Hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis

Hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis | 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 Hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis Nigora Fayzullayeva, Abrorjon Kayumov, Tamara Aripova, Alisher Raufov, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8601812/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 Background Drug-induced liver injury (DILI) is a major cause of acute liver failure, highlighting the need for effective hepatoprotective agents. Natural products with antioxidant properties represent promising therapeutic options. This study aimed to evaluate the hepatoprotective, antioxidant, and anti-inflammatory effects of cold-pressed safflower oil in a rat model of paracetamol-induced acute toxic hepatitis. Methods Acute toxic hepatitis was induced in male Wistar rats by intragastric administration of paracetamol at a dose of 1000 mg/kg for two consecutive days. This dosing regimen was selected based on established experimental models of acute toxic hepatitis that reliably induce liver injury while maintaining animal survival. Safflower oil was administered orally at a dose of 10 mL/kg for 14 days. Hepatic injury was evaluated by assessing serum biochemical markers, including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase; oxidative stress parameters (malondialdehyde); antioxidant enzyme activities (superoxide dismutase and catalase); inflammatory cytokine levels (IL-1β, IL-6, IL-4, and TNF-α); and histopathological changes in liver tissue. Statistical analyses were performed using parametric or non-parametric methods, as appropriate. Results Paracetamol administration caused significant liver injury, evidenced by elevated serum aminotransferase and alkaline phosphatase activities, increased malondialdehyde levels, suppression of antioxidant enzyme activity, and marked histopathological damage. Treatment with safflower oil significantly reduced aspartate aminotransferase and alkaline phosphatase levels and attenuated oxidative stress, as indicated by decreased malondialdehyde concentrations and restoration of superoxide dismutase and catalase activities (p < 0.05). Inflammatory cytokine levels were partially normalised following safflower oil treatment, with significant reductions in IL-1β, IL-6, and IL-4, while TNF-α remained elevated. Histological examination confirmed reduced hepatocellular degeneration, necrosis, inflammatory infiltration, and early fibrotic changes in treated animals compared with the untreated model group. Conclusions Cold-pressed safflower oil exhibits significant hepatoprotective, antioxidant, and anti-inflammatory effects in paracetamol-induced acute toxic hepatitis. These effects are associated with suppression of oxidative stress, modulation of inflammatory responses, normalisation of liver enzyme activity, and preservation of hepatic histoarchitecture. Safflower oil may represent a promising natural adjunctive agent for the prevention and management of toxic liver injury. Rat toxic hepatitis safflower oil hepatoprotective activity oxidative stress antioxidant enzymes inflammatory cytokines liver histology. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Liver diseases remain one of the leading causes of mortality worldwide, claiming approximately 2 million lives annually, which accounts for about 4% of all deaths [1]. These pathologies are characterised by hepatocyte injury, inflammatory infiltration, and disruption of organ architecture, leading to a progressive loss of liver function [2]. According to data from the Global Burden of Disease study, in 2019, 1.26 million deaths were attributed to cirrhosis and other chronic liver diseases (a 13% increase compared with 1990), indicating a steady rise in disease burden [3]. Among these conditions, drug-induced liver injury (DILI) occupies a significant place and has become increasingly significant in recent decades [4]. DILI is a serious complication of pharmacotherapy and represents one of the leading causes of acute liver failure in clinical practice [5,6]. To date, more than 1,000 substances with potential hepatotoxicity have been identified [7]. In addition to medications, hepatotropic toxins include industrial chemicals such as chloroform and phosphorus [8]. Despite the existence of general treatment approaches, including lifestyle modification and pharmacotherapy [9], no specific therapy for DILI has been developed due to the heterogeneity of its pathogenesis and individual differences in xenobiotic metabolism [10]. Given the limitations of existing approaches, increasing attention is being directed toward the investigation of natural compounds with antioxidant and hepatoprotective activity. Herbal preparations are considered a promising avenue for the prevention and treatment of toxic liver injuries [11]. One such plant is safflower (Carthamus tinctorius L.), which has been traditionally used in Eastern medicine. Its extracts have been shown to possess anti-inflammatory, antioxidant, immunomodulatory, and vascular effects [12]. However, most available data concern aqueous or alcoholic extracts of the flowers, whereas the hepatoprotective potential of cold-pressed safflower oil has been studied only to a limited extent. Existing experimental studies in animal models indicate that safflower oil may reduce hepatic transaminase activity, attenuate histological signs of liver damage, and exert antioxidant effects comparable to those of silymarin [13,14]. At the same time, there is a lack of large comparative studies evaluating its efficacy in models of drug-induced toxic hepatitis, as well as insufficient data on optimal dosages, mechanisms of action, and bioavailability. Thus, the effects of safflower oil as a potential hepatoprotective agent in drug-induced liver injury remain insufficiently investigated. Further in-depth research on this topic is of considerable interest for the development of new therapeutic strategies aimed at the prevention and treatment of DILI. Although safflower extracts have been investigated in various experimental models, data on the hepatoprotective efficacy of cold-pressed safflower oil in drug-induced toxic hepatitis remain limited. Therefore, this study aimed to evaluate the hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis. Methods This experimental study was conducted in collaboration with the Institute of Immunology and Human Genomics of the Academy of Sciences of the Republic of Uzbekistan and the Institute of Bioorganic Chemistry named after Academician O.S.Sodikov, Academy of Sciences of the Republic of Uzbekistan. Study design Male Wistar rats (n = 10 per group) were randomly assigned to three experimental groups: a Control group, a toxic hepatitis model group (Model TH), and a safflower oil–treated group (Model TH + SO). Acute toxic hepatitis was induced in the Model TH and Model TH + SO groups by intragastric administration of paracetamol for two consecutive days. From the third day of the experiment, rats in the Model TH + SO group received safflower oil orally at a dose of 10 mL/kg once daily for 14 days, whereas rats in the Control and Model TH groups received an equivalent volume of physiological saline. On day 17 of the experiment, all animals were euthanised, and blood and liver tissue samples were collected for biochemical, oxidative stress, inflammatory, and histopathological analyses (Fig. 1 ). Animals Thirty healthy male Wistar rats, aged 8.02 [7.75–8.25] weeks and weighing 203.07 [198.5–205.5] g, were used in this study. Animals were housed under specific pathogen-free (SPF) laboratory conditions (temperature 20.2 ± 5°C, relative humidity 55 ± 10%, 12:12 h light–dark cycle) with free access to standard laboratory chow and water. Before the experiment, all animals underwent a 10–14-day quarantine period. After the quarantine period, rats were randomly allocated to three experimental groups (n = 10 per group). Randomisation was performed using a computer-generated random number sequence. The experimental groups were as follows: Control group (n = 10) healthy rats receiving no treatment. Model TH group (n = 10) rats with paracetamol-induced acute toxic hepatitis. Treatment group (Model TH + SO, n = 10): rats with paracetamol-induced acute toxic hepatitis were treated with safflower oil. Toxic hepatitis induction Animals in the Model TH (n = 10) and Treatment (Model TH + SO ) (n = 10) groups were induced to acute toxic hepatitis by intragastric administration of an aqueous solution of paracetamol at a dose of 1000 mg/kg using a gavage once daily for two consecutive days [Mossanen & Tacke, 2015; Jaeschke et al., 2021]. Safflower Oil Treatment Beginning on the third day of the experiment, animals in the Model TH + SO treatment group (n = 10) received safflower oil, cold-pressed from the seeds of Carthamus tinctorius and manufactured by Botanic Herbs LLC, once daily at a dose of 10 mL/kg via oral gavage using a dosing syringe; animals in the Control and Model TH groups received an equivalent volume of physiological saline, and treatment was continued for 14 consecutive days. Liver function assessment Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP) were determined using standard biochemical methods. Analyses were performed with commercially available assay kits (Cypress Diagnostics BV, Belgium) according to the manufacturer’s instructions. Antioxidant Status Assessment Antioxidant status was evaluated by measuring serum levels of malondialdehyde (MDA) and the activities of catalase and superoxide dismutase (SOD). The analysis was performed using an enzyme-linked immunosorbent assay with reagent kits (Wuhan Fine Biotech Co., Ltd., China). Morphological Analysis Liver samples were fixed in 10% neutral buffered formalin for 24 hours. Following fixation, tissues were dehydrated in a graded series of ethanol and xylene, and then embedded in paraffin according to standard protocols. Histological sections were prepared from paraffin blocks and stained with hematoxylin and eosin (H&E). Microscopic examination and acquisition of digital images of representative areas were performed using a Cilika BT-P microscope (MedPrime Technologies Pvt. Ltd., India) with the corresponding software. Histology was assessed qualitatively. Histological evaluation was performed by two independent observers blinded to group allocation. Statistical Analysis Statistical analyses were performed using Microsoft Excel and JASP (version 0.19.3). Normality of data distribution was assessed using the Shapiro–Wilk test. Normally distributed data (ALT) are presented as mean ± standard deviation (SD) and were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test, whereas non-normally distributed data are presented as median with interquartile range (IQR) and were analysed using the Kruskal–Wallis test followed by Dunn’s post hoc test with Holm correction. A p-value < 0.05 was considered statistically significant. Results Comparison of liver enzymes Administration of paracetamol resulted in significant alterations in biochemical parameters in the model group compared with the control (healthy) group, confirming the successful induction of acute toxic hepatitis. As summarised in Table 1, animals in the model group showed a marked increase in serum ALT activity (47.8 ± 10.82 U/L), AST activity (52.5 [37.63–55.13] U/L), and ALP levels (117.47 [98.81–124.38] U/L) relative to the control group (32.2 ± 5.17 U/L, 35.0 [32.38–40.25] U/L, and 62.19 [55.28–69.10] U/L, respectively) (Fig. 2 ). Overall group differences were statistically significant (p < 0.05) (Table 1). Treatment with safflower oil significantly attenuated paracetamol-induced biochemical disturbances. In the treatment with safflower oil group, serum AST (35.0 [31.50– Table 1. Comparison of liver enzyme levels among experimental groups Parameter (unit) Control (n = 10) Model TH (n = 10) Model TH + SO (n = 10) p value ALT (U/L) 32.2 ± 5.17ᵃ 47.8 ± 10.82ᵇ 39.4 ± 3.80ᵃ < .001 AST (U/L) 35.0 [32.38–40.25]ᵃᵇ 52.5 [37.63–55.13]ᵇ 35.0 [31.50–35.0]ᵃ 0.011 ALP (U/L) 62.19 [55.28–69.10]ᵃ 117.47 [98.81–124.38]ᵇ 62.19 [55.28–75.32] < .001 GGT (U/L) 0.596 [0.522–0.596] 0.792 [0.566–0.795] 0.713 [0.606–0.775] 0.171 Note. Data are presented as mean ± standard deviation (SD) for ALT and as median with interquartile range (IQR) for AST, ALP, and GGT. Normality of data distribution was assessed using the Shapiro–Wilk test. Because ALT data were normally distributed, they were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test. Because AST, ALP, and GGT data were not normally distributed, they were analysed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Values sharing at least one common superscript letter are not significantly different. A value of p < 0.05 was considered statistically significant. 35.0] U/L) and ALP (62.19 [55.28–75.32] U/L) levels were significantly reduced compared with the untreated model group and were comparable to control values (Table 1; Fig. 2 ). Similarly, ALT levels in the treatment group (39.4 ± 3.80 U/L) were significantly lower than those observed in the model group (Table 1, Fig. 3 ). In contrast, GGT levels did not differ significantly among groups (p = 0.171). The toxic effect of paracetamol led to the development of pronounced oxidative stress in the model group (Table 2). Serum malondialdehyde (MDA) levels, a key marker of lipid peroxidation, were significantly increased in the Model TH group (3.71 [3.66–3.76] nmol/mL) compared with the control group (2.91 [2.85–3.02] nmol/mL) (p < 0.05). In contrast, treatment with safflower oil significantly attenuated lipid peroxidation, as evidenced by a reduction in MDA levels in the Model TH + SO group (3.13 [3.03–3.20] nmol/mL) relative to the untreated Model TH group (p < 0.05). Table 2. Oxidative stress marker (MDA) and antioxidant enzyme activities (SOD and catalase) in experimental groups Parameter (unit) Control (n = 10) Model TH (n = 10) Model TH + SO (n = 10) p value (Kruskal–Wallis) MDA (nmol/mL) 2.91 [2.85–3.02]ᵃ 3.71 [3.66–3.76]ᵇ 3.13 [3.03–3.20]ᵃ < 0.001 Catalase (µkat/mg) 34.64 [34.21–35.59]ᵃ 22.12 [21.17–25.26]ᵇ 30.68 [29.32–31.78]ᶜ < 0.001 SOD (U/mg protein) 1.33 [1.29–1.35]ᵃ 1.10 [1.08–1.12]ᵇ 1.26 [1.24–1.27]ᶜ < 0.001 Note. Data are presented as median with interquartile range (IQR). Group differences were assessed using the Kruskal–Wallis test, followed by Dunn’s post-hoc test. Values sharing the same superscript letter within a row are not significantly different, whereas values with different superscript letters are significantly different (p < 0.05). Concomitantly, the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) were significantly decreased in the Model TH group compared with controls (p < 0.05), indicating impairment of the endogenous antioxidant defence system (Table 2). Administration of safflower oil contributed to the correction of paracetamol-induced oxidative stress (Table 2). In the Model TH + SO group, serum MDA levels were significantly reduced (3.13 [3.03–3.20] nmol/mL) compared with the untreated Model TH group (3.71 [3.66–3.76] nmol/mL; p < 0.05), indicating attenuation of lipid peroxidation. Concomitantly, the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) were significantly increased in safflower oil–treated animals (30.68 [29.32–31.78] µkat/mg and 1.26 [1.24–1.27] U/mg protein, respectively) relative to the Model TH group (22.12 [21.17–25.26] µkat/mg and 1.10 [1.08–1.12] U/mg protein; p < 0.05, Table 2). Notably, oxidative stress parameters in the Model TH + SO group approached control values, suggesting a substantial restoration of antioxidant defence. Comparison of inflammatory marker levels Table 3 Comparison of serum inflammatory cytokine levels in experimental groups Parameter (unit) Control (n = 10) Model TH (n = 10) Model TH + SO (n = 10) p value (Kruskal–Wallis) IL-1β pg/mL 7.18 [6.64–7.73]ᵃ 15.75 [11.46–27.21]ᵇ 11.88 [10.54–12.29]ᵇ < .001 IL-6 pg/mL 2.53 [2.25–2.57]ᵃ 4.33 [3.97–4.49]ᵇ 3.52 [3.48–3.62]ᵇ < .001 IL-4 pg/mL 1.19 [1.02–1.24]ᵃ 3.65 [3.18–5.10]ᵇ 2.37 [2.20–2.87]ᵃᵇ < .001 TNF-α pg/mL 9.67 [8.77–10.28]ᵃ 34.90 [26.78–45.60]ᵇ 23.25 [19.60–29.00]ᵇ < .001 Note. Data are presented as median with interquartile range (IQR). Group differences were assessed using the Kruskal–Wallis test, followed by Dunn’s post-hoc test with Holm correction. Values sharing at least one common superscript letter are not significantly different, whereas values with different superscript letters are significantly different (p < 0.05). Paracetamol administration induced a pronounced inflammatory response, as evidenced by significant alterations in serum cytokine levels in the Model TH group compared with the control group (Table 3 ). On day 17, serum concentrations of IL-1β, IL-6, IL-4, and TNF-α were significantly elevated in the Model TH group (15.75 [11.46–27.21], 4.33 [3.97–4.49], 3.65 [3.18–5.10], and 34.90 [26.78–45.60], respectively) relative to control values (7.18 [6.64–7.73], 2.53 [2.25–2.57], 1.19 [1.02–1.24], and 9.67 [8.77–10.28], respectively; p < 0.05, Table 3 ). Treatment with safflower oil significantly attenuated the paracetamol-induced inflammatory response. In the Model TH + SO group, serum levels of IL-1β (11.88 [10.54–12.29]) and IL-6 (3.52 [3.48–3.62]) were significantly reduced compared with the untreated Model TH group (p < 0.05), although they remained higher than control values. IL-4 levels in the treatment group (2.37 [2.20–2.87]) did not differ significantly from either the control or Model TH groups, indicating partial normalisation. In contrast, TNF-α concentrations remained significantly elevated in both the Model TH and Model TH + SO groups compared with controls, with no significant difference between the two groups. Overall, these findings demonstrate that safflower oil partially suppresses paracetamol-induced inflammatory activation, with a more pronounced modulatory effect on IL-1β, IL-6, and IL-4 than on TNF-α. Morphological assessment Morphological examination of the liver confirmed the biochemical findings. In animals of the model group (without treatment), pronounced pathological changes were observed, including fatty degeneration, hepatocyte necrosis, and moderate inflammatory infiltration (Fig. 4 . B, F). The use of safflower oil as a hepatoprotective agent significantly reduced the severity of dystrophic and inflammatory processes. The structure of hepatocytes was close to normal, and fatty degeneration was observed only sporadically (Fig. 4 . C, D, G, H). With Masson’s trichrome staining, collagen structures in the control group were detected only as a normal component of vascular walls (Fig. 4 I), whereas in the model group, the formation of thin fibrotic strands in the intercellular matrix around the central vein structures was noted (Fig. 4 J). In the treatment group, the severity of fibrotic changes was markedly lower and was represented by single thin fibrous fibres between hepatocytes (Fig. 4 . H, I ). Discussion In the present study, safflower oil demonstrated pronounced hepatoprotective and antioxidant effects in a rat model of paracetamol-induced acute toxic hepatitis. Paracetamol administration resulted in the development of acute liver injury, as evidenced by significant alterations in biochemical markers in the model group (Fig. 5 ). These findings are consistent with previous studies that have reported the hepatoprotective effects of various safflower-derived preparations. For instance, red safflower pigment administered at doses of 10 and 20 mg/kg significantly reduced ALT and AST activity in rats with CCl₄-induced hepatitis [13]. Similarly, safflower seed oil (200 mg/kg) normalised ALT, AST, and ALP levels in alloxan-induced diabetic rats [14], while aqueous safflower extracts dose-dependently reduced transaminase activity in alcohol-induced liver injury models [18]. Oxidative stress represents a key pathogenetic mechanism in paracetamol-induced hepatotoxicity. In the present study, oxidative damage was reflected by a significant increase in malondialdehyde (MDA) levels to 3.71 [3.66–3.76] nmol/mL and a concomitant suppression of superoxide dismutase (SOD) activity to 1.10 [1.08–1.12] U/mg protein in the model group. Treatment with safflower oil effectively counteracted these changes, significantly reducing MDA levels to 3.13 [3.03–3.20] nmol/mL and restoring SOD activity to 1.26 [1.24–1.27] U/mg protein (p < 0.05). These observations are in agreement with the findings of Alshareef et al., who demonstrated that methanolic extracts of safflower flowers (300 mg/kg) reduced hepatic lipid peroxidation and enhanced SOD and glutathione activity in a rat model of type 2 diabetes [19]. The authors attributed these effects to activation of the Nrf2-dependent antioxidant signalling pathway, a mechanism that has also been reported for red safflower pigment in CCl₄-induced liver injury [13]. The high therapeutic efficacy of safflower oil may be attributed to its unique chemical composition. Safflower oil contains up to 75–80% polyunsaturated linoleic acid, along with oleic, palmitic, and stearic acids. In addition, it is rich in biologically active compounds, including polyphenols, flavonoids (such as quercetin and kaempferol), quinochalcones, tocopherols (vitamin E), serotonin derivatives, and plant sterols. This complex phytochemical profile confers potent antioxidant, anti-inflammatory, and membrane-stabilising properties [14, 20–22]. Notably, the exceptionally high linoleic acid content and the distinctive polyphenolic composition of safflower oil may underlie its pronounced hepatoprotective activity [21,22]. Histological examination further corroborated the biochemical findings. In the model group, paracetamol exposure induced pronounced fatty degeneration, hepatocyte necrosis, and early fibrotic changes. In contrast, safflower oil treatment markedly reduced the severity of dystrophic and necrotic alterations. In the protective group, hepatic architecture was largely preserved, with minimal fatty infiltration and only mild fibrotic changes, confirming the structural basis of the observed functional improvements. Limitations This study has several limitations. Only male Wistar rats were included, which may limit generalisability given known sex-related differences in drug metabolism. In addition, only a single dose of safflower oil and short treatment durations were evaluated, precluding assessment of dose–response relationships and long-term efficacy. Although multiple functional and histological endpoints were examined, molecular mechanisms were not fully explored. Finally, as the study was based on a paracetamol-induced acute toxic hepatitis model, extrapolation to other forms of drug-induced liver injury and to humans should be made with caution. Conclusion Safflower oil demonstrates significant hepatoprotective, antioxidant, and anti-inflammatory effects in a rat model of paracetamol-induced acute toxic hepatitis. Its therapeutic action involves suppression of oxidative stress, modulation of inflammatory cytokines, normalisation of liver enzyme activity, and preservation of hepatic histoarchitecture. These findings support the potential of safflower oil as a natural adjunctive agent for the prevention and treatment of toxic liver injury. Abbreviations ALP, Alkaline phosphatase ALT, Alanine aminotransferase AST, Aspartate aminotransferase DILI, Drug-induced liver injury GGT, Gamma-glutamyltransferase IQR, Interquartile range MDA, Malondialdehyde Model TH, Toxic hepatitis model group Model TH + SO, Toxic hepatitis model treated with safflower oil SOD, Superoxide dismutase Declarations Ethics approval and consent to participate All experimental procedures involving animals were reviewed and approved by the Bioethics Committee of the Immunology and Human Genomics Institution, Uzbekistan (protocol IRB №2025-0001). The study was conducted in accordance with “Rules for the Care and Use of Laboratory Animals” of the Republic of Uzbekistan and the European Directive 2010/63/EU on the protection of animals used for scientific purposes. This study is reported in accordance with the ARRIVE 2.0 guidelines for reporting animal research. Clinical trial registration Not applicable Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study did not receive any specific funding. Authors’ contributions NF, AK, and AR conceptualised the study. KB and AK conducted the experiments. ST and AK performed the biochemical analyses. ON, AK, and AR drafted the manuscript. TU, ShM, TA, UP, ME, AYa, and NR reviewed the manuscript. NO, AK, and AR edited the manuscript. NF supervised the study. Acknowledgements The authors express their sincere gratitude to Professor Tursunov Abbasxon Sabirxonovich, Director of the O.S. Sodikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, and to Academician Aripova Tamara Uktamovna, Director of the Institute of Immunology and Human Genomics, Academy of Sciences of the Republic of Uzbekistan, for their scientific guidance, institutional support, and for providing the facilities necessary to conduct this research. The authors also thank the leadership of the Academy of Sciences of the Republic of Uzbekistan, as well as the staff of the participating laboratories and research groups, for their technical assistance and valuable contributions, which were essential for the successful completion of this study. References Devarbhavi H, Asrani SK, Arab JP, Nartey YA, Pose E, Kamath PS. Global burden of liver disease: 2023 update. J Hepatol. 2023;79(2):516–537. Han H, Desert R, Das S, Song Z, Athavale D, Ge X, et al. Danger signals in liver injury and restoration of homeostasis. J Hepatol. 2020;73(4):933–951. 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1","display":"","copyAsset":false,"role":"figure","size":77535,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design and timeline of paracetamol-induced acute toxic hepatitis and safflower oil treatment in rats\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/baedebbd8c83234c3092b812.png"},{"id":100415908,"identity":"5643ae62-171e-4f3d-9aac-352b247e5e10","added_by":"auto","created_at":"2026-01-16 13:22:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffects of safflower oil on serum AST and ALP activities in rats with paracetamol-induced acute toxic hepatitis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/336b45b7abd66cd3ac2885db.png"},{"id":100416122,"identity":"08f05048-1217-4c4d-8d71-e4395fc3d2a6","added_by":"auto","created_at":"2026-01-16 13:23:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41573,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of safflower oil on serum ALT levels in a paracetamol-induced toxic hepatitis model\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/0b37d09b932ccbf1a16d670e.png"},{"id":100416152,"identity":"6117b6a5-6025-44fb-9a76-baf0be804329","added_by":"auto","created_at":"2026-01-16 13:23:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":994191,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative histopathological changes in the liver of rats from the control, model, and treatment groups. Haematoxylin and eosin staining, ×200 (A–H); Masson’s trichrome staining, ×200 (I–L).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/9a3bd2b2033dd6711356d08c.png"},{"id":100415961,"identity":"30edb157-1e47-482e-8731-588b7c9586d1","added_by":"auto","created_at":"2026-01-16 13:22:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99915,"visible":true,"origin":"","legend":"\u003cp\u003eProposed hepatoprotective mechanisms of safflower oil in paracetamol-induced acute toxic hepatitis.\u003c/p\u003e\n\u003cp\u003eParacetamol overdose induces oxidative stress, inflammation, and hepatocellular damage. Safflower oil reduces ROS and lipid\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/ca62f4aa2079359ccc0c1f3d.png"},{"id":105275901,"identity":"357930ce-0c24-4246-a236-eee46f25e247","added_by":"auto","created_at":"2026-03-24 09:28:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2085493,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8601812/v1/0dfd669b-de81-4862-858c-a3f605f250b9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis","fulltext":[{"header":"Background","content":"\u003cp\u003eLiver diseases remain one of the leading causes of mortality worldwide, claiming approximately 2\u0026nbsp;million lives annually, which accounts for about 4% of all deaths [1]. These pathologies are characterised by hepatocyte injury, inflammatory infiltration, and disruption of organ architecture, leading to a progressive loss of liver function [2]. According to data from the Global Burden of Disease study, in 2019, 1.26\u0026nbsp;million deaths were attributed to cirrhosis and other chronic liver diseases (a 13% increase compared with 1990), indicating a steady rise in disease burden [3]. Among these conditions, drug-induced liver injury (DILI) occupies a significant place and has become increasingly significant in recent decades [4]. DILI is a serious complication of pharmacotherapy and represents one of the leading causes of acute liver failure in clinical practice [5,6]. To date, more than 1,000 substances with potential hepatotoxicity have been identified [7]. In addition to medications, hepatotropic toxins include industrial chemicals such as chloroform and phosphorus [8].\u003c/p\u003e \u003cp\u003eDespite the existence of general treatment approaches, including lifestyle modification and pharmacotherapy [9], no specific therapy for DILI has been developed due to the heterogeneity of its pathogenesis and individual differences in xenobiotic metabolism [10]. Given the limitations of existing approaches, increasing attention is being directed toward the investigation of natural compounds with antioxidant and hepatoprotective activity. Herbal preparations are considered a promising avenue for the prevention and treatment of toxic liver injuries [11]. One such plant is safflower (Carthamus tinctorius L.), which has been traditionally used in Eastern medicine. Its extracts have been shown to possess anti-inflammatory, antioxidant, immunomodulatory, and vascular effects [12]. However, most available data concern aqueous or alcoholic extracts of the flowers, whereas the hepatoprotective potential of cold-pressed safflower oil has been studied only to a limited extent.\u003c/p\u003e \u003cp\u003eExisting experimental studies in animal models indicate that safflower oil may reduce hepatic transaminase activity, attenuate histological signs of liver damage, and exert antioxidant effects comparable to those of silymarin [13,14]. At the same time, there is a lack of large comparative studies evaluating its efficacy in models of drug-induced toxic hepatitis, as well as insufficient data on optimal dosages, mechanisms of action, and bioavailability. Thus, the effects of safflower oil as a potential hepatoprotective agent in drug-induced liver injury remain insufficiently investigated. Further in-depth research on this topic is of considerable interest for the development of new therapeutic strategies aimed at the prevention and treatment of DILI.\u003c/p\u003e \u003cp\u003eAlthough safflower extracts have been investigated in various experimental models, data on the hepatoprotective efficacy of cold-pressed safflower oil in drug-induced toxic hepatitis remain limited. Therefore, this study aimed to evaluate the hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis experimental study was conducted in collaboration with the Institute of Immunology and Human Genomics of the Academy of Sciences of the Republic of Uzbekistan and the Institute of Bioorganic Chemistry named after Academician O.S.Sodikov, Academy of Sciences of the Republic of Uzbekistan.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eMale Wistar rats (n\u0026thinsp;=\u0026thinsp;10 per group) were randomly assigned to three experimental groups: a Control group, a toxic hepatitis model group (Model TH), and a safflower oil\u0026ndash;treated group (Model TH\u0026thinsp;+\u0026thinsp;SO). Acute toxic hepatitis was induced in the Model TH and Model TH\u0026thinsp;+\u0026thinsp;SO groups by intragastric administration of paracetamol for two consecutive days. From the third day of the experiment, rats in the Model TH\u0026thinsp;+\u0026thinsp;SO group received safflower oil orally at a dose of 10 mL/kg once daily for 14 days, whereas rats in the Control and Model TH groups received an equivalent volume of physiological saline. On day 17 of the experiment, all animals were euthanised, and blood and liver tissue samples were collected for biochemical, oxidative stress, inflammatory, and histopathological analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eThirty healthy male Wistar rats, aged 8.02 [7.75\u0026ndash;8.25] weeks and weighing 203.07 [198.5\u0026ndash;205.5] g, were used in this study. Animals were housed under specific pathogen-free (SPF) laboratory conditions (temperature 20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C, relative humidity 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, 12:12 h light\u0026ndash;dark cycle) with free access to standard laboratory chow and water. Before the experiment, all animals underwent a 10\u0026ndash;14-day quarantine period. After the quarantine period, rats were randomly allocated to three experimental groups (n\u0026thinsp;=\u0026thinsp;10 per group). Randomisation was performed using a computer-generated random number sequence.\u003c/p\u003e \u003cp\u003eThe experimental groups were as follows:\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eControl group (n\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e \u003cp\u003ehealthy rats receiving no treatment.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eModel TH group (n\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e \u003cp\u003erats with paracetamol-induced acute toxic hepatitis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment group\u003c/b\u003e (Model TH\u0026thinsp;+\u0026thinsp;SO, n\u0026thinsp;=\u0026thinsp;10): rats with paracetamol-induced acute toxic hepatitis were treated with safflower oil.\u003c/p\u003e\n\u003ch3\u003eToxic hepatitis induction\u003c/h3\u003e\n\u003cp\u003eAnimals in the Model TH (n\u0026thinsp;=\u0026thinsp;10) and Treatment (Model TH\u0026thinsp;+\u0026thinsp;SO ) (n\u0026thinsp;=\u0026thinsp;10) groups were induced to acute toxic hepatitis by intragastric administration of an aqueous solution of paracetamol at a dose of 1000 mg/kg using a gavage once daily for two consecutive days [Mossanen \u0026amp; Tacke, 2015; Jaeschke et al., 2021].\u003c/p\u003e\n\u003ch3\u003eSafflower Oil Treatment\u003c/h3\u003e\n\u003cp\u003eBeginning on the third day of the experiment, animals in the Model TH\u0026thinsp;+\u0026thinsp;SO treatment group (n\u0026thinsp;=\u0026thinsp;10) received safflower oil, cold-pressed from the seeds of \u003cem\u003eCarthamus tinctorius\u003c/em\u003e and manufactured by Botanic Herbs LLC, once daily at a dose of 10 mL/kg via oral gavage using a dosing syringe; animals in the Control and Model TH groups received an equivalent volume of physiological saline, and treatment was continued for 14 consecutive days.\u003c/p\u003e\n\u003ch3\u003eLiver function assessment\u003c/h3\u003e\n\u003cp\u003eSerum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP) were determined using standard biochemical methods. Analyses were performed with commercially available assay kits (Cypress Diagnostics BV, Belgium) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Status Assessment\u003c/h2\u003e \u003cp\u003eAntioxidant status was evaluated by measuring serum levels of malondialdehyde (MDA) and the activities of catalase and superoxide dismutase (SOD). The analysis was performed using an enzyme-linked immunosorbent assay with reagent kits (Wuhan Fine Biotech Co., Ltd., China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMorphological Analysis\u003c/h3\u003e\n\u003cp\u003eLiver samples were fixed in 10% neutral buffered formalin for 24 hours. Following fixation, tissues were dehydrated in a graded series of ethanol and xylene, and then embedded in paraffin according to standard protocols. Histological sections were prepared from paraffin blocks and stained with hematoxylin and eosin (H\u0026amp;E). Microscopic examination and acquisition of digital images of representative areas were performed using a Cilika BT-P microscope (MedPrime Technologies Pvt. Ltd., India) with the corresponding software. Histology was assessed qualitatively. Histological evaluation was performed by two independent observers blinded to group allocation.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Microsoft Excel and JASP (version 0.19.3). Normality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test. Normally distributed data (ALT) are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were analysed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s HSD post hoc test, whereas non-normally distributed data are presented as median with interquartile range (IQR) and were analysed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc test with Holm correction. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparison of liver enzymes\u003c/h2\u003e \u003cp\u003eAdministration of paracetamol resulted in significant alterations in biochemical parameters in the model group compared with the control (healthy) group, confirming the successful induction of acute toxic hepatitis. As summarised in Table\u0026nbsp;1,\u003c/p\u003e \u003cp\u003eanimals in the model group showed a marked increase in serum ALT activity (47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.82 U/L), AST activity (52.5 [37.63\u0026ndash;55.13] U/L), and ALP levels (117.47 [98.81\u0026ndash;124.38] U/L) relative to the control group (32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17 U/L, 35.0 [32.38\u0026ndash;40.25] U/L, and 62.19 [55.28\u0026ndash;69.10] U/L, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overall group differences were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;1). Treatment with safflower oil significantly attenuated paracetamol-induced biochemical disturbances. In the treatment with safflower oil group, serum AST (35.0 [31.50\u0026ndash;\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eTable\u0026nbsp;1. Comparison of liver enzyme levels among experimental groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter (unit)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eControl (n\u0026thinsp;=\u0026thinsp;10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eModel TH (n\u0026thinsp;=\u0026thinsp;10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eModel TH\u0026thinsp;+\u0026thinsp;SO (n\u0026thinsp;=\u0026thinsp;10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ep value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.82ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.80ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.0 [32.38\u0026ndash;40.25]ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.5 [37.63\u0026ndash;55.13]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.0 [31.50\u0026ndash;35.0]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALP (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.19 [55.28\u0026ndash;69.10]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e117.47 [98.81\u0026ndash;124.38]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.19 [55.28\u0026ndash;75.32]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.596 [0.522\u0026ndash;0.596]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.792 [0.566\u0026ndash;0.795]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.713 [0.606\u0026ndash;0.775]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eNote.\u003c/b\u003e Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) for ALT and as median with interquartile range (IQR) for AST, ALP, and GGT. Normality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test. Because ALT data were normally distributed, they were analysed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s HSD post hoc test. Because AST, ALP, and GGT data were not normally distributed, they were analysed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc test. Values sharing at least one common superscript letter are not significantly different. A value of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e35.0] U/L) and ALP (62.19 [55.28\u0026ndash;75.32] U/L) levels were significantly reduced compared with the untreated model group and were comparable to control values (Table\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, ALT levels in the treatment group (39.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.80 U/L) were significantly lower than those observed in the model group (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, GGT levels did not differ significantly among groups (p\u0026thinsp;=\u0026thinsp;0.171).\u003c/p\u003e \u003cp\u003eThe toxic effect of paracetamol led to the development of pronounced oxidative stress in the model group (Table\u0026nbsp;2). Serum malondialdehyde (MDA) levels, a key marker of lipid peroxidation, were significantly increased in the Model TH group (3.71 [3.66\u0026ndash;3.76] nmol/mL) compared with the control group (2.91 [2.85\u0026ndash;3.02] nmol/mL) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, treatment with safflower oil significantly attenuated lipid peroxidation, as evidenced by a reduction in MDA levels in the Model TH\u0026thinsp;+\u0026thinsp;SO group (3.13 [3.03\u0026ndash;3.20] nmol/mL) relative to the untreated Model TH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eTable\u0026nbsp;2. Oxidative stress marker (MDA) and antioxidant enzyme activities (SOD and catalase) in experimental groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter (unit)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModel TH (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModel TH\u0026thinsp;+\u0026thinsp;SO (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep value (Kruskal\u0026ndash;Wallis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA (nmol/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.91\u003c/p\u003e \u003cp\u003e[2.85\u0026ndash;3.02]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.71\u003c/p\u003e \u003cp\u003e[3.66\u0026ndash;3.76]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.13\u003c/p\u003e \u003cp\u003e[3.03\u0026ndash;3.20]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase (\u0026micro;kat/mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.64\u003c/p\u003e \u003cp\u003e[34.21\u0026ndash;35.59]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.12\u003c/p\u003e \u003cp\u003e[21.17\u0026ndash;25.26]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.68\u003c/p\u003e \u003cp\u003e[29.32\u0026ndash;31.78]ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD (U/mg protein)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003cp\u003e[1.29\u0026ndash;1.35]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003cp\u003e[1.08\u0026ndash;1.12]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003cp\u003e[1.24\u0026ndash;1.27]ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eNote.\u003c/b\u003e Data are presented as median with interquartile range (IQR). Group differences were assessed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s post-hoc test. Values sharing the same superscript letter within a row are not significantly different, whereas values with different superscript letters are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConcomitantly, the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) were significantly decreased in the Model TH group compared with controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating impairment of the endogenous antioxidant defence system (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eAdministration of safflower oil contributed to the correction of paracetamol-induced oxidative stress (Table\u0026nbsp;2). In the Model TH\u0026thinsp;+\u0026thinsp;SO group, serum MDA levels were significantly reduced (3.13 [3.03\u0026ndash;3.20] nmol/mL) compared with the untreated Model TH group (3.71 [3.66\u0026ndash;3.76] nmol/mL; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating attenuation of lipid peroxidation. Concomitantly, the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD) were significantly increased in safflower oil\u0026ndash;treated animals (30.68 [29.32\u0026ndash;31.78] \u0026micro;kat/mg and 1.26 [1.24\u0026ndash;1.27] U/mg protein, respectively) relative to the Model TH group (22.12 [21.17\u0026ndash;25.26] \u0026micro;kat/mg and 1.10 [1.08\u0026ndash;1.12] U/mg protein; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eNotably, oxidative stress parameters in the Model TH\u0026thinsp;+\u0026thinsp;SO group approached control values, suggesting a substantial restoration of antioxidant defence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparison of inflammatory marker levels\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of serum inflammatory cytokine levels in experimental groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModel TH (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModel TH\u0026thinsp;+\u0026thinsp;SO (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep value (Kruskal\u0026ndash;Wallis)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-1β pg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.18 [6.64\u0026ndash;7.73]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.75 [11.46\u0026ndash;27.21]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.88 [10.54\u0026ndash;12.29]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-6 pg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.53 [2.25\u0026ndash;2.57]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.33 [3.97\u0026ndash;4.49]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.52 [3.48\u0026ndash;3.62]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-4 pg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.19 [1.02\u0026ndash;1.24]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.65 [3.18\u0026ndash;5.10]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.37 [2.20\u0026ndash;2.87]ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α pg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.67 [8.77\u0026ndash;10.28]ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.90 [26.78\u0026ndash;45.60]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.25 [19.60\u0026ndash;29.00]ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026nbsp;.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eNote.\u003c/b\u003e Data are presented as median with interquartile range (IQR). Group differences were assessed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s post-hoc test with Holm correction. Values sharing at least one common superscript letter are not significantly different, whereas values with different superscript letters are significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eParacetamol administration induced a pronounced inflammatory response, as evidenced by significant alterations in serum cytokine levels in the Model TH group compared with the control group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn day 17, serum concentrations of IL-1β, IL-6, IL-4, and TNF-α were significantly elevated in the Model TH group (15.75 [11.46\u0026ndash;27.21], 4.33 [3.97\u0026ndash;4.49], 3.65 [3.18\u0026ndash;5.10], and 34.90 [26.78\u0026ndash;45.60], respectively) relative to control values (7.18 [6.64\u0026ndash;7.73], 2.53 [2.25\u0026ndash;2.57], 1.19 [1.02\u0026ndash;1.24], and 9.67 [8.77\u0026ndash;10.28], respectively; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment with safflower oil significantly attenuated the paracetamol-induced inflammatory response. In the Model TH\u0026thinsp;+\u0026thinsp;SO group, serum levels of IL-1β (11.88 [10.54\u0026ndash;12.29]) and IL-6 (3.52 [3.48\u0026ndash;3.62]) were significantly reduced compared with the untreated Model TH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although they remained higher than control values. IL-4 levels in the treatment group (2.37 [2.20\u0026ndash;2.87]) did not differ significantly from either the control or Model TH groups, indicating partial normalisation. In contrast, TNF-α concentrations remained significantly elevated in both the Model TH and Model TH\u0026thinsp;+\u0026thinsp;SO groups compared with controls, with no significant difference between the two groups.\u003c/p\u003e \u003cp\u003eOverall, these findings demonstrate that safflower oil partially suppresses paracetamol-induced inflammatory activation, with a more pronounced modulatory effect on IL-1β, IL-6, and IL-4 than on TNF-α.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMorphological assessment\u003c/h2\u003e \u003cp\u003eMorphological examination of the liver confirmed the biochemical findings. In animals of the model group (without treatment), pronounced pathological changes were observed, including fatty degeneration, hepatocyte necrosis, and moderate inflammatory infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. B, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe use of safflower oil as a hepatoprotective agent significantly reduced the severity of dystrophic and inflammatory processes. The structure of hepatocytes was close to normal, and fatty degeneration was observed only sporadically (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. C, D, G, H). With Masson\u0026rsquo;s trichrome staining, collagen structures in the control group were detected only as a normal component of vascular walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), whereas in the model group, the formation of thin fibrotic strands in the intercellular matrix around the central vein structures was noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). In the treatment group, the severity of fibrotic changes was markedly lower and was represented by single thin fibrous fibres between hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. H, I ).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, safflower oil demonstrated pronounced hepatoprotective and antioxidant effects in a rat model of paracetamol-induced acute toxic hepatitis. Paracetamol administration resulted in the development of acute liver injury, as evidenced by significant alterations in biochemical markers in the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese findings are consistent with previous studies that have reported the hepatoprotective effects of various safflower-derived preparations. For instance, red safflower pigment administered at doses of 10 and 20 mg/kg significantly reduced ALT and AST activity in rats with CCl₄-induced hepatitis [13]. Similarly, safflower seed oil (200 mg/kg) normalised ALT, AST, and ALP levels in alloxan-induced diabetic rats [14], while aqueous safflower extracts dose-dependently reduced transaminase activity in alcohol-induced liver injury models [18]. Oxidative stress represents a key pathogenetic mechanism in paracetamol-induced hepatotoxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the present study, oxidative damage was reflected by a significant increase in malondialdehyde (MDA) levels to 3.71 [3.66\u0026ndash;3.76] nmol/mL and a concomitant suppression of superoxide dismutase (SOD) activity to 1.10 [1.08\u0026ndash;1.12] U/mg protein in the model group. Treatment with safflower oil effectively counteracted these changes, significantly reducing MDA levels to 3.13 [3.03\u0026ndash;3.20] nmol/mL and restoring SOD activity to 1.26 [1.24\u0026ndash;1.27] U/mg protein (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThese observations are in agreement with the findings of Alshareef et al., who demonstrated that methanolic extracts of safflower flowers (300 mg/kg) reduced hepatic lipid peroxidation and enhanced SOD and glutathione activity in a rat model of type 2 diabetes [19]. The authors attributed these effects to activation of the Nrf2-dependent antioxidant signalling pathway, a mechanism that has also been reported for red safflower pigment in CCl₄-induced liver injury [13].\u003c/p\u003e \u003cp\u003eThe high therapeutic efficacy of safflower oil may be attributed to its unique chemical composition. Safflower oil contains up to 75\u0026ndash;80% polyunsaturated linoleic acid, along with oleic, palmitic, and stearic acids. In addition, it is rich in biologically active compounds, including polyphenols, flavonoids (such as quercetin and kaempferol), quinochalcones, tocopherols (vitamin E), serotonin derivatives, and plant sterols. This complex phytochemical profile confers potent antioxidant, anti-inflammatory, and membrane-stabilising properties [14, 20\u0026ndash;22]. Notably, the exceptionally high linoleic acid content and the distinctive polyphenolic composition of safflower oil may underlie its pronounced hepatoprotective activity [21,22].\u003c/p\u003e \u003cp\u003eHistological examination further corroborated the biochemical findings. In the model group, paracetamol exposure induced pronounced fatty degeneration, hepatocyte necrosis, and early fibrotic changes. In contrast, safflower oil treatment markedly reduced the severity of dystrophic and necrotic alterations. In the protective group, hepatic architecture was largely preserved, with minimal fatty infiltration and only mild fibrotic changes, confirming the structural basis of the observed functional improvements.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThis study has several limitations. Only male Wistar rats were included, which may limit generalisability given known sex-related differences in drug metabolism. In addition, only a single dose of safflower oil and short treatment durations were evaluated, precluding assessment of dose\u0026ndash;response relationships and long-term efficacy. Although multiple functional and histological endpoints were examined, molecular mechanisms were not fully explored. Finally, as the study was based on a paracetamol-induced acute toxic hepatitis model, extrapolation to other forms of drug-induced liver injury and to humans should be made with caution.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSafflower oil demonstrates significant hepatoprotective, antioxidant, and anti-inflammatory effects in a rat model of paracetamol-induced acute toxic hepatitis. Its therapeutic action involves suppression of oxidative stress, modulation of inflammatory cytokines, normalisation of liver enzyme activity, and preservation of hepatic histoarchitecture. These findings support the potential of safflower oil as a natural adjunctive agent for the prevention and treatment of toxic liver injury.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALP, Alkaline phosphatase\u003c/p\u003e\n\u003cp\u003eALT, Alanine aminotransferase\u003c/p\u003e\n\u003cp\u003eAST, Aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eDILI, Drug-induced liver injury\u003c/p\u003e\n\u003cp\u003eGGT, Gamma-glutamyltransferase\u003c/p\u003e\n\u003cp\u003eIQR, Interquartile range\u003c/p\u003e\n\u003cp\u003eMDA, Malondialdehyde\u003c/p\u003e\n\u003cp\u003eModel TH, Toxic hepatitis model group\u003c/p\u003e\n\u003cp\u003eModel TH + SO, Toxic hepatitis model treated with safflower oil\u003c/p\u003e\n\u003cp\u003eSOD, Superoxide dismutase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animals were reviewed and approved by the Bioethics Committee of the Immunology and Human Genomics Institution, Uzbekistan (protocol IRB №2025-0001). The study was conducted in accordance with \u0026ldquo;Rules for the Care and Use of Laboratory Animals\u0026rdquo; of the Republic of Uzbekistan and the European Directive 2010/63/EU on the protection of animals used for scientific purposes. This study is reported in accordance with the ARRIVE 2.0 guidelines for reporting animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not receive any specific funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNF, AK, and AR conceptualised the study. KB and AK conducted the experiments. ST and AK performed the biochemical analyses. ON, AK, and AR drafted the manuscript. TU, ShM, TA, UP, ME, AYa, and NR reviewed the manuscript. NO, AK, and AR edited the manuscript. NF supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to Professor Tursunov Abbasxon Sabirxonovich, Director of the O.S. Sodikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, and to Academician Aripova Tamara Uktamovna, Director of the Institute of Immunology and Human Genomics, Academy of Sciences of the Republic of Uzbekistan, for their scientific guidance, institutional support, and for providing the facilities necessary to conduct this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors also thank the leadership of the Academy of Sciences of the Republic of Uzbekistan, as well as the staff of the participating laboratories and research groups, for their technical assistance and valuable contributions, which were essential for the successful completion of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDevarbhavi H, Asrani SK, Arab JP, Nartey YA, Pose E, Kamath PS. Global burden of liver disease: 2023 update. 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Pharmacogn Mag. 2022;18(80):1082\u0026ndash;1088.\u003c/li\u003e\n\u003cli\u003eAlshareef NS, AlSedairy SA, Al-Harbi LN, Alshammari GM, Yahya MA. \u003cem\u003eCarthamus tinctorius\u003c/em\u003e L. flower extract attenuates hepatic injury and steatosis in a rat model of type 2 diabetes mellitus via Nrf2-dependent effects. Antioxidants (Basel). 2024;13(9):1098.\u003c/li\u003e\n\u003cli\u003eKhalid N, Khan RS, Hussain MI, Farooq M, Ahmad A, Ahmed I. A comprehensive characterisation of safflower oil for its potential applications as a bioactive food ingredient: a review. Trends Food Sci Technol. 2017;66:176\u0026ndash;186.\u003c/li\u003e\n\u003cli\u003eFristiohady A, Al-Ramadan W, Asasutjarit R, Julian LOM. Phytochemistry, pharmacology and medicinal uses of \u003cem\u003eCarthamus tinctorius\u003c/em\u003e Linn: an updated review. Biointerface Res Appl Chem. 2023;13:441.\u003c/li\u003e\n\u003cli\u003eCoşkun Uğurlu R, Ayar A. Effects of herbal safflower oil on longevity and oxidative stress. Commagene J Biol. 2024;8(2).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","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":"Rat, toxic hepatitis, safflower oil, hepatoprotective activity, oxidative stress, antioxidant enzymes, inflammatory cytokines, liver histology.","lastPublishedDoi":"10.21203/rs.3.rs-8601812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8601812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDrug-induced liver injury (DILI) is a major cause of acute liver failure, highlighting the need for effective hepatoprotective agents. Natural products with antioxidant properties represent promising therapeutic options. This study aimed to evaluate the hepatoprotective, antioxidant, and anti-inflammatory effects of cold-pressed safflower oil in a rat model of paracetamol-induced acute toxic hepatitis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAcute toxic hepatitis was induced in male Wistar rats by intragastric administration of paracetamol at a dose of 1000 mg/kg for two consecutive days. This dosing regimen was selected based on established experimental models of acute toxic hepatitis that reliably induce liver injury while maintaining animal survival. Safflower oil was administered orally at a dose of 10 mL/kg for 14 days. Hepatic injury was evaluated by assessing serum biochemical markers, including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase; oxidative stress parameters (malondialdehyde); antioxidant enzyme activities (superoxide dismutase and catalase); inflammatory cytokine levels (IL-1β, IL-6, IL-4, and TNF-α); and histopathological changes in liver tissue. Statistical analyses were performed using parametric or non-parametric methods, as appropriate.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eParacetamol administration caused significant liver injury, evidenced by elevated serum aminotransferase and alkaline phosphatase activities, increased malondialdehyde levels, suppression of antioxidant enzyme activity, and marked histopathological damage. Treatment with safflower oil significantly reduced aspartate aminotransferase and alkaline phosphatase levels and attenuated oxidative stress, as indicated by decreased malondialdehyde concentrations and restoration of superoxide dismutase and catalase activities (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inflammatory cytokine levels were partially normalised following safflower oil treatment, with significant reductions in IL-1β, IL-6, and IL-4, while TNF-α remained elevated. Histological examination confirmed reduced hepatocellular degeneration, necrosis, inflammatory infiltration, and early fibrotic changes in treated animals compared with the untreated model group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCold-pressed safflower oil exhibits significant hepatoprotective, antioxidant, and anti-inflammatory effects in paracetamol-induced acute toxic hepatitis. These effects are associated with suppression of oxidative stress, modulation of inflammatory responses, normalisation of liver enzyme activity, and preservation of hepatic histoarchitecture. Safflower oil may represent a promising natural adjunctive agent for the prevention and management of toxic liver injury.\u003c/p\u003e","manuscriptTitle":"Hepatoprotective and antioxidant effects of safflower oil in a rat model of paracetamol-induced acute toxic hepatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 11:56:43","doi":"10.21203/rs.3.rs-8601812/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"13b6c643-3d1c-4034-bb97-33a569d31a2c","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T09:27:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 11:56:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8601812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8601812","identity":"rs-8601812","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}