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Research Article

The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model

[version 1; peer review: 1 approved, 2 approved with reservations]
Shahad Hassan Hadi
https://orcid.org/0009-0009-0983-9412
1, Mohammed Qasim Yahya Malallah A. Al-Atrakji
https://orcid.org/0000-0001-5047-5854
2
Shahad Hassan Hadi
https://orcid.org/0009-0009-0983-9412
1, Mohammed Qasim Yahya Malallah A. Al-Atrakji
https://orcid.org/0000-0001-5047-5854
2
PUBLISHED 27 Jan 2025
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Background

Methotrexate (MTX) is an antifolate medication indicated to treat an array of tumors and autoinflammatory maladies. MTX may exhibit harmful impacts on multiple organs, especially liver injury and cirrhosis. Juniperus macrocarpa is a medicinal herb enriched with polyphenols and flavonoids featuring robust anti-inflammatory and antioxidative benefits.

Objective

To evaluate the hepatoprotective effects of Juniperus macrocarpa aqueous extract on MTX-aggravated liver toxicity.

Methods

The study involved 20 male middle-aged albino rats, arbitrarily allocated into 4 groups of 5 animals each. Group 1 (control) were given distilled water (DW) once daily for two weeks. Group 2 (MTX) got an intraperitoneal single dose of MTX (20 mg/kg) for two weeks. Rats in groups 3 and 4 were given daily dosages of 100 mg and 200 mg of Juniperus macrocarpa aqueous extract, respectively, for two weeks before receiving a single intraperitoneal MTX injection.

Results

Juniperus macrocarpa extracts at both low and high doses substantially alleviated the MTX-provoked biochemical alterations, as evidenced by decreased levels of inflammatory parameters including TNF-α and IL-6 and hepatic enzymes including ALT, AST, and ALP. Juniperus macrocarpa also significantly boosted levels of the anti-oxidant enzymes like SOD and GPX. Moreover, Juniperus macrocarpa extract attenuated congestive and degenerative hepatic changes, as indicated by improved histopathological findings.

Conclusion

The anti-oxidative and anti-inflammatory activities of Juniperus macrocarpa extract are a promising approach for ameliorating MTX-aggravated hepatotoxicity.

Keywords

Juniperus Macrocarpa; Methotrexate; hepatotoxicity; Oxidative stress; Apoptosis; Cytokines

Corresponding author: Shahad Hassan Hadi
Competing interests: No competing interests were disclosed.
Grant information: The author(s) declared that no grants were involved in supporting this work.
Copyright:  © 2025 Hadi SH and Al-Atrakji MQYMA. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Hadi SH and Al-Atrakji MQYMA. The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:131 (https://doi.org/10.12688/f1000research.158839.1) First published: 27 Jan 2025, 14:131 (https://doi.org/10.12688/f1000research.158839.1) Latest published: 27 Jan 2025, 14:131 (https://doi.org/10.12688/f1000research.158839.1)

Introduction

Drug-induced liver Injury (DILI) is a generic term encompassing the spectrum of pathological responses that occur in the liver as a result of exposure to chemical compounds that have the potential to be destructive to the liver.1–3 DILI is a serious risk that is a subject of great concern, and it is a risk that is present in both the preclinical drug development process and as a primary source of candidate drug attrition.2,4–6 One of the most common reasons for acute liver failure (ALF) in developed countries is thought to be pharmaceutical intoxication, which is then followed by acute hepatitis B.7,8

Methotrexate (MTX) is a potent anticancer drug that acts as an antagonist to folic acid. It has proven to be highly effective in treating various types of cancer, including lymphoma, leukemia, breast cancer, and osteosarcoma.9–11 Moreover, MTX is extensively utilized in the management of autoimmune conditions such as rheumatoid arthritis and psoriasis.12–15 Despite its clinical efficacy, the use of MTX has been associated with hepatotoxicity, which is a major adverse effect that manifests as tissue destruction, liver dysfunction, fibrosis, and cirrhosis, along with notable modifications in the function of the liver, biochemical indicators, and histological structure.16–18 The exact mechanism by which MTX-induced liver damage is not understood; however, several researchers attributed MTX-induced hepatotoxicity to oxidative stress, apoptosis, and inflammation.19–21

Several disorders have been successfully treated with herbal treatment, which involves the use of items derived from plants. When compared to synthetic medication, these natural remedies are safer and have fewer adverse effects than the synthetic medicine.22–32 Therefore, several researchers tried to evaluate the effectiveness of herbal products such as Thymus Vulgaris,19 Origanum onites essential oil,33 Crocus sativus stigma,21 Syzygium aromaticum essential oil extract,34 and Moringa oleifera leaf extract35 in MTX-induced hepatotoxicity.

Juniperus Macrocarpa is a. plant growing in Turkey and used for various medicinal purposes in traditional folk medicine.36–38 Phytochemical identification of Juniperus Macrocarpa extract revealed that it is rich in phenolic acids (such as protocatechuic acid and gallic acid), flavonoids (such as rutin, catechin, quercitrin, epicatechin, luteolin, and naringenin), and coumarins such as umbelliferone. The relative amount of active ingredient in the extract is dependent on the plant species and cultivation area.39–41

To the best of our knowledge, there have been no previous studies determined the potential effects of Juniperus Macrocarpa extract in the attenuation of MTX-exacerbated hepatotoxicity. The objective of this research was to assess whether or not pre-treatment with Juniperus Macrocarpa extract is effective in amelioration of MTX-induced hepatotoxicity via investigating the effects of the extract on the serum levels of the antioxidant enzymes, inflammatory markers, hepatic enzymes, and histological findings.

Methods

Experimental animals

Twenty male albino rats aged between 6 and 12 months with an average weight of 220 ± 30 g, were used in this study. The animals were acquired from the animal house of the Iraqi Center for Cancer Research and Medical Genetics– Baghdad. They were placed in polyethylene cages with stainless steel covers and kept for acclimatization for one week before the experiment. They were maintained in standard laboratory conditions (232°F, 12-hour light-dark cycle) and had free access to food from a chow pallet and tap water. The study was started from December 31, 2023, to July 1, 2024. This study was approved by the ethical committee for experimental studies at the College of Medicine/University of Baghdad.

Drugs and reagents

Juniperus Macrocarpa plant was obtained from the Department of Pharmacognosy /College of Pharmacy/Baghdad University, Iraq. Methotrexate Methotrexate (MTX) was purchased from Medwise Healthcare Limited, 843 Finchley Road, London, UK. Catalog Number: M1435. Other substances were bought from well-known manufacturers.

Methods

Drug preparations

We used the dose of MTX required to induce hepatotoxicity in rats as 20 mg/kg (the quantity around 4.4 mg per rat), which was estimated according to previous studies.42 The dose was calculated according to the rat’s body weight and given intraperitoneally.

Furthermore, an oral solution of Juniperus macrocarpa was prepared by estimating the quantity of Juniperus macrocarpa based on the findings of an oral acute toxicity study in which rats were given a dosage of 2000 mg/kg and exhibited no indication of toxicity in their bodies. Therefore, the study utilized one-tenth of this dose, which is equivalent to 200 mg/kg as a high dose, and half of the one-tenth as a low-level dose, to validate the safety of the substance, as stated by another study.43,44 An oral solution of the powder was made by combining powder Juniperus Macrocarpa with distilled water to provide a concentration of 40 mg/mL, for experimental rats with an average weight of 220 ± 30 g, a volume of 1 ml is necessary to provide a dose of 200 mg/ mL and 0.5 mL to provide a dose of 200 mg/mL according to the prescribed protocol.

Experimental design

The preset study was performed at the Department of Pharmacology, College of Medicine, University of Baghdad, and the Iraqi Center for Cancer and Medical Genetic Research. The twenty rats were randomly divided into four groups with a total of five rats in each group. Participants who were a part of the experimental groups included the following individuals:

  • • Group I (apparently healthy) group(n=5): consisted of normal, healthy rats that were kept under normal laboratory conditions and received 1 mL of distilled water orally for 13 days by oral gavage.

  • • Group II (Induction) group(n=5): rats were adminstrated pre-treatment with 1 mL of distilled water orally for 13 days by oral gavage and 20mg/kg of Methotrexate intraperitoneally on day 13 of the study.42

  • • Group III (low-dose Juniperus Macrocarpa extract-treated group)(n=5) rats were given Juniperus Macrocarpa extract (100 mg/kg) for 13 days by oral gavage and MTX intraperitoneally (as in Group II) on day 13 of the study.

  • • Group IV (high-dose Juniperus Macrocarpa extract-treated group)(n=5), rats were given Juniperus Macrocarpa extract (200 mg/kg) for 13 days by oral gavage and MTX intraperitoneally (as in Group II) on day 13 of the study.

Anesthetic/Method of euthanasia

All precautions were taken to guarantee the wellbeing of the rats being studied and to limit any and reduce any pain, grief, or suffering. Attempts involved proper ventilation, typical cage disinfection, wood husk replacement every two days, attentive animal transportation. After 24 hours from the last MTX administration, the animals were euthanized using intraperitoneal anesthesia with 87 mg/kg of ketamine and 13 mg/kg of xylazine.45–49

Tissue and blood sample Collection

The blood samples were obtained using direct cardiac puncture using and transferred into gel tubes. Subsequently, the tubes were centrifuged at a speed of 3000 revolutions per minute for a duration of 10 minutes.50–53 Following complete blood separation, the serum was extracted, placed into 2 mL non-treated plastic tubes, and stored at a temperature of -20 °C for subsequent analysis.54–56

Following the collection of the blood sample, the liver tissues were then isolated and rinsed with distilled water and preserved in a 10% formalin solution to improve and maintain the tissue structure while preventing degradation by lysosomal enzymes. This was done in preparation for a histopathological examination.57–61

The serum concentrations of superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were measured for all experimental groups (1-4). The ELISA kits used in this study were commercially available and followed the instructions provided by the manufacturer (Cloud-Clone Crop ® Laboratory, China). The initial phase in the experiment is to add anti-biomarker antibodies to a 96-well plate. Samples and standards were added to the wells, and the wrapping antibodies attracted any circulatory TNF-α, IL-β, SOD, or GPx to the wells. After extracting the unattached biotin-linked antibody, streptavidin and horseradish peroxidase (HRP) were carefully placed onto the plate.62–65 The number of several bio-markers in every specimen was calculated via comparison of optical density to conventional curves. The ceramic plates were washed again, and then TMB-substrate mixtures were applied to indicate the paired indicator amounts using the color produced. The absorbance of the samples was quantified using a microplate reader spectrophotometer. The color amplitude is computed at 450 nm as the color transitions from blue to yellow with a stopping solution.66–69

Assessment of hepatic marker enzymes

The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were quantified using the automated Flexor-EL80 system manufactured by Vitalab. 500μL of serum was added and incubated for 30 minutes before being read.3,5,70–72

Assessment of histopathological changes

The liver for all experimental groups (1-4) were collected 24 hours after MTX injection on day 14 of the experiment. Following the administration of ketamine and xylazine anesthesia, the liver was extracted and preserved in 10% formalin for histological inspection.57,73–75 The study utilized the conventional paraffin-embedded approach to prepare liver tissue for microscopic analysis.76–78 Afterward, the samples were immersed in paraffin, cut into slices that were 5 μm in thickness, and then treated with hematoxylin and eosin (H&E) stain. The histological slides were analyzed using standard light microscopy techniques at GENEX Laboratories in the USA. An experienced pathologist examined the slides without any prior knowledge, and only one sample slide was selected for each group.79–82 The semi-quantitative score compromises several categories including hepatocyte regeneration and necrosis, central and portal veins dilation and congestion, degree of fibrosis, infiltration of MNCs, and proliferation of cholangiocytes was evaluated by ranking the severity of hepatocyte necrosis, fibrosis (collagen deposition), cellular infiltration, apoptosis, and fatty alteration from liver sections were graded from 1 (minimum), 2 (mild), 3 (moderate), and 4 (marked).83–86 The total score for each slide was calculated and the median score with interquartile range was obtained.

Statistical analysis

Graph Pad Prism 9 was used to conduct the statistical analysis. The median and interquartile ranges were utilized by the histolpathogical scoring system; however, the mean and standard deviation were calculated for all other data. The analysis of variance (ANOVA) and the post hoc Tukey’s tests were utilized to ascertain the group relationships. To achieve statistical significance, the P-value needed to be lower than 0.05. The non-parametric Kruskal-Wallis test was followed by Dunn’s multiple comparisons test to analyze the scores obtained from the histological groups.87

Results

According to the results of the current study, the serum levels of antioxidant enzymes (SOD and GPx) in the MTX induction group (G2) were significantly lower (p<0.05) than the control(G1). Furthermore, the serum levels of SOD and GPx in Juniperus Macrocarpa extract-treated groups G3 and G4 were significantly higher (p<0.05) than the MTX induction group (G2). Also, the serum levels of both SOD and GPx in G4 were significantly higher (p<0.05) than in G3. Additionally, the serum levels of both SOD and GPx in G4 were significantly higher (p<0.05) than the control group (G1) as shown in Figure 1.

0cd9c2f8-da3d-40dc-bf3c-6985d4a94149_figure1.gif

Figure 1. Effects of studied medications on serum levels of antioxidant enzymes SOD and GPx.

Data are indicated as mean ±SD; ns (non-significant) = p > 0.05; ****= significant differences (p < 0.0001).

According to the results of the current study, the serum levels of TNF-α and IL-6 in the MTX induction group (G2) were significantly higher (p<0.05) than the control (G1). Furthermore, the serum levels of TNF-α and IL-6 in Juniperus Macrocarpa extract-treated groups G3 and G4 were significantly lower (p<0.05) than the MTX induction group (G2). Also, the serum levels of both TNF-α and IL-6 in Juniperus Macrocarpa extract-treated groups G4 (200 mg/kg) were significantly lower (p<0.05) than in Macrocarpa extract-treated groups G3(100 mg/kg). However, the serum levels of both TNF-α and IL-6 in G3 and G4 were still significantly higher (p<0.05) than the control group (G1) as seen in Figure 2.

0cd9c2f8-da3d-40dc-bf3c-6985d4a94149_figure2.gif

Figure 2. Effects of studied medications on serum levels of inflammatory cytokines TNF-α and IL-6.

Data are indicated as mean ±SD; **= significant differences (p < 0.01); ****= significant differences (p < 0.0001).

According to the results of the current study, the serum levels of AST, ALT, and ALP in the MTX induction group (G2) were significantly higher (p<0.05) than the control (G1). Furthermore, the serum levels of AST, ALT, and ALP in Juniperus Macrocarpa extract-treated groups G3 and G4 were significantly lower (p<0.05) than the MTX induction group (G2). Also, the serum levels of ALT in Juniperus Macrocarpa extract-treated groups G4 (200 mg/kg) were significantly lower (p<0.05) than in Macrocarpa extract-treated groups G3(100 mg/kg) While there was an insignificant difference (p>0.05) between G3 and G4 in the serum levels of AST and ALP. However, the serum levels of ALT in both G3 and G4 were still significantly higher (p<0.05) than in the control group (G1) as illustrated in Figure 3.

0cd9c2f8-da3d-40dc-bf3c-6985d4a94149_figure3.gif

Figure 3. Effects of studied medications on hepatic enzymes AST, ALT, and ALP.

Data are indicated as mean ±SD; ns (non-significant) = p > 0.05; **= significant differences (p < 0.01); ****= significant differences (p < 0.0001).

According to Figure 4, liver sections from the control group (G1) had normal central veins, hepatic cords, sinusoids, hepatocytes, and kupffer cells. In the MTX-induction group (G2), liver histopathology showed mild dilatation with central venous congestion, cellular and sinusoids, and localized necrosis with MNC aggregations. Furthermore, severe sinusoidal congestion with marked cellular swelling of hepatocytes and marked disarrangement of the hepatic cords coupled with fibroplasia and marked proliferation of cholangiocytes ( Figure 4). Pretreatment with low-dose Juniperus Macrocarpa extract 100 mg/kg/day (G3) preserved the normal liver structure as histopathological sections revealed a normal central vein with a normal arrangement of the hepatic cords. However, marked zonal cellular swelling with nuclear pyknosis and little necrosis are seen. Furthermore, pretreatment with Juniperus Macrocarpa extract 200 mg/kg/day (G4) resulted in sections with mild congestion of the central vein and hepatic sinusoids, a normal appearance of the hepatocytes arrangement that revealed a feature of bi-nucleated. The portal triad revealed normal cytoarchitecture and fibrous tissue. Other figures were similar to those of the control group.

0cd9c2f8-da3d-40dc-bf3c-6985d4a94149_figure4.gif

Figure 4. Effects of studied medications on hepatic histopathological changes.

Histological photomicrographs obtained by processing rats’ livers from control (A), MTX induction (B), Juniperus Macrocarpa pretreatment (100 mg/kg) (C), and Juniperus Macrocarpa treatment (200 mg/kg) (D) groups. The control group shows the normal appearance of central vein (V), sinusoid (Black arrows) & normal arrangement of hepatic cords (Asterisk). The MTX induction group shows severe sinusoidal congestion with marked cellular swelling of hepatocytes with little necrosis (Arrows) and markedly disarrangement of hepatic cords. Regarding low-dose Juniperus Macrocarpa pretreatment, liver sections showed normal central vein (V), a normal arrangement of hepatic cords with marked zonal cellular swelling with little necrosis (Asterisks). Regarding high-dose Juniperus Macrocarpa pretreatment, liver sections showed mild congestion of the central vein (V) and a normal appearance of the portal triad (Arrow), with normally arranged hepatic cords (Asterisk), H&E stain.100×.

Discussion

MTX is the main treatment for various leukemia and lymphoma subtypes.88,89 Adverse responses limit the therapeutic use of this medication.90 MTX was also utilized to control a range of inflammatory illnesses, notably psoriasis, vasculitis, and ulcerating colitis, as well as to terminate ectopic pregnancies on specific occasions.91–97 MTX’s most common and dangerous side effect is hepatotoxicity.98 Previous research demonstrates that MTX produces several forms of harm in both humans and rats, including hepatitis, cirrhosis, elevated liver enzyme levels, gastrointestinal toxicity, and nephrotoxicity.99 The primary center of metabolism, the liver performs a variety of tasks including producing biochemical enzymes, detoxifying the body, and producing vital proteins for blood clotting.100

Numerous investigations have shown that MTX and its metabolites cause hepatocyte inflammation, oxidative stress, fibrosis, and apoptosis.35,101 The current study investigated whether Juniperus Macrocarpa extract may reduce MTX’s cytotoxic effect on liver cells. We measured blood antioxidant enzymes, inflammatory indicators, hepatic enzymes, and liver tissue histology to achieve our goal. We measured blood antioxidant enzymes, inflammatory indicators, hepatic enzymes, and liver tissue histology to achieve our goal.

According to the results of the current study, the level of antioxidant enzymes (SOD and GPx) in the serum is significantly reduced by MTX administration as compared to the control group. This finding agrees with previously published studies.102 Nrf2 regulates antioxidant enzyme synthesis and is released from Kaap1, transcripted in the nucleus, and increases antioxidant enzyme synthesis during oxidative stress.102,103 Multiple studies demonstrated that Nrf2 mRNA levels are significantly downregulated in MTX-induced hepatotoxicity compared to the normal control group.104 Treatment with 100 mg/kg and 200 mg/kg of Juniperus Macrocarpa extract as in groups G3 and G4 showed increased levels of antioxidant enzyme as compared to the MTX-induction group (G2). These effects of Juniperus Macrocarpa are attributed to its high polyphenols, flavonoid, and catechins compounds as indicated in the preliminary screening study.39 These compounds are well known for their antioxidant potential which is described by several published articles.105

According to the literature, the principal flavonoids present in Juniperus Macrocarpa extract were rutin, catechin, quercitrin, epicatechin, luteolin, and naringenin.39 While polyphenolic compounds present were protocatechuic acid and gallic acid.39 The principal mechanisms of flavonoid’s antioxidant activity are scavenging oxygen radicals, protecting against lipid peroxidation, and chelating metal ions.105 Furthermore, it has been reported that these phytochemicals can induce Nrf2 expression or inhibit its proteasomal degradation by modifying the Nrf2–Keap1 complex which in turn increases antioxidant enzyme (SOD and GPx) synthesis.106,107

Additionally, G4 (200 mg/kg/day) exhibited statistically significantly greater SOD and GPx levels than G3 (100 mg/kg/day). The extract includes more pharmacologically active phytochemicals; therefore higher doses should be more helpful. Besides, in a study conducted to explore the protective effects of luteolin in MTX-induced hepatotoxicity, results showed that levels of SOD and GPx in the hepatic tissue were significantly increased coupled with upregulation in the Nrf2 gene expression.108 Furthermore, to investigate the protective effects of gallic acid against MTX-induced hepatotoxicity, it was found that the tissue levels of antioxidant enzymes (SOD and GPx) were significantly elevated compared to untreated groups owing to their ROS scavenging ability.109,110

Results also demonstrated that MTX therapy increased pro-inflammatory cytokines in the blood. Another study found similar results.111 MTX-induced inflammation is caused by increased ROS generation, which activates NF-κB in Kupffer cells.112 NF-κB activation leads to the transcription of pro-inflammatory cytokines such as TNF-α and IL-6. Multiple hepatotoxic medications have been linked to elevated liver NF-κB expression and TNF- α levels.113,114 While the Juniperus Macrocarpa treatment groups G3 and G4 showed reduced levels of inflammatory cytokines as compared to the MTX-induction group (G2); these findings are attributed to the phytochemical composition of Juniperus Macrocarpa as it is abundant in polyphenols and flavonoid compounds which have antioxidant and anti-inflammatory effects.39 The antioxidant properties of Juniperus Macrocarpa extract mitigates the oxidative stress induced by MTX leading to a reduction in the activation of oxidative stress response of NF-κB and reduced the expression of pro-inflammatory cytokines.115 Additionally, it was found that G4 had lower TNF-α and IL-6 levels compared to G3 which is statistically significant. As the extract contains more pharmacologically active phytochemicals, bigger dosages should have more therapeutic potential.

As mentioned previously, MTX-induced hepatic damage is associated with cellular death and necrosis coupled with the release of hepatic enzymes into the circulation.116 This resulted in a significant elevation of the AST, ALT, and ALP serum levels in MTX-induction (G2) as compared to the normal control group. However, Juniperus Macrocarpa pretreatment groups G3 and G4 had lower AST, ALT, and ALP levels than the MTX-induction group (G2). This indicates less damage to the hepatocyte and the hepatoprotective effects of Juniperus Macrocarpa extract. An explanation for this finding could be made based on the previous result represented by the ability of the extract to enhance the level of antioxidant enzymes and counteract the state of oxidative stress. This leads to a decrease in lipid peroxidation and the proposed damage to the cell membrane. Controlling oxidative stress diminishes NF-κB pathway activation, TNF-α, and IL-6 production, leading to mitigated inflammatory response. These actions are attributed to the phytochemical composition of the Juniperus Macrocarpa extract represented by polyphenols and flavonoid content leading collectively to less damage to the hepatocytes and reduced serum levels of AST, ALT, and ALP.39 Furthermore, G4 had lower AST, ALT, and ALP levels compared to G3 which is only significant in the case of ALT. This is expected as higher doses of the extract have more concentration of pharmacologically active phytochemicals leading to enhanced therapeutic potential. Regarding the hepatic enzymes, ALT is solely produced by the liver so it is more reliable to assess the magnitude of liver damage.117 In research on apigenin protective effects against MTX-induced hepatotoxicity, serum AST, ALT, and ALP had dramatically decreased upon pretreatment. These findings were attributed to the antioxidant effects of apigenin and its protection against lipid peroxidation which preserves cell membrane and hepatic cells from necrosis.118 Similarly, epicatechin pretreatment significantly reduces serum levels of AST, ALT, and ALP owing to their anti-inflammatory and antioxidant properties which protect hepatocytes from damage.119

The histopathological damage score for G2 was significantly higher (p<0.05) than the control group (G1). Hepatic sections stained with H and E revealed that the MTX induction group (G2) suffered from several histopathological alterations including dilatation and congestion of the hepatic sinusoids, inflammation, hepatocyte degeneration, vacuolization, and fibrosis. The observed changes agree with previously obtained biochemical results represented by a reduction in the levels of antioxidant enzymes, elevation of the circulatory levels of hepatic enzymes (AST, ALT, and ALP), and proinflammatory cytokinins. These results agree with previous studies which declared that MTX causes focal necrosis, dilatation in the central vein, accompanied by inflammatory cells, many bodies of apoptosis with dense cytoplasm, and peripheralized pyknotic nuclei.120,121 Furthermore, MTX-induction (G2) showed a high degree of cholangiocyte infiltration indicating damage to the bile duct which is compatible with elevated levels of ALP previously obtained. This effect was also obtained in another research.122 This finding is explained by damage to the hepatic cells caused by MTX resulting in oxidative stress and ROS coupled with MTX-induced downregulation in the Nrf2 gene expression, eventually leading to a reduction in the activities of the antioxidant enzymes.35,102 ROS causes damage to the cell membrane and induces an inflammatory response by activation of NF- κB and increased expression of proinflammatory cytokines (TNF-α and IL-6).123–125

In apoptosis, MTX increased Bax and decreased Bcl-2.126 In MTX hepatotoxicity, oxidative stress causes Bax translocation to the outer mitochondrial membrane, which increases mitochondrial permeability and cytochrome c release into the cytosol, activating downstream effector caspases.127 HSC activation is also caused by oxidative stress and ROS.128 HSC activation promotes substantial portal collagen and fibrosis deposition.129 To test whether pretreatment with Juniperus Macrocarpa extract (100 and 200 mg/kg) lowers MTX toxicity, we gave rats MTX after treatment. The G3 and G4 groups had considerably reduced histopathological damage scores (p<0.05) than the MTX-induction group (G2). The fact that the Juniperus Macrocarpa extract-pretreated groups experienced less MTX-induced liver damage suggests their ability to maintain a respectable level of hepatic integrity. Additionally, there was less inflammatory cell infiltration and fibrosis than the MTX-treated group with the median hepatic damage score for both G3 and G4 significantly less than the MTX-induction group G2. The histopathological data presented in this section are consistent with earlier studies that showed elevated activity of antioxidant enzymes and a decrease in serum levels of liver enzymes. However, it was insignificantly different between G3 and G4 where the animals were given low and high doses of Juniperus Macrocarpa extract despite the lower mean value of the hepatic damage score in G3 which reflects low sample size. The found hepatoprotective activity may be attributed to their antioxidant potential against reactive oxygen and nitrogen species, which inhibit lipid peroxidation and liver cell necrosis or apoptosis. The phenol-rich ethyl acetate fraction of the ethanolic extract of J. communis leaves showed a strong hepatoprotective effect against paracetamol-induced hepatotoxicity in rats without cytotoxicity, according to prior research.130 The Limitations of the current study could be summarized by a small number of animals used and the lack of estimation of the exact molecular mechanism through which Juniperus Macrocarpa extract exerts its hepatoprotective effect. Estimation of Malondialdehyde (MDA) tissue levels, being the product of lipid peroxidation and the expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA is usually used to predict the pathways of enhancing antioxidant activity while the nuclear factor kappa (NF-kB) mRNA to estimate the anti-inflammatory activity of the extract. Additionally, the expression levels of Bax and caspase-3 as a pro-apoptotic protein and Bcl-2 as an anti-apoptotic mRNA are recommended for further investigation to estimate the exact mechanism by which Juniperus Macrocarpa extract exerts its antiapoptotic effects.

Conclusions

The present study showed that pretreatment with Juniperus Macrocarpa extract can attenuate severe alterations in hepatic biochemical markers and disruptions of its histological structure.

Ethics statement

The ethics council at the College of Medicine/University of Baghdad gave permission for this study. The obligations and standards outlined in the Declaration of Helsinki were rigorously followed when developing this research. On December 3, 2023, a local ethical authority verified the necessary papers and client information to validate the experiment’s techniques (document authorization number UoB.Med.36). All efforts were made to ameliorate the total number of animals used in the testing and their suffering by housing them in separate sterile containers with an expanded wire-meshed ground underneath suitable locations, ensuring a period of twelve hours of light and darkness, and administering anesthetic drugs to alleviate any encountered discomfort or pain.

Author contributions

Shahad Hassan Hadi prepared the final copy of the paper, which encouraged participation in the project’s approach, gave funding, monitored the examination, and funded supplies. Mohammed Qasim Yahya Malallah A. Al-atrakji accomplished the roles of invention, validation, and supervision and provided the executable language of the updated paperwork as well as statistical data calculation and electronic reinforcement.

Data and Software availability

Underlying data

Figshare: Ameliorative impact of Juniperus Macrocarpa extract on methotrexate-induced hepatic damage in rats, https://doi.org/10.6084/m9.figshare.27629925.v2.131

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Reporting guidelines

The article adheres to the ARRIVE guidelines. Figshare: Ameliorative impact of Juniperus Macrocarpa extract on methotrexate -induced hepatic damage in rats (10.6084/m9.figshare.27629925).132

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgment

The authors are grateful to the College of Medicine at the University of Baghdad for providing facilities for the duration of this project. The authors also would like to thank Dr. Adnan Alsawak from the Research Centre for Cancer Research and Medical Genetics for his assistance with the induction step, as well as Assistant Prof. Dr. Dhyaa Abdul Hussein Abood from the College of Veterinary Medicine, University of Baghdad, for interpreting the histopathological section.

References

  • 1.  Allison R, Guraka A, Shawa IT, et al.: Drug induced liver injury–a 2023 update. J. Toxic. Environ. Health, Part B. 2023; 26(8): 442–467. PubMed Abstract | Publisher Full Text
  • 2.  Ke L, Lu C, Shen R, et al.: Knowledge mapping of drug-induced liver injury: a scientometric investigation (2010–2019). Front. Pharmacol. 2020; 11: 842. PubMed Abstract | Publisher Full Text | Free Full Text
  • 3.  Ali KH, Al-Jawad FH, Kadhim HM: The possible hepatoprotective effects of combination of an oral krill oil and silymarin against carbon tetrachloride (Ccl4)-induced liver fibrosis/injury in white albino rats: Histopathological, and biochemical studies. Int. J. Drug Deliv. Technol. 2021; 11(3): 827–833.
  • 4.  Korver S, Bowen J, Pearson K, et al.: The application of cytokeratin-18 as a biomarker for drug-induced liver injury. Arch. Toxicol. 2021; 95: 3435–3448. PubMed Abstract | Publisher Full Text | Free Full Text
  • 5.  Ali KH, Al-Jawad FH, Kadhim HM: The possible hepatoprotecive effects of “krill oil and silymarin against carbon tetrachloride (CCL4)-induced rats model of liver fibrosis: in vivo study”. Res. J. Pharm. Technol. 2021; 14(11): 5953–5958.
  • 6.  Abu-Raghif AR, Sahib AS, Hasan SA: Hepatoprotective effects of thyme extract in Cisplatin-induced liver toxicity in rabbits. Pharm. Lett. 2016; 8(18): 22–26.
  • 7.  Bechmann L, Manka P, Best J, et al.: Drug-induced liver injury as predominant cause of acute liver failure in a monocenter study. Dtsch. Med. Wochenschr. (1946). 2014; 139(17): 878–882.
  • 8.  Hassan MF, Kadhim HM, Jawad E: Effects of Emodin on CCl4 Induced Liver Fibrosis in Mice Model. J. Glob. Pharma Technol. 2020; 12(2): 745–760.
  • 9.  Attarbashee RK, Hamodat HF, Mammdoh JK, et al.: The Possible effect of Bosentan on the methotrexate-induced salivary gland changes in male rats: histological and Immunohistochemical study. Toxicol. Res. 2025; 14(1). PubMed Abstract | Publisher Full Text | Free Full Text
  • 10.  Elgemeie GH, Mohamed-Ezzat RA: Chapter 2 - Antifolate-based anticancer drugs.Elgemeie GH, Mohamed-Ezzat RA, editors. New Strategies Targeting Cancer Metabolism. Elsevier; 2022; pp. 35–67.
  • 11.  Inose R, Hashimoto N, Hosomi K, et al.: Association between malignancy and methotrexate and biological disease-modifying antirheumatic drugs in patients with rheumatoid arthritis. Int. J. Clin. Pharmacol. Ther. 2020 Mar; 58(3): 131–138. Epub 2019/12/20. eng. PubMed Abstract | Publisher Full Text
  • 12.  Ridha-Salman H, Shihab EM, Hasan HK, et al.: Mitigative Effects of Topical Norfloxacin on an Imiquimod-Induced Murine Model of Psoriasis. ACS Pharmacol. Transl. Sci. 2024 2024/08/02; 7(9): 2739–2754. Publisher Full Text
  • 13.  Elango T, Dayalan H, Gnanaraj P, et al.: Impact of methotrexate on oxidative stress and apoptosis markers in psoriatic patients. Clin. Exp. Med. 2014; 14: 431–437. PubMed Abstract | Publisher Full Text
  • 14.  Abbas AH, Abbas ZH, Ridha-Salman H, et al.: The attenuated effects of Topical Empagliflozin on Imiquimod-induced Model of Psoriasis in Mice. J. Trop. Life Sci. 2024; 14(3): 459–468. Publisher Full Text
  • 15.  Koźmiński P, Halik PK, Chesori R, et al.: Overview of Dual-Acting Drug Methotrexate in Different Neurological Diseases, Autoimmune Pathologies and Cancers. Int. J. Mol. Sci. 2020 May 14; 21(10). Epub 2020/05/20. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 16.  Kızıl HE, Caglayan C, Darendelioğlu E, et al.: Morin ameliorates methotrexate-induced hepatotoxicity via targeting Nrf2/HO-1 and Bax/Bcl2/Caspase-3 signaling pathways. Mol. Biol. Rep. 2023 Apr; 50(4): 3479–3488. Epub 2023/02/14. eng. PubMed Abstract | Publisher Full Text
  • 17.  Howard SC, McCormick J, Pui CH, et al.: Preventing and Managing Toxicities of High-Dose Methotrexate. Oncologist. 2016 Dec; 21(12): 1471–1482. Epub 2016/08/09. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 18.  Dogra A, Gupta D, Bag S, et al.: Glabridin ameliorates methotrexate-induced liver injury via attenuation of oxidative stress, inflammation, and apoptosis. Life Sci. 2021; 278: 119583. PubMed Abstract | Publisher Full Text
  • 19.  Schmidt S, Messner CJ, Gaiser C, et al.: Methotrexate-induced liver injury is associated with oxidative stress, impaired mitochondrial respiration, and endoplasmic reticulum stress in vitro. Int. J. Mol. Sci. 2022; 23(23): 15116. PubMed Abstract | Publisher Full Text | Free Full Text
  • 20.  Fadel MA, Abdullah MA, Al-Mahmood SS, et al.: Protective effect of propolis on liver and kidney injury caused by methotrexate in chicks. Iraqi J. Vet. Sci. 2022; 36(4): 1061–1067. Publisher Full Text
  • 21.  Hoshyar R, Sebzari A, Balforoush M, et al.: The impact of Crocus sativus stigma against methotrexate-induced liver toxicity in rats. J. Complement. Integr. Med. 2020; 17(2): 20190201. PubMed Abstract | Publisher Full Text
  • 22.  Jihad A, Attawi JAA, Hussein UA-R, et al.: Investigation of adsorption of Fluorouracil as anticancer drug on C82, Si82, Ti-C82 and Ti-Si82 nanocages. Inorg. Chem. Commun. 2023 2023/09/01/; 155: 111115. Publisher Full Text
  • 23.  Raheem AK, Abu-Raghif AR, Abd-alakhwa SZ: Irbesartan Attenuates Sepsis-Induced Renal Injury In Mice Models. J. Pharm. Negat. Results. 2022: 662–669.
  • 24.  Manna MJ, Abu-Raghif A, Al-Saree O, et al.: The value of doxycycline in acetic acid induce ulcerative colitis in rats. Int. J. Pharm. Sci. Res. 2018; 9(8): 3567–3572.
  • 25.  Ali KA, Abu-Raghif AR, Ridha-Salman H: Evaluation of common topical therapeutic agents of plane warts. Arch. Dermatol. Res. 2025; 317(1): 246. PubMed Abstract | Publisher Full Text
  • 26.  Jasim SA, Al-Lami MS, Ameer AJ, et al.: Nanostructures of boron nitride: A promising nanocarrier for anti-cancer drug delivery. Micro Nanostructures. 2024; 185: 207708. Publisher Full Text
  • 27.  Fayed AM, Abdelzaher MA, Hassoni Mahdi N, et al.: Effect of ginger, chamomile, and green tea extracts on prostate cancer cells. J. Genet. Eng. Biotechnol. 2024; 22(3): 100395. PubMed Abstract | Publisher Full Text | Free Full Text
  • 28.  Khaleel BJ, Ridha-Salman H, Kadhim HM, et al.: Anti-angiogenic and anti-oxidant effects of 2-NTI indole derivative vs. suramin in ex vivo, in vivo, and in vitro studies. Cytotechnology. 2025; 77(1): 38. PubMed Abstract | Publisher Full Text | Free Full Text
  • 29.  Almohmadi NH, Aldhalmi AK, Zahran M, et al.: Hepatoprotective efficacy of Lagenaria siceraria seeds oil against experimentally carbon tetrachloride-induced toxicity. Open. Vet. J. 2024; 14(8): 2016–2028. PubMed Abstract | Publisher Full Text | Free Full Text
  • 30.  Oubaid EN, Abu-Raghif AR, Al-Sudani IM: Phytochemical Screening and Antioxidant Activity of Uncaria tomentosa Extract: In Vitro: and: In Vivo: Studies. Med. J. Babylon. 2023; 20(1): 136–142. Publisher Full Text
  • 31.  Khaleq MAA, Abu-Raghif AR, Kadhim SR: Antibacterial activity of aloe vera essential oil against skin infection with Staphylococcus aureus: in vitro and in vivo studies. Int. J. Pharm. Sci. Rev. Res. 2015; 33(2): 192–197.
  • 32.  Abu-Raghif AR, Sahib HB, Abbas SN: Anti-hyperlipidemic effect of Vitex agnus castus Extracts in Mice.
  • 33.  Aykaç A, Becer E, Özbeyli D, et al.: Protective effects of Origanum onites essential oil in the methotrexate-induced rat model: role on apoptosis and hepatoxicity. Rec. Nat. Prod. 2020; 14(6): 395–404. Publisher Full Text
  • 34.  Al-Azem DA, Al-Derawi KH, Malik Al-Saadi SA: The protective effects of syzygium aromaticum essential oil extract against methotrexate induced hepatic and renal toxicity in rats. J. Pure Appl. Microbiol. 2019; 13(1): 505–515. Publisher Full Text
  • 35.  Soliman MM, Aldhahrani A, Alkhedaide A, et al.: The ameliorative impacts of Moringa oleifera leaf extract against oxidative stress and methotrexate-induced hepato-renal dysfunction. Biomed. Pharmacother. 2020; 128: 110259. PubMed Abstract | Publisher Full Text
  • 36.  Gök HN, Orhan N, Özüpek B, et al.: Standardization of Juniperus macrocarpa Sibt. & Sm. and Juniperus excelsa M. Bieb. extracts with carbohydrate digestive enzyme inhibitory and antioxidant activities. Iran. J. Pharm. Res. 2021; 20(3): 441–455. PubMed Abstract | Publisher Full Text
  • 37.  Orhan N, Aslan M, Demirci B, et al.: A bioactivity guided study on the antidiabetic activity of Juniperus oxycedrus subsp. oxycedrus L. leaves. J. Ethnopharmacol. 2012 2012/03/27/; 140(2): 409–415. PubMed Abstract | Publisher Full Text
  • 38.  Marino A, Bellinghieri V, Nostro A, et al.: In vitro effect of branch extracts of Juniperus species from Turkey on Staphylococcus aureus biofilm. FEMS Immunol. Med. Microbiol. 2010; 59(3): 470–476. PubMed Abstract | Publisher Full Text
  • 39.  Lesjak MM, Beara IN, Orčić DZ, et al.: Phytochemical composition and antioxidant, anti-inflammatory and antimicrobial activities of Juniperus macrocarpa Sibth. et Sm. J. Funct. Foods. 2014 2014/03/01/; 7: 257–268. Publisher Full Text
  • 40.  Tiranakwit T, Puangpun W, Tamprasit K, et al.: Phytochemical Screening on Phenolic, Flavonoid Contents, and Antioxidant Activities of Six Indigenous Plants Used in Traditional Thai Medicine. Int. J. Mol. Sci. 2023 Aug 30; 24(17). Epub 2023/09/09. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 41.  Meringolo L, Bonesi M, Sicari V, et al.: Essential Oils and Extracts of Juniperus macrocarpa Sm. and Juniperus oxycedrus L.: Comparative Phytochemical Composition and Anti-Proliferative and Antioxidant Activities. Plants. 2022; 11(8): 1025. PubMed Abstract | Publisher Full Text | Free Full Text
  • 42.  Yanaşoğlu E, Büyükavcı M, Çetinkaya A, et al.: Silibinin effect on methotrexate-induced hepatotoxicity in rats. Eurasian J. Med. 2022; 54(3): 264–269. PubMed Abstract | Publisher Full Text | Free Full Text
  • 43.  Abid R, Mahmood R: Acute and sub-acute oral toxicity of ethanol extract of Cassia fistula fruit in male rats. Avicenna J. Phytomed. 2019; 9(2): 117–125. PubMed Abstract
  • 44.  Singh H, Prakash A, Kalia A, et al.: Synergistic hepatoprotective potential of ethanolic extract of Solanum xanthocarpum and Juniperus communis against paracetamol and azithromycin induced liver injury in rats. J. Tradit. Complement. Med. 2016; 6(4): 370–376. PubMed Abstract | Publisher Full Text | Free Full Text
  • 45.  Pierozan P, Jernerén F, Ransome Y, et al.: The Choice of Euthanasia Method Affects Metabolic Serum Biomarkers. Basic Clin. Pharmacol. Toxicol. 2017 Aug; 121(2): 113–118. Epub 2017/03/01. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 46.  Van Pelt L: Ketamine and xylazine for surgical anesthesia in rats. J. Am. Vet. Med. Assoc. 1977; 171(9): 842–844. PubMed Abstract
  • 47.  Manna MJ, Abu-raghif A, Muhsin HY: The effect of Niclosamide in acetic acid induce colitis: an experimental study. Prensa méd argent. 2019; 309–316.
  • 48.  Swayeh N, Kadhim H: Effects of methanol extract of Corchorus olitorius cultivated in Iraq on high fat diet plus streptozotocin-induced type ii diabetes in rats. Int. J. Drug Deliv. Technol. 2022; 12(2): 754–759. Publisher Full Text
  • 49.  Atarbashe RK, Abu-Raghif A: The therapeutic effects of ambrisentan on experimentally induced colitis in a male rat’s models. Ann. Trop. Med. Public Health. 2020; 23(4): 90–99. Publisher Full Text
  • 50.  Fareed NY, Kassab HJ: A comparative study of oral diacerein and transdermal diacerein as Novasomal gel in a model of MIA induced Osteoarthritis in rats. Pharmacia. 2023; 70(4): 1363–1371. Publisher Full Text
  • 51.  Oubaid EN, Abu-Raghif A, Al-Sudani IM: Ibudilast ameliorates experimentally induced colitis in rats via down-regulation of proinflammatory cytokines and myeloperoxidase enzyme activity. Pharmacia. 2023; 70(1): 187–195. Publisher Full Text
  • 52.  Kadhim H, Gatea F, Raghif AA, et al.: Role of Topical Ritodrine Hydrochloride in Experimentally Induced Hypertrophic Scar in Rabbits. Iraqi J. Pharm. Sci. 2022; 31(2): 260–70. (P-ISSN 1683-3597 E-ISSN 2521-3512).
  • 53.  Hsu CY, Al-Yasiri SAM, Shather AH, et al.: The capability of pure and modified boron carbide nanosheet as a nanocarrier for dacarbazine anticancer drug delivery: DFT study. Pramana J. Phys. 2024; 98(2). Publisher Full Text
  • 54.  Al-Kenany SA, Al-Shawi NN: Protective effect of cafestol against doxorubicin-induced cardiotoxicity in rats by activating the Nrf2 pathway. Front. Pharmacol. 2023; 14: 1206782. PubMed Abstract | Publisher Full Text | Free Full Text
  • 55.  Kamal S, Khadhim H: Effects of Irbesartan in induced Parkinson’s disease in mice. Int. J. Pharmaceut. Qual. Assur. 2021; 12(1): 31–39.
  • 56.  Hassan Z, Hassan T, Abu-Rghif A: Evaluation the Effectiveness of Phenolic Compound of Salvia frigida on Induced Atopic Dermatitis in Experimental Mice. Iraqi J. Pharm. Sci. 2022; 31(1): 154–66. (P-ISSN 1683-3597 E-ISSN 2521-3512).
  • 57.  Shafiq SA, Al-Joofy AK: Histopathological and enzymatic study on the effect of Aspergillus fumigatus in mice. J. Fac. Med. Baghdad. 2010; 52(4). Publisher Full Text
  • 58.  Yahiya YI, Hadi NR, Abu Raghif A, et al.: Role of Iberin as an anti-apoptotic agent on renal ischemia-reperfusion injury in rats. J. Med. Life. 2023 Jun; 16(6): 915–919. Epub 2023/09/07. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 59.  Raheem AKK, Abu-Raghif AR, Zigam QA: Cilostazol Protects Against Sepsis-Induced Kidney Impairment in a Mice Model. J. Med. Chem. Sci. 2023; 6(5): 1193–1203.
  • 60.  Khorsheed SM, Abu-Raghif A, Ridha-Salman H: Alleviative Effects of Combined Topical Melatonin and Rutin on Imiquimod-Induced Psoriasis Mouse Model. Pharmacia. 2024; 71: 1–13. Publisher Full Text
  • 61.  Kadhim HM, Al-Mosawi AM: Effects of Emodin and Salvianolic Acid on Carbon Tetrachloride (CCl4)-induced Lung Fibrosis in Mice Model. Int. J. Drug Deliv. Technol. 2021; 11(4): 1269–1274.
  • 62.  Salman HR, Alzubaidy AA, Abbas AH, et al.: Attenuated effects of topical vinpocetine in an imiquimod-induced mouse model of psoriasis. J. Taibah. Univ. Med. Sci. 2024 2024/02/01/; 19(1): 35–53. PubMed Abstract | Publisher Full Text | Free Full Text
  • 63.  Kadhim HM: Antiinflmmatory and antihyperlipidemic effect of adjuvant cinnamon in type 2 diabetic patients. Int. J. Pharm. Sci. Rev. Res. 2016; 41: 88–98.
  • 64.  Al-Humadi FW, Qassim HW, Hameed AM, et al.: Comparative assessment of the effects of two intrauterine systems for long-term contraception on some haematological, biochemical, and immunological markers. Review of Clinical Pharmacology and Pharmacokinetics, International Edition. 2024; 38: 63–67. Publisher Full Text
  • 65.  Aldhalmi AK, Al-Athari AJH: The Association between Alfacalcidol (1-Alphahydroxyvitamin D3) and Oxidative Stress in Patients with Type II Diabetic Nephropathy. Sys. Rev. Pharm. 2020; 11(11): 918–927.
  • 66.  Mohammed MT: Biochemical studies on the effect of Crataegus aqueous extract on oxidative stress during ischemia/reperfusion induced myocardial injuries. J. Fac. Med. Baghdad. 2015; 57(3): 248–253. Publisher Full Text
  • 67.  Shareef BQ, Al Qadhi HI, Shayma’a AJ: Antioxidant Effects of Selenium Nanoparticles Prepared from Eruca Sativa Extract on Ketoconazole–Induced Testicular Oxidative Damage in Male Rats. J. Fac. Med. Baghdad. 2024; 66(1): 58–66. Publisher Full Text
  • 68.  Khafaji AWM, Al-Zubaidy AAK, Farhood IG, et al.: Ameliorative effects of topical ramelteon on imiquimod-induced psoriasiform inflammation in mice. Naunyn Schmiedeberg’s Arch. Pharmacol. 2024 2024/03/06; 397(8): 6231–6248. PubMed Abstract | Publisher Full Text
  • 69.  Yahiya YI, Hadi NR, Abu Raghif A, et al.: Protective effect of IAXO-102 on renal ischemia-reperfusion injury in rats. J. Med. Life. 2023 Apr; 16(4): 623–630. Epub 2023/06/12. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 70.  Sharquie KE, Turki KM, Abu-Raghif AR, et al.: Oxidative stress in patients with premature hair grayness. Saudi Med. J. 2005; 26(8): 1310–1311. PubMed Abstract
  • 71.  Abu-Raghif AR, Qasim BJ, Abady AH, et al.: Effects of aqueous thyme extract against cisplatin induced nephrotoxicity in rabbits. Int. J. Pharm. Sci. Rev. Res. 2015; 30(1): 190–194.
  • 72.  Saghir SAM, Al Hroob AM, Al-Tarawni AH, et al.: Effect of Lactiplantibacillus plantarum on the growth, hemato-biochemical, inflammation, apoptosis, oxidative stress markers, involved gens and histopathological alterations in growing rabbits challenged with aflatoxin B1. Poult. Sci. 2024; 103(9): 104002. PubMed Abstract | Publisher Full Text | Free Full Text
  • 73.  Jaafer H, Al-Kinani KK: Preparation of Idebenone as a Thermosetting Nasal Gel for Better Bioavailability and Histopathological Effect. J. Fac. Med. Baghdad. 2023; 65(3): 234–240. Publisher Full Text
  • 74.  Hassan RF, Kadhim HM: Comparative Effects of Phenolic Extract As an Ointment Dosage Form in Inducing Wound Healing in Mice and β-sitosterol in Experimentally Induced Acute Wound Healing in Mice. J. Pharm. Negat. Results. 2022; 13(3): 194–203.
  • 75.  Abed-Mansoor A, Abu-Raghif AR: Attenuated effects of rivastigmine in induced cytokine storm in mice. Journal of Emergency Medicine, Trauma & Acute Care. 2022; 2022(3): 12. Publisher Full Text
  • 76.  Al-Sabawy HB, Rahawy AM, Al-Mahmood SS: Standard techniques for formalin-fixed paraffin-embedded tissue: a pathologist’s perspective.2021.
  • 77.  Ridha-Salman H, Al-Zubaidy AA, Abbas AH, et al.: The alleviative effects of canagliflozin on imiquimod-induced mouse model of psoriasis-like inflammation. Naunyn Schmiedeberg’s Arch. Pharmacol. 2024 2024/09/10; 398: 1–21. Publisher Full Text
  • 78.  Hassan RF, Kadhim HM: Exploring the role of phenolic extract as an ointment dosage form in inducing wound healing in mice. J. Pharm. Negat. Results. 2022; 13(3): 186–193.
  • 79.  Ghazy DN, Abu-Raghif AR: Effects of Apremilast on Induced Hypertrophic Scar of Rabbits. Arch. Razi Inst. 2021 Dec; 76(6): 1803–1813. Epub 2022/05/14. eng. PubMed Abstract | Free Full Text
  • 80.  Hassan ZY, Hassan TY, AbuRaghif AR: Evaluation the Effect of Phytosterol Fraction of Chenopodium Muralein Comparison with Tacrolimus on Mice Induced Atopic Dermatitis. Iraqi J. Pharm. Sci. 2023; 32(1): 84–91. Publisher Full Text
  • 81.  Shihab EM, Kadhim HM: The Impact of Carvedilol on Organ Index, Inflammatory Mediators, Oxidative Stress Parameters and Skin Markers in D-Galactose-Induced Aging Mice. Int. J. Drug Deliv. Technol. 2023; 13(3): 1017–1023. Publisher Full Text
  • 82.  Mekkey SM, Abu Raghif AR, Alkafaji HA-R, et al.: The anti-Parkinson effects of Liraglutide in rat model of Rotenone induced Parkinsonism. International Journal of Pharmaceutical Research. 2020; 12(2): 3695–706. (09752366).
  • 83.  Pietras ES: Chronic hepatitis: an update on terminology and reporting. Radiology. 1996; 200(3): 784. Publisher Full Text
  • 84.  Al-Bairmani RJ, Kadhim HM: Evaluation of Anti-aging Effects of Gemfibrozil on D-galactose induced Aging Mouse Model. Int. J. Drug Deliv. Technol. 2023; 13(3): 1011–1016. Publisher Full Text
  • 85.  Thwaini MH, Abu Ragif AR, Hadi NR: Effects of sulforaphane in brain ischemic reperfusion injury in rats. Int. J. Pharm. Res. 2020; 12(4): 3687–3694.
  • 86.  Aldhalmi AK, Sahib HB, Hassan OM, et al.: Anti-Angiogenic and Anti-Proliferative Activity of 4-2-(5-bromo-1H-indol-2-carbonyl)-N-(4-methoxyphenyl) Hydrazine-1-carbothioamide: Ex-vivo and in vitro Study. Asian Pac. J. Cancer Prev. 2024 Jul 1; 25(7): 2509–2513. Epub 2024/07/28. eng. PubMed Abstract | Publisher Full Text | Free Full Text
  • 87.  Nundy S, Kakar A, Bhutta ZA, et al.: Understanding Medical Biostatistics. How to Practice Academic Medicine and Publish from Developing Countries? A Practical Guide.2022; 95–116.
  • 88.  Hasani Z, Hassan AF: Evaluation of the protective effect of Omega-7 against Methotrexate Genotoxicity in bone marrow Cells of Mice. J. Fac. Med. Baghdad. 2023; 65(4). Publisher Full Text
  • 89.  Ali AH, Ali SM: A short-term comparison between the effect of two different doses of methotrexate on ovarian tissue and function in female albino rats. J. Fac. Med. Baghdad. 2022; 64(4). Publisher Full Text
  • 90.  Koźmiński P, Halik PK, Chesori R, et al.: Overview of dual-acting drug methotrexate in different neurological diseases, autoimmune pathologies and cancers. Int. J. Mol. Sci. 2020; 21(10): 3483. PubMed Abstract | Publisher Full Text | Free Full Text
  • 91.  Wahed M, Louis-Auguste J, Baxter L, et al.: Efficacy of methotrexate in Crohn’s disease and ulcerative colitis patients unresponsive or intolerant to azathioprine/mercaptopurine. Aliment. Pharmacol. Ther. 2009; 30(6): 614–620. Publisher Full Text
  • 92.  Cecchino GN, Araujo Júnior E, Elito Júnior J: Methotrexate for ectopic pregnancy: when and how. Arch. Gynecol. Obstet. 2014; 290: 417–423. Publisher Full Text
  • 93.  Nielsen OH, Steenholdt C, Juhl CB, et al.: Efficacy and safety of methotrexate in the management of inflammatory bowel disease: a systematic review and meta-analysis of randomized, controlled trials. EClinicalMedicine. 2020; 20: 100271. Publisher Full Text
  • 94.  Dawood JO, Abu-Raghif A: Labetalol Ameliorates Experimental Colitis in Rat Possibly Through its Effect on Proinflammatory Mediators and Oxidative Stress. Clin. Lab. 2024; 70(2): 353–362.
  • 95.  Spies C, Burmester G, Buttgereit F: Methotrexate treatment in large vessel vasculitis and polymyalgia rheumatica. Clin. Exp. Rheumatol. 2010; 28(5 Suppl 61): S172–S177. PubMed Abstract
  • 96.  Salman HR, Al-Zubaidy AA, Abbas AH, et al.: The ameliorative effects of topical gemifloxacin alone or in combination with clobetasol propionate on imiquimod-induced model of psoriasis in mice. Naunyn Schmiedeberg’s Arch. Pharmacol. 2024 2024/01/01; 397(1): 599–616. PubMed Abstract | Publisher Full Text
  • 97.  Manna MJ, Abu-Raghif A, Alsaraf KM: Therapeutic effect of sildenafil in experimental colitis through anti-oxidative stress and inhibition of adhesion molecules. J. Pharm. Sci. Res. 2017; 9(9): 1615–1623.
  • 98.  Hamed KM, Dighriri IM, Baomar AF, et al.: Overview of methotrexate toxicity: a comprehensive literature review. Cureus. 2022; 14(9). Publisher Full Text
  • 99.  Marin G-E, Neag M-A, Burlacu C-C, et al.: The Protective Effects of Nutraceutical Components in Methotrexate-Induced Toxicity Models—An Overview. Microorganisms. 2022; 10(10): 2053. PubMed Abstract | Publisher Full Text | Free Full Text
  • 100.  Mitra V, Metcalf J: Metabolic functions of the liver. Anaesthesia & Intensive Care Medicine. 2012; 13(2): 54–55. Publisher Full Text
  • 101.  Abdel-Wahab BA, Ali FE, Alkahtani SA, et al.: Hepatoprotective effect of rebamipide against methotrexate-induced hepatic intoxication: role of Nrf2/GSK-3β, NF-κβ-p65/JAK1/STAT3, and PUMA/Bax/Bcl-2 signaling pathways. Immunopharmacol. Immunotoxicol. 2020; 42(5): 493–503. PubMed Abstract | Publisher Full Text
  • 102.  Abd El-Ghafar OA, Hassanein EH, Ali FE, et al.: Hepatoprotective effect of acetovanillone against methotrexate hepatotoxicity: Role of Keap-1/Nrf2/ARE, IL6/STAT-3, and NF-κB/AP-1 signaling pathways. Phytother. Res. 2022; 36(1): 488–505. PubMed Abstract | Publisher Full Text
  • 103.  Kang T-C: Nuclear factor-erythroid 2-related factor 2 (Nrf2) and mitochondrial dynamics/mitophagy in neurological diseases. Antioxidants. 2020; 9(7): 617. PubMed Abstract | Publisher Full Text | Free Full Text
  • 104.  He F, Ru X, Wen T: NRF2, a transcription factor for stress response and beyond. Int. J. Mol. Sci. 2020; 21(13): 4777. PubMed Abstract | Publisher Full Text | Free Full Text
  • 105.  Shen N, Wang T, Gan Q, et al.: Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. Food Chem. 2022; 383: 132531. PubMed Abstract | Publisher Full Text
  • 106.  Xu W, Lu H, Yuan Y, et al.: The antioxidant and anti-inflammatory effects of flavonoids from propolis via Nrf2 and NF-κB pathways. Foods. 2022; 11(16): 2439. PubMed Abstract | Publisher Full Text | Free Full Text
  • 107.  Suraweera TL, Rupasinghe HV, Dellaire G, et al.: Regulation of Nrf2/ARE pathway by dietary flavonoids: a friend or foe for cancer management? Antioxidants. 2020; 9(10): 973. PubMed Abstract | Publisher Full Text | Free Full Text
  • 108.  Dar A, Fehaid A, Alkhatani S, et al.: The protective role of luteolin against the methotrexate-induced hepato-renal toxicity via its antioxidative, anti-inflammatory, and anti-apoptotic effects in rats. Hum. Exp. Toxicol. 2021; 40(7): 1194–1207. PubMed Abstract | Publisher Full Text
  • 109.  Safaei F, Mehrzadi S, Khadem Haghighian H, et al.: Protective effects of gallic acid against methotrexate-induced toxicity in rats. Acta Chir. Belg. 2018; 118(3): 152–160. PubMed Abstract | Publisher Full Text
  • 110.  Abd El-Hack ME, Alabdali AYM, Aldhalmi AK, et al.: Impacts of Purslane (Portulaca oleracea) extract supplementation on growing Japanese quails’ growth, carcass traits, blood indices, nutrients digestibility and gut microbiota. Poult. Sci. 2022; 101(11): 102166. PubMed Abstract | Publisher Full Text | Free Full Text
  • 111.  Santhakumar P, Roy A, Mohanraj KG, et al.: Ethanolic extract of capparis decidua fruit ameliorates methotrexate-induced hepatotoxicity by activating Nrf2/HO-1 and PPARγ mediated pathways. Ind. J. Pharm. Educ. 2021; 55(1s): s265–s274. Publisher Full Text
  • 112.  Matata BM, Galiñanes M: Peroxynitrite is an essential component of cytokines production mechanism in human monocytes through modulation of nuclear factor-κB DNA binding activity. J. Biol. Chem. 2002; 277(3): 2330–2335. PubMed Abstract | Publisher Full Text
  • 113.  Iqbal N, Zubair HM, Almutairi MH, et al.: Hepatoprotective effect of Cordia rothii extract against CCl4-induced oxidative stress via Nrf2–NFκB pathways. Biomed. Pharmacother. 2022; 156: 113840. PubMed Abstract | Publisher Full Text
  • 114.  Shen X-L, Guo Y-N, Lu M-H, et al.: Acetaminophen-induced hepatotoxicity predominantly via inhibiting Nrf2 antioxidative pathway and activating TLR4-NF-κB-MAPK inflammatory response in mice. Ecotoxicol. Environ. Saf. 2023; 252: 114590. PubMed Abstract | Publisher Full Text
  • 115.  Mahmoud AM, Hozayen WG, Ramadan SM: Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARγ, and suppressing oxidative stress and apoptosis in rats. Biomed. Pharmacother. 2017; 94: 280–291. PubMed Abstract | Publisher Full Text
  • 116.  Moghadam AR, Tutunchi S, Namvaran-Abbas-Abad A, et al.: Pre-administration of turmeric prevents methotrexate-induced liver toxicity and oxidative stress. BMC Complement. Altern. Med. 2015; 15: 1–13.
  • 117.  Tag HM: Hepatoprotective effect of mulberry (Morus nigra) leaves extract against methotrexate induced hepatotoxicity in male albino rat. BMC Complement. Altern. Med. 2015; 15: 1–9. Publisher Full Text
  • 118.  Goudarzi M, Kalantar M, Sadeghi E, et al.: Protective effects of apigenin on altered lipid peroxidation, inflammation, and antioxidant factors in methotrexate-induced hepatotoxicity. Naunyn Schmiedeberg’s Arch. Pharmacol. 2021; 394: 523–531. PubMed Abstract | Publisher Full Text
  • 119.  Azadnasab R, Kalantar H, Khorsandi L, et al.: Epicatechin ameliorative effects on methotrexate-induced hepatotoxicity in mice. Hum. Exp. Toxicol. 2021; 40(12_suppl): S603–S610. PubMed Abstract | Publisher Full Text
  • 120.  Kamel WH, Ali MF, Afifi SH: Histopathological and Biochemical Studies of Methotrexate Hepatotoxicity on Albino Rats. Assiut Vet. Med. J. 2023; 69(179): 60–68.
  • 121.  Abdel-kawy SH, Mohamed DH, Lotfy M, et al.: Possible Hepatoprotective Role of Berberine versus Silymarin on Methotrexate Toxicity: Histological and Biochemical Study. Egypt. J. Histol. 2023; 46(1): 435–447.
  • 122.  Khalifa MM, Bakr AG, Osman AT: Protective effects of phloridzin against methotrexate-induced liver toxicity in rats. Biomed. Pharmacother. 2017; 95: 529–535. PubMed Abstract | Publisher Full Text
  • 123.  Al-khawalde AA-mA, Abukhalil MH, Jghef MM, et al.: Punicalagin protects against the development of methotrexate-induced hepatotoxicity in mice via activating Nrf2 signaling and decreasing oxidative stress, inflammation, and cell death. Int. J. Mol. Sci. 2022; 23(20): 12334. PubMed Abstract | Publisher Full Text | Free Full Text
  • 124.  Ridha NM, Taher MA: Oxidative stress and some liver functions parameters in patients with symptomatic Cholelithiasis. J. Fac. Med. Baghdad. 2013; 55(1): 73–76. Publisher Full Text
  • 125.  Aldhalmi AK, Al-Athari AJH, Al-Hindy HAAM: Association of Tumor Necrosis Factor-α and Myeloperoxidase enzyme with Severe Asthma: A comparative study. Rep. Biochem. Mol. Biol. 2022; 11(2): 238–245. PubMed Abstract | Publisher Full Text
  • 126.  Mukherjee S, Ghosh S, Choudhury S, et al.: Pomegranate reverses methotrexate-induced oxidative stress and apoptosis in hepatocytes by modulating Nrf2-NF-κB pathways. J. Nutr. Biochem. 2013; 24(12): 2040–2050. PubMed Abstract | Publisher Full Text
  • 127.  Abo-Haded HM, Elkablawy MA, Al-Johani Z, et al.: Hepatoprotective effect of sitagliptin against methotrexate induced liver toxicity. PLoS One. 2017; 12(3): e0174295. PubMed Abstract | Publisher Full Text | Free Full Text
  • 128.  Ghatak S, Biswas A, Dhali GK, et al.: Oxidative stress and hepatic stellate cell activation are key events in arsenic induced liver fibrosis in mice. Toxicol. Appl. Pharmacol. 2011; 251(1): 59–69. PubMed Abstract | Publisher Full Text | Free Full Text
  • 129.  Duval F, Moreno-Cuevas JE, González-Garza MT, et al.: Liver fibrosis and protection mechanisms action of medicinal plants targeting apoptosis of hepatocytes and hepatic stellate cells. Adv. Pharmacol. Pharm. Sci. 2014; 2014(1): 373295.
  • 130.  Ved A, Gupta A, Rawat AKS: Antioxidant and hepatoprotective potential of phenol-rich fraction of Juniperus communis Linn. leaves. Pharmacogn. Mag. 2017; 13(49): 108–113. PubMed Abstract | Publisher Full Text
  • 131.  Hassan Hadi S, Mohammed QYMAA-A: Ameliorative impact of Juniperus Macrocarpa extract on methotrexate-induced hepatic damage in rats. Dataset. figshare. 2024. Publisher Full Text
  • 132.  Hassan Hadi S, Mohammed QYMAA-A: Ameliorative impact of Juniperus Macrocarpa extract on methotrexate-induced hepatic damage in rats. Dataset. figshare. 2024. Publisher Full Text

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Hadi SH and Al-Atrakji MQYMA. The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:131 (https://doi.org/10.12688/f1000research.158839.1)
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
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Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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Reviewer Report 10 Mar 2025
Yaarub Sadiq, Pharmacology and pharmaceutical sciences, University of Sharjah, Sharjah, Sharjah, United Arab Emirates 
Approved
VIEWS 5
https://doi.org/10.5256/f1000research.174485.r368333
Congratulations on the outstanding effort accomplished by the author team. My main concern I want to highlight is that the authors mentioned in the abstract that Group 2 (MTX) got an intraperitoneal single dose of MTX (20 mg/kg) for two weeks. ... Continue reading
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Sadiq Y. Reviewer Report For: The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:131 (https://doi.org/10.5256/f1000research.174485.r368333)
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https://f1000research.com/articles/14-131/v1#referee-response-368333
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 08 Mar 2025
Hayder Ridha-Salman, Al-Mustaqbal University, College of Pharmacy, Babylon, Hilla, 51001, Iraq 
Approved with Reservations
VIEWS 11
https://doi.org/10.5256/f1000research.174485.r368325
1.The authors could enhance their introduction by incorporating more details pertaining to the rationale behind the selection of Juniperus macrocarpa extract as an alternative to other herbal remedies.
2. Please provide details regarding pro-inflammatory and oxidative stress biomarkers. This could ... Continue reading
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Ridha-Salman H. Reviewer Report For: The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:131 (https://doi.org/10.5256/f1000research.174485.r368325)
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https://f1000research.com/articles/14-131/v1#referee-response-368325
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 04 Mar 2025
Ahemed Abu-Raghif, Al-Nahrain University, Baghdad, Iraq;  Pharmacology, Al-Nahrain University, College of Medicine, Baghdad, Baghdad, Iraq 
Approved with Reservations
VIEWS 10
https://doi.org/10.5256/f1000research.174485.r368331
• The species name of the medicinal plant is not written correctly and must adhere to proper scientific nomenclature as follows: Juniperus macrocarpa.
• The Materials and Methods section should start with a brief description of the study, including ... Continue reading
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HOW TO CITE THIS REPORT
Abu-Raghif A. Reviewer Report For: The role of Juniperus Macrocarpa extract as anti-inflammatory and antioxidant on methotrexate-induced acute liver injury in rat model [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2025, 14:131 (https://doi.org/10.5256/f1000research.174485.r368331)
The direct URL for this report is:
https://f1000research.com/articles/14-131/v1#referee-response-368331
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Alongside their report, reviewers assign a status to the article:

Approved
The paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations
A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved
Fundamental flaws in the paper seriously undermine the findings and conclusions

Reviewer Reports

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Version 1
27 Jan 25 		read read read
  1. Ahemed Abu-Raghif, Al-Nahrain University, Baghdad, Iraq; Al-Nahrain University, College of Medicine, Baghdad, Iraq
  2. Hayder Ridha-Salman, Al-Mustaqbal University, College of Pharmacy, Babylon, Iraq
  3. Yaarub Sadiq, University of Sharjah, Sharjah, United Arab Emirates

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    Alongside their report, reviewers assign a status to the article:
    Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
    Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
    Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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