{"paper_id":"10513b5c-9b98-4c22-970b-e0ae2637e19f","body_text":"6-Gingerol inhibition of NF-kB expression in diethylnitrosamine initiated, 2-acetylaminofluorene-promoted liver dysfunction and inflammation involves induction of antioxidant enzymes and suppression of pro-inflammatory cytokines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 6-Gingerol inhibition of NF-kB expression in diethylnitrosamine initiated, 2-acetylaminofluorene-promoted liver dysfunction and inflammation involves induction of antioxidant enzymes and suppression of pro-inflammatory cytokines Ikenna C. Maduako, Ebenezer O. Farombi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6792607/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the hepatoprotective effects of [6]-gingerol against liver dysfunction and inflammation induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) in mice. BALB/c mice were divided into four groups and treated with DEN, 2-AAF, [6]-gingerol, or a combination thereof. Biochemical, histological, and immunohistochemical analyses were conducted to evaluate liver function, oxidative stress, and inflammation. Mice exposed to DEN and 2-AAF exhibited significant hepatic injury, characterized by elevated serum liver enzymes (ALT, AST, ALP, γ-GT), reduced antioxidant enzyme activities (SOD, CAT, GPx, GST), increased lipid peroxidation, nitric oxide production, and expression of pro-inflammatory markers (NF-κB, iNOS, COX-2). Histopathological examination confirmed substantial liver damage. Pre-treatment with [6]-gingerol significantly mitigated these effects, restoring antioxidant enzyme activities, reducing oxidative and nitrosative stress, and suppressing the expression of inflammatory mediators. Liver histoarchitecture also improved markedly in [6]-gingerol-pretreated mice. These findings suggest that [6]-gingerol confers protective effects via antioxidant and anti-inflammatory mechanisms, primarily through modulation of the NF-κB signaling pathway. The results highlight the therapeutic potential of [6]-gingerol in preventing liver diseases associated with xenobiotic-induced oxidative stress and inflammation. Toxicology Biochemical Research Methods Molecular Biology Diethylnitrosamine 2-acetylaminofluorene liver toxicity NF-kB oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Persistent liver inflammation arises from a combination of chronic effects triggered by various harmful substances. 1 – 3 This condition can ultimately progress to cirrhosis, characterized by the formation of fibrous tissue and hepatocyte nodules. However, the intricate processes leading to liver dysfunction and inflammation are not fully understood. The characteristics of this condition include alterations in biochemical pathways, programmed cell death, activation of hepatic stellate cells (HSCs), free radical production, necrosis, and oxidative stress, all of which are essential factors in these mechanisms. 4 – 6 Exposure to environmental and occupational contaminants, such as diethylnitrosamine (DEN), 2-acetylaminofluorene (2-AAF), specific dietary factors, viruses, and agrochemicals, is associated with a heightened risk of developing liver preneoplasia. 7 – 10 DEN, a potent carcinogen and hepatotoxic agent, is commonly employed in animal studies to investigate the onset and progression of hepatic cancer. It induces liver lesions ranging from degenerative to neoplastic changes, offering insights into the development of hepatic carcinogenesis. 11 – 13 When combined with other toxins like 2-AAF, phenobarbital, or carbon tetrachloride, DEN's ability to induce liver dysfunction has garnered significant scientific interest. 14 These substances often stimulate the overproduction of chemical mediators, including cytokines, chemokines, NF-kB associated proteins. While these molecules play roles in pathogen elimination and tissue repair, their dysregulated expression can lead to chronic inflammatory disorders such as endotoxemia and fulminant hepatitis. 15 – 17 Efforts to mitigate these harmful effects have turned to natural nutritional phytochemicals, which are gaining recognition as a foundation for chemoprevention. Among these, the ginger rhizome (Zingiber officinale), used for its antioxidant and anti-inflammatory properties, has been a staple in traditional medicine and culinary practices worldwide. 18 , 19 Its chemical constituents, notably [6]-gingerol and [6]-shogaol, have shown to exhibit various biological effects, including the inhibition of tumor growth and cell proliferation. 20 , 21 Numerous studies indicate that [6]-gingerol suppresses NF-κB-regulated proteins linked to cell proliferation and angiogenesis, while also inhibiting inflammatory mediators like TNF-α and IL-1β. 22 Preclinical and experimental studies further highlight its potential to reduce nitric oxide synthesis and counteract inflammation. 23 – 26 Despite these findings, the specific mechanisms by which [6]-gingerol exerts its hepatoprotective effects are still uncertain. This study is designed to uncover the mechanism by which [6]-gingerol prevents sub-chronic liver inflammation and oxidative damage in a DEN + 2-AAF-induced mouse model. 2. Materials and methods 2.1. Chemicals Diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) were bought from Sigma-Aldrich Co., St. Louis, MO, USA. Polyclonal antibodies targeting nuclear factor (NF-kB), cyclooxygenase-2, and inducible nitric oxide synthase (iNOS) procured from Elabscience, China. Other analytical reagents were purchased from British Drug Houses, located in Poole, Dorset, UK. 2.2. Authentication and Extraction of [6]-gingerol Fresh ginger roots and foliage were purchased at Bodija Market in Ibadan, Nigeria, from a local vendor. The collected plant samples were authenticated at the herbarium (voucher number: UIH-22390) at the Department of Botany, University of Ibadan. The extraction of [6]-gingerol, with a purity exceeding 91%, was conducted using a method described by Ajayi et al. 27 2.3. Animals Forty BALB/c mice (16-18g) were sourced from the University of Ibadan's Faculty of Veterinary Medicine. Prior to the experiment, the animals underwent a one-week acclimatization period in suspended plastic cages. The mice were given free access to standard laboratory feed and water and kept on a 12-hour light-dark cycle throughout the study. Animal care and handling adhered to internationally recognized guidelines, ensuring humane treatment and compliance with institutional regulations. The research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/122. 2.4. Treatment schedule The experimental animals were divided into four groups (I–IV), each comprising ten mice (n = 10). Group I: received an oral treatment of corn oil at a dose of 2 mL/kg. Group II: was administered a single intraperitoneal (i.p.) dose of DEN (75 mg/kg) in normal saline, followed by a 7-day recovery period, and subsequently fed a diet formulated with 2-AAF (200 mg/kg). Group III: was treated daily with [6]-gingerol (100 mg/kg) in corn oil. Group IV: underwent a 7-day pre-treatment with [6]-gingerol in corn oil, after which they were subjected to the same regimen as Group II. The selection of doses for [6]-gingerol (100 mg/kg), 2-AAF (200 mg/kg in feed), and DEN (75 mg/kg) was guided by findings from a pilot study conducted in our laboratory, as well as data from previous study. 28 – 30 All treatments were administered via intraperitoneal injection and oral delivery for a period of four weeks. 2.5. Preparation of homogenates After the final treatment, all animals were weighed and euthanized. Following a 24-hour interval, they were sacrificed, and their livers were promptly excised. Part of the liver tissue was fixed in 4% buffered formalin for histological analysis while the other remaining liver tissue rinsed in cold phosphate-buffered saline, carefully blotted dry using filter paper, and weighed to facilitate immunohistochemical procedures and analysis of biochemical parameters. 2.6. Determination of liver function indices The serum activities of AST, ALT, ALP, and GGT were analyzed using Randox's commercially available diagnostic kits. 2.7. Determination of hepatic oxidative damage Liver tissues were homogenized in ten volumes of 0.1 M phosphate buffer (pH 7.4) using a Teflon homogenizer from Thomas Scientific, Swedesboro, NJ, USA. The homogenates were subsequently centrifuged at 15,000 g for 10 minutes at a temperature of 4°C using Thermo Scientific Sorvall TM WX Floor Ultra Centrifuges. The resulting supernatant was collected and stored for biochemical analysis. Protein concentration was determined using Bradford 31 , a spectroscopic method that measures protein concentration based on the binding of Coomassie Brilliant Blue dye to protein molecules at 595 nm. The activity of superoxide dismutase (SOD) was evaluated by monitoring the inhibition of adrenaline autoxidation under alkaline conditions at pH 10.2, as described in the method by Misra and Fridovich 32 , this method measures the enzyme's ability to convert superoxide radicals into hydrogen peroxide and oxygen, while catalase (CAT) activity was determined using hydrogen peroxide as a substrate, following the protocol by Clairborne 33 , this assay quantifies the enzyme's efficiency in breaking down hydrogen peroxide into water and oxygen. The hepatic activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were evaluated using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, following the methods by Rotruck et al. 34 and Habig et al. 35 , respectively. Also, reduced glutathione (GSH) levels were measured by quantifying the chromophoric complex formed between 5’,5’-dithiobis-2-nitrobenzoic acid (DTNB) and endogenous liver glutathione, as described by Jollow et al. 36 Lipid peroxidation (LPO) was assessed using a modified protocol, as described by Farombi et al. 29 This method involves measuring the formation of malondialdehyde (MDA), a pink-colored end product generated when 2-thiobarbituric acid (TBA) reacts with lipid peroxides. Hepatic hydrogen peroxide (H₂O₂) levels were evaluated following the method outlined by Wolff 37 , which is based on the oxidation of ferrous ions (Fe²⁺) coupled with sorbitol, leading to color development. Xanthine oxidase (XO) activity was assessed following Stripe and Della Corte method. 38 This assay is based on the catalytic conversion of xanthine to uric acid, with the reaction being measured spectrophotometrically at 290 nm. Nitric oxide (NO) levels were quantified by determining hepatic nitrite end-products using the Griess reagent, as described by Green et al. 39 Myeloperoxidase (MPO) activity was evaluated by monitoring the oxidation of o-Dianisidine in the presence of hydrogen peroxide, following the method of Granell et al. 40 with slight modifications. 2.8. Histological examination Liver samples were freshly excised and fixed in 10% formalin diluted with phosphate-buffered saline. Following 48 hours of fixation, the tissues were dehydrated, washed in xylene, and processed for infiltration and embedding. Subsequently, they were sectioned into slices of 5 µm thickness using a rotary microtome (Leica RM2125 RTS, Germany). The sections were stained with hematoxylin and eosin, and a pathologist, who remained unaware of the study groups, performed a detailed light microscopic examination of the liver tissue. 2.9. Immunohistochemistry Liver tissue samples embedded in paraffin wax on poly-L-lysine-coated slides were rehydrated by immersion in xylene and a graded series of ethanol concentrations (100–50%). Heat-induced epitope recovery was achieved by treating the sections with citrate buffer (pH 6) for 20 minutes, followed by immersion in cold water for 10 minutes. To block the activity of endogenous peroxidase, the tissue sections marked with a PAP pen were treated with 5% hydrogen peroxide in a dark environment. The slides were then incubated overnight at 4°C with primary polyclonal antibodies targeting anti-iNOS, anti-NF-kB, and anti-COX-2. After thorough washing with tris-buffered saline, the sections were incubated with horseradish peroxidase-labeled anti-mouse polyclonal secondary antibodies (Dako, Agilent Technologies, USA). The immune complexes formed were visualized by treating the sections with 0.05% 3,3-diaminobenzidine (DAB) for 5 minutes, followed by counterstaining with hematoxylin. Subsequent dehydration of the slides was performed using increasing concentrations of ethanol, followed by a brief immersion in xylene before mounting. Liver images were captured using a Leica DM 500 digital light microscope (Germany) equipped with a detachable ICC50 digital camera (Germany). The images were processed and analyzed using ImageJ-Fiji software (National Institutes of Health, USA). 2.10. Statistical analysis Data analysis utilized single-factor ANOVA via SPSS (version 17), with Bonferroni post hoc tests at a significance level of p < 0.05. 3. Results 3.1. Effects of DEN and 2-AAF on body weight gain, liver weight, and relative liver weight Administration of 75 mg/kg DEN followed by exposure to a diet containing 2-AAF (200 mg/kg) markedly reduced the final body weight in the treated mice in comparison to the control group, as illustrated in Table 1. Additionally, treatment with DEN and 2-AAF caused a statistically significant (p < 0.05) increase in liver weight and/or relative liver weight in comparison to the control. Pre-treatment with [6]-gingerol significantly (p < 0.05) mitigated these effects, reversing the impact of DEN and 2-AAF on both the final body weight and organ weight of the mice. Notably, the administration of [6]-gingerol alone did not alter the organ weight of the mice in comparison to the control group, as shown in Table 1. These findings align with observations reported by Sultana et al. 41 Table 1: Chemo preventive effect of 6-gingerol on body and organ weight (g) and relative organ weight (g/100g bw) of mice treated with DEN+2-AAF for 4 weeks. Data expressed as mean ± SD of 10 mice/group. a Values are significantly different when compared to control (p<0.05) and bValues are significantly different from DEN+2-AAF (p<0.05). 3.2. Effects of [6]-gingerol pretreatment on the activities of serum hepatic enzymes following DEN and 2-AAF exposure Figure 1 demonstrates that administering DEN (75 mg/kg) and 2-AAF (200 mg/kg in the diet) significantly (p < 0.05) increased serum levels of ALT, AST, ALP, and γ-GT in comparison to the control group. However, pre-treatment with [6]-gingerol (100 mg/kg) led to a notable decrease (p < 0.05) in the levels of these hepatic enzymes. 3.3. Effects of [6]-gingerol pre-treatment on liver redox status biomarkers Figure 2 reveals that mice exposed to 75 mg/kg DEN and a formulated diet of 2-AAF (200 mg/kg) exhibited a marked decline within the functionality of antioxidant enzymes, such as SOD, CAT, and GST relative to the control group. However, [6]-gingerol (100 mg/kg) administration effectively prevented the reduction in these enzyme activities in DEN and 2-AAF-treated mice (p < 0.05). Furthermore, [6]-gingerol restored hepatic glutathione (GSH) concentrations, reversing the depletion caused by DEN and 2-AAF exposure. Mice subjected to DEN and dietary 2-AAF treatment showed a marked decline in glutathione peroxidase (GPx) activity, alongside a rise in lipid peroxidation, indicated by heightened levels of malondialdehyde. Conversely, the administration of [6]-gingerol significantly mitigated oxidative stress (p < 0.05) in these mice. Additionally, DEN and 2-AAF exposure substantially increased hepatic myeloperoxidase (MPO) activity and nitric oxide (NO) levels compared to the control group. Pre-treatment with [6]-gingerol proved to be significantly effective (p < 0.05) in reducing MPO activity and NO levels, restoring them to near-normal values in DEN and 2-AAF-exposed mice, as shown in Fig. 3 . 3.4. Effects of [6]-gingerol pre-treatment on mice liver architecture Hematoxylin and eosin staining of liver sections from both the control and the [6]-gingerol-treated mice revealed normal histological features, including intact central veins, organized liver cords, healthy hepatocytes, and thin sinusoidal spaces. In contrast, mice exposed to DEN (75 mg/kg) and a diet containing 2-AAF (200 mg/kg) exhibited significant histopathological abnormalities. These included disrupted central veins, distorted portal spaces infiltrated with inflammatory cells such as lymphocytes and macrophages, lipid-laden swollen hepatocytes, and dilated sinusoidal spaces. Pre-treatment with [6]-gingerol (100 mg/kg) followed by DEN (75 mg/kg) and dietary 2-AAF exposure led to a notable improvement in liver histology. Treated mice displayed mild sinusoidal distortions, moderate hepatocyte hypertrophy, slight neutrophil infiltration, and a moderately intact central vein (Fig. 4 ). 3.5. Effects of [6]-gingerol on the expression of pro-inflammatory enzymes The pro-inflammatory biomarkers iNOS, COX-2, and NF-kB have been linked to stimulation caused by DEN + 2-AAF exposure. The anti-inflammatory effects of [6]-gingerol were assessed by analyzing its impact on DEN + 2-AAF-induced iNOS and COX-2 expression (Figs. 5 A and 5 B) through immunohistochemical examination of liver tissue in the treated mice. Mice challenged with DEN (75 mg/kg) and subsequently exposed to 200 mg/kg 2-AAF in their diet exhibited a significant (p < 0.05) expression of pro-inflammatory cytokines within the inflammatory foci of the liver relative to control mice. Nonetheless, pretreatment with [6]-gingerol successfully suppressed the immune expression of these inflammatory biomarkers in the DEN + 2-AAF-treated mice. 4. Discussion The toxicological impact of DEN combined with 2-AAF in the diet has been associated with its production of highly reactive intermediates and reactive oxygen species (ROS) during the process of bioactivation. Recent studies have highlighted its potential to induce hepatotoxicity, causing liver damage, inflammation, oxidative stress, and abnormal cell proliferation. 42 In this experimental model, a notable decrease in the final body weight, accompanied by an increase in liver weight, was observed in mice treated with an initial single dose of DEN and fed a 2-AAF-supplemented diet, highlighting the harmful effects of DEN + 2-AAF metabolites on growth and liver function (Table 1). Interestingly, the hepatoprotective properties of [6]-gingerol were evident without any associated changes in body weight, suggesting that [6]-gingerol administration exhibits minimal toxicity. A previous study has demonstrated [6]-gingerol tolerance with minimal or no toxicity. 43 A key indicator of hepatic injury is the release of intracellular enzymes into the plasma, resulting from chemical disruptions in hepatocyte transport functions. 44 Assessing serum enzymes, including AST, ALT, ALP, and γ-GT, provides a dependable indicator of liver health. Elevated levels of these enzymes in the blood are often indicative of impaired hepatic function or damage. 45 Based on our findings (Fig. 1 ), exposure to DEN and 2-AAF had detrimental effects on liver function markers in mice. This was demonstrated by a marked rise in serum activities of AST and ALT, along with increased levels of ALP and γ-GT in DEN + 2-AAF-treated mice relative to the control. Numerous studies attribute hepatoprotective properties of both pharmaceutical drugs and herbal agents to their ability to combat oxidative stress through antioxidant activity and free radical scavenging mechanisms. 46 – 48 However, the administration of [6]-gingerol effectively mitigated these effects, promoting liver health by significantly suppressing the elevated liver function enzymes. To further validate our serum findings, Fig. 4 highlights the histopathological evaluation of liver tissue. Exposure to DEN + 2-AAF in mice resulted in notable structural damage, including hyperchromatic nuclei, infiltration of inflammatory cells, and severe congestion, indicative of compromised hepatic integrity. However, the administration of [6]-gingerol significantly alleviated these hepatic lesions in DEN + 2-AAF-treated mice, restoring the liver tissue architecture to near-normal conditions comparable to that of the sham-treated control group. The observed restoration of serum transaminase levels, accompanied by hepatic parenchymal repair and hepatocyte regeneration, aligns with the findings of Salihu et al. 26 The hepatoprotective effects of [6]-gingerol can be ascribed to its antioxidant characteristics and its capacity to stabilize cellular membranes, thereby preventing the release of intracellular enzymes. Superoxide dismutase is a crucial enzymatic antioxidant responsible for the rapid conversion of superoxide ions into hydrogen peroxide, while catalase detoxifies reactive peroxide radicals, converting them into harmless water and oxygen. 49 Multiple studies recognize these enzymes as a robust protective duo, functioning as a defense system against oxidative damage. 50 – 52 This study demonstrated a decline in superoxide dismutase and catalase activities in mice exposed to DEN and 2-AAF, indicating the suppression of these enzymes. As a result, this leads to a buildup of deleterious superoxide radicals and hydrogen peroxide within the liver tissue of the treated mice. However, the administration of [6]-gingerol significantly restored SOD and CAT activities in the liver of DEN + 2-AAF-challenged mice. This finding indicates the potential antioxidant properties of [6]-gingerol, as evidenced in Figs. 2 A and 2 B. Many hepatoprotective agents, including Sylimarin 53 , Commiphora berryi 54 , and Phyllanthus niruri 55 , effectiveness in inhibiting lipid peroxidation induced by ROS has been well-established, owing to their strong free radical neutralizing capabilities. Glutathione (GSH) plays a crucial role in protecting against oxidative stress by directly neutralizing hydroxyl radicals and singlet oxygen, facilitating drug detoxification, and restoring antioxidant vitamins C and E to their functional states. In contrast, glutathione peroxidase (GPx), a family of selenium-dependent enzymes, interacts with GSH to break down hydrogen peroxide and lipid hydroperoxides. 56 , 57 Both GSH and GPx play crucial roles in maintaining the body's antioxidant defense mechanisms. Meanwhile, glutathione-S-transferase is instrumental in catalyzing the conjugation of toxic electrophilic compounds with GSH, functioning as a key enzyme in phase II drug detoxification processes. 58 Non-enzymatic antioxidants like glutathione and ascorbic acid safeguard against xenobiotic toxicity by neutralizing free radicals, a process that results in their oxidation. 59 Consequently, the hepatotoxic effects of DEN and 2-AAF are largely attributed to the free radicals generated during their metabolic activation. Our study revealed a substantial decrease in GSH levels, along with diminished activities of GPx and GST, in mice treated with DEN + 2-AAF (Figs. 2 C, 2 D, and 3 C). However, the administration of [6]-gingerol effectively counteracted these reductions, revitalizing antioxidant enzyme functions and replenishing the levels of GSH in treated mice. The observed elevation in hepatic GSH levels in mice exposed to DEN + 2-AAF-exposed mice suggests that [6]-gingerol plays a role in facilitating the neutralization of reactive oxygen species generated during the P450 bioactivation of DEN + 2-AAF. The DEN + 2-AAF+[6]-gingerol-treated groups exhibited nearly normal levels in enzymic and non-enzymic antioxidants, underscoring the significance of polyphenols in preserving the integrity of the endogenous cellular antioxidant defense system. Moreso, the antioxidant properties exhibited by [6]-gingerol in this study align with previous research, which has consistently highlighted its efficacy against reactive oxygen-mediated damage. 60 , 61 Lipid peroxidation serves as a key indicator of oxidative damage triggered by ROS, leading to compromised membrane functionality, reduced fluidity, and the inactivation of membrane-bound receptors and enzymes. 62 It also leads to the production of toxic by-products, such as malondialdehyde (MDA) and 4-hydroxynonenal, which can target cellular components like DNA, causing mutagenesis and cancer. 63 , 64 In this study, an elevated level of liver MDA was observed in mice treated with DEN + 2-AAF, indicating heightened ROS production and significant lipid peroxidation (Fig. 3 A). However, the administration of [6]-gingerol effectively reduced liver MDA levels in DEN + 2-AAF-challenged mice, demonstrating its antioxidant and free radical scavenging potential. These findings align with prior reports 27 , 28 , 65 , which further emphasized [6]-gingerol liver protecting capacity on lipid peroxidation. The oxidation reaction driven by xanthine oxidase mainly yields hydrogen peroxide, making it a crucial reactive oxygen species of interest when investigating xanthine oxidase involvement in inflammation and cellular communication. 66 In this study, treatment with DEN + 2-AAF in mice led to a substantial increase in H₂O₂ production, accompanied by elevated XO activity (Figs. 3 B and 3 F). However, the administration of [6]-gingerol effectively suppressed the heightened XO activity and H₂O₂ generation in the liver of DEN + 2-AAF-treated mice, attributed to its anti-inflammatory properties. These findings are consistent with the observations 67 , who reported increased XO activity along with enhanced H₂O₂ production in the liver of rats. To understand the role of nitrosative stress, we measured iNOS and NO levels in DEN + 2-AAF-induced liver inflammation. Evidence suggests that NO contributes to nitrosative stress and subsequent cellular damage by directly or indirectly interacting with biomolecules, especially in cases where the antioxidant defense system has been depleted. 68 Furthermore, inflammatory markers such as NO and MPO are upregulated via the expression of iNOS and COX-2. 24 , 25 , 69 Infiltrated neutrophils and macrophages exhibit elevated MPO activity, a key enzymatic marker of inflammation. Activated neutrophils also release additional pro-inflammatory cytokines, amplifying the inflammatory response. 70 Mounting scientific evidence strongly supports the intrinsic link between inflammation and liver dysfunction. 71 , 72 A key mechanism linking inflammation to hepatic disease is the transcription factor NF-κB, which is rapidly activated by cytokines, viruses, or chemotherapeutic agents and plays a regulatory role in COX-2 expression. 73 , 74 During prostaglandin biosynthesis, COX-2 has demonstrated significant potential as a molecular target for various anti-inflammatory and therapeutic agents. 75 Elevated NO and MPO levels, coupled with increased iNOS, COX-2, and NF-κB expression (Figs. 3 D, 3 E, and 5 A and B), confirm liver inflammation in DEN + 2-AAF-treated mice. Our result indicated that [6]-gingerol’s anti-inflammatory actions might involve downregulating iNOS and COX-2 by inhibiting NF-κB. Collectively, our data revealed that [6]-gingerol confers protection by downregulating iNOS and COX-2 via NF-κB pathway suppression. This emphasizes that its liver protection is tightly associated with its potent anti-inflammatory abilities. 5. Conclusion In conclusion, [6]-gingerol has been identified in several studies as an effective hepatoprotective agent, with its beneficial properties largely attributed to antioxidant and anti-inflammatory effects. 49 , 76 , 77 Furthermore, the findings from our study highlight the significance of [6]-gingerol in combating liver inflammatory conditions. Evidence from this study demonstrated that [6]-gingerol successfully mitigated DEN + 2-AAF-induced hepatic inflammation associated with preneoplasia in mice by inhibiting the excessive release of inflammatory cytokines, enhancing non-enzymatic and enzymatic antioxidant activity, and effectively suppressing oxidative stress and tissue damage. Consequently, [6]-gingerol shows promise as a therapeutic agent for the prevention and management of liver diseases induced by toxic xenobiotics. Contributing authors Ikenna Maduako : Investigation and analysis, drafting original manuscript, editing, and review. Olatunde Farombi : Concept idea, supervision, review, and editing Ethical approval /consent The research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/022. Conflict declaration We declared that there were no conflicting interests between the authors with regard to this publication. Abbreviations 2-AAF 2-Acetylaminofluorene ALP Alkaline Phosphatase ALT Alanine Transaminase AST Aspartate Aminotransferase CAT Catalase COX-2 Cyclooxygenase-2 DEN Diethylnitrosamine GSH Reduced glutathione GST Glutathione S-transferase H2O2 Hydrogen peroxide iNOS Inducible nitric oxide synthase LPO Lipid peroxidation MPO Myeloperoxidase NF-kB Nuclear Factor kappa-B NO Nitric oxide SOD Superoxide dismutase XO Xanthine oxidase y-GT Gamma-Glutamyl Transferase Declarations Contributing authors Ikenna Maduako: Investigation and analysis, drafting original manuscript, editing, and review. Olatunde Farombi: Concept idea, supervision, review, and editing Ethical approval/consent The research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/022. Conflict declaration We declared that there were no conflicting interests between the authors with regard to this publication. Acknowledgements The authors are grateful to Dr Babajide Ajayi and Dr Ifeoluwa Awogbindin for their assistance in the extraction of 6-gingerol used in this study and immunohistochemistry imaging techniques. Declaration of generative AI and AI-assisted technologies During the preparation of this work, the author(s) used Grammarly to improve language and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and took full responsibility for the publication's content. References Friedman S (2003) Liver fibrosis from bench to bedside. J Hepatol 38:38–53 Pinzani M, Rombouts K (2004) Liver fibrosis: from the bench to clinical targets. Dig Liver Dis 36:231–242 Sakeran MI, Zidan N, Rehman H, Aziz AT, Saggu S (2014) Abrogation by trifolium alexandrium root extract on hepatotoxicity induced by acetaminophen in rats. Redox Rep 19(1):26–33 Canbay A, Friedman S, Gores GJ (2004) Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39:273–278 Poli G (2000) Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med 21:49–98 Janani P, Sivakumari K, Parthasarathy C (2009) Hepatoprotective activity of bacoside A against N-nitrosodiethylamine-induced liver toxicity in adult rats. Cell Biol Toxicol 25:425–434 Ciemniak A (2006) A comparison of N–nitrosodimethylamine contents in selected meat products. Rocz Panstw Zakl Hig 57:341–346 Garcea G, Dennison AR, Steward WP, Berry DP (2005) Role of inflammation in pancreatic carcinogenesis and the implications for future therapy. Pancreatol 5(6):514–529 Mossman BT, Kamp DW, Weitzman SA (1996) Mechanisms of carcinogenesis and clinical features of asbestos associated cancers. Cancer Invest 14(5):466–480 Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radical and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84 Al-Rejaie SS, Alesia AM, Al-Yahya AA (2009) Progression of diethylnitrosamine-induced hepatic carcinogenesis in carnitine-depleted rats. World J Gastroenterol 15:1373–1380 Prakash B, Motiwale L, Ignle AD (2002) Protective effect of Rhodotorula glutinis NCIM3353 on the development of hepatic preneoplastic lesions. Curr Sci 83:303–308 Tamano I, Shirai ST (2003) A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 94:3–8 Claria J, Lopez-Parra M, Titos E, Gonzalez-Perez A (2011) The role of inflammatory mediators in liver failure. In: Gines P. Kamath P. Arroyo V. (eds), Chronic liver failure, Clin. Gastroenterol. ; pp 131–153. https://doi.org/10.1007/978-1-60761-866-9_7 Nah SS, Choi IY, Lee CK, Oh JS, Kim YG, Moon HB, Yoo B (2008) Effects of advanced glycation end products on the expression of COX-2, PGE2 and NO in human osteoarthritic chondrocytes. Rheumatol 47(4):425–431 Tabernero A, Schneider F, Potenza MA, Randriamboavonjy V, Chasserot S, Wolf P, Mitolo-Chieppa D, Stoclet JC, Andriantsitohaina R (2003) Cycloxygenase-2 and inducible nitric oxide synthase in omental arteries harvested from patients with severe liver diseases: immuno-localization and influence on vascular tone. Intensive Care Med 29(2):262–270 Lantz RC, Chen GJ, Sarihan M, Sólyom AM, Jolad SD, Timmermann BN (2007) The effect of extracts from ginger rhizome on inflammatory mediator production. Phytomedicine 14(2–3):123–128 Surh YJ (2003) Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 3:768–780 Surh Y (1999) Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat Res 428(1–2):305–327 Surh YJ, Lee SS (1994) Enzymic reduction of [6]-gingerol, a major pungent principle of ginger, in the cell-free preparation of rat liver. Life Sci 54(19):321–326 Nonn L, Duong D, Peehl DM (2007) Chemopreventive anti-inflammatory activities of curcumin and other phytochemicals mediated by MAP kinase phosphatase-5 in prostate cells. Carcinogenesis 28:1188–1196 Tripathi S, Maier KG, Bruch D, Kittur DS (2007) Effect of 6-gingerol on proinflammatory cytokine production and co-stimulatory molecule expression in murine peritoneal macrophages. J Surg Res 138:209–213 Pan MH, Hsieh MC, Hsu PC, Ho SY, Lai CS, Wu H, Sang S, Ho CT (2008) 6-Shogaol suppressed lipopolysaccharide induced up-expression of iNOS and COX-2 in murine macrophages. Mol Nutr Food Res 52:1467–1477 Aktan F, Henness S, Tran VH, Duke CC, Roufogalis BD, Ammit AJ (2006) Gingerol metabolite and a synthetic analogue Capsarol inhibit macrophage NF-kappa B-mediated iNOS gene expression and enzyme activity. Planta Med 72(8):727–734 Tjendraputra E, Tran VH, Liu-Brennan D, Roufogalis BD, Duke C (2001) Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells. Bioorg Chem 29:156–163 Salihu M, Ajayi BO, Adedara IA, Farombi EO (2016) 6-gingerol-rich fraction from zingiber officinale prevents hematotoxicity and oxidative damage in kidney and liver of rats exposed to carbendazim. J Diet Suppl 13(4):433–448 Ajayi BO, Adedara IA, Farombi EO (2015) Pharmacological activity of 6-Gingerol in dextran sulphate sodium- induced ulcerative colitis in balb/c mice. Phytother Res. 10.1002/ptr.5286 Ajayi BO, Adedara IA, Farombi EO (2016) Benzo[a]pyrene induces oxidative stress, pro-inflammatory cytokines, expression of nuclear factor-kappaB and deregulation of wnt/beta-catenin signaling in colons of BALB/c mice. Food Chem Toxicol 95:42–51 Farombi EO, Tahnteng JG, Agboola AO, Nwankwo JO, Emerole GO (2000) Chemoprevention of 2-acetylaminofluorene- induced hepatotoxicity and lipid peroxidation in rats by kolaviron – a garcinia kola seed extract. Food Chem Toxicol 38:535–541 Klinman NR, Erslev AJ (1963) Cellular response to partial hepatectomy. Proceedings of the Society for Experimental Biology and medicine. ; 112: 338–40 Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175 Clairborne A (1995) Catalase activity. In: Greewald AR (ed) Handbook of Methods for Oxygen Radical Research. CRC, pp 237–242 Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590 Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first orange for measurement of hydroperoxides. Methods Enzymol 233:182–189 Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR (1974) Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4 bromobenzene oxide as the hepatotoxic metabolite. Pharmacol 11:151–169 Wolff SP (1994) Ferrous ion oxidation in the presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol 233:182–189 Stripe F, Della-Corte E (1969) The regulation of rat liver xanthine oxidase conversion in vitro of enzyme activity from dehydrogenase (type D) to oxidase (type O). J Biol Chem 244:3855–3863 Green LC, Wagner DA, Glogowski J, Skiper PL, Wishnock JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite and [15 N] nitrate in biological fluids. Anal Biochem 126:131–138 Granell S, Gironella M, Bulbena O, Panes J, Mauri M, Sabater L, Aparisi L, Gelpí (2003) E. Closa D. Heparin mobilizes xanthine oxidase and induces lung inflammation in acute pancreatitis. Crit. Care Med. ; 31: 525–530 Sultana S, Nafees S, Khan AQ (2013) Perillyl alcohol as a protective modulator against rat hepatocarcinogenesis via amelioration of oxidative damage and cell proliferation. Hum Experimental Toxicol 32(11):1179–1192 Tolba R, Kraus T, Liedtke C, Schwarz M, Weiskirchen R (2015) Diethylnitrosamine (DEN)-induced carcinogenic liver injury in mice. Lab Anim 49(1 Suppl):59–69. https://doi.org/10.1177/0023677215570086 Ajayi BO, Adedara IA, Farombi EO (2019) 6-Gingerol abates benzo[a]pyrene-induced colonic injury via suppression of oxido-inflammatory stress responses in BALB/c mice. Chemico-Biol Interact 307:1–7 Sabir SM, Rocha JB (2008) Antioxidant and hepatoprotective activity of aqueous extract of solanum fastigiatum against paracetamol-induced liver damage in mice. J Ethnopharmacol 120(2):226–232 Kaplan MM (1993) Laboratory test. In: Schiff L, Schiff ER (eds) Diseases of the liver. JB Lippincott, Seventh Philadelphia, pp 108–144 Rafie R, Hosseini SA, Hajiani E, Malehi AS, Mard SA (2020) Effect of Ginger Powder Supplementation in Patients with Non-Alcoholic Fatty Liver Disease: A Randomized Clinical Trial. Clin Exp Gastroenterol 13:35–45 Naik SR, Panda VS (2007) Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodents. Liver Int 27:393–399 Kodai S, Takemura S, Minamiyama Y, Hai S, Yamamoto S, Kubo S, Yoshida Y, Niki E, Okada S, Hirohashi K, Suehiro S (2007) S-allyl cysteine prevents CCl induced acute liver injury in rats. Free Radic Res 41:489–497 Halliwell B (1990) How to characterize a biological antioxidant. Free Radic Res Comm 9:1–32 Halliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1–85 Reiter RJ, Tan D, Osuna C, Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 7:444–458 Wang BJ, Lien YH, Yu ZR (2004) Supercritical fluid extractive fractionation: study of the antioxidant activities of propolis. Food Chem 86:237–243 Manokaran S, Jaswanth A, Sengottuvelu S, Nandhakumar J, Duraisamy R, Karthikeyan D (2008) Hepatoprotective activity of Aerva lanata Linn. Against paracetamol induced hepatotoxicity in rats. Res J Pharm Tech 1:398–400 Gowri-Shanka NL, Manavalan R, Venkappayya D, David RC (2008) Hepatoprotective and antioxidant effects of Commiphora berryi (Arn) Engl bark extract against CCl -induced oxidative damage in rats. Food Chem Toxicol 46:3182–3185 Bhattacharjee R, Sil PC (2007) Protein isolate from the herb, Phyllanthus niruri L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties. Food Chem Toxicol 45:817–826 Masella R, Di BR, Vari R, Filesi C, Giovannini C (2005) Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione related enzymes. J Nutr Biochem 16:577–586 Arthur JR (2000) The gluthatione peroxidase. Cell Mol Life Sci 57(13–14):1825–1835 Valko M, Jomova K, Rhodes CJ, Kuca K, Musílek (2016) K. Redox- and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol. ; 90: 1–37 Kadiska MB, Gladen BC, Baird DD, Dikalov AE, Sohal RS, Hatch GB, Jones DP, Mason RP, Barret JC (2000) Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl. poisoning? J Free Rad Biol Med 28:838–845 Ippoushi K, Azuma K, Ito H, Horie H, Higashio H (2023) [6]-Gingerol inhibits nitric oxide synthesis in activated 1774.1 mouse macrophages and prevents peroxynitrite induced oxidation and nitration reactions. Life Sci 73(26):3427–3437 Lee TY, Lee KC, Chen SY, Chang HH (2019) 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-KB pathways in lipopolysaccharide-stimulated mouse macrophages. Biochem Biophys Res Commun 382(1):134–113 Viani P, Cervato G, Fiorilli A, Cestaro B (1991) Age-related differences in synaptosomal peroxidative damage and membrane properties. J Neurochem 56:253–258 Zawart LL, Meerman JH, Commandeur JN, Vermeulen NP (2009) Biomarkers of free radical damage applications in experimental animals and humans. Free Radic Biol Med 26:202–226 Banaker MC, Paramacivan SK, Chattopadhyay MB, Datta S, Chakraborty P, Chatterjee M, Kannan K, Thygarajan E (2014) 1α, 25-dihydroxyvitamin D3 prevents DNA damage and restores antioxidant enzymes in rat hepatocarcinogenesis induced by diethylnitrosamine and promoted by phenobarbital. World J Gastroenterol 10:1268–1275 Sanchez-Perez C, Carrasco-Legleu C, Garcia-Cuellar J, Perez-Carreon S, Hernandez-Garcia M, Salcido-Neyoy L, Aleman-Lazarini S (2015) Oxidative stress in carcinogenesis. Correlation between lipid peroxidation and induction of preneoplastic lesions in rat hepatocarcinogenesis. Cancer Lett 217:25–32 Kelly EE, Khoo NKH, Hindley NJ, Malik UZ, Freeman BA, Tarpey MM (2019) Hydrogen peroxide is the major oxidant product of xanthine oxidase. Free Radic Biol Med 48(4):493–499 Gu Z, Nakamura T, Lipton SA (2019) Redox reactions induced by nitrosative stress mediate protein misfolding and mitochondrial dysfunction in neurodegenerative diseases. Mol Neurobiol 41:55–72 Kiuchi F, Iwakami S, Shibuya M, Hanaoka F, Sankawa U (2022) Inhibition of prostaglandin and leukotriene biosynthesis by gingerols and diarylheptanoids. Chem Pharm Bull 40(2):387–391 Stanley PL, Steiner S, Havens M, Tramposch KM (2018) Mouse skin inflammation induced by multiple topical application of 12 –O- tetra decanoylphorbol-13-acetate. Ski Pharmacol 4:262–271 Balkwill F, Coussens LM (2004) The NF-KB protein is a key player in inflammation. It now seems that it might also activate signalling pathways, in both cancer cells and tumour-associated inflammatory cells that promote malignancy. Nature 431:405–406 Greten RF, Karin M (2004) The IKK/NF-kB activation pathway—a target for prevention and treatment of cancer. Cancer Lett 206:193–199 Garg A, Aggarwal BB (2002) Nuclear transcription factor-kappaB as a target for cancer drug development. Leukemia 16:1053–1068 Mohan S, Epstein JB (2003) Carcinogenesis and cyclooxygenase: the potential role of COX-2 inhibition in upper aerodigestive tract in cancer. Oral Oncol 39:537–546 Williams CS, Shattuck-Brandt RL, DuBois RN (2009) The role of COX-2 in intestinal cancer. Expert Opin Investig Drugs 8:1–12 El Sharaky AS, Newairy AA, Kamel MA, Eweda SM (2009) Protective effect of ginger extract against bromobenzene-induced hepatotoxicity in male rats. Food Chem Toxicol 47:1584–1590 Aeschbach R, Loliger J, Scott BC, Murcia A, Butler J, Halliwell B, Aruoma OI (1994) Antioxidant actions of thymol, carcacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem Toxicol 2:31–36 Additional Declarations The authors declare no competing interests. Supplementary Files GraphicalAbstract.ppt Graphical Abstract ConflictofInterest.doc Conflict declaration Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6792607\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":464635598,\"identity\":\"c0fc61e0-3d96-40e4-a53e-2e49b537cea0\",\"order_by\":0,\"name\":\"Ikenna C. Maduako\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACNiCWYGBIYOCXYGwAsplJ0CI5g1gtDDAtBjfAbCK08IkdfnibpyZNzvh2c5sEQ4V1YoN0+wX8DpNOM7bmOZZjbHbnIFDLmfTEBpkzBQS0JJhJ87BVJG67kdgmwdh2OLFBIieBgJb0b9I8/yoSN88AaflHlJYcM2netpzEDRIgLQ0gLekHCGkptpzbl2Yscedgs0XCsXTjNokcvDoY5Genb7zx5luyHP/s9oc3PtRYy/ZLpD/ArwcFgDzBxsBjQIIWCGAnxZZRMApGwSgYAQAApJ9C//wzAS4AAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0002-1001-4866\",\"institution\":\"Benson Idahosa University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Ikenna\",\"middleName\":\"C.\",\"lastName\":\"Maduako\",\"suffix\":\"\"},{\"id\":464635666,\"identity\":\"35b57f2f-fa30-44d0-be3e-fd7982d7fd78\",\"order_by\":1,\"name\":\"Ebenezer O. Farombi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Ibadan\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ebenezer\",\"middleName\":\"O.\",\"lastName\":\"Farombi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-05-31 18:55:50\",\"currentVersionCode\":1,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":true,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":true},\"doi\":\"10.21203/rs.3.rs-6792607/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6792607/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":83828573,\"identity\":\"ff41edcf-260a-47c8-a5d4-271238e47f81\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":105781,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of 6-Gingerol on serum enzymes markers AST, ALT, GGT and ALP in DEN+2-AAF exposed mice for 4 weeks. Each bar represents mean± SD of 10 animals. a Values differ significantly from control (p\\u0026lt;0.05). b Values differ significantly from DEN+2-AAF (p\\u0026lt;0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/d04a8d6badf912bce4d8fc6d.png\"},{\"id\":83828570,\"identity\":\"143bff16-49f4-471a-a19c-4ae677f3eda5\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":95077,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of 6-Gingerol on the activities of SOD, CAT, GSH and GST in liver of DEN+2AAF-treated mice respectively for 4 weeks. Each bar represents mean ± SD of 10 mice. a: Values differ significantly from control (p \\u0026lt; 0.05). b: Values differ significantly from DEN+2AAF group (p \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/5301d1791f531afd8210e999.png\"},{\"id\":83828571,\"identity\":\"2497a40c-a933-49c4-bfe0-b7b88d5d8085\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":77794,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect of 6-Gingerol on MDA, H2O2, GPX, NO, MPO and XO levels in liver of DEN+2AAF-treated mice for 4 weeks. Each bar represents mean ± SD of 10 mice. a: Values differ significantly from control (p \\u0026lt; 0.05). b: Values differ significantly from DEN+2AAF group (p \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/803614e9c1da8be8504e6ebb.png\"},{\"id\":83828575,\"identity\":\"cdae6596-2230-40d1-a5e0-987f9d42d39e\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":538068,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRepresentative photomicrographs of histopathological examination of experimental mice liver after 4 weeks treatment. H\\u0026amp;E (X400). (A) Control orally administered corn oil (2mL/kg): showed normal liver architecture. (B) DEN+2-AAF: showed fibrosis and inflammatory cells, mainly lymphocytes and macrophages in the distorted portal space. (C) 6[G]: showed normal liver architecture. (D) 6[G]+DEN+2-AAF: showed restored integrity of the liver with mild hepatic congestion.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/82cbb44471b614ea8e15ec18.png\"},{\"id\":83828798,\"identity\":\"2e19b2cc-4ffa-4190-b902-f09b354c3a6c\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 11:06:34\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1332573,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA: Influence of 6G on hepatic NF-κB protein expression in mice exposed to DEN+2AAF for 4 weeks. Brown colour indicates positive staining for NF-κB antibody on 5 μm section of paraffin fixed mice liver. (x40). Each bar represents mean ± SD of 10 mice. a: Values differ significantly from control (p \\u0026lt; 0.05).b: Values differ significantly from DEN+2AAF group (p \\u0026lt; 0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eB: Influence of 6G on hepatic iNOS (A) and COX-2 (B) protein expression in mice exposed to DEN+2AAF for 4 weeks. Brown colour indicates positive staining for iNOS and COX-2 antibody on 5 μm section of paraffin fixed mice liver. (x40). Each bar represents mean ± SD of 10 mice. a: Values differ significantly from control (p \\u0026lt; 0.05).b: Values differ significantly from DEN+2AAF group (p \\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Slide5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/af1973cd8504b72a31a297d3.png\"},{\"id\":83829514,\"identity\":\"0eee899d-0234-4d9f-93a1-1cb34002eaf2\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 11:14:35\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3119971,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/eaadf618-09f6-494f-92e4-4ea72f6086cf.pdf\"},{\"id\":83828577,\"identity\":\"c2c12efa-8815-4455-b996-11a887882e7c\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"ppt\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1546752,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGraphical Abstract\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"GraphicalAbstract.ppt\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/98b06f207dfa7d8bfb2dff5a.ppt\"},{\"id\":83828572,\"identity\":\"7dbde8c1-a60f-4d91-b66b-88ea694bf196\",\"added_by\":\"auto\",\"created_at\":\"2025-06-03 10:58:34\",\"extension\":\"doc\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":29184,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eConflict declaration\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"ConflictofInterest.doc\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6792607/v1/433ac764cbc723fe894e4093.doc\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"\\u003cp\\u003e6-Gingerol inhibition of NF-kB expression in diethylnitrosamine initiated, 2-acetylaminofluorene-promoted liver dysfunction and inflammation involves induction of antioxidant enzymes and suppression of pro-inflammatory cytokines\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003e \\u003c/p\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003ePersistent liver inflammation arises from a combination of chronic effects triggered by various harmful substances.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e This condition can ultimately progress to cirrhosis, characterized by the formation of fibrous tissue and hepatocyte nodules. However, the intricate processes leading to liver dysfunction and inflammation are not fully understood. The characteristics of this condition include alterations in biochemical pathways, programmed cell death, activation of hepatic stellate cells (HSCs), free radical production, necrosis, and oxidative stress, all of which are essential factors in these mechanisms.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR5\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e Exposure to environmental and occupational contaminants, such as diethylnitrosamine (DEN), 2-acetylaminofluorene (2-AAF), specific dietary factors, viruses, and agrochemicals, is associated with a heightened risk of developing liver preneoplasia.\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e – \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eDEN, a potent carcinogen and hepatotoxic agent, is commonly employed in animal studies to investigate the onset and progression of hepatic cancer. It induces liver lesions ranging from degenerative to neoplastic changes, offering insights into the development of hepatic carcinogenesis.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR12\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e When combined with other toxins like 2-AAF, phenobarbital, or carbon tetrachloride, DEN's ability to induce liver dysfunction has garnered significant scientific interest.\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e These substances often stimulate the overproduction of chemical mediators, including cytokines, chemokines, NF-kB associated proteins. While these molecules play roles in pathogen elimination and tissue repair, their dysregulated expression can lead to chronic inflammatory disorders such as endotoxemia and fulminant hepatitis.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eEfforts to mitigate these harmful effects have turned to natural nutritional phytochemicals, which are gaining recognition as a foundation for chemoprevention. Among these, the ginger rhizome (Zingiber officinale), used for its antioxidant and anti-inflammatory properties, has been a staple in traditional medicine and culinary practices worldwide.\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e Its chemical constituents, notably [6]-gingerol and [6]-shogaol, have shown to exhibit various biological effects, including the inhibition of tumor growth and cell proliferation.\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e Numerous studies indicate that [6]-gingerol suppresses NF-κB-regulated proteins linked to cell proliferation and angiogenesis, while also inhibiting inflammatory mediators like TNF-α and IL-1β.\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e Preclinical and experimental studies further highlight its potential to reduce nitric oxide synthesis and counteract inflammation.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR24 CR25\\\" citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eDespite these findings, the specific mechanisms by which [6]-gingerol exerts its hepatoprotective effects are still uncertain. This study is designed to uncover the mechanism by which [6]-gingerol prevents sub-chronic liver inflammation and oxidative damage in a DEN + 2-AAF-induced mouse model.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cp\\u003e\\u003c/p\\u003e \"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003ch2\\u003e2.1. Chemicals\\u003c/h2\\u003e\\n\\u003cp\\u003eDiethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) were bought from Sigma-Aldrich Co., St. Louis, MO, USA. Polyclonal antibodies targeting nuclear factor (NF-kB), cyclooxygenase-2, and inducible nitric oxide synthase (iNOS) procured from Elabscience, China. Other analytical reagents were purchased from British Drug Houses, located in Poole, Dorset, UK.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.2. \\u003cstrong\\u003eAuthentication and Extraction of [6]-gingerol\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eFresh ginger roots and foliage were purchased at Bodija Market in Ibadan, Nigeria, from a local vendor. The collected plant samples were authenticated at the herbarium (voucher number: UIH-22390) at the Department of Botany, University of Ibadan. The extraction of [6]-gingerol, with a purity exceeding 91%, was conducted using a method described by Ajayi et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003ch2\\u003e2.3. Animals\\u003c/h2\\u003e\\n\\u003cp\\u003eForty BALB/c mice (16-18g) were sourced from the University of Ibadan\\u0026apos;s Faculty of Veterinary Medicine. Prior to the experiment, the animals underwent a one-week acclimatization period in suspended plastic cages. The mice were given free access to standard laboratory feed and water and kept on a 12-hour light-dark cycle throughout the study. Animal care and handling adhered to internationally recognized guidelines, ensuring humane treatment and compliance with institutional regulations. The research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/122.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.4. Treatment schedule\\u003c/h2\\u003e\\n\\u003cp\\u003eThe experimental animals were divided into four groups (I\\u0026ndash;IV), each comprising ten mice (n\\u0026thinsp;=\\u0026thinsp;10).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGroup I:\\u0026nbsp;\\u003c/strong\\u003ereceived an oral treatment of corn oil at a dose of 2 mL/kg.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGroup II:\\u0026nbsp;\\u003c/strong\\u003ewas administered a single intraperitoneal (i.p.) dose of DEN (75 mg/kg) in normal saline, followed by a 7-day recovery period, and subsequently fed a diet formulated with 2-AAF (200 mg/kg).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGroup III:\\u0026nbsp;\\u003c/strong\\u003ewas treated daily with [6]-gingerol (100 mg/kg) in corn oil.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGroup IV:\\u0026nbsp;\\u003c/strong\\u003eunderwent a 7-day pre-treatment with [6]-gingerol in corn oil, after which they were subjected to the same regimen as Group II.\\u003c/p\\u003e\\n\\u003cp\\u003eThe selection of doses for [6]-gingerol (100 mg/kg), 2-AAF (200 mg/kg in feed), and DEN (75 mg/kg) was guided by findings from a pilot study conducted in our laboratory, as well as data from previous study.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u0026ndash;\\u003cspan class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e All treatments were administered via intraperitoneal injection and oral delivery for a period of four weeks.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.5. Preparation of homogenates\\u003c/h2\\u003e\\n\\u003cp\\u003eAfter the final treatment, all animals were weighed and euthanized. Following a 24-hour interval, they were sacrificed, and their livers were promptly excised. Part of the liver tissue was fixed in 4% buffered formalin for histological analysis while the other remaining liver tissue rinsed in cold phosphate-buffered saline, carefully blotted dry using filter paper, and weighed to facilitate immunohistochemical procedures and analysis of biochemical parameters.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.6. Determination of liver function indices\\u003c/h2\\u003e\\n\\u003cp\\u003eThe serum activities of AST, ALT, ALP, and GGT were analyzed using Randox\\u0026apos;s commercially available diagnostic kits.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.7. Determination of hepatic oxidative damage\\u003c/h2\\u003e\\n\\u003cp\\u003eLiver tissues were homogenized in ten volumes of 0.1 M phosphate buffer (pH 7.4) using a Teflon homogenizer from Thomas Scientific, Swedesboro, NJ, USA. The homogenates were subsequently centrifuged at 15,000 g for 10 minutes at a temperature of 4\\u0026deg;C using Thermo Scientific Sorvall TM WX Floor Ultra Centrifuges. The resulting supernatant was collected and stored for biochemical analysis. Protein concentration was determined using Bradford\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e, a spectroscopic method that measures protein concentration based on the binding of Coomassie Brilliant Blue dye to protein molecules at 595 nm. The activity of superoxide dismutase (SOD) was evaluated by monitoring the inhibition of adrenaline autoxidation under alkaline conditions at pH 10.2, as described in the method by Misra and Fridovich\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e, this method measures the enzyme\\u0026apos;s ability to convert superoxide radicals into hydrogen peroxide and oxygen, while catalase (CAT) activity was determined using hydrogen peroxide as a substrate, following the protocol by Clairborne\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, this assay quantifies the enzyme\\u0026apos;s efficiency in breaking down hydrogen peroxide into water and oxygen. The hepatic activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were evaluated using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, following the methods by Rotruck et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e and Habig et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e, respectively. Also, reduced glutathione (GSH) levels were measured by quantifying the chromophoric complex formed between 5\\u0026rsquo;,5\\u0026rsquo;-dithiobis-2-nitrobenzoic acid (DTNB) and endogenous liver glutathione, as described by Jollow et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLipid peroxidation (LPO) was assessed using a modified protocol, as described by Farombi et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e This method involves measuring the formation of malondialdehyde (MDA), a pink-colored end product generated when 2-thiobarbituric acid (TBA) reacts with lipid peroxides. Hepatic hydrogen peroxide (H₂O₂) levels were evaluated following the method outlined by Wolff \\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e\\u003c/sup\\u003e, which is based on the oxidation of ferrous ions (Fe\\u0026sup2;⁺) coupled with sorbitol, leading to color development. Xanthine oxidase (XO) activity was assessed following Stripe and Della Corte method.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e This assay is based on the catalytic conversion of xanthine to uric acid, with the reaction being measured spectrophotometrically at 290 nm. Nitric oxide (NO) levels were quantified by determining hepatic nitrite end-products using the Griess reagent, as described by Green et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u003c/sup\\u003e Myeloperoxidase (MPO) activity was evaluated by monitoring the oxidation of o-Dianisidine in the presence of hydrogen peroxide, following the method of Granell et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e with slight modifications.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.8. Histological examination\\u003c/h2\\u003e\\n\\u003cp\\u003eLiver samples were freshly excised and fixed in 10% formalin diluted with phosphate-buffered saline. Following 48 hours of fixation, the tissues were dehydrated, washed in xylene, and processed for infiltration and embedding. Subsequently, they were sectioned into slices of 5 \\u0026micro;m thickness using a rotary microtome (Leica RM2125 RTS, Germany). The sections were stained with hematoxylin and eosin, and a pathologist, who remained unaware of the study groups, performed a detailed light microscopic examination of the liver tissue.\\u003c/p\\u003e\\n\\u003ch2\\u003e2.9. Immunohistochemistry\\u003c/h2\\u003e\\n\\u003cp\\u003eLiver tissue samples embedded in paraffin wax on poly-L-lysine-coated slides were rehydrated by immersion in xylene and a graded series of ethanol concentrations (100\\u0026ndash;50%). Heat-induced epitope recovery was achieved by treating the sections with citrate buffer (pH 6) for 20 minutes, followed by immersion in cold water for 10 minutes. To block the activity of endogenous peroxidase, the tissue sections marked with a PAP pen were treated with 5% hydrogen peroxide in a dark environment. The slides were then incubated overnight at 4\\u0026deg;C with primary polyclonal antibodies targeting anti-iNOS, anti-NF-kB, and anti-COX-2. After thorough washing with tris-buffered saline, the sections were incubated with horseradish peroxidase-labeled anti-mouse polyclonal secondary antibodies (Dako, Agilent Technologies, USA). The immune complexes formed were visualized by treating the sections with 0.05% 3,3-diaminobenzidine (DAB) for 5 minutes, followed by counterstaining with hematoxylin. Subsequent dehydration of the slides was performed using increasing concentrations of ethanol, followed by a brief immersion in xylene before mounting. Liver images were captured using a Leica DM 500 digital light microscope (Germany) equipped with a detachable ICC50 digital camera (Germany). The images were processed and analyzed using ImageJ-Fiji software (National Institutes of Health, USA).\\u003c/p\\u003e\\n\\u003ch2\\u003e2.10. Statistical analysis\\u003c/h2\\u003e\\n\\u003cp\\u003eData analysis utilized single-factor ANOVA via SPSS (version 17), with Bonferroni post hoc tests at a significance level of p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1. Effects of DEN and 2-AAF on body weight gain, liver weight, and relative liver weight\\u003c/h2\\u003e\\n \\u003cp\\u003eAdministration of 75 mg/kg DEN followed by exposure to a diet containing 2-AAF (200 mg/kg) markedly reduced the final body weight in the treated mice in comparison to the control group, as illustrated in Table\\u0026nbsp;1. Additionally, treatment with DEN and 2-AAF caused a statistically significant (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) increase in liver weight and/or relative liver weight in comparison to the control. Pre-treatment with [6]-gingerol significantly (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) mitigated these effects, reversing the impact of DEN and 2-AAF on both the final body weight and organ weight of the mice. Notably, the administration of [6]-gingerol alone did not alter the organ weight of the mice in comparison to the control group, as shown in Table\\u0026nbsp;1. These findings align with observations reported by Sultana et al.\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003eTable 1: Chemo\\u003c/span\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003epreventive effect of 6-gingerol on body and organ weight (g) and relative organ weight (g/100g bw) of mice treated with DEN+2-AAF for 4 weeks.\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cimg src=\\\"data:image/png;base64,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\\\" style=\\\"width: 1000px;\\\"\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003eData expressed as mean \\u0026plusmn; SD of 10 mice/group. a Values are significantly different when compared to control (p\\u0026lt;0.05) and bValues are significantly different from DEN+2-AAF (p\\u0026lt;0.05).\\u003c/span\\u003e\\u003csup\\u003e\\u003cspan class=\\\"CitationRef\\\"\\u003e\\u003cbr\\u003e\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003ch2\\u003e3.2. \\u003cstrong\\u003eEffects of [6]-gingerol pretreatment on the activities of serum hepatic enzymes following DEN and 2-AAF exposure\\u003c/strong\\u003e\\u003c/h2\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eFigure \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e demonstrates that administering DEN (75 mg/kg) and 2-AAF (200 mg/kg in the diet) significantly (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) increased serum levels of ALT, AST, ALP, and \\u0026gamma;-GT in comparison to the control group. However, pre-treatment with [6]-gingerol (100 mg/kg) led to a notable decrease (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) in the levels of these hepatic enzymes.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.3. Effects of [6]-gingerol pre-treatment on liver redox status biomarkers\\u003c/h2\\u003e\\n \\u003cp\\u003eFigure \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e reveals that mice exposed to 75 mg/kg DEN and a formulated diet of 2-AAF (200 mg/kg) exhibited a marked decline within the functionality of antioxidant enzymes, such as SOD, CAT, and GST relative to the control group. However, [6]-gingerol (100 mg/kg) administration effectively prevented the reduction in these enzyme activities in DEN and 2-AAF-treated mice (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Furthermore, [6]-gingerol restored hepatic glutathione (GSH) concentrations, reversing the depletion caused by DEN and 2-AAF exposure. Mice subjected to DEN and dietary 2-AAF treatment showed a marked decline in glutathione peroxidase (GPx) activity, alongside a rise in lipid peroxidation, indicated by heightened levels of malondialdehyde. Conversely, the administration of [6]-gingerol significantly mitigated oxidative stress (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) in these mice. Additionally, DEN and 2-AAF exposure substantially increased hepatic myeloperoxidase (MPO) activity and nitric oxide (NO) levels compared to the control group. Pre-treatment with [6]-gingerol proved to be significantly effective (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) in reducing MPO activity and NO levels, restoring them to near-normal values in DEN and 2-AAF-exposed mice, as shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4. Effects of [6]-gingerol pre-treatment on mice liver architecture\\u003c/h2\\u003e\\n \\u003cp\\u003eHematoxylin and eosin staining of liver sections from both the control and the [6]-gingerol-treated mice revealed normal histological features, including intact central veins, organized liver cords, healthy hepatocytes, and thin sinusoidal spaces. In contrast, mice exposed to DEN (75 mg/kg) and a diet containing 2-AAF (200 mg/kg) exhibited significant histopathological abnormalities. These included disrupted central veins, distorted portal spaces infiltrated with inflammatory cells such as lymphocytes and macrophages, lipid-laden swollen hepatocytes, and dilated sinusoidal spaces. Pre-treatment with [6]-gingerol (100 mg/kg) followed by DEN (75 mg/kg) and dietary 2-AAF exposure led to a notable improvement in liver histology. Treated mice displayed mild sinusoidal distortions, moderate hepatocyte hypertrophy, slight neutrophil infiltration, and a moderately intact central vein (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.5. Effects of [6]-gingerol on the expression of pro-inflammatory enzymes\\u003c/h2\\u003e\\n \\u003cp\\u003eThe pro-inflammatory biomarkers iNOS, COX-2, and NF-kB have been linked to stimulation caused by DEN\\u0026thinsp;+\\u0026thinsp;2-AAF exposure. The anti-inflammatory effects of [6]-gingerol were assessed by analyzing its impact on DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-induced iNOS and COX-2 expression (Figs. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB) through immunohistochemical examination of liver tissue in the treated mice. Mice challenged with DEN (75 mg/kg) and subsequently exposed to 200 mg/kg 2-AAF in their diet exhibited a significant (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) expression of pro-inflammatory cytokines within the inflammatory foci of the liver relative to control mice. Nonetheless, pretreatment with [6]-gingerol successfully suppressed the immune expression of these inflammatory biomarkers in the DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-treated mice.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eThe toxicological impact of DEN combined with 2-AAF in the diet has been associated with its production of highly reactive intermediates and reactive oxygen species (ROS) during the process of bioactivation. Recent studies have highlighted its potential to induce hepatotoxicity, causing liver damage, inflammation, oxidative stress, and abnormal cell proliferation.\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e In this experimental model, a notable decrease in the final body weight, accompanied by an increase in liver weight, was observed in mice treated with an initial single dose of DEN and fed a 2-AAF-supplemented diet, highlighting the harmful effects of DEN\\u0026thinsp;+\\u0026thinsp;2-AAF metabolites on growth and liver function (Table\\u0026nbsp;1). Interestingly, the hepatoprotective properties of [6]-gingerol were evident without any associated changes in body weight, suggesting that [6]-gingerol administration exhibits minimal toxicity. A previous study has demonstrated [6]-gingerol tolerance with minimal or no toxicity.\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eA key indicator of hepatic injury is the release of intracellular enzymes into the plasma, resulting from chemical disruptions in hepatocyte transport functions.\\u003csup\\u003e\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e\\u003c/sup\\u003e Assessing serum enzymes, including AST, ALT, ALP, and γ-GT, provides a dependable indicator of liver health. Elevated levels of these enzymes in the blood are often indicative of impaired hepatic function or damage.\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e Based on our findings (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), exposure to DEN and 2-AAF had detrimental effects on liver function markers in mice. This was demonstrated by a marked rise in serum activities of AST and ALT, along with increased levels of ALP and γ-GT in DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-treated mice relative to the control. Numerous studies attribute hepatoprotective properties of both pharmaceutical drugs and herbal agents to their ability to combat oxidative stress through antioxidant activity and free radical scavenging mechanisms.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR47\\\" citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e However, the administration of [6]-gingerol effectively mitigated these effects, promoting liver health by significantly suppressing the elevated liver function enzymes.\\u003c/p\\u003e \\u003cp\\u003eTo further validate our serum findings, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e highlights the histopathological evaluation of liver tissue. Exposure to DEN\\u0026thinsp;+\\u0026thinsp;2-AAF in mice resulted in notable structural damage, including hyperchromatic nuclei, infiltration of inflammatory cells, and severe congestion, indicative of compromised hepatic integrity. However, the administration of [6]-gingerol significantly alleviated these hepatic lesions in DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-treated mice, restoring the liver tissue architecture to near-normal conditions comparable to that of the sham-treated control group. The observed restoration of serum transaminase levels, accompanied by hepatic parenchymal repair and hepatocyte regeneration, aligns with the findings of Salihu et al.\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e The hepatoprotective effects of [6]-gingerol can be ascribed to its antioxidant characteristics and its capacity to stabilize cellular membranes, thereby preventing the release of intracellular enzymes.\\u003c/p\\u003e \\u003cp\\u003eSuperoxide dismutase is a crucial enzymatic antioxidant responsible for the rapid conversion of superoxide ions into hydrogen peroxide, while catalase detoxifies reactive peroxide radicals, converting them into harmless water and oxygen.\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e Multiple studies recognize these enzymes as a robust protective duo, functioning as a defense system against oxidative damage.\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR51\\\" citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e This study demonstrated a decline in superoxide dismutase and catalase activities in mice exposed to DEN and 2-AAF, indicating the suppression of these enzymes. As a result, this leads to a buildup of deleterious superoxide radicals and hydrogen peroxide within the liver tissue of the treated mice. However, the administration of [6]-gingerol significantly restored SOD and CAT activities in the liver of DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-challenged mice. This finding indicates the potential antioxidant properties of [6]-gingerol, as evidenced in Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB. Many hepatoprotective agents, including Sylimarin\\u003csup\\u003e\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e, Commiphora berryi\\u003csup\\u003e\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e\\u003c/sup\\u003e, and Phyllanthus niruri\\u003csup\\u003e\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e, effectiveness in inhibiting lipid peroxidation induced by ROS has been well-established, owing to their strong free radical neutralizing capabilities.\\u003c/p\\u003e \\u003cp\\u003eGlutathione (GSH) plays a crucial role in protecting against oxidative stress by directly neutralizing hydroxyl radicals and singlet oxygen, facilitating drug detoxification, and restoring antioxidant vitamins C and E to their functional states. In contrast, glutathione peroxidase (GPx), a family of selenium-dependent enzymes, interacts with GSH to break down hydrogen peroxide and lipid hydroperoxides.\\u003csup\\u003e\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e\\u003c/sup\\u003e Both GSH and GPx play crucial roles in maintaining the body's antioxidant defense mechanisms. Meanwhile, glutathione-S-transferase is instrumental in catalyzing the conjugation of toxic electrophilic compounds with GSH, functioning as a key enzyme in phase II drug detoxification processes.\\u003csup\\u003e\\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e\\u003c/sup\\u003e Non-enzymatic antioxidants like glutathione and ascorbic acid safeguard against xenobiotic toxicity by neutralizing free radicals, a process that results in their oxidation.\\u003csup\\u003e59\\u003c/sup\\u003e Consequently, the hepatotoxic effects of DEN and 2-AAF are largely attributed to the free radicals generated during their metabolic activation. Our study revealed a substantial decrease in GSH levels, along with diminished activities of GPx and GST, in mice treated with DEN\\u0026thinsp;+\\u0026thinsp;2-AAF (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, \\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). However, the administration of [6]-gingerol effectively counteracted these reductions, revitalizing antioxidant enzyme functions and replenishing the levels of GSH in treated mice. The observed elevation in hepatic GSH levels in mice exposed to DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-exposed mice suggests that [6]-gingerol plays a role in facilitating the neutralization of reactive oxygen species generated during the P450 bioactivation of DEN\\u0026thinsp;+\\u0026thinsp;2-AAF. The DEN\\u0026thinsp;+\\u0026thinsp;2-AAF+[6]-gingerol-treated groups exhibited nearly normal levels in enzymic and non-enzymic antioxidants, underscoring the significance of polyphenols in preserving the integrity of the endogenous cellular antioxidant defense system. Moreso, the antioxidant properties exhibited by [6]-gingerol in this study align with previous research, which has consistently highlighted its efficacy against reactive oxygen-mediated damage.\\u003csup\\u003e\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eLipid peroxidation serves as a key indicator of oxidative damage triggered by ROS, leading to compromised membrane functionality, reduced fluidity, and the inactivation of membrane-bound receptors and enzymes.\\u003csup\\u003e\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e\\u003c/sup\\u003e It also leads to the production of toxic by-products, such as malondialdehyde (MDA) and 4-hydroxynonenal, which can target cellular components like DNA, causing mutagenesis and cancer.\\u003csup\\u003e\\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e\\u003c/sup\\u003e In this study, an elevated level of liver MDA was observed in mice treated with DEN\\u0026thinsp;+\\u0026thinsp;2-AAF, indicating heightened ROS production and significant lipid peroxidation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). However, the administration of [6]-gingerol effectively reduced liver MDA levels in DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-challenged mice, demonstrating its antioxidant and free radical scavenging potential. These findings align with prior reports \\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e\\u003c/sup\\u003e, which further emphasized [6]-gingerol liver protecting capacity on lipid peroxidation. The oxidation reaction driven by xanthine oxidase mainly yields hydrogen peroxide, making it a crucial reactive oxygen species of interest when investigating xanthine oxidase involvement in inflammation and cellular communication.\\u003csup\\u003e\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e66\\u003c/span\\u003e\\u003c/sup\\u003e In this study, treatment with DEN\\u0026thinsp;+\\u0026thinsp;2-AAF in mice led to a substantial increase in H₂O₂ production, accompanied by elevated XO activity (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF). However, the administration of [6]-gingerol effectively suppressed the heightened XO activity and H₂O₂ generation in the liver of DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-treated mice, attributed to its anti-inflammatory properties. These findings are consistent with the observations \\u003csup\\u003e\\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e\\u003c/sup\\u003e, who reported increased XO activity along with enhanced H₂O₂ production in the liver of rats.\\u003c/p\\u003e \\u003cp\\u003eTo understand the role of nitrosative stress, we measured iNOS and NO levels in DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-induced liver inflammation. Evidence suggests that NO contributes to nitrosative stress and subsequent cellular damage by directly or indirectly interacting with biomolecules, especially in cases where the antioxidant defense system has been depleted.\\u003csup\\u003e\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e\\u003c/sup\\u003e Furthermore, inflammatory markers such as NO and MPO are upregulated via the expression of iNOS and COX-2.\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e\\u003c/sup\\u003e Infiltrated neutrophils and macrophages exhibit elevated MPO activity, a key enzymatic marker of inflammation. Activated neutrophils also release additional pro-inflammatory cytokines, amplifying the inflammatory response.\\u003csup\\u003e\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e\\u003c/sup\\u003e Mounting scientific evidence strongly supports the intrinsic link between inflammation and liver dysfunction.\\u003csup\\u003e\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e\\u003c/sup\\u003e A key mechanism linking inflammation to hepatic disease is the transcription factor NF-κB, which is rapidly activated by cytokines, viruses, or chemotherapeutic agents and plays a regulatory role in COX-2 expression.\\u003csup\\u003e\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e74\\u003c/span\\u003e\\u003c/sup\\u003e During prostaglandin biosynthesis, COX-2 has demonstrated significant potential as a molecular target for various anti-inflammatory and therapeutic agents.\\u003csup\\u003e\\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e75\\u003c/span\\u003e\\u003c/sup\\u003e Elevated NO and MPO levels, coupled with increased iNOS, COX-2, and NF-κB expression (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE, and \\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and B), confirm liver inflammation in DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-treated mice. Our result indicated that [6]-gingerol\\u0026rsquo;s anti-inflammatory actions might involve downregulating iNOS and COX-2 by inhibiting NF-κB. Collectively, our data revealed that [6]-gingerol confers protection by downregulating iNOS and COX-2 via NF-κB pathway suppression. This emphasizes that its liver protection is tightly associated with its potent anti-inflammatory abilities.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eIn conclusion, [6]-gingerol has been identified in several studies as an effective hepatoprotective agent, with its beneficial properties largely attributed to antioxidant and anti-inflammatory effects.\\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e76\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR76\\\" class=\\\"CitationRef\\\"\\u003e77\\u003c/span\\u003e\\u003c/sup\\u003e Furthermore, the findings from our study highlight the significance of [6]-gingerol in combating liver inflammatory conditions. Evidence from this study demonstrated that [6]-gingerol successfully mitigated DEN\\u0026thinsp;+\\u0026thinsp;2-AAF-induced hepatic inflammation associated with preneoplasia in mice by inhibiting the excessive release of inflammatory cytokines, enhancing non-enzymatic and enzymatic antioxidant activity, and effectively suppressing oxidative stress and tissue damage. Consequently, [6]-gingerol shows promise as a therapeutic agent for the prevention and management of liver diseases induced by toxic xenobiotics.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eContributing authors\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIkenna Maduako\\u003c/b\\u003e: Investigation and analysis, drafting original manuscript, editing, and review. \\u003cb\\u003eOlatunde Farombi\\u003c/b\\u003e: Concept idea, supervision, review, and editing\\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eEthical approval\\u003c/strong\\u003e \\u003cp\\u003e \\u003cb\\u003e/consent\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003e The research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/022.\\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eConflict\\u003c/strong\\u003e \\u003cp\\u003e \\u003cb\\u003edeclaration\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe declared that there were no conflicting interests between the authors with regard to this publication.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003e2-AAF\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;2-Acetylaminofluorene\\u003c/p\\u003e\\n\\u003cp\\u003eALP\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Alkaline Phosphatase\\u003c/p\\u003e\\n\\u003cp\\u003eALT\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Alanine Transaminase\\u003c/p\\u003e\\n\\u003cp\\u003eAST\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Aspartate Aminotransferase\\u003c/p\\u003e\\n\\u003cp\\u003eCAT\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Catalase\\u003c/p\\u003e\\n\\u003cp\\u003eCOX-2\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Cyclooxygenase-2\\u003c/p\\u003e\\n\\u003cp\\u003eDEN\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Diethylnitrosamine\\u003c/p\\u003e\\n\\u003cp\\u003eGSH\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Reduced glutathione\\u003c/p\\u003e\\n\\u003cp\\u003eGST\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Glutathione S-transferase\\u003c/p\\u003e\\n\\u003cp\\u003eH2O2\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Hydrogen peroxide\\u003c/p\\u003e\\n\\u003cp\\u003eiNOS\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Inducible nitric oxide synthase\\u003c/p\\u003e\\n\\u003cp\\u003eLPO\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Lipid peroxidation\\u003c/p\\u003e\\n\\u003cp\\u003eMPO\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Myeloperoxidase\\u003c/p\\u003e\\n\\u003cp\\u003eNF-kB\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;Nuclear Factor kappa-B\\u003c/p\\u003e\\n\\u003cp\\u003eNO\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Nitric oxide\\u003c/p\\u003e\\n\\u003cp\\u003eSOD\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Superoxide dismutase\\u003c/p\\u003e\\n\\u003cp\\u003eXO\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;Xanthine oxidase\\u003c/p\\u003e\\n\\u003cp\\u003ey-GT \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; Gamma-Glutamyl Transferase\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eContributing authors\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIkenna Maduako:\\u003c/strong\\u003e Investigation and analysis, drafting original manuscript, editing, and review. \\u003cstrong\\u003eOlatunde Farombi:\\u003c/strong\\u003e Concept idea, supervision, review, and editing\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval/consent\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe research proposal involving animal subjects was granted ethical approval by the Animal Care and Use Research Ethics Committee at the University of Ibadan, under reference number UI-ACUREC/21/022.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe declared that there were no conflicting interests between the authors with regard to this publication.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors are grateful to Dr Babajide Ajayi and Dr Ifeoluwa Awogbindin for their assistance in the extraction of 6-gingerol used in this study and immunohistochemistry imaging techniques.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of generative AI and AI-assisted technologies\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDuring the preparation of this work, the author(s) used Grammarly to improve language and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and took full responsibility for the publication\\u0026apos;s content.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eFriedman S (2003) Liver fibrosis from bench to bedside. J Hepatol 38:38\\u0026ndash;53\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePinzani M, Rombouts K (2004) Liver fibrosis: from the bench to clinical targets. Dig Liver Dis 36:231\\u0026ndash;242\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSakeran MI, Zidan N, Rehman H, Aziz AT, Saggu S (2014) Abrogation by trifolium alexandrium root extract on hepatotoxicity induced by acetaminophen in rats. Redox Rep 19(1):26\\u0026ndash;33\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCanbay A, Friedman S, Gores GJ (2004) Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39:273\\u0026ndash;278\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePoli G (2000) Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med 21:49\\u0026ndash;98\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJanani P, Sivakumari K, Parthasarathy C (2009) Hepatoprotective activity of bacoside A against N-nitrosodiethylamine-induced liver toxicity in adult rats. Cell Biol Toxicol 25:425\\u0026ndash;434\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCiemniak A (2006) A comparison of N\\u0026ndash;nitrosodimethylamine contents in selected meat products. Rocz Panstw Zakl Hig 57:341\\u0026ndash;346\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGarcea G, Dennison AR, Steward WP, Berry DP (2005) Role of inflammation in pancreatic carcinogenesis and the implications for future therapy. Pancreatol 5(6):514\\u0026ndash;529\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMossman BT, Kamp DW, Weitzman SA (1996) Mechanisms of carcinogenesis and clinical features of asbestos associated cancers. Cancer Invest 14(5):466\\u0026ndash;480\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eValko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007) Free radical and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44\\u0026ndash;84\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAl-Rejaie SS, Alesia AM, Al-Yahya AA (2009) Progression of diethylnitrosamine-induced hepatic carcinogenesis in carnitine-depleted rats. World J Gastroenterol 15:1373\\u0026ndash;1380\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePrakash B, Motiwale L, Ignle AD (2002) Protective effect of Rhodotorula glutinis NCIM3353 on the development of hepatic preneoplastic lesions. Curr Sci 83:303\\u0026ndash;308\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTamano I, Shirai ST (2003) A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 94:3\\u0026ndash;8\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eClaria J, Lopez-Parra M, Titos E, Gonzalez-Perez A (2011) The role of inflammatory mediators in liver failure. In: Gines P. Kamath P. Arroyo V. (eds), Chronic liver failure, Clin. Gastroenterol. ; pp 131\\u0026ndash;153. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/978-1-60761-866-9_7\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/978-1-60761-866-9_7\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNah SS, Choi IY, Lee CK, Oh JS, Kim YG, Moon HB, Yoo B (2008) Effects of advanced glycation end products on the expression of COX-2, PGE2 and NO in human osteoarthritic chondrocytes. Rheumatol 47(4):425\\u0026ndash;431\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTabernero A, Schneider F, Potenza MA, Randriamboavonjy V, Chasserot S, Wolf P, Mitolo-Chieppa D, Stoclet JC, Andriantsitohaina R (2003) Cycloxygenase-2 and inducible nitric oxide synthase in omental arteries harvested from patients with severe liver diseases: immuno-localization and influence on vascular tone. Intensive Care Med 29(2):262\\u0026ndash;270\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLantz RC, Chen GJ, Sarihan M, S\\u0026oacute;lyom AM, Jolad SD, Timmermann BN (2007) The effect of extracts from ginger rhizome on inflammatory mediator production. Phytomedicine 14(2\\u0026ndash;3):123\\u0026ndash;128\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSurh YJ (2003) Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 3:768\\u0026ndash;780\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSurh Y (1999) Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat Res 428(1\\u0026ndash;2):305\\u0026ndash;327\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSurh YJ, Lee SS (1994) Enzymic reduction of [6]-gingerol, a major pungent principle of ginger, in the cell-free preparation of rat liver. Life Sci 54(19):321\\u0026ndash;326\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNonn L, Duong D, Peehl DM (2007) Chemopreventive anti-inflammatory activities of curcumin and other phytochemicals mediated by MAP kinase phosphatase-5 in prostate cells. Carcinogenesis 28:1188\\u0026ndash;1196\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTripathi S, Maier KG, Bruch D, Kittur DS (2007) Effect of 6-gingerol on proinflammatory cytokine production and co-stimulatory molecule expression in murine peritoneal macrophages. J Surg Res 138:209\\u0026ndash;213\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePan MH, Hsieh MC, Hsu PC, Ho SY, Lai CS, Wu H, Sang S, Ho CT (2008) 6-Shogaol suppressed lipopolysaccharide induced up-expression of iNOS and COX-2 in murine macrophages. Mol Nutr Food Res 52:1467\\u0026ndash;1477\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAktan F, Henness S, Tran VH, Duke CC, Roufogalis BD, Ammit AJ (2006) Gingerol metabolite and a synthetic analogue Capsarol inhibit macrophage NF-kappa B-mediated iNOS gene expression and enzyme activity. Planta Med 72(8):727\\u0026ndash;734\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTjendraputra E, Tran VH, Liu-Brennan D, Roufogalis BD, Duke C (2001) Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells. Bioorg Chem 29:156\\u0026ndash;163\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSalihu M, Ajayi BO, Adedara IA, Farombi EO (2016) 6-gingerol-rich fraction from zingiber officinale prevents hematotoxicity and oxidative damage in kidney and liver of rats exposed to carbendazim. J Diet Suppl 13(4):433\\u0026ndash;448\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAjayi BO, Adedara IA, Farombi EO (2015) Pharmacological activity of 6-Gingerol in dextran sulphate sodium- induced ulcerative colitis in balb/c mice. Phytother Res. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1002/ptr.5286\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/ptr.5286\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAjayi BO, Adedara IA, Farombi EO (2016) Benzo[a]pyrene induces oxidative stress, pro-inflammatory cytokines, expression of nuclear factor-kappaB and deregulation of wnt/beta-catenin signaling in colons of BALB/c mice. Food Chem Toxicol 95:42\\u0026ndash;51\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFarombi EO, Tahnteng JG, Agboola AO, Nwankwo JO, Emerole GO (2000) Chemoprevention of 2-acetylaminofluorene- induced hepatotoxicity and lipid peroxidation in rats by kolaviron \\u0026ndash; a garcinia kola seed extract. Food Chem Toxicol 38:535\\u0026ndash;541\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKlinman NR, Erslev AJ (1963) Cellular response to partial hepatectomy. Proceedings of the Society for Experimental Biology and medicine. ; 112: 338\\u0026ndash;40\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBradford MM (1976) A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\\u0026ndash;254\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMisra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170\\u0026ndash;3175\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eClairborne A (1995) Catalase activity. In: Greewald AR (ed) Handbook of Methods for Oxygen Radical Research. CRC, pp 237\\u0026ndash;242\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588\\u0026ndash;590\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHabig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first orange for measurement of hydroperoxides. Methods Enzymol 233:182\\u0026ndash;189\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJollow DJ, Mitchell JR, Zampaglione N, Gillette JR (1974) Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4 bromobenzene oxide as the hepatotoxic metabolite. Pharmacol 11:151\\u0026ndash;169\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWolff SP (1994) Ferrous ion oxidation in the presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol 233:182\\u0026ndash;189\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eStripe F, Della-Corte E (1969) The regulation of rat liver xanthine oxidase conversion in vitro of enzyme activity from dehydrogenase (type D) to oxidase (type O). J Biol Chem 244:3855\\u0026ndash;3863\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGreen LC, Wagner DA, Glogowski J, Skiper PL, Wishnock JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite and [15 N] nitrate in biological fluids. Anal Biochem 126:131\\u0026ndash;138\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGranell S, Gironella M, Bulbena O, Panes J, Mauri M, Sabater L, Aparisi L, Gelp\\u0026iacute; (2003) E. Closa D. Heparin mobilizes xanthine oxidase and induces lung inflammation in acute pancreatitis. Crit. Care Med. ; 31: 525\\u0026ndash;530\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSultana S, Nafees S, Khan AQ (2013) Perillyl alcohol as a protective modulator against rat hepatocarcinogenesis via amelioration of oxidative damage and cell proliferation. Hum Experimental Toxicol 32(11):1179\\u0026ndash;1192\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTolba R, Kraus T, Liedtke C, Schwarz M, Weiskirchen R (2015) Diethylnitrosamine (DEN)-induced carcinogenic liver injury in mice. Lab Anim 49(1 Suppl):59\\u0026ndash;69. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1177/0023677215570086\\u003c/span\\u003e\\u003cspan address=\\\"10.1177/0023677215570086\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAjayi BO, Adedara IA, Farombi EO (2019) 6-Gingerol abates benzo[a]pyrene-induced colonic injury via suppression of oxido-inflammatory stress responses in BALB/c mice. Chemico-Biol Interact 307:1\\u0026ndash;7\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSabir SM, Rocha JB (2008) Antioxidant and hepatoprotective activity of aqueous extract of solanum fastigiatum against paracetamol-induced liver damage in mice. J Ethnopharmacol 120(2):226\\u0026ndash;232\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKaplan MM (1993) Laboratory test. In: Schiff L, Schiff ER (eds) Diseases of the liver. JB Lippincott, Seventh Philadelphia, pp 108\\u0026ndash;144\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRafie R, Hosseini SA, Hajiani E, Malehi AS, Mard SA (2020) Effect of Ginger Powder Supplementation in Patients with Non-Alcoholic Fatty Liver Disease: A Randomized Clinical Trial. Clin Exp Gastroenterol 13:35\\u0026ndash;45\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNaik SR, Panda VS (2007) Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodents. Liver Int 27:393\\u0026ndash;399\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKodai S, Takemura S, Minamiyama Y, Hai S, Yamamoto S, Kubo S, Yoshida Y, Niki E, Okada S, Hirohashi K, Suehiro S (2007) S-allyl cysteine prevents CCl induced acute liver injury in rats. Free Radic Res 41:489\\u0026ndash;497\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHalliwell B (1990) How to characterize a biological antioxidant. Free Radic Res Comm 9:1\\u0026ndash;32\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHalliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1\\u0026ndash;85\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eReiter RJ, Tan D, Osuna C, Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 7:444\\u0026ndash;458\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang BJ, Lien YH, Yu ZR (2004) Supercritical fluid extractive fractionation: study of the antioxidant activities of propolis. Food Chem 86:237\\u0026ndash;243\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eManokaran S, Jaswanth A, Sengottuvelu S, Nandhakumar J, Duraisamy R, Karthikeyan D (2008) Hepatoprotective activity of Aerva lanata Linn. Against paracetamol induced hepatotoxicity in rats. Res J Pharm Tech 1:398\\u0026ndash;400\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGowri-Shanka NL, Manavalan R, Venkappayya D, David RC (2008) Hepatoprotective and antioxidant effects of Commiphora berryi (Arn) Engl bark extract against CCl -induced oxidative damage in rats. Food Chem Toxicol 46:3182\\u0026ndash;3185\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBhattacharjee R, Sil PC (2007) Protein isolate from the herb, Phyllanthus niruri L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties. Food Chem Toxicol 45:817\\u0026ndash;826\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMasella R, Di BR, Vari R, Filesi C, Giovannini C (2005) Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione related enzymes. J Nutr Biochem 16:577\\u0026ndash;586\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eArthur JR (2000) The gluthatione peroxidase. Cell Mol Life Sci 57(13\\u0026ndash;14):1825\\u0026ndash;1835\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eValko M, Jomova K, Rhodes CJ, Kuca K, Mus\\u0026iacute;lek (2016) K. Redox- and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol. ; 90: 1\\u0026ndash;37\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKadiska MB, Gladen BC, Baird DD, Dikalov AE, Sohal RS, Hatch GB, Jones DP, Mason RP, Barret JC (2000) Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl. poisoning? J Free Rad Biol Med 28:838\\u0026ndash;845\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eIppoushi K, Azuma K, Ito H, Horie H, Higashio H (2023) [6]-Gingerol inhibits nitric oxide synthesis in activated 1774.1 mouse macrophages and prevents peroxynitrite induced oxidation and nitration reactions. Life Sci 73(26):3427\\u0026ndash;3437\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLee TY, Lee KC, Chen SY, Chang HH (2019) 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-KB pathways in lipopolysaccharide-stimulated mouse macrophages. Biochem Biophys Res Commun 382(1):134\\u0026ndash;113\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eViani P, Cervato G, Fiorilli A, Cestaro B (1991) Age-related differences in synaptosomal peroxidative damage and membrane properties. J Neurochem 56:253\\u0026ndash;258\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZawart LL, Meerman JH, Commandeur JN, Vermeulen NP (2009) Biomarkers of free radical damage applications in experimental animals and humans. Free Radic Biol Med 26:202\\u0026ndash;226\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBanaker MC, Paramacivan SK, Chattopadhyay MB, Datta S, Chakraborty P, Chatterjee M, Kannan K, Thygarajan E (2014) 1α, 25-dihydroxyvitamin D3 prevents DNA damage and restores antioxidant enzymes in rat hepatocarcinogenesis induced by diethylnitrosamine and promoted by phenobarbital. World J Gastroenterol 10:1268\\u0026ndash;1275\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSanchez-Perez C, Carrasco-Legleu C, Garcia-Cuellar J, Perez-Carreon S, Hernandez-Garcia M, Salcido-Neyoy L, Aleman-Lazarini S (2015) Oxidative stress in carcinogenesis. Correlation between lipid peroxidation and induction of preneoplastic lesions in rat hepatocarcinogenesis. Cancer Lett 217:25\\u0026ndash;32\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKelly EE, Khoo NKH, Hindley NJ, Malik UZ, Freeman BA, Tarpey MM (2019) Hydrogen peroxide is the major oxidant product of xanthine oxidase. Free Radic Biol Med 48(4):493\\u0026ndash;499\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGu Z, Nakamura T, Lipton SA (2019) Redox reactions induced by nitrosative stress mediate protein misfolding and mitochondrial dysfunction in neurodegenerative diseases. Mol Neurobiol 41:55\\u0026ndash;72\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKiuchi F, Iwakami S, Shibuya M, Hanaoka F, Sankawa U (2022) Inhibition of prostaglandin and leukotriene biosynthesis by gingerols and diarylheptanoids. Chem Pharm Bull 40(2):387\\u0026ndash;391\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eStanley PL, Steiner S, Havens M, Tramposch KM (2018) Mouse skin inflammation induced by multiple topical application of 12 \\u0026ndash;O- tetra decanoylphorbol-13-acetate. Ski Pharmacol 4:262\\u0026ndash;271\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBalkwill F, Coussens LM (2004) The NF-KB protein is a key player in inflammation. It now seems that it might also activate signalling pathways, in both cancer cells and tumour-associated inflammatory cells that promote malignancy. Nature 431:405\\u0026ndash;406\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGreten RF, Karin M (2004) The IKK/NF-kB activation pathway\\u0026mdash;a target for prevention and treatment of cancer. Cancer Lett 206:193\\u0026ndash;199\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGarg A, Aggarwal BB (2002) Nuclear transcription factor-kappaB as a target for cancer drug development. Leukemia 16:1053\\u0026ndash;1068\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMohan S, Epstein JB (2003) Carcinogenesis and cyclooxygenase: the potential role of COX-2 inhibition in upper aerodigestive tract in cancer. Oral Oncol 39:537\\u0026ndash;546\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWilliams CS, Shattuck-Brandt RL, DuBois RN (2009) The role of COX-2 in intestinal cancer. Expert Opin Investig Drugs 8:1\\u0026ndash;12\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEl Sharaky AS, Newairy AA, Kamel MA, Eweda SM (2009) Protective effect of ginger extract against bromobenzene-induced hepatotoxicity in male rats. Food Chem Toxicol 47:1584\\u0026ndash;1590\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAeschbach R, Loliger J, Scott BC, Murcia A, Butler J, Halliwell B, Aruoma OI (1994) Antioxidant actions of thymol, carcacrol, 6-gingerol, zingerone and hydroxytyrosol. Food Chem Toxicol 2:31\\u0026ndash;36\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"University of Ibadan, Ibadan Nigeria\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Diethylnitrosamine, 2-acetylaminofluorene, liver toxicity, NF-kB, oxidative stress\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6792607/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6792607/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThis study investigates the hepatoprotective effects of [6]-gingerol against liver dysfunction and inflammation induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) in mice. BALB/c mice were divided into four groups and treated with DEN, 2-AAF, [6]-gingerol, or a combination thereof. Biochemical, histological, and immunohistochemical analyses were conducted to evaluate liver function, oxidative stress, and inflammation. Mice exposed to DEN and 2-AAF exhibited significant hepatic injury, characterized by elevated serum liver enzymes (ALT, AST, ALP, γ-GT), reduced antioxidant enzyme activities (SOD, CAT, GPx, GST), increased lipid peroxidation, nitric oxide production, and expression of pro-inflammatory markers (NF-κB, iNOS, COX-2).\\u003c/p\\u003e\\n\\u003cp\\u003eHistopathological examination confirmed substantial liver damage. Pre-treatment with [6]-gingerol significantly mitigated these effects, restoring antioxidant enzyme activities, reducing oxidative and nitrosative stress, and suppressing the expression of inflammatory mediators. Liver histoarchitecture also improved markedly in [6]-gingerol-pretreated mice. These findings suggest that [6]-gingerol confers protective effects via antioxidant and anti-inflammatory mechanisms, primarily through modulation of the NF-κB signaling pathway.\\u003c/p\\u003e\\n\\u003cp\\u003eThe results highlight the therapeutic potential of [6]-gingerol in preventing liver diseases associated with xenobiotic-induced oxidative stress and inflammation.\\u003c/p\\u003e\",\"manuscriptTitle\":\"6-Gingerol inhibition of NF-kB expression in diethylnitrosamine initiated, 2-acetylaminofluorene-promoted liver dysfunction and inflammation involves induction of antioxidant enzymes and suppression of pro-inflammatory cytokines\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-06-03 10:58:29\",\"doi\":\"10.21203/rs.3.rs-6792607/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"b7a25fc1-13d4-4772-8adf-003e53245af8\",\"owner\":[],\"postedDate\":\"June 3rd, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":49333388,\"name\":\"Toxicology\"},{\"id\":49333389,\"name\":\"Biochemical Research Methods\"},{\"id\":49333390,\"name\":\"Molecular Biology\"}],\"tags\":[],\"updatedAt\":\"2025-06-03T10:58:29+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-06-03 10:58:29\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6792607\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6792607\",\"identity\":\"rs-6792607\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}