein complex. The docking results were evaluated using AutoDock Vina’s scoring function, which considers key interactions such as hydrogen bonding, hydrophobic contacts, and binding affinity to predict the stability and strength of ligand–protein binding38,39.

Study design

Following a one-week acclimatization period, mice were randomly divided into six groups (n = 6) and treated for eight consecutive days. For seven days, the vehicle control group received 0.9% normal saline +  < 0.1% DMSO i.p., the positive control group was administered SIL (100 mg/kg i.p.), while the experimental groups were treated with three different doses of M3F (100, 200, and 400 µg/kg i.p.). To enhance and standardize PCM-induced liver injury, mice were fasted overnight (~ 12–16 h) prior to PCM administration40,41. The next day (day 8), PCM was administered as a single dose of 300 mg/kg i.p. to all groups except the vehicle control, which received 0.9% saline +  < 0.1% DMSO i.p. only42. The schematic representation of the study design is shown in Fig. 2.

Fig. 2
figure 2

Schematic representation of hepatoprotective model plan created using Microsoft PowerPoint. The visual elements (such as images of mice, liver, etc.) were sourced from Google Images for illustrative purposes.

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Sample collection

Twenty-four hours post-PCM administration, animals were deeply anesthetized using a combination of xylazine (30 mg/kg body weight; 0.15 ml/100 g) and ketamine (300 mg/kg body weight; 0.3 ml/100 g) i.p. to induce deep anesthesia, followed by cervical dislocation to ensure humane euthanasia, in accordance with institutional ethical guidelines43. Blood samples were collected via heart puncture and analyzed for complete blood cell count. The blood was left undisturbed at room temperature for 60 min to allow clot formation, followed by centrifugation at 120 × g for 5 min at low speed to obtain the straw-colored serum. One portion of liver is excised, washed with saline, and fixed in 10% formalin. Remaining portion is used to prepare homogenate by centrifuging liver tissues at 1000 × g for 10 min at 4 °C. The supernatant was collected after centrifugation and was used for biochemical assays.

Biochemical examination

Serum was used to study markers of liver damage, including Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), total protein and bilirubin were determined as mentioned by44. Additionally, the lipid profile, including Triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and high-density lipoprotein (HDL), applying the technique of45; while the Atherogenic Index of Plasma (AIP) was expressed as log (TG/HDL-C), and the Coronary Risk Index (CRI) was determined using the ratio TC/HDL-C46. Complete blood count was performed as described by47 and serum electrolyte levels, including sodium, potassium, and bicarbonate, were determined as described by43. These parameters have previously been reported to reflect systemic effects and secondary metabolic disturbances associated with acetaminophen-induced hepatotoxicity, as evident from the previous literature48,49,50.

Antioxidant assay

The liver homogenate supernatant was analyzed for catalase (CAT), glutathione (GSH), glutathione-S-transferase (GST), and nitric oxide (NO) levels following the method described by51,52.

Liver weight to body weight assessment

To evaluate the hepatoprotective effects of the M3F, liver weight was determined and presented as a proportion of total body weight, serving as a marker for liver hypertrophy or atrophy, as reported in earlier findings48,49,50.

Histopathological analysis

Isolated tissue samples were fixed in a 10% formalin solution, followed by dehydration and embedding in paraffin blocks. Thin sections of 5 µm were prepared and stained with hematoxylin and eosin (H&E). Histopathological alterations among the experimental groups were examined under a light microscope. Microscopic slides were examined, and images were captured using a microscope (Olympus CX41). To minimize experimenter bias during image collection and analysis, the experimenter was blinded to the treatment groups throughout all phases of the study53,54.

Pro-inflammatory cytokines

ELISA was conducted in accordance with the instructions provided by the manufacturer. Standards and liver supernatants were added to pre-coated 96-well plates, followed by incubation with biotinylated antibodies and streptavidin-HRP. After adding TMB substrate, the reaction was stopped with sulfuric acid, and optical density was read at 450 nm. Cytokine levels (IL-6, IL-1β, and TNF-α) were quantified by analyzing all samples in triplicate47, as supported by prior literature55.

Real-time polymerase chain reaction

RNA extraction protocol involved processing the sample with RNA Digestion Buffer and Proteinase K at 55 °C for 15 min, followed by centrifugation. The supernatant was mixed with ethanol, loaded onto a HiPure RNA Mini Column, and centrifuged to bind RNA. The column was washed with Buffer RNA Wash Concentrate (RWC), treated with DNase I for 15 min to remove DNA, and washed again with Buffer RWC. Two additional washes with Buffer RW2 were performed, and the column was dried by centrifugation. RNA was eluted by adding RNase-free water to the membrane, incubating for 2 min, and centrifuging. Extracted RNA was kept at − 80 °C, with an optional re-elution step for higher yields. Each reaction was conducted in triplicate to maintain reliability and precision. Following expression analysis, the 2−ΔΔCT method was applied56. Primer sequences are listed in Table 1.

Table 1 Primers for real-time PCR analysis.
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Statistical evaluation

Data was analysed using SPSS version 27 for Windows (IBM Corp, Armonk, NY, USA) to compute the means. Data were presented as mean ± SEM. One-way ANOVA followed by the Least Significant Difference (LSD), a post-hoc test was used to determine statistically significant differences between group means. This test was used to assess statistical significance, with p-values less than 0.05 considered significant.

Results

In silico analysis

Drug-likeness

M3F exhibits a molecular weight of 428.48 g/mol, a topological polar surface area of 88.87 Å2, one hydrogen bond donor, and four hydrogen bond acceptors, as shown in Fig. 3.

Fig. 3
figure 3

M3F drug likeness and pharmacokinetic properties.

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Toxicity prediction

M3F exhibits an LD₅₀ value of 1000 mg/kg (Fig. 4).

Fig. 4
figure 4

Evaluation of M3F toxicity by ProTox.

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Molecular docking

Molecular docking studies revealed strong binding affinities ( − 7.1 to − 11.2 kcal/mol) of M3F to key inflammatory and oxidative stress-related targets. The binding affinity followed the order: TLR4 > HO-1 > NF-κB > TNF-α > IL-17 > IL-6 > IL-1β > Nrf2. Detailed interactions, including binding energy, hydrogen bonding, and hydrophobic interactions, are summarized in Table 2. The 3D interactions of M3F with target proteins are visualized in Fig. 5.

Table 2 Binding affinities and interaction profiles of M3F with target proteins from docking studies.
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Fig. 5
figure 5

3D interaction of M3F against selected macromolecule IL-6 (a), IL-1β (b), TNF-α (c) IL-17 (d), NF-κB (e), Nrf2 (f), HO-1 (g) and TLR4 (h) by PyRx.

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Liver biochemistry

Vehicle control group showed ALT, AST, ALP, total protein and bilirubin levels as 37 u/l, 85 u/l, 108 u/l, 4.8 g/dl and 0.24 mg/dl. PCM significantly altered levels as 1796 u/l, 2439 u/l, 438 u/l, 9.6 g/dl and 0.95 mg/dl. SIL 100 mg/kg + PCM group showed values of 828 u/l, 1676 u/l, 304 u/l, 5.06 g/dl, and 0.52 mg/dl. At a dose of 100 µg/kg + PCM the values were 1463 u/l, 1884 u/l, 285 u/l, 6.4 g/dl, and 0.64 mg/dl, respectively. At a dose of 200 µg/kg + PCM the values were recorded as 1428 u/l, 1371 u/l, 259 u/l, 5.48 g/dl, and 0.54 mg/dl, respectively. M3F 400 µg/kg + PCM) showed values of 278 u/l, 326 u/l, 118 u/l, 5.2 g/dl, 0.43 mg/dl (Fig. 6).

Fig. 6
figure 6

Effect of M3F on ALT (a), AST (b), ALP (c), Total Protein (d), and Total Bilirubin (e) levels in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where M3F, test drug; PCM, Paracetamol; SIL, Silymarin.

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Lipid profile

In the vehicle control group, TG, TC, LDL, VLDL, HDL, CRI and AIP levels are 110.12 mg/dl, 110.10 mg/dl, 33.53 mg/dl, 23.19 mg/dl, 61.42 mg/dl, 2.09 and 0.31, respectively. PCM significantly altered the lipid profile as 171.57 mg/dl, 187.44 mg/dl, 126.15 mg/dl, 34.06 mg/dl, 27.15 mg/dl, 7.46 and 0.81, respectively. SIL 100 mg/kg + PCM showed values of 129.44 mg/dl, 139.40 mg/dl, 72.20 mg/dl, 26.06 mg/dl, 41.88 mg/dl, 3.4 and 0.49, respectively. In the M3F 100 µg/kg + PCM group, the values were 139.06 mg/dl, 149.88 mg/dl, 86.0 mg/dl, 27.63 mg/dl, 36.44 mg/dl, 4.36 and 0.59, respectively. The M3F 200 µg/kg + PCM group showed values of 129.62 mg/dl, 131.42 mg/dl, 60.8 mg/dl, 27.51 mg/dl, 43.62 mg/dl, 3.10 and 0.47, respectively. The M3F 400 µg/kg + PCM group exhibited the most significant changes, with values of 119.19 mg/dl, 122.98 mg/dl, 40.8 mg/dl, 24.17 mg/dl, 58.16 mg/dl, 2.1 and 0.3, respectively, as demonstrated in Fig. 7.

Fig. 7 
figure 7

Effect of M3F on Lipid Profile: TG (a), TC (b), Low Density Lipoprotein LDL (c), very low-density lipoprotein VLDL (d), high-density lipoprotein HDL (e), Coronary Risk Index (f), and Atherogenic Index (g) levels in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where HDL, high-density lipoprotein; LDL, Low Density Lipoprotein; M3F, test drug; PCM, Paracetamol; SIL, Silymarin; TC, Total Cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein.

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Hematological parameters

In the vehicle control group, hematological parameters including RBC, Hb, platelet and WBC are: 10.90 ± 0.79 × 106/μl, 15.79 ± 0.41 g/dl, 901.2 ± 25.97 × 103/μl and 20 ± 1.30 × 103/μl, respectively. PCM significantly altered values as 6.82 ± 0.58 × 106/μl, 11.27 ± 0.61 g/dl, 598.2 ± 32.65 × 103/μl, and 29.2 ± 0.86 × 103/μl, respectively. SIL 100 mg/kg + PCM group has shown values as follows: 10.78 ± 0.58 × 106/μl, 14.77 ± 0.89 g/dl, 784.6 ± 40.24 × 103/μl, and 22.2 ± 1.46 × 103/μl, respectively. Treatment with M3F (100, 200, and 400 µg/kg) + PCM showed a dose-dependent improvement. At 100 µg/kg, the values are 9.31 ± 0.70 × 106/μl, 14.09 ± 0.62 g/dl, 648.6 ± 27.42 × 103/μl and 23.2 ± 1.56 × 103/μl, respectively. At M3F 200 µg/kg, the values are 10.60 ± 0.55 × 106/μl, 15.14 ± 0.84 g/dl, 757.8 ± 32.88 × 103/μl and 22 ± 2 × 103/μl, respectively. The high-dose M3F (400 µg/kg) demonstrated significant results as 10.43 ± 0.91 × 106/μl, 15.80 ± 0.64 g/dl, 796.6 ± 57.08 × 103/μl, and 21.8 ± 1.46 × 103/μl, respectively, as depicted in Table 3.

Table 3 Effect of M3F on hematological parameters in PCM-treated mice.
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Serum electrolytes

Serum electrolytes such as sodium, potassium and bicarbonate levels are shown in Table 4.

Table 4 Effect of M3F on electrolyte profile in PCM-treated mice.
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Antioxidant markers

Vehicle control group showed CAT, GSH, GST and NO levels as 29.56 mmol/L/min/0.1 g tissue, 61.61 mmol/L/min/0.1 g tissue, 70.71 mmol/L/min/0.1 g tissue and 93.06 nmol/g of the liver tissue, respectively. PCM group showed values as 9.49 mmol/L/min/0.1 g tissue, 17.72 mmol/L/min/0.1 g tissue, 23.20 mmol/L/min/0.1 g tissue and 145.46 nmol/g of the liver tissue, respectively. SIL 100 mg/kg + PCM group showed values of 23.27 mmol/L/min/0.1 g tissue, 60.75 mmol/L/min/0.1 g tissue, 62.93 mmol/L/min/0.1 g tissue, and 128.8 nmol/g of the liver tissue, respectively. However, M3F (100 µg/kg) + PCM group showed values as 19.60 mmol/L/min/0.1 g tissue, 45.29 mmol/L/min/0.1 g tissue, 49.41 mmol/L/min/0.1 g tissue and 133.33 nmol/g of the liver tissue, respectively. M3F (200 µg/kg) + PCM group, as 20.88 mmol/L/min/0.1 g tissue, 52.19 mmol/L/min/0.1 g tissue, 55.12 mmol/L/min/0.1 g tissue and 133.46 nmol/g of the tissue, respectively, and M3F (400 µg/kg) + PCM group, as 25.65 mmol/L/min/0.1 g tissue, 54.87 mmol/L/min/0.1 g tissue, 68.01 mmol/L/min/0.1 g tissue and 127.20 nmol/g of the liver tissue, respectively, as shown in Fig. 8.

Fig. 8 
figure 8

Effect of M3F on Antioxidant Activity. Catalase (a), GSH (b), GST (c), and NO (d) activity in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where GSH, Glutathione; GST, Glutathione S-transferase; M3F, test drug; NO, Nitric Oxide; PCM, Paracetamol; SIL, Silymarin.

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Liver weight to body weight variation

In the vehicle control group, the liver weight to body weight percentage was noted as 2.86% which is significantly increased in PCM group as 5.94%. In the SIL 100 mg/kg + PCM group, the percentage was reduced to 4.11%. Treatment with M3F (100, 200, and 400 µg/kg) showed decreased liver weight to body weight percentage as 4.63%, 4.41%, and 3.93%, respectively, as presented in Fig. 9.

Fig. 9 
figure 9

Effect of M3F on Percent change in Liver Weight/Body Weight in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where M3F, test drug; PCM, Paracetamol; SIL, Silymarin.

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Histopathological findings

Vehicle control group displayed normal liver architecture. In contrast, PCM (300 mg/kg) induced severe hepatic damage, including widespread degeneration, hepatocyte necrosis, hemorrhage, and neutrophil infiltration. Treatment with M3F (100, 200, and 400 µg/kg) reduced necrosis, restored structural integrity, and promoted hepatocyte regeneration, with the highest dose yielding the most significant improvements, as illustrated in Fig. 10.

Fig. 10
figure 10

Histopathological Examination: H&E Staining of Liver Tissue. (a) Vehicle Control group shows the normal histological structure (b) PCM group shows extensive cellular damage with hemorrhage (red arrow), and neutrophil infiltration (white arrow). (c) Silymarin-treated group shows partial restoration of hepatic architecture. (d, e, f) M3F treatment at 100 µg/kg, 200 µg/kg, and 400 µg/kg, respectively demonstrates hepatocyte regeneration (grey arrows). Where H&E, Hematoxylin and Eosin; M3F, Test Drug; PCM, Paracetamol.

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ELISA assessment

IL-6, IL-1β and TNF-α levels in vehicle control group were 21%, 19%, and 12% respectively. The pro‐inflammatory cytokine showed a significant increase in the PCM group, however SIL 100 mg/kg + PCM group has inflammatory cytokine levels of 74%, 68% and 62%, respectively. Treatment with M3F 100 µg/kg + PCM further lowered the levels to 80%, 78%, and 67%, respectively. The M3F 200 µg/kg + PCM group demonstrated greater reductions to 64%, 67%, and 55%, respectively. Notably, the M3F 400 µg/kg + PCM group achieved the most significant decrease, with levels dropping to 42%, 44%, and 40%, respectively, as illustrated in Fig. 11.

Fig. 11
figure 11

Effect of M3F on Pro-inflammatory Cytokine Analysis by ELISA. IL-6% (a), IL-1β % (b), and TNF-α % (c) in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where IL-1β, Interleukin-1 beta; IL-6, Interleukin-6; M3F, test drug; PCM, Paracetamol; SIL, Silymarin; TNF-α, Tumor Necrosis Factor-alpha.

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PCR analysis

PCM significantly increased IL-17, NF-κB, Nrf2, HO-1 and TLR4 expression (3.08, 2.55, 0.52, 0.61, and 1.99). Treatment with M3F (M3F 400 µg/kg + PCM group) resulted in a significant reduction in IL-17, NF-κB and TLR4 expression (1.61, 1.60 and 1.49), and increase in Nrf2 and HO-1 expression (1.46 and 1.50) (Fig. 12).

Fig. 12
figure 12

Effect of M3F on PCR Analysis. Gene expression of IL-17 (a), NF-κB (b), Nrf2 (c), HO-1 (d) and TLR4 (e) in PCM-treated mice. Each value represents mean ± S.E.M (n = 5), analyzed by One Way ANOVA using the LSD test for multiple comparisons. Level of significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 compared to PCM group and ###p < 0.001 compared to vehicle control group. Mice received a 7-day pretreatment with vehicle, silymarin, and M3F in respective groups. On day 8, hepatotoxicity was induced with PCM in all groups except vehicle control, and samples were collected 24 h post-PCM. Where IL-17 Interleukin-17 (a); M3F, test drug; Nrf2 Nuclear Factor Erythroid 2-Related Factor 2; NF-κB Nuclear Factor Kappa B, (b); PCM, Paracetamol; SIL, Silymarin.

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Discussion

PCM is a widely used non-prescription analgesic and antipyretic agent; however, excessive consumption is a major contributor to acute liver failure globally57. The hepatotoxic effects of PCM arise from its metabolic conversion by cytochrome P450 enzymes into NAPQI, a highly reactive intermediate. Excessive NAPQI depletes hepatic glutathione reserves, triggering oxidative stress, lipid peroxidation, mitochondrial dysfunction, and subsequent hepatocyte necrosis58. Therapeutic interventions, such as antioxidant supplementation and anti-inflammatory agents, work by replenishing glutathione levels, scavenging harmful oxygen radicals and suppressing inflammatory pathways to mitigate PCM-induced hepatotoxicity. This study examined the hepatoprotective effect of M3F in vivo, with a focus on its modulation of NF-κB and Nrf2 signaling pathways.

ADME analysis of M3F revealed favorable pharmacokinetic properties, including efficient absorption, distribution, metabolism, and excretion, along with high lipophilicity, suggesting its potential for effective systemic delivery. Additionally, M3F complies with Lipinski’s rule of five and exhibits a high LD₅₀ value (1000 mg/kg), indicating good drug-likeness and low toxicity (Class 4)59. Structural stability was confirmed with the 3D conformation ensuring optimal functional group arrangement for biological interactions, essential for docking studies. Molecular docking revealed strong binding affinities ( − 7.1 to − 11.2 kcal/mol) to inflammatory and oxidative stress-related targets in the following order: TLR4 > HO-1 > NF-κB > TNF-α > IL-17 > IL-6 > IL-1β > Nrf2. The molecular docking predictions regarding target proteins were biologically validated through ELISA and qPCR assays, which demonstrated significant modulation of these markers at both protein and mRNA levels, thus reinforcing the relevance of the predicted target interactions. These results underscore the M3F’s potential to modulate inflammation and oxidative stress pathways, highlighting its potential as a therapeutic candidate.

M3F’s hepatoprotective effect was assessed in in vivo studies using biochemical, molecular, and histopathological analyses. Fasting prior to PCM administration is a well-established practice in hepatotoxicity models, as it depletes GSH reserves and enhances the susceptibility of hepatocytes to NAPQI-induced oxidative injury41. This improves the reproducibility and severity of the liver damage, allowing clearer differentiation of hepatoprotective effects. In this study, mice were fasted overnight (~ 12–16 h) before PCM administration, consistent with standard protocols reported in the literature40,42. This approach ensured effective induction of hepatotoxicity and avoided variability in hepatic metabolism that can occur in fed states.

Despite complexity of mechanism of action, it is evident from literature that PCM depletes hepatic glutathione, leading to oxidative stress, cellular damage, and hepatocyte necrosis58. Our findings indicate that M3F significantly alleviated oxidative stress in PCM-induced hepatotoxicity. Membrane disruption and oxidative damage leads to hepatocyte leakage and liver injury that elevated liver enzyme levels in the PCM group as reported in the previous literature60,61. M3F reduced these enzyme levels dose-dependently, particularly at 400 µg/kg, by enhancing antioxidant defenses and stabilizing hepatocyte membranes, thereby reduced hepatic injury. The study revealed that PCM elevated bilirubin and total protein levels evident from the earlier research62,63, indicating impaired liver clearance, inflammation, and possible renal damage. Treatment with the M3F showed reduction in bilirubin levels which reflected improved hepatic clearance and bile flow, while normalization of total protein levels suggested restored lowering systemic toxicity and reduced inflammation64. Hepatic injury and oxidative damage led to dysregulation of lipid-processing pathways in the PCM group, as evidenced by elevated TG, TC, LDL, VLDL, and reduced HDL levels65. M3F, which incorporates pharmacophores such as imidazole known for their antihyperlipidemic properties66, demonstrated a therapeutic effect. Supported by prior studies on benzimidazole derivatives17, M3F at 400 µg/kg ameliorated lipid abnormalities by reducing oxidative stress and restoring liver parameters. Furthermore, M3F treatment significantly improved the AI and CRI, highlighting its potential in mitigating cardiovascular risks associated with liver damage67.

Hematological abnormalities, including decreased RBCs, platelets, and hemoglobin levels, along with increased WBCs, were observed in the PCM group. These findings are also reported in past literature60 reflecting systemic inflammation, impaired erythropoiesis, and possible bone marrow suppression68. Treatment with the M3F improved these parameters, suggesting its role in reducing inflammation and promoting hematopoiesis. PCM also disrupted serum electrolytes, as demonstrated by altered sodium, potassium and bicarbonate levels due to hepatic and renal toxicity affecting fluid balance and ionic regulation as reported by earlier findings69. Structurally similar agents like azilsartan medoxomil and candesartan cilexetil—both benzimidazole-based—have shown reno-protective effects70,71. The M3F restored these electrolyte levels, likely by reducing hepatic oxidative stress and improving renal handling of electrolytes. Oxidative stress and cellular necrosis induced by PCM trigger inflammation and hepatic injury, leading to an increased liver-to-body weight ratio72. Treatment with the M3F significantly reduced this ratio by mitigating inflammation and restoring cellular integrity.

PCM triggers massive cellular degeneration, necrotic hepatocytes, hemorrhage, and neutrophil infiltration as evident in earlier findings60. These changes can be attributed to oxidative stress and inflammatory responses triggered by excessive NAPQI production and subsequent hepatocyte injury73. In contrast, the M3F-treated groups displayed restoration of liver architecture, with reduced necrosis and enhanced hepatocyte regeneration, particularly with highest dose 400 µg/kg. This is attributed to M3F anti-inflammatory and antioxidant effects, that helped reduce oxidative damage and promote tissue regeneration.

PCM induced hepatocyte necrosis which resulted in the release of intracellular components, that activates TLR4 leading to NF-κB signaling pathways, ultimately driving the upregulation of inflammatory cytokines, as noted in earlier findings9,74. These cytokines are mediators of hepatic inflammation, contributing to hepatocyte damage and the progression of oxidative stress75,76. Treatment with the M3F demonstrated a dose-dependent reduction in these cytokines, with the highest dose (400 µg/kg) achieving significant result. The anti-inflammatory effect is supposed to be mediated by modulation of TLR4 and suppression of NF-κB pathway, as reported in literature77. The decrease in IL-6 and IL-1β levels suggests the M3F’s potential role in attenuating inflammation by modulating immune cell activation, while the reduction in TNF-α indicates its possible efficacy in minimizing the cascade of inflammation, as this cascade of antiinflammation is reported in literature10,11,78. These findings emphasize the anti-inflammatory potential of the M3F in mitigating liver damage and restoring immune balance in hepatotoxic conditions.

Previous studies have reported that numerous hepatoprotective compounds alleviate PCM-induced hepatotoxicity by activating Nrf2-regulated pathways11,79. Nrf2 serves as a key regulatory factor in the body’s defense against oxidative stress. It enhances the expression of antioxidant enzymes like HO-1, contributing to cellular protection80. We found that M3F could activate the Nrf2-HO-1 signaling pathway. These findings highlight the potential of M3F 400 µg/kg as a hepatoprotective agent by modulating oxidative stress and inflammatory pathways, suggesting its therapeutic application in hepatotoxicity.

Notably, our current findings show M3F treatment exhibited a dose-dependent reduction in inflammatory cytokines (IL-6, IL-1β, TNF-α) compared to the PCM group (though levels remained elevated versus vehicle group), suggesting selective modulation of inflammatory pathways without complete suppression of PCM’s metabolic cascade. Future studies are warranted to conduct in vitro CYP activity assays using liver microsomes, pre-PCM GSH levels, protein adduct quantification and to analyze early-phase plasma and hepatic levels of PCM and its metabolites using HPLC or LC–MS to conclusively determine whether M3F influences, if at all, the metabolic activation cascade of PCM.

Conclusion

Based on the observed antioxidant and anti-inflammatory effects, M3F demonstrates preliminary hepatoprotective potential in a PCM-induced liver injury model, supporting its consideration for further pharmacological evaluation.