Vitamin K1 attenuates acetaminophen-induced ferroptotic hepatic damage in mice via targeting keap1/Nrf2/HO-1 pathway

Abstract

Excessive acetaminophen (APAP) intake is a major cause of acute liver injury, primarily through its conversion to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), which induces oxidative stress and ferroptosis, a form of iron-dependent, lipid peroxidation-mediated cell death. This study investigated the hepatoprotective effects of vitamin K1(Vit K1) and its role in modulating ferroptosis via the Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) antioxidant pathway. Male mice were pretreated with Vit K1 (1, 2, or 3 mg/kg) prior to APAP injection (200 mg/kg). Liver damage was assessed by serum biomarkers Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and albumin, oxidative stress markers including reduced glutathione (GSH), malondialdehyde (MDA) and Nitric Oxide (NO), and ferroptosis indicators glutathione peroxidase 4 (GPX4), hepatic iron, acyl-CoA synthetase Long-chain family member 4 (ACSL4). APAP significantly increased ALT, AST, MDA, NO, and iron, while reducing albumin, GSH, and GPX4 levels, indicating oxidative injury and ferroptosis. Vit K1 pretreatment ameliorated these effects dose-dependently by restoring antioxidant balance, suppressing ACSL4 and Keap1 expression, and upregulating Nrf2 and HO-1. These results suggest that Vit K1 may protect against APAP-induced hepatotoxicity by inhibiting ferroptosis and activating antioxidant responses through the Keap1-Nrf2/HO-1 pathway, supporting its potential as a therapeutic candidate for drug-induced liver injury. 

Introduction

Acetaminophen (APAP) represents one of the most frequently prescribed medications for pain relief and fever reduction. However, despite its popularity and effectiveness, APAP is considered one of the prominent triggers of acute hepatic damage linked to drug-related liver failure, which may eventually lead to patient death (Alhaddad et al. 2025; Abdel-Hamid et al. 2022). APAP-induced liver toxicity predominantly results from the development of its harmful metabolite, N-acetyl-p-benzoquinone imine (NAPQI), reduces intracellular glutathione (GSH) and overwhelms the liver’s antioxidant defenses (McGill and Jaeschke 2013). NAPQI rapidly reduces GSH level, overwhelms the liver’s antioxidant defenses and binds to cellular macromolecules, triggering oxidative stress, mitochondrial dysfunction, and cell death pathways including ferroptosis (Jaeschke et al. 2021). While the underlying mechanisms of APAP-mediated acute hepatic damage are complex, current treatment options are still limited. Until now, the only clinically approved antidote is N-acetylcysteine, highlighting a crucial necessity for new, more effective therapeutic strategies (Liao et al. 2023). 

Ferroptosis, an iron-dependent lipid peroxidation-induced way of cellular death, has gained growing interest due to its involvement in various pathological conditions. Emerging studies recommend that targeting ferroptosis may offer a hopeful therapeutic potential, particularly in diseases such as drug-mediated hepatic damage (Shi et al. Sep. 2024). Moreover, ferroptosis has been involved in several critical disorders, including acute kidney injury (Ni et al. 2022) and hepatic ischemia–reperfusion damage (Han and Zhai 2025).

A crucial regulator of the ferroptosis progression is glutathione peroxidase 4 (GPX4), a glutathione (GSH)-dependent enzyme that protects cells by reducing lipid peroxides and preventing their toxic accumulation (Xing et al. 2025). Furthermore, the nuclear factor erythroid 2–related factor 2 (Nrf2) is a critical transcription component that participates in mitigating oxidative stress by stimulating different antioxidant defense systems, such as genes involved in GSH synthesis (Bayo Jimenez et al. 2022). Due to its regulatory influence on redox homeostasis, Nrf2 also emerges as a key component of ferroptosis modulation. As reactive oxygen species (ROS) overproduction is a hallmark of APAP-induced hepatotoxicity (Xiang and Yang 2025). The stimulation of Nrf2 serves as a protective response by enhancing the expression of antioxidant enzymes, together with heme oxygenase-1 (HO-1) (Hu et al. 2020). Moreover, Nrf2 activation is strongly controlled by the redox-sensitive Kelch-like ECH-associated protein 1 (Keap1) (Hu et al. 2020), underscoring its crucial role in protecting against drug-mediated hepatic injury (Wang et al. 2017).

Antioxidant compounds can effectively suppress ferroptosis (Conlon et al. 2021); however, no compound has yet been approved for clinical use as a ferroptosis inhibitor. Consequently, finding a stable, safe, and clinically viable ferroptosis inhibitor holds significant therapeutic promise, particularly for treating acute liver injury (Mohamed et al. 2022). In this context, Vit K1, a fat-soluble vitamin, has emerged as a promising therapeutic candidate due to its well-established safety profile and long-standing clinical use. It has been effectively administered for decades in preventing vitamin K deficiency bleeding in neonates (Ardell et al. 2018) besides its use in various adult conditions with no reported toxicity even at high doses, highlighting its potential for broader therapeutic use (Kaesler et al. 2022; Brandenburg et al. 2017). Several studies have reported that Vit K1 possesses effective lipid peroxidation-inhibiting properties, with emerging evidence supporting its role in oxidative stress regulation (Sai Varsha et al. 2015; Tasatargil et al. 2007).

Further studies demonstrated that Vit K1 can inhibit oxidative cell loss in neurons and oligodendrocytes by inhibiting glutathione depletion and lipoxygenase activity, mechanisms closely resembling those underlying ferroptosis (Li et al. 2009). Furthermore, a recent study reported that Vit K1 antagonist significantly promoted ferroptosis in vitro and negatively affected the progression of acute kidney injury in vivo (Kolbrink et al. Jul. 2022).

These findings led us to hypothesize that Vit K1, already widely used in clinical practice for other indications, could serve as a potent and safe ferroptosis inhibitor with potential therapeutic benefits for acute liver toxicity.

Materials and methods

Chemical compounds and drugs

Acetaminophen (APAP) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Vitamin K1 (phylloquinone) was obtained from a local pharmacy (El Ezaby Pharmacy, Cairo, Egypt). It was dispersed in 1% carboxymethylcellulose (CMC), which was obtained from the EL-Gomhoria Egyptian Company (Cairo, Egypt). All remaining compounds are of superior analytical quality.

Animals

The animal treatment was approved by the Committee of Institutional Animal Care and Use (KFS-IACUC) at Kafr Elsheikh University, Egypt, in agreement with the "Principles of Laboratory Animal Care" (National Institutes of Health Publication No. 85–23, updated 1985). Approval Number: KFS-IACUC/240/2025. Thirty-six Male albino mice, weighing from 16 to 28 g, were utilized. The animals were obtained from the National Research Center in Giza, Egypt. In this treatment, all animals were Maintained under standardized conditions of temperature at 25± 2 °C, with a 12-h light/12-h darkness cycle and had unrestricted access to food and water. All experiments were carried out in the Faculty of Pharmacy's animal unit at Kafr Elsheikh University, Kafr Elsheikh, Egypt.

Induction of liver toxicity

The animals were administered 1% CMC at a dose of 4 mg/kg daily for one week, followed by an intraperitoneal (IP) injection of APAP (200 mg/kg) on the last day. The model's efficacy was validated by obtaining serum samples for subsequent investigation of liver enzyme levels (ALT and AST), followed by the euthanasia of randomly selected mice administered APAP to isolate liver tissue for histological evaluation (Urrunaga et al. 2015).

Design of experiment

After the acclimation phase, the mice were categorized into six groups, each including 6 mice, as illustrated in Fig. 1. 

• Control group (Cnt, n = 6): received no treatment, set as healthy control animals, received 4 ml/kg 1% CMC daily during the experimental period, and 0.5 ml saline on the last day.

• Control Vit K1 (3 mg) (Cnt Vit K1 3 mg, n = 6): received 3 mg/kg BW of vitamin K1 by gavage once daily during the experimental period (Haraikawa et al. 2011).

• APAP group (n = 6): received a 4 ml/kg dose of 1% CMC one time per day for one week and APAP at a dose of 200 mg/kg) IP on the last day (Urrunaga et al. 2015).

• APAP + 1 mg Vit K1 group (n = 6): received 1 mg/kg BW of vitamin K1 by gavage once per day for one week and APAP (200 mg/kg IP) on the final day (Xiao et al. 2024).

• APAP + 2 mg Vit K1 group (n = 6): received 2 mg/kg Body Weight of vitamin K1 by gavage once per day for one week and APAP at a dose (200 mg/kg IP) on the last day.

• APAP + 3 mg Vit K1 group (n = 6): received 3 mg/kg Body Weight of vitamin K1 by gavage once per day for one week and APAP at a dose (200 mg/kg IP) on the final day (Haraikawa et al. 2011).

• The administration of CMC or 3 mg/kg vitamin K1 only in animals for one week exhibited no noteworthy impact on the primary biological markers; hence, their outcomes were omitted from the results for clarity.

Fig. 1.

Schematic presentation of experimental outline

Collection of the samples

Blood and liver specimens were obtained following an overnight fast after anesthesia utilizing pentobarbital (50 mg/kg) intraperitoneally. After that, blood samples were centrifuged for 15 min at 4 °C at 2147 g to prepare serum and kept frozen at −80 °C for further assessment of serum biological markers. Subsequently, all animals were euthanized, and the entire liver was excised and sectioned into three segments. For the quantitative real-time polymerase chain reaction (qRT-PCR), the first sample was immediately dipped in liquid nitrogen. The second section was conserved in a buffered formalin solution (pH 7.2) for histological analysis. The third was homogenized in ice-cold phosphate-buffered saline (pH 7.4), centrifuged, and stored at −80 °C for additional biochemical analysis.

Hepatic tissue investigation by histopathological analysis

Liver tissues that had been formalin-fixed with phosphate were embedded in paraffin. For histological analysis under a Light microscope, 4μm-thick slices were removed and stained with hematoxylin and eosin (H&E). A digital camera attached to an Olympus BX51 optical microscope (Olympus Corporation, Tokyo, Japan) was used to record histopathological changes (Bancroft and Gamble 2007). Grading of the histological lesions within the different groups was done according to presence of the vascular and necro-inflammatory lesions as follow; score 0 revealed normal hepatic parenchyma, score 1 revealed mild vascular congestion and with mild centrolobular eosinophilic degenerative changes, score 2 showed increase congestion of central vein and blood sinusoids and with centrolobular and midzonal area of eosinophilic degenerative changes, score 3 showed Marked congestion of the hepatic blood vasculature and confluent area of hepatic degeneration with focal hepatic necrosis associated with foci of inflammatory cells infiltration and score 4 demonstrated severe congestion including the portal blood vessels with diffuse piecemeal degeneration and necrosis with extensive inflammatory cells infiltration.

Immunohistochemical staining of ACSL4 and NRF2

After deparaffinization and rehydration, the mice's polyclonal ACSL4 antibody (Invitrogen, USA, Cat # PA5-27,137) and Nrf2 (Invitrogen, USA, Cat # 17,791,444) were applied to the tissue slides at 4 °C for the entire night. After that, PBS was used for washing the slides, which were then incubated at room temperature with a goat anti-rabbit secondary antibody (Cat# K4003, EnVision + ™ System Horseradish Peroxidase Labelled Polymer; Dako) for two hours before being observed using the DAB kit. A light microscope was then used to examine the sections. Quantitative estimation of the immunostaining of ACSL4 was detected through the determination of the percentage of positive images using Image J analysis software (NIH, USA).

Investigating biochemical markers by colorimetric procedure

By measuring the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes (Spectrum Diagnostics, Egypt) and measuring albumin levels (BioMed Company, Heliopolis, Egypt).

Malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (NO) levels were detected in the homogenates of liver samples to evaluate oxidative stress (Biodiagnostic Company, Dokki, Giza, Egypt). Furthermore, GPX4, a marker of ferroptosis, was quantified using liver homogenates by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Based on the manufacturer's instructions, the homogenate was then utilized to measure the iron level using an iron colorimetric assay kit (Biodiagnostic Company, Dokki, Giza, Egypt).

Quantitative evaluation of mRNA expression of Keap1, Nrf2, and HO-1 by (qRT-PCR)

The TRIzol reagent (Life Technologies, Camarillo, CA, USA) was utilized for isolating total Ribonucleic acid (RNA) from the liver samples. The RNA amount was assessed using Maxima SYB Green/Fluorescein qPCR Master Mix (Fermentas, Waltham, MA, USA). 1 μg of total RNA was then reverse transcribed into single-stranded complementary DNA (cDNA) utilizing the QuantiTect reverse transcription kit (Qiagen, Valencia, CA, USA) using a random primer hexamer in a two-step RT-PCR process. During this process, genomic DNA (gDNA) contamination was initially eliminated using the gDNA Wipeout Buffer. The housekeeping gene utilized was β-actin from mice, serving as an internal reference standard. The sequence of the PCR primer is presented in Table 1. The thermal cycling conditions consisted of an initial incubation at 95 °C for 5 min, followed by 40 cycles of 94 °C for 20 s and 60 °C for 1 min. Data was acquired during the extension phase. Melting curve analysis was employed to verify the authenticity and specificity of PCR products. Data were automatically gathered using the Rotor-Gene Q (Qiagen, Valencia, CA, USA), which assessed the threshold cycle (Ct) value. The relative expression levels of Keap1, Nrf2, HO-1, and β-actin mRNA in mice were determined using the 2^−DDCt technique.

Table 1.

Forward (F) and reverse primer (R) sequence of Keap1, Nrf2, HO-1, and β-actin

Primer	Sequence	Annealing temperature oC
Keap1	F 5´-TGGGACGGAGGCCCCTAA- 3´	60
	R 5´-TCGTTCATGACGCCAAAAGC- 3´	60
Nrf2	F 5´- ATGGACTTGGAGTTGCCACC- 3´	60
	R 5´- CCTGTTCCTTCTGGAGTTGCT- 3´	60
HO-1	F 5´- CCTCACAGATGGCGTCACTT- 3´	60
	R 5´- TGGGGGCCAGTATTGCATTT- 3´	60
β-actin	F 5´- GACTGTTACTGAGCTGCGTTT- 3´	60
	R 5´- AGGGTGAGGGACTTCCTGTA- 3´	60

Statistical exploration

Results are presented as mean ± standard deviation (SD). Descriptive statistics for quantitative variables were conducted using One-way analysis of variance (ANOVA), followed by Tukey's post-hoc test, utilizing GraphPad Prism Software version 8 (San Diego, CA, USA). A p-value of less than 0.05 was found to be statistically significant for all tests.

Results

Impact of Vit K1 on hepatic injury induced by APAP

The evaluation of serum markers for liver injury

To investigate the therapeutic impact of Vit K1, various concentrations (1, 2, and 3 mg/kg) were administered as treatment, followed by 200 mg/kg APAP by intraperitoneal injection. Paracetamol significantly elevated the indicators of hepatic damage ALT, AST, and reduced the albumin level in contrast with the Cnt group (p < 0.0001). However, the administration of Vit K1 markedly reduced the levels of ALT and AST and elevated albumin level, compared to the untreated APAP groups (p < 0.05), as exhibited in Fig. 2I-III. The hepatoprotective role of Vit K1 on the APAP-induced hepatic damage was dose-dependent. Our results were validated by histopathological examinations of the liver tissues of mice, as detailed below.

Fig. 2.

Effects of Vitamin K1(Vit K1) on APAP-induced acute liver damage. The serum levels of (I) ALT, (II) AST, and (III) Albumin in different animal groups (n = 6 per group)

The impact of Vit K1 on APAP-induced liver injury was evaluated through histopathological analyses

The liver of control animals exhibited normal hepatic parenchyma, characterized by a normal lobular pattern and normal cellular components consisted mostly of normal hepatocytes arranged in cord-like pattern around the central vein. Hepatocytes demonstrated normal eosinophilic cytoplasm and a normal vesicular nucleus and with prominent nucleolus. The livers of Vit K1 (3mg) animals showed normal hepatic tissue and mostly were within normal. The diseased animal administered APAP showed marked degenerative and apoptotic changes within the hepatic tissues, mostly centrilobular and extended to midzonal area associated with marked cytoplasmic eosinophilia, marked nuclear pyknosis, in addition to vascular congestion of portal blood vessels and central vein, and marked mononuclear inflammatory cells mainly consisted of lymphocytes and macrophages around the portal area. The APAP groups treated with Vit K1 showed a marked decrease in the degenerative and apoptotic changes within the hepatic tissues in a dose-dependent manner, Fig. 3-I. Quantitative scoring of the hepatic lesions revealed a marked increase in the hepatic lesion scoring in the diseased group, compared to the control group (p<0.0001), accompanied by a notable decrease within the treated groups Fig. 3-II.

Fig. 3.

Effects of Vitamin K1(Vit K1) on APAP-induced acute liver damage. (I) Hepatic sections of different treated groups; A) liver of control animal (arrow indicates normal hepatocytes with normal eosinophilic cytoplasm and normal vesicular nucleus (white arrow), B) liver of normal animal treated with high dose of Vit K1(3 mg/kg) showing normal hepatic parenchyma (white arrow), C) liver of APAP treated animal showing marked congestion of portal vein (green arrow), portal inflammation associated with mononuclear cells infiltration (blue arrow), and severe degree of eosinophilic degenerative changes within the hepatocytes (red arrow), D) liver of APAP-administered animal treated with Vit K1 (1 mg/kg) showing decrease portal inflammation and hepatic degeneration (blue and red arrows respectively), E) liver of APAP-administered animal treated with Vit K1 (2 mg/kg) showing remarkable decrease portal inflammation (blue arrow) and limited periportal hepatic degeneration (red arrow) and F) liver of APAP-administered animal treated with Vit K1 (3 mg/kg) showing single hepatic cell degeneration (red arrow), H&E stain bar= 50 µm. (II) The hepatic lesion score within the different groups. Records were compared by one-way ANOVA followed by post-hoc Tukey–Kramer tests. Significant difference vs.^* Cnt, ^# APAP, ^& APAP+ 1 mg Vit K1, each at p < 0.05

Impact of vitamin K1(Vit K1) on APAP-induced oxidative stress

Oxidative stress is considered as one of the main causes of endoplasmic reticulum stress (Ong and Logue 2023). Researches indicate that too much APAP results in excessive production of the toxic metabolite NAPQI, causing serious damage to the liver tissue by causing oxidative stress (Chowdhury et al. 2020). In this investigation, we measured the lipid peroxidation product MDA as well as the hepatic antioxidant GSH and nitric oxides concentrations. The levels of hepatic MDA were noticeably elevated in the APAP group compared to the Cnt group (p<0.0001). MDA levels were significantly decreased upon Vit K1 treatments (p<0.0001), as exhibited in Fig. 4-I. Additionally, the hepatic GSH concentration was markedly lowered in the APAP group, contrasting with the Cnt group (p<0.0001). Furthermore, GSH levels exhibited a significant elevation after Vit K1 treatments when compared to the APAP group (p<0.0001) as shown in Fig. 4-II. Nitric oxide (NO) levels were noticeably greater in the APAP treatment than in the Cnt group (p<0.0001). When compared with the APAP group, Vit K1 treatments exhibited a marked reduction in nitric oxide concentration (p <0.0001), as revealed in Fig. 4-III.

Fig. 4.

Effect of Vitamin K1(Vit K1) on APAP-Induced Oxidative Stress Markers. The hepatic levels of (I) GSH, (II) MDA, and (III) NO (n = 6 per group). Data were compared by one-way ANOVA followed by post-hoc Tukey–Kramer tests. Significant difference vs.^* Cnt, ^# APAP, ^& APAP+ 1 mg Vit K1, ^@ APAP+ 2 mg Vit K1, each at p < 0.05

These oxidative stress indicators were markedly dose-dependently reversed by Vit K1 treatments.

The effect of Vitamin K1(Vit K1) on APAP -Induced ferroptotic markers

Ferroptosis is defined as a non-apoptotic type of cellular death distinguished by iron-dependent lipid peroxidation. A vital inhibitor of this process is GPX4, which acts by altering lipid hydroperoxides into non-toxic alcohols using GSH. When GPX4 activity is suppressed, lipid peroxides accumulate, an essential hallmark of ferroptosis (Ma et al. Dec. 2022). We observed a significant rise in the level of GPX4, a crucial regulator in the ferroptosis signaling and linked to oxidative stress. In the mice liver samples, we identified a decline in the protein level of GPX4 due to APAP administration in relation to the Cnt group (p < 0.0001). On the contrary, Vit K1 dose-dependently prevented the APAP-induced change in GPX4, contrasting with the APAP group (p < 0.0001) as illustrated in Fig. 5-I. These observations indicate that Vit K1 induces the expression of GPX4 to mitigate the oxidative stress caused by APAP that is closely linked to ferroptosis.

Fig. 5.

Impact of Vitamin K1(Vit K1) on APAP-Induced Ferroptosis. The hepatic levels of (I) GPX4, and (II) iron in different groups (n= 6 per group). (III) Liver sections from the different experimental groups showing the immunoexpression of ACSL4 antibody, A) control group, B) control vitamin K (3 mg/kg) group, C) APAP group, D, E, F; APAP animals treated with Vit K1 at doses of 1 mg, 2 mg, and 3 mg respectively. Arrows indicate positive expressions. The figure showed marked expression in the APAP group, and a remarkable decrease in ACSL4 expression in the Vit K1-treated groups. (IV) Percent hepatic immunostaining of ACSL4. Bar 50 µm. G shows the quantitative scoring of ACSL4 immunoexpression. A one-way ANOVA and post-hoc Tukey-Kramer tests were used to compare the data. Significant difference vs.^* Cnt, ^# APAP, ^& APAP+ 1 mg Vit K1, ^@ APAP+ 2 mg Vit K1, each at p < 0.05

Ferroptosis is described by iron accumulation, along with increased reactive oxygen indicators (Chen et al. 2021). Accordingly, we measured the total iron content in the hepatic tissues. The hepatic iron level was considerably increased in the untreated APAP group, contrasting with the Cnt group (p < 0.0001). On the other hand, APAP mice received Vit K1 showed a markedly decreased hepatic iron level, compared to APAP in a dose-dependent way (p < 0.0001), Fig. 5-II.

ACSL4 acts as a key point of cellular sensitivity to ferroptosis. Ferroptosis is linked explicitly with ACSL4, rather than other participants of the ACSL family. ACSL4 exhibited a vital function in modifying the lipid composition necessary for the execution of ferroptosis (Doll et al. 2017). Likewise, the hepatic tissue of the Vit K1 groups and the control groups exhibited mild cytoplasmic immunostaining of ACSL4. However, animals given paracetamol exhibited a noticeable increase in ACSL4 antibody cytoplasmic expression. Figure 5-III and IV illustrates the dose-dependent reduction in ACSL4 cytoplasmic staining observed in diseased animals receiving Vit K1 treatments (p < 0.0001).

The impact of vitamin K1 (Vit K1) on the expressions of Keap1/Nrf2/HO1 signaling pathway in APAP-treated mice

One crucial treatment approach for several illnesses is thought to activate the Keap1/Nrf2 signaling pathway (Yu and Xiao Jan. 2021). This pathway is regarded as a vital defense mechanism of the cell towards oxidative stress and various harmful stimuli, especially in the brain and liver (Luo et al. 2022). Accordingly, we investigate the possible mechanistic role of vitamin K1 in attenuating APAP-mediated liver toxicity by targeting Keap1/Nrf2/HO1 signaling pathway. The hepatic expression of Keap1 was detected using qRT-PCR techniques, which revealed that the hepatic expression of Keap1 mRNA was noticeably upregulated in the untreated APAP group against the Cnt group (p < 0.0001). Furthermore, the treatment with Vit K1 significantly exhibited a dose-dependent decrease in hepatic Keap1 mRNA expression contrasting with the untreated APAP group (Fig. 6-I).

Fig. 6.

The relative expression of mRNA of (I) Keap1, (II) Nrf2, (III) HO1 in the liver homogenate Liver, and (IV) sections from the different experimental groups showing the immunoexpression of Nrf2 antibody, A) control, B) Control vitamin K (3 mg/kg), C) APAP, D, E, F; APAP animals treated with Vit K1 at doses of 1 mg, 2 mg, and 3 mg respectively. Arrows indicate the positive expression of Nrf2. The figure shows marked decrease in the expression of Nrf2 antibody within the liver of APAP group, and a remarkable increase of Nrf2 expression in the Vit K1-treated groups in dose-dependent manner. (V) Percent of Nrf2 hepatic immunostaining. Data were compared by one-way ANOVA followed by post-hoc Tukey–Kramer tests. Significant difference vs.^* Cnt, ^# APAP, ^& APAP+ 1 mg Vit K1, APAP+ 2 mg Vit K1, each at p < 0.05

Furthermore, the hepatic expressions of Nrf2 and HO-1 were also measured using the RT-PCR technique. APAP administration induced a notable reduction in Nrf2 in contrast with the Cnt group (p < 0.0001). Remarkably, comparing with the untreated APAP group, Vit K1 treatments enhanced Nrf2 expression and its target subsequent proteins, including HO-1, while inhibiting Keap1 expression, as revealed in Fig. 6-II and III. Thus, the findings show that Vit K1 may have a protective effect towards APAP-mediated hepatic damage by stimulating the Keap1/Nrf2 pathway, and that these effects were dose dependent.

Moreover, immunostaining of Nrf2 antibody showed marked expression in Cnt and vitamin K (3 mg/kg) groups (p < 0.0001). The APAP group demonstrated marked decrease in Nrf2 hepatic immunostaining compared to Cnt and vitamin K (3 mg/kg) groups (p < 0.0001). APAP groups pre-treated with Vit K1 exhibited notable increase in Nrf2 immunostaining in a dose-dependent manner compared to APAP group (p < 0.0001) (Fig. 6-IV and V).

Discussion

Acetaminophen (APAP), a common analgesic drug used globally, is safe in general if taken within the recommended therapeutic range. However, APAP is of great scientific and therapeutic interest since it is an intrinsic hepatotoxin that always causes liver damage after an overdose in people and animals (Liao et al. 2023). The hepatic damage caused by APAP is due to the production of its toxic metabolite, NAPQI, which is produced by cytochrome P450 2E1 (CYP2E1). Thus, pathological conditions increasing CYP2E1 activity can increase APAP-induced liver injury, which is characterized by hepatic cellular death and inflammation (Massart et al. 2021). In therapeutic doses, NAPQI is neutralized by GSH, but under overdose conditions, GSH stores are rapidly depleted. This leads to covalent binding of NAPQI to cellular proteins, triggering mitochondrial dysfunction, oxidative stress, and ultimately, regulated forms of cell death as ferroptosis (Kim et al. 2021).

In the current investigation, we explored the potential role of vitamin K1 (Vit K1) on liver damage mediated by APAP using a mouse model. Our findings indicated that Vit K1 can alleviate hepatic injury by targeting ferroptosis and oxidative stress pathways. Additionally, the antioxidant benefits of vitamin K1 are linked to the activation of the Keap1/Nrf2/HO-1 signaling pathway. To our knowledge, this is the first in vivo study demonstrating the dose-dependent protective effect of Vit K1 against APAP-induced hepatotoxicity via modulation of the Keap1/Nrf2/HO-1-ferroptosis axis.

In the present study, APAP administration significantly increases the ALT and AST activities and decreases albumin level in mice serum samples, contrasting with the control group, suggesting hepatic damage. Furthermore, the histological analysis confirmed these findings, showing a severe centrilobular necrotic change in the hepatic tissues of animals treated with APAP, which is indicative of hepatotoxicity caused by APAP. These results align with previously published studies (Latchoumycandane et al. 2007, Lai et al., 2025, Long et al., 2025, Yamada et al., 2020, Gao et al. 2022). Interestingly, different doses of Vit K1 significantly reduced hepatic necrosis and the APAP-triggered elevation in blood activities of ALT and AST, while restoring serum albumin levels to a level near the control line.

APAP-mediated hepatic damage is mainly caused by increased oxidative stress. An indicator of the antioxidant capability of the body is GSH, a type of low molecular weight scavenger (Zhou et al. 2023). Since GSH depletion is the harmful pathway of oxidative stress caused by APAP, measuring the amount of GSH in the liver is especially crucial. Lipid peroxidation produces MDA, which can indirectly indicate the extent of cell damage (Całyniuk et al. 2016). Consequently, GSH and MDA could be used as indicators to assess oxidative damage to the liver (Wu et al. 2024). In earlier research, Vit K1 has been shown to have antioxidant properties and help reduce oxidative stress, particularly in lipid cell membranes. Reactive oxygen species concentrations are reduced by Vit K1 (Nuszkiewicz et al. 2023). In line with this, our findings demonstrated that Vit K1 administration reduced oxidative stress by raising GSH and lowering MDA and NO levels in a dose-dependent way.

Considerable studies have been made to understand the mechanisms underlying liver injury and cellular death. Ferroptosis, a distinct form of regulated cell death induced by iron accumulation and lipid peroxidation, has recently emerged as a key contributor to APAP-induced hepatocyte death (Jaeschke et al. 2021). It is characterized by excessive amounts of iron, increased ROS generation, decreased GSH levels, and increased lipid peroxidation. Excessive NAPQI production from APAP metabolism depletes GSH and impairs GPX4 activity, both central regulators of ferroptosis (Yarmohammadi et al. 2021). On the other hand, ACSLs are a family of enzymes that stimulate the conversion of saturated and unsaturated fatty acids, ranging from 8 to 22 carbon atoms in length, into fatty acyl-CoA esters. They have a critical role in fatty acid metabolism and are extensively involved in processes such as ferroptosis and endoplasmic reticulum stress. Among them, ACSL4 is especially important, as it facilitates the production of lipid peroxides, thus driving ferroptosis in cells (Jia et al. 2023). As ACSL4 can stimulate ferroptosis, it serves as a biomarker for it (Jia et al. 2023). This explains how different drugs and chemicals can cause organ toxicity. For specific organ toxicities, blocking ferroptosis may offer a novel approach to prevention and treatment.

Numerous investigations have demonstrated that acute liver injury can be improved by inhibiting ferroptosis (Shi et al. Sep. 2024; Xu et al. 2023). The current study investigated the effect of ferroptosis on acute hepatic damage. The untreated APAP animals exhibited elevated ferroptosis, as confirmed by elevated ACSL4 and total iron and decreased GPX4 levels. On the other hand, pretreatment with vitamin K1 at different doses reduced ferroptosis markers by increasing GPX4 and decreasing ACSL4 and total iron, compared with the untreated APAP groups, suggesting the effect of Vitamin K1 in preventing ferroptosis and lowering oxidative stress. Consistent with our findings, previous investigations also revealed the protective role of vitamin K1 towards ferroptosis but in different conditions (Kolbrink et al. Jul. 2022; Hirschhorn and Stockwell 2022; Mishima et al. 2022). Compared to synthetic ferroptosis inhibitors such as Ferrostatin-1, Vit K1 offers the advantage of established clinical safety, wide availability, and low cost, making it an attractive candidate for translational applications (Mladěnka et al. 2022).

Nuclear Factor Erythroid 2–related Factor 2 (Nrf2) is the transcriptional principal regulator of cellular reactions. It modulates the expression of numerous antioxidant-linked genes or enzymes, with Keap1 acting as a negative regulator (Ngo and Duennwald 2022). The crucial function that Nrf2 plays in ferroptosis is becoming increasingly evident as our knowledge of the process grows, especially regarding the way it modulates iron homeostasis, the cell's antioxidant capability, and its metabolic state via its target genes (Dodson et al. 2019). Keap1-mediated degradation often maintains Nrf2, which regulates small amounts of the essential ferroptosis inhibitor, GPX4 (Guo et al. 2024). Nrf2 is activated by ferroptosis, which causes it to activate many protective genes related to oxidative defense and iron metabolism (Wang et al. 2023). However, in disease conditions with low Nrf2, ferroptosis and overall protein-lipid oxidation are significantly improved by subsequent Nrf2 target inactivation and elevated lipid oxidation, which further promotes disease progression (Rosa et al. 2021).

The pathway mediated by Nrf2 is essential for combating acute liver injury. Nrf2 dissociates from Keap1 and travels to the nucleus in acute liver injury, where it binds to the components that respond to antioxidants, causing different antioxidant proteins and enzymes to be expressed, involved in detoxification, improving the ability of hepatic cells to counteract NAPQI and diminishing damage and death of hepatic cells (Bellezza et al. 2018). Consequently, to avoid and treat acute hepatic damage, several studies have investigated how medications or other methods can activate the Nrf2 signaling pathway (Wang et al. 2023)–(Zhang et al. 2024). Furthermore, a previous study has demonstrated that by controlling the ferroptosis process, Nrf2 signaling pathway activation reduces liver damage (Salama et al. 2022; Wu et al. 2022).

In the same line, a previous study exhibited that the expression of Nrf2 was reduced by APAP therapy and increased ferroptosis in mice hepatic tissues. Moreover, kaempferol administration increased Nrf2 gene expression and decreased ferroptosis to ameliorate the hepatic damage (Li et al. 2023). Furthermore, other investigations found that the inhibition of the Nrf2 signaling pathway led to an elevation in the severity of the liver damage (Cai et al. 2025). Our findings, consistent with previous studies, revealed that APAP treatment in the mice liver inhibited Nrf2 signaling (Shen et al. 2023). Nevertheless, Vit K1 treatment markedly enhanced Nrf2 signaling activation by elevating Nrf2 and HO-1 expression while inhibiting Keap1 expression in a dose-dependent manner, thus promoting nuclear translocation of Nrf2 in the mice liver following APAP damage. These results suggest that Vit K1 may mitigate liver damage caused by APAP by mediating the Nrf2 signaling via the ferroptosis cascade.

The protective effects were also dose-dependent; to our knowledge, this is the first investigation to illustrate this relationship. While this study focused on ferroptosis and antioxidant effects, it remains unknown whether Vit K1 modulates the biotransformation of APAP via CYP-mediated NAPQI formation. Several studies have reported that antioxidants can downregulate or inhibit cytochrome P450 enzymes, particularly CYP2E1, which is pivotal in APAP bioactivation (Carrasco et al. 2021). For instance, previous studies performed on resveratrol, curcumin, and quercetin have been shown to suppress the activity of CYP2E1, reducing NAPQI formation, thereby reflecting their hepatoprotective effects (Abbasi-Oshaghi 2019). As vitamin K1 also exhibits strong antioxidant properties, it is plausible that it may exert a similar modulatory effect on CYP2E1. Further work is therefore needed to elucidate whether vitamin K1 inhibits CYP2E1 activity or reduces NAPQI adduct accumulation, revealing an additional protective mechanism beyond ferroptosis inhibition.

Collectively, our results demonstrate that Vit K1 mitigates APAP-induced hepatotoxicity primarily by inhibiting ferroptosis through the suppression of ACSL4 and iron accumulation, restoration of GPX4 activity, and activation of the Keap1/Nrf2/HO-1 signaling pathway Fig. 7. This complex antioxidant mechanism may represent a novel therapeutic approach to drug-induced liver injury. Furthermore, future investigations could explore the hepatoprotective effects of Vit K1 in human liver in clinical cohorts.

Fig. 7.

Schematic representation of the mechanistic role of vitamin K1 (Vit K1) in ameliorating the hepatotoxic effect of paracetamol overdose. APAP, Acetaminophen; NAPQI, N-acetyl-p-benzoquinone imine; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; GSH, Reduced glutathione; MDA, Malondialdehyde; NO, Nitric Oxide; GPX4, Glutathione Peroxidase 4; ACSL4, Acyl-CoA Synthetase Long-Chain Family Member 4; Keap1, Kelch-like ECH-associated Protein 1; Nrf2, Nuclear Factor Erythroid 2–related Factor 2; HO-1, Heme Oxygenase-1

Conclusion

Our study demonstrated that ferroptosis plays a major role in hepatotoxicity mediated by APAP. Lipid peroxidation, combined with oxidative stress, causes ferroptosis of hepatocytes, resulting in acute hepatic injury. Additionally, the use of vitamin K1 supplements helped prevent acute hepatic injury triggered by APAP, and this defensive process may be associated with the enhancement of ferroptosis suppression and the activation of Nrf2 signaling pathway. These results offer recent revelations regarding the mechanisms behind hepatotoxicity caused by APAP and indicate that ferroptosis could represent a novel target for treatment of APAP-induced acute hepatic injury. 

Abbreviations

ACSL4

Acyl-CoA Synthetase Long-Chain Family Member 4

ALT

Alanine Aminotransferase

APAP

Acetaminophen

AST

Aspartate Aminotransferase

CMC

Carboxymethylcellulose

Ct

Threshold cycle

GPX4

Glutathione Peroxidase 4

GSH

Reduced glutathione

HO-1

Heme Oxygenase-1,

Keap1

Kelch-like ECH-associated Protein 1

MDA

Malondialdehyde

NAPQI

N-acetyl-p-benzoquinone imine

NO

Nitric Oxide

Nrf2

Nuclear Factor Erythroid 2–related Factor 2

qRT-PCR

Quantitative Real-Time Polymerase Chain Reaction

Vit K1

Vitamin K1

Author contributions

Shimaa A. Abass, Ahmed A. Mohamed, Ahmed H. Abd El - Slam, and Mona Mohammed Abdel Hamid: Conceptualization, Methodology, analysis, and writing. Basma Salah: Data Curation, writing manuscript draft, and investigation. Abdalrahman Mohammed Yousef and Mohammed karim Ayoub: Validation, writing, editing and Methodology. Walid Abdo: Methodology, analysis, writing & editing, and Supervision. All authors have read and approved the content of the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

Data are available upon request from the corresponding author.

Declarations

Clinical trial number 

Not applicable.

Conflict of interest

The authors declare no competing interests.