Comparative study on the protective effects of Silybum marianum L. seeds and leaves against nickel chloride induced anemia, liver and kidney injury: an in vivo and in silico approach

Abstract

While nickel is required in trace amounts for human health, excessive exposure can be harmful. It can cause significant damage to the liver, kidneys, and other organs, depending on the route of exposure. The aim of this study is to investigate the protective effect of the Silybum marianum L. seeds and leaves on the liver and kidneys as well as blood parameters. The purpose is to examine whether a one-month dietary supplementation with Silybum marianum L. seeds and leaves could prevent and prepare the body to resist nickel-induced damage. Seed and leaf extracts were administered orally to albino rats for one month. The rats were then exposed intraperitoneally to nickel chloride NiCl₂ for 10 days. Hematological and biochemical parameters as well as biomarkers for oxidative stress were measured in this investigation. Injection of Nickel-chloride showed significant alterations along the period of exposure. Seed extract exerted a marked protective effect on the biochemical, hematological, and oxidative stress parameters. In contrast, the leaf extract showed only partial protection for some parameters, while other parameters showed no significant protection, which highlights the differences in efficacy among plant parts. These results were supported by histological analysis which demonstrated that exposure to NiCl₂ resulted in notable renal and hepatic lesions, such as hydropic degeneration, necrosis, sinusoidal congestion, and tubular damage. Seed extract has completely prevented these alterations, whereas leaf extract has conferred only partial protection. Molecular docking analysis confirms that Silybin predominantly modulates AMPK and that Silydianin preferentially engages SIRT1, whereas Silychristin exhibits a dual-target modulatory activity.

Introduction

“Heavy metals” are metallic elements distinguished by their relatively high density generally above 4 g/cm³ or at least five times greater than that of water. These elements are broadly distributed throughout the Earth’s crust and are often linked to environmental contamination and toxic effects on human health. Human exposure to heavy metals often arises from industrial processes, such as metal casting, oil refining, petrochemical operations, pesticide formulation, and chemical production facilities¹. In addition, heavy metals can enter the body through multiple routes, including consumption of contaminated food and water, inhalation of polluted air, dermal contact, as well as through medical interventions, such as vaccines, injections, and implants². Most heavy metals are xenobiotics with no beneficial role in human physiology. Unable to be metabolized, they tend to accumulate in specific tissues and can be highly toxic even at trace levels, impairing the function of organs such as the central nervous system, hematopoietic system, liver, and kidneys^3,2. Heavy metals like lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As) are considered non-essential, as they serve no beneficial function in biological systems. In contrast, metals such as copper (Cu), iron (Fe), zinc (Zn), cobalt (Co), manganese (Mn), magnesium (Mg), molybdenum (Mo), and trivalent chromium (Cr³⁺) are essential micronutrients required for various metabolic processes⁴. However, any essential or non-essential trace element present in amounts exceeding safe levels can cause physiological or morphological abnormalities, genetic mutations, and other adverse effects, such as impaired growth or developmental defects⁵.

Nickel and its compounds are naturally widespread in the Earth’s crust, ranking as the 24th most abundant element and are continually released into the atmosphere at low levels through natural processes. The toxicity of nickel depends on its bioavailability, which is influenced by its physicochemical properties, as well as the dose, duration, and route of exposure⁶. Nickel and its compounds induce oxidative stress by enhancing the production of reactive oxygen species (ROS) and weakening antioxidant defense mechanisms. ROS generation is a critical factor in nickel toxicity and contributes to its carcinogenic properties. The resulting oxidative imbalance disrupts cellular homeostasis and mediates toxicity, which is closely associated with a significant reduction in major antioxidant enzymes, indicating impairment of the cellular antioxidant system^7,8. This antioxidant system consists of enzymatic components, such as superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST), as well as non-enzymatic components including reduced glutathione (GSH) and vitamins. The inhibition of these defense mechanisms further amplifies oxidative damage, leading to lipid peroxidation and cellular dysfunction⁹. Nickel-induced oxidative stress is not only related to increased ROS production but also to mitochondrial dysfunction, leading to electron leakage and superoxide generation¹⁰. In addition, Ni²⁺ ions can bind to thiol groups and interfere with antioxidant enzymes, thereby reducing their activity¹¹.

In humans, the main exposure pathways are inhalation, ingestion, and skin contact. Diets high in nickel and prolonged occupational exposure can lead to significant accumulation in the body, causing serious chronic health effects¹². The main organs where nickel accumulates include the bones, kidneys, lungs, liver, and heart⁸. Workers in nickel metallurgy and refining industries experience particularly high levels of exposure to nickel. Approximately 2% of employees in these sectors are exposed to airborne nickel particles at concentrations ranging from 0.1 to 1 mg/m³¹³. During nickel electrolysis, workers are commonly exposed to compounds such as nickel chloride ( NiCl₂) and nickel sulfate (NiSO₄), which serve as key electrolytes in the plating and purification of nickel⁶. A fundamental process in living organisms involves the production of reactive oxygen species (ROS), which can cause lipid peroxidation, harm vital biomolecules like proteins and DNA, and ultimately lead to cell damage or death¹. Considering this, Numerous studies have investigated the protective effects of medicinal plants against heavy metal toxicity.

Research is ongoing to discover natural compounds and herbal medicines that can effectively prevent or mitigate the toxic effects of heavy metals on human health. One of the herbal medicines that has been widely used for centuries to protect the liver against toxins is Silybum marianum L. (Milk thistle), valued for its antioxidant and anti-inflammatory effects which are attributed to silymarin, which is a complex of flavonolignans mixture that includes silybin A and B, isosilybin A and B, silychristin A and silydianin with silybin B typically being slightly more abundant¹⁴. Silybum marianum extracts and silymarin have been used for centuries in the treatment of liver diseases, valued for their ability to protect liver cells, promote regeneration, and reduce inflammation caused by toxins, infections, or metabolic disorder^15,16. Silybum marianum is considered to be well tolerated, with no significant side effects reported, supporting their safety profile for potential therapeutic use¹⁷. In the current work, we aimed to investigate whether a one-month dietary supplementation with Silybum marianum seeds and leaves could prevent and prepare the body to resist nickel-induced damage to blood, liver, and kidneys. To the best of our knowledge, a comparative study assessing the effects of Silybum marianum seeds and leaves on nickel-induced blood, liver, and kidney toxicity has not been performed. This was conducted by evaluating biochemical, hematological markers, oxidative stress parameters, and histological examination of liver and kidney organs. Additionally, molecular docking simulations were performed for the silymarin mixture with AMPK and SIRT1 to explore their potential interactions and binding affinities.

Materials and methods

Plant material

The extracts used in this study were obtained from the seeds and leaves of Silybum marianum L., collected in May 2023, in areas where it grows spontaneously on the roadsides in the Rabat–Salé–Kénitra region of Morocco, in the city of Tamesna 34.1728° N, 6.2376° W. The collection was done on public land, where no specific permission was required. The species was identified and authenticated by Professor Karima Selmaoui, Laboratory of Botany, Biotechnology and Plant Protection, Department of Biology. No voucher specimen was deposited in a publicly accessible herbarium. Leaves were gathered during the mid-flowering stage, while seeds were harvested at full maturity. After collection, the plant materials were air-dried at room temperature for 20 days. Once dried, all samples were ground into a fine powder.

Preparation of the plant treatment extracts

The extraction procedure as well as the yield are found in¹⁸.

Animals

Two-month-old Wistar albino rats, weighing 155.62 ± 1.34 g, were sourced from the animal breeding unit of the Faculty of Sciences, Ibn Tofaïl University, Kenitra. The rats are placed in a cage and are kept at 22 ± 1 °C in a controlled environment with a 12-hour light/dark cycle and had free access to food and water during the whole duration of the study. The animals were maintained under appropriate living conditions, and all necessary welfare measures were ensured. Every effort was made to reduce the number of rats used and to minimize their suffering. All animal-related procedures were conducted with prior approval from the Institutional Animal Ethics Committee of the Faculty of Science, Ibn Tofail University - Kenitra, in accordance with the “Guide for the Care and Use of Laboratory Animals.”

Experimental design and treatments

The experimental procedure followed the method of¹⁹. Nickel chloride (NiCl₂) and the extracts were dissolved in saline solution (0.9% NaCl) and 4% dimethyl sulfoxide (DMSO) respectively. At the beginning of the experiment, the animals were randomly divided into three groups, with 10 rats in each group receiving seeds and leaves extracts for one month.

• Group 1 (n = 10): Rats were receiving by oral gavage 100 mg/kg of distilled water containing 4% of dimethyl sulfoxide (DMSO).

• Group 2 (n = 10): Rats were receiving by oral gavage 100 mg/kg seed extract.

• Group 3 (n = 10): Rats were receiving by oral gavage 100 mg/kg of leaf extract.

Afterward, each group was divided into two subgroups (n = 5) and each subgroup received a daily injection of 4 mg/kg per body weight of nickel chloride (NiCl₂) or (0.9% NaCl) for 10 days. At the end of the experimental period, rats were anesthetized with Pentobarbital, administered via intraperitoneal injection at a dose of 200 mg/kg body weight in accordance with the AVMA Guidelines for the Euthanasia of animals (2020)^20,21. Blood sampling was performed by cardiac puncture under deep anesthesia. Death was confirmed by respiratory arrest and absence of cardiac activity before organ collection (liver and kidneys) for subsequent analyses. The tissues were then excised for further analysis (Fig. 1).

Fig. 1.

Schematic representation of the experimental protocol (created with BioRender).

Hematological and biochemical parameters

At the end of the experiment, blood samples were collected by cardiac puncture under anesthesia. Whole blood was separated into EDTA-treated tubes for hematological analysis and plain tubes for serum separation by centrifugation (3000 rpm, 10 min) for biochemical analysis. Hematological parameters include white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), platelet count (PLT), red cell distribution width - standard deviation (RDW-SD), platelet distribution width (PDW), mean platelet volume (MPV), and platelet-large cell ratio (P-LCR). Serum biochemical parameters include (urea, creatinine, total cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

Body weight gain and relative organ weight

Initial body weight was measured on Day 0 prior to any treatment. After 30 days of oral administration (extract or distilled water), body weight was recorded again (Day 30), before the intraperitoneal phase (NaCl or NiCl₂). Final body weight was measured on Day 40. Two indices were calculated: the total body weight gain over 40 days and the specific gain during the intraperitoneal phase (Day 30 to 40).

At the end of the experiment, the liver and kidneys were carefully excised, rinsed with cold physiological saline, blotted dry, and weighed. The relative weight of each organ was expressed as a percentage of the final body weight using the following formula²²:

Histopathology

Tissue samples were sectioned at a thickness of 5 μm, mounted on clean microscope slides, and stained with hematoxylin and eosin (H&E) for microscopic examination. Histopathological alterations in both organs were assessed by a qualified pathologist blinded to the treatment groups.

Tissue homogenate preparation

2 g of liver and kidneys of each animal were fixed in 2 ml of 4% Phosphate-Buffered Saline (PBS). The samples were then sonicated (10 s, twice) and centrifuged at 5000 g for 30 min. Supernatants were collected and preserved at − 80 °C for subsequent analysis of oxidative stress markers.

Analysis of oxidative stress biomarkers

Enzymatic antioxidants (Catalase CAT, Superoxide dismutase SOD, Glutathione S-transferase GST), non-enzymatic antioxidant (reduced glutathione GSH, lipid peroxidation marker MDA), and total protein content were evaluated. All samples were assigned random codes and evaluated blindly to ensure unbiased analysis.

Determination of malondialdehyde (MDA)

Lipid peroxidation was assessed following the Ohkawa method. In a test tube, 200 µL of liver and kidney tissue homogenate were mixed with 200 µL of 8.1% Sodium Dodecyl Sulfate solution, 1.5 mL of 20% acetic acid, 1.5 mL of 0.8% thiobarbituric acid, and 0.7 mL of water. This mixture was placed in boiling water for one hour, then cooled on ice for 10 min to stop the reaction. Next, an extraction was performed using a butanol-pyridine mixture (15:1 v/v). After adding 1 mL of water, the tubes were centrifuged at 4000 rpm for 10 min at 4 °C. Absorbance was measured at 532 nm, and the results were compared to a standard curve prepared with 1,1,3,3-tetramethoxypropane under the same conditions. The MDA concentration was expressed as nanomoles per milligram of protein (nmol/mg protein)²³.

Total protein assay

Protein content in each sample was measured using the Bradford assay. This method relies on Coomassie Brilliant Blue, which binds to proteins and causes an increase in absorbance at 595 nm. Bovine serum albumin (BSA) was used to generate the calibration curve²⁴.

Reduced glutathione (GSH) assay

GSH levels in liver and renal tissues were measured according to the method of Rahman et al. Liver homogenates were deproteinized using perchloric acid and then centrifuged at 4 °C. GSH was detected through its reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), producing a yellow-colored compound, which was quantified at 412 nm²⁵.

Glutathione S-transferase (GST) assay

GST activity was determined using kit #CS0410 (Sigma-Aldrich). Absorbance at 340 nm was recorded during the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione and increased absorbance reflecting GST activity, expressed as µmol/min/mg protein.

Catalase (CAT) assay

Catalase activity in the liver and kidney was determined using H₂O₂ as the substrate. Fifty microliters of sample were added to 1 ml of 30% H₂O₂ in a spectrophotometer cuvette, and the absorbance at 240 nm was monitored for 1 min²⁶.

Superoxide dismutase (SOD)

Superoxide dismutase (SOD) activity in liver tissue was determined by measuring the inhibition of NBT reduction at 560 nm, according to Beauchamp and Fridovich. Homogenates were prepared in phosphate buffer (pH 7.8) with 50 mM tissue homogenate, 0.1 mM EDTA, 13 mM L-methionine, 2 µM riboflavin, and 75 µM NBT. Results were expressed as units per mg protein²⁷.

Statistical analysis

The results were presented as mean ± standard error of the mean (SEM), with n = 5 animals per group. Statistical analysis was conducted using GraphPad Prism version 10.5.0. After verifying the normality of the data (Shapiro-Wilk test) and the homogeneity of variances (Bartlett and Brown-Forsythe tests), a one-way analysis of variance (ANOVA) was used to compare the experimental groups. In the case of significant differences (p < 0.05), a post hoc Tukey’s test was conducted for multiple comparisons. Groups sharing the same letter do not show significant differences from each other (Tukey’s test, p > 0.05).

Molecular docking

Molecular docking studies were performed using AutoDock Vina version 1.5.6^28,29 to evaluate the possible interaction modes between the selected ligands of Silybum marianum seeds and their respective biological targets. The main compounds identified in the extract is silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B and silydianin¹³. To explore the mechanism of SIRT1 inhibition, the crystal structure of human SIRT1 in complex with resveratrol and an AMC-containing peptide (PDB ID: 5BTR) was used. This structure allows the identification of potential binding sites that can modulate or inhibit the NAD⁺-dependent deacetylase activity of SIRT1.

Similarly, to investigate the inhibition of AMPK activity, the full-length human AMPK structure in complex with the thienopyridone activator A-769,662 (PDB ID: 4CFF) was selected. This structure highlights the regulatory and catalytic domains of AMPK that are critical for ligand binding, providing insight into potential inhibitory interactions. Protein structures were retrieved in PDB format from the RCSB Protein Data Bank (www.rcsb.org), while the studied ligands were downloaded from the ZINC database³⁰. Protein preparation involved the addition of Gasteiger charges and polar hydrogens, the removal of water molecules and all irrelevant heteroatoms, and conversion into PDBQT format. Energy minimization of the protein structures was conducted using AutoDock tools. The chemical structures of the ligands were drawn, optimized, and saved in mol2 format, which was subsequently converted to PDBQT format for docking simulations. To identify binding sites, the grid was generated around the active site of each protein using the grid center of x = − 20.00, y = − 64.15, z = 13.25 for the 5BTR and x = − 23.63, y = 1.70, z = 203.41 for 4CFF with a common grid size: X = 20, Y = 20, Z = 20 with a spacing of 0.375 Å. Ligand binding affinity with a kcal/mol unit was scored as a negative classification. With the help of Biovia Discovery Studio Visualizer 4.0 (DSV 4.0), the protein-ligand interactions were viewed in 2 and 3D and studied.

Results

Effects on body weight and relative organ weights

The evolution of body weight and the relative weights of the liver and kidneys are summarized in Tables 1 and 2. Over the entire experimental period (Day 0–40), the total body weight gain did not differ significantly among the groups, and all animals exhibited a comparable overall increase in body weight. However, the specific body weight gain (Day 30–40) showed significant differences. Rats treated with NiCl₂ alone displayed almost no weight gain (+ 0.18%), which was significantly lower than that observed in the control group (+ 5.68%) as well as in the groups receiving seed extract (+ 5.66%) and leaf extract (+ 5.26%). Co-administration of extracts with NiCl₂ partially improved this parameter, with the seed + NiCl₂ (+ 4.51%) and leaf + NiCl₂ (+ 3.27%) groups showing intermediate values. These results indicate that the extracts partially protected the animals from nickel-induced reduction in body weight gain.

Table 1.

Evolution of body weight across the experimental protocol in the different treatment groups.

Parameters	Control	NiCl2	Seeds	Seeds + NiCl2	Leaves	Leaves + NiCl2
Initial body weight (Day 0) (g)	153.8 ± 6.6	161.4 ± 5.2	159.6 ± 6.6	156.6 ± 7.1	156.8 ± 7.1	159.0 ± 7.9
Body weight (Day 30) (g)	199.8 ± 5.2	210.6 ± 7.0	202.8 ± 10.0	201.4 ± 7.8	201.6 ± 9.1	194.2 ± 11.5
Final body weight (Day 40) (g)	211.2 ± 6.4	210.8 ± 5.8	214.4 ± 11.3	210.6 ± 9.0	212.2 ± 9.7	200.2 ± 10.8
Total body weight gain (%)	+ 37.71 ± 2.98a	+ 30.76 ± 2.15a	+ 34.45 ± 4.83a	+ 34.54 ± 0.66a	+ 35.47 ± 3.13a	+ 25.87 ± 2.67a
Body weight gain (Day 30–40) (%)	+ 5.68 ± 1.14a	+ 0.18 ± 0.72b	+ 5.66 ± 0.97a	+ 4.51 ± 1.06ab	+ 5.26 ± 0.55a	+ 3.27 ± 1.68ab

Data are presented as mean ± SEM, n = 5 rats per group. Different superscript letters (a, b, c) within the same row indicate statistically significant differences between groups, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a significance level of p < 0.05.

Table 2.

Absolute and relative liver and kidney weights in the experimental groups.

Parameters	Control	NiCl2	Seeds	Seeds + NiCl2	Leaves	Leaves + NiCl2
Absolute liver weight (g)	9.37 ± 0.45	10.60 ± 0.08	9.42 ± 0.22	9.24 ± 0.29	8.93 ± 0.44	9.15 ± 0.63
Relative liver weight (%)	4.43 ± 0.13b	5.04 ± 0.11a	4.43 ± 0.20b	4.40 ± 0.06b	4.21 ± 0.05b	4.57 ± 0.18ab
Absolute kidney weight (g)	0.85 ± 0.05	0.80 ± 0.03	0.84 ± 0.08	0.80 ± 0.02	0.81 ± 0.05	0.77 ± 0.05
Relative kidney weight (%)	0.40 ± 0.01a	0.38 ± 0.02a	0.39 ± 0.03a	0.38 ± 0.02a	0.38 ± 0.01a	0.38 ± 0.02a

Data are presented as mean ± SEM, n = 5 rats per group. Different superscript letters (a, b, c) within the same row indicate statistically significant differences between groups, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a significance level of p < 0.05.

For the relative liver weight, the NiCl₂ group (5.04%) was significantly higher compared to the control (4.43%). This alteration was completely corrected in the seed + NiCl₂ group (4.40%), which showed values identical to the control. The leaf + NiCl₂ group (4.57%) exhibited an intermediate value, without significant differences compared to NiCl₂ alone or the control. In contrast, the relative kidney weight was not significantly different between the experimental groups.

Changes in hematological parameters of different groups

According to the Table 3, hematological parameters revealed several alterations following nickel exposure and their amelioration due to pre-treatments of Silybum marianum extracts. Notably, 10 days exposure of 4 mg/kg of NiCl₂, showed a significant decrease in RBC and HGB suggesting the development of anemia. In contrast, groups receiving one month of dietary supplementation with Silybum marianum seeds and leaf extracts, whether alone or before administration of NiCl₂, maintained RBC and HGB levels comparable to the control, suggesting a protective effect of these extracts against Ni-induced hematological toxicity. Notably, the pre-treatment with seeds provided almost complete protection, while the leaf extract showed partial attenuation of hemoglobin. An increase in MCV and RDW-SD was observed in Ni exposure group compared to the control group which can effectively indicate a macrocytic anemia caused Ni toxicity³¹. The group receiving one month of dietary supplementation with Silybum marianum seeds prior to nickel exposure showed no significant change in MCV compared to the control. However, the group receiving Silybum marianum leaves extract did not differ significantly from those of either the control or Ni group which indicates that the leaf extract did not fully protect the change of MCV. Along with an increase in MCV in Ni exposure group, MCH also tended to increase, consistent with the larger volume of red cells.

Table 3.

Hematological profile of the experimental groups.

Hematological parameters	Control	NiCl2	Seeds	Seeds + NiCl2	Leaves	Leaves + NiCl2
WBC (103 /µL)	3.79 ± 0.60a	3.26 ± 1.02a	3.99 ± 0.65a	3.43 ± 0.49a	3.73 ± 0.54a	3.36 ± 0.58a
RBC (106 /µL)	7.27 ± 0.41a	4.84 ± 0.29b	7.55 ± 0.34a	6.85 ± 0.53a	8.00 ± 0.41a	6.97 ± 0.63a
HGB (g /dL)	13.84 ± 0.69a	10.80 ± 0.70b	14.06 ± 0.47a	14.04 ± 0.29a	14.23 ± 0.43a	13.02 ± 0.69ab
HCT (%)	42.18 ± 2.88a	32.41 ± 2.78a	42.34 ± 1.94a	40.95 ± 2.67a	44.97 ± 2.38a	35.91 ± 4.76a
MCV (fL)	54.76 ± 1.16b	66.26 ± 3.05a	55.68 ± 1.16b	58.20 ± 1.21b	55.32 ± 0.98b	59.44 ± 2.35ab
MCH (pg)	18.10 ± 0.38b	21.94 ± 1.43a	18.36 ± 0.48b	18.12 ± 0.82b	17.36 ± 0.48b	18.80 ± 0.83ab
PLT (103 /µL)	789.0 ± 115.0a	736.6 ± 269.2a	735.2 ± 121.3a	704.2 ± 57.7a	754.8 ± 99.6a	745.0 ± 152.7a
RDW-SD (fL)	26.20 ± 2.42b	39.46 ± 3.17a	25.92 ± 1.60b	29.58 ± 0.64b	26.74 ± 2.25b	31.12 ± 0.87ab
PDW (fL)	7.36 ± 0.31c	10.92 ± 0.49a	7.60 ± 0.53bc	7.78 ± 0.42bc	7.42 ± 0.32bc	9.17 ± 0.35ab
MPV (fL)	7.56 ± 0.19b	9.38 ± 0.25a	7.54 ± 0.24b	7.86 ± 0.20b	7.70 ± 0.19b	7.78 ± 0.12b
P-LCR (%)	8.16 ± 1.23b	21.68 ± 1.61a	8.92 ± 2.42b	10.62 ± 1.88b	7.94 ± 0.99b	10.58 ± 1.37b

Data are presented as mean ± SEM, n = 5 rats per group. Different superscript letters (a, b, c) within the same row indicate statistically significant differences between groups, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a significance level of p < 0.05. Hematological parameters measured include white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), platelet count (PLT), red cell distribution width - standard deviation (RDW-SD), platelet distribution width (PDW), mean platelet volume (MPV), and platelet-large cell ratio (P-LCR).

The administered dose of NiCl₂ during the exposure period did not induce any observable changes in PLT compared to the control. However, there is a significant increase in MPV along with P-LCR in the Ni-exposure group, which indicates a change in platelet. The groups treated with Silybum marianum seeds and leaf extract did not show any change compared to the control. There is an increase of PDW in Ni- exposure group compared to the control, whereas the group receiving dietary supplementation with Silybum marianum seeds - whether alone or after Ni-exposure - showed no significant change in PDW. In contrast, the group receiving dietary supplementation with Silybum marianum leaf extract did not show any significant change in PDW compared to the Ni-exposed group, suggesting that the leaf extract supplementation alone was insufficient to mitigate the effects of nickel exposure on platelet size variability. On the other hand, the administered dose of NiCl₂ during the exposure period did not induce any observable changes in WBC and HCT in any group.

Changes in serum biochemical parameters

From Table 4, the serum biochemical parameters showed variations among the different groups in certain measures. The urea level was significantly elevated in the Ni-exposure group, suggesting kidney damage. Groups receiving one month of dietary supplementation with Silybum marianum seeds, whether alone or prior to nickel exposure, had urea levels similar to those in the control group, indicating protection. Notably, groups receiving one month of dietary supplementation with Silybum marianum leaves prior to nickel exposure showed an intermediate urea level between control and Ni exposure group, suggesting partial protection. The significant increase in ALT and AST biomarkers, triglycerides and a decrease in HDL level, following intraperitoneal NiCl₂ administration, reflects liver dysfunction and potential hepatocellular damage. Notably, pretreatment with one month of seeds extract significantly mitigated these alterations, suggesting its hepatoprotective potential. In contrast, the leaf extract was not significantly different from either the control or NiCl₂ group, indicating only partial protection and suggesting a less pronounced effect on NiCl₂-induced liver damage. There were no statistically significant differences in creatinine level, total cholesterol and LDL levels across all groups, which indicates that a dose of 4 mg/kg administered over 10 days did not affect these biomarkers.

Table 4.

Serum biochemical parameters analysis of the experimental groups.

Biochemical parameters	Control	NiCl2	Seeds	Seeds + NiCl2	Leaves	Leaves + NiCl2
Urea (g/L)	0.30 ± 0.030b	0.49 ± 0.029a	0.29 ± 0.037b	0.32 ± 0.035b	0.33 ± 0.035b	0.41 ± 0.038ab
Creatinine (mg/L)	3.80 ± 0.466a	4.56 ± 0.768a	3.82 ± 0.485a	4.03 ± 0.568a	4.07 ± 0.864a	4.51 ± 0.686a
Total cholesterol (g/L)	1.07 ± 0.083a	1.37 ± 0.119a	1.02 ± 0.088a	1.02 ± 0.092a	0.99 ± 0.090a	1.12 ± 0.092a
Triglycerides (g/L)	0.54 ± 0.023b	0.85 ± 0.066a	0.56 ± 0.031b	0.60 ± 0.038b	0.55 ± 0.057b	0.70 ± 0.079ab
HDL (mmol/L)	0.75 ± 0.054a	0.43 ± 0.021b	0.71 ± 0.075a	0.69 ± 0.043a	0.69 ± 0.065a	0.60 ± 0.070ab
LDL (mmol/L)	0.060 ± 0.003a	0.068 ± 0.004a	0.060 ± 0.003a	0.062 ± 0.004a	0.060 ± 0.003a	0.062 ± 0.004a
AST (U/L)	119.2 ± 8.7b	188.0 ± 10.3a	121.4 ± 8.5b	142.4 ± 4.0b	131.2 ± 11.5b	151.0 ± 13.7ab
ALT (U/L)	48.2 ± 4.1b	95.4 ± 8.7a	53.0 ± 11.0b	55.4 ± 8.4b	57.0 ± 9.5b	73.8 ± 9.0ab

Data are presented as mean ± SEM, n = 5 rats per group. Different superscript letters (a, b, c) within the same row indicate statistically significant differences between groups, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a significance level of p < 0.05. Biochemical parameters measured include glucose, urea, creatinine, total cholesterol, triglycerides, HDL (high-density lipoprotein), LDL (low-density lipoprotein), AST (aspartate aminotransferase), and ALT (alanine aminotransferase).

Changes in oxidative stress parameters

As illustrated in (Fig. 2), Nickel exposure caused an increase in MDA levels in liver and kidneys compared to the control group, serving as a key marker of lipid peroxidation. One month of dietary supplement of Silybum marianum seeds prior to nickel exposure appears to confer protection against lipid peroxidation. However, the liver appears to be more severely affected than the kidneys. One month of dietary supplement of leaf extract did not appear to protect the liver against lipid peroxidation, whereas a slight protective effect was observed in the kidneys. In addition, NiCl₂ exposure resulted in a significant reduction in both CAT and SOD activities in the liver compared to the control group. In contrast, no significant differences were observed in the kidney, indicating that kidney antioxidant enzyme activities were less affected.

Fig. 2.

Oxidative stress biomarker analysis in liver (A–E) and kidney (F–J) of the experimental groups. Data are presented as mean ± SEM, n = 5 rats per group. MDA, malondialdehyde; GSH, reduced glutathione; GST, glutathione S-transferase; CAT, catalase; SOD, superoxide dismutase. Different superscript letters (a, b, c) indicate statistically significant differences between groups, as determined by one-way ANOVA followed by Tukey’s post hoc test, with a significance level of p < 0.05.

Silybum marianum seed supplementation for one month, with or without nickel exposure, appears to prevent the decrease of SOD and CAT biomarkers in liver caused by nickel exposure. On the other hand, Silybum marianum leaf supplementation for one month did not reduce liver CAT levels, as observed by a non-significant difference compared to the nickel-exposed group, but it showed partial protection of the SOD enzyme. In contrast, the kidneys appeared unaffected, as there was no significant difference in CAT activity between the control and NiCl₂-exposed groups. As for GSH and GST, there was a significant difference between the control group and the Ni-exposed group in both the liver and kidneys. However, administration of 100 mg/kg of Silybum marianum seeds prior to nickel exposure helped preserve GSH and GST levels in liver and kidney. In contrast, the Silybum marianum leaf extract did not preserve these enzymes.

Histopathological changes

Liver

Liver sections from the control group displayed normal architecture, with well-organized hepatic cords and preserved cellular morphology (Fig. 3A). Similarly, rats treated with seed or leaf extracts alone, in addition to those co-treated with seed extract and NiCl₂, showed largely normal liver morphology, indicating a notable protective effect of the seed extract (Fig. 3D–F).

Fig. 3.

Histological sections of rat liver tissue: Control (A), NiCl₂ (B and C), Seeds (D), Seeds + NiCl₂ (E), Leaves (F), and Leaves + NiCl₂ (G). Hematoxylin and eosin staining (H&E), ×400. Symbols indicate specific histopathological changes: circle, hepatocellular necrosis; gray arrow, hydropic degeneration; black arrow, pyknotic nuclei; red arrow, inflammatory infiltrate; green star, centrilobular vein congestion.

In contrast, NiCl₂-treated rats exhibited marked histopathological alterations. Hepatic cords were disorganized, with partial loss of the normal radial arrangement. Many hepatocytes showed hydropic degeneration, characterized by clear, ballooned cytoplasm, along with pyknotic nuclei indicative of cellular stress. Sinusoidal congestion was also observed. Foci of hepatocellular necrosis were present in some areas. In the portal tracts, thickening of the portal vein walls was accompanied by periportal inflammatory cell infiltration and mild connective tissue proliferation, suggesting early portal fibrosis (Fig. 3B, C).

In the NiCl₂ + leaf extract group, moderate alterations were observed. Marked congestion was observed in the centrilobular veins, with peri-centrilobular hepatocytes showing a granular cytoplasm. The hepatic cords lost their normal radial arrangement, and pyknotic nuclei were present, suggesting that the leaf extract offered only limited protection against nickel-induced liver injury (Fig. 3G).

Table 5 shows that NiCl₂ caused moderate to severe liver lesions, that seed extract provided complete protection, and that leaf extract offered only partial protection.

Table 5.

Severity of histopathological alterations in liver tissues of different rat groups.

Group	Hepatic cord disorganization	Hydropic degeneration	Pyknotic nuclei	Hepatocellular necrosis	Immune cell infiltration	Sinusoidal congestion
Control	−	−	−	−	−	−
NiCl2	++	++	+++	++	+++	++
Seeds	−	−	−	−	−	−
Seeds + NiCl2	−	−	−	−	+	−
Leaves	−	−	−	−	−	−
Leaves + NiCl2	+	+	+	++	+	++

Scoring system: none (−), mild (+), moderate (++), and severe (+++)

Kidney

The renal tissue of control rats showed preserved architecture, characterized by intact glomeruli and regularly arranged tubules (Fig. 4A). Similarly, rats treated with seed or leaf extracts alone, as well as those co-treated with seed extract and NiCl₂, maintained an overall normal renal morphology, indicating a protective effect of the seed extract (Fig. 4C–E).

Fig. 4.

Histological sections of rat kidney tissue: Control (A), NiCl₂ (B), Seeds (C), Seeds + NiCl₂ (D), Leaves (E), and Leaves + NiCl₂ (F). Hematoxylin and eosin staining (H&E), ×400. Symbols indicate specific histopathological changes: circle, degenerated cells; red arrow, glomerular degeneration; black arrow, tubular degeneration; red star, tubular dilation

In contrast, kidneys from NiCl₂-intoxicated rats displayed marked histopathological alterations. The glomeruli appeared congested with increased cellularity, suggestive of early hypercellularity. Tubular structures exhibited pronounced degenerative changes, including cytoplasmic vacuolization (hydropic degeneration), irregular luminal dilation, and a granular, disorganized cytoplasm in tubular epithelial cells. Although extensive necrosis was not observed, the lesions were diffuse and consistent with significant tubular degeneration (Fig. 4B). In the NiCl₂ + leaf extract group, only partial protection was observed. Renal sections showed evident glomerular degeneration and disorganization of the tubular epithelium, with necrotic and degenerated cells disrupting the normal architecture. These results suggest that, unlike the seed extract, the leaf extract provided only limited protection against nickel-induced nephrotoxicity (Fig. 4F).

Table 6 presents the histopathological scores, providing a quantitative overview of tissue alterations observed. They confirm that NiCl₂ alone induces moderate to severe glomerular and tubular lesions, while the seed extract offers near complete protection, and the leaf extract provides only partial protection, consistent with the microscopic findings.

Table 6.

Severity of histopathological alterations in kidney tissues of different rat groups.

Group	Glomerular congestion	Cytoplasmic vacuolization/hydropic degeneration	Tubular dilation	Disorganized tubular cytoplasm	Necrosis/degenerated cells
Control	−	−	−	−	−
NiCl2	++	++	+++	++	+++
Seeds	−	−	−	−	−
Seeds +NiCl2	−	−	−	−	+
Leaves	−	−	−	−	−
Leaves +NiCl2	+	+	+	++	+

Scoring system: none (−), mild (+), moderate (++), and severe (+++).

Molecular docking

Nicotinamide adenine dinucleotide (NAD⁺) is a central metabolic coenzyme linking redox balance to cellular longevity. Through the NAD⁺-dependent deacetylase SIRT1, it modulates histones and metabolic regulators to sustain homeostasis under stress. SIRT1, in turn, activates AMPK (5′-AMP-activated protein kinase), a major energy sensor responsible for balancing ATP synthesis, energy expenditure, and cellular adaptation to stress³². The concerted activity of the NAD⁺/SIRT1/AMPK axis promotes energy efficiency, enhances antioxidant defenses, and contributes to cellular longevity. This pathway has been widely linked to the prevention of metabolic disorders and the maintenance of healthy aging³³.

Molecular docking simulations were conducted to investigate the binding potential of four flavonolignans “Silybin, Isosilybin, Silydianin, and Silychristin” with two key metabolic regulators: AMPK (PDB ID: 4CFF) and SIRT1 (PDB ID: 5BTR). The obtained binding energies, expressed in kcal/mol, reflect the predicted affinities between the ligands and their respective targets, with more negative values indicating stronger interactions (Table 7). In all cases, the predominant interaction type observed was hydrophobic, consistent with the structural nature of the binding pockets in both proteins. Silybin exhibited the highest binding affinity toward AMPK, with a docking score of − 9.5 kcal/mol, suggesting a strong and stable association with the allosteric binding pocket of the enzyme. This result implies that Silybin may act as a potent AMPK modulator, potentially influencing the regulation of cellular energy homeostasis. (Fig. 5) illustrates the binding mode of Silybin with AMPK, highlighting key hydrophobic interactions with residues such as LEU22, ALA156, and MET93, as well as hydrogen bonds with GLU494 and LYS45, which contribute to the ligand’s orientation and stabilization within the active site. In contrast, docking to SIRT1 revealed that Silydianin showed the highest predicted affinity (− 9.2 kcal/mol), indicating a potential for interfering with or modulating the NAD⁺-dependent deacetylase activity of the enzyme. Figure 6 illustrates the binding of Silydianin to SIRT1. Hydrophobic contacts with residues PHE414, PRO212, and THR209, together with hydrogen bonds involving ARG276 and GLN294, stabilize the ligand within the regulatory domain of the enzyme. Silychristin, with a docking score of − 9.0 kcal/mol for both AMPK and SIRT1, stands out as a potential dual-target inhibitor, showing balanced affinity for both enzymes. Such dual activity could prove useful in therapeutic approaches targeting both energy metabolism and epigenetic control. Isosilibinin, while slightly less potent, still demonstrated favorable docking scores (− 8.3 kcal/mol for AMPK and − 8.7 kcal/mol for SIRT1), positioning it as a viable candidate for further exploration.

Table 7.

Docking scores and predominant interaction types of flavonolignan compounds with AMPK (PDB ID: 4CFF) and SIRT1 (PDB ID: 5BTR).

Compound	PDB code: 4CFF		PDB code: 5BTR
	Docking score (kcal/mol)	predominant type of interaction	Docking score (kcal/mol)	predominant type of interaction
Silibin	− 9.5	Hydrophobic	− 9.0	Hydrophobic
Isosilibin	− 8.3	Hydrophobic	− 8.7	Hydrophobic
Silydianin	− 8.7	Hydrophobic	− 9.2	Hydrophobic
Silychristin	− 9.0	Hydrophobic	− 9.0	Hydrophobic

Fig. 5.

The molecular docking results of Silibinin with 4CFF protein, surfaces around ligand and 2D forms.

Fig. 6.

The molecular docking results of Silydianin with 5BTR protein, surfaces around ligand and 2D forms.

In general, docking analyses demonstrated that Silybin has a preferential binding to AMPK, while Silydianin has higher affinity to SIRT1. Silychristin appears capable of modulating both targets. The predominance of hydrophobic contacts in all complexes is consistent with the lipophilic nature of their binding pockets.

Discussion

The primary routes of nickel entry into the human body are dietary intake and inhalation of nickel-containing particles. Among these, food represents the most significant source of exposure for the general population. Consequently, nickel deficiency is rare due to its widespread presence in the environment; therefore, maintaining a nickel-deficient diet is challenging³⁴. Higher nickel intake may result from consuming nickel-rich foods like oatmeal, green beans, broccoli, cocoa, peas, chocolate, canned fruit, canned vegetables, nuts, and soy products^35,36. Reactive oxygen species (ROS) are highly reactive, short-lived molecules that contain oxygen⁶. Oxidative stress plays a crucial role in both the initiation and progression of liver and kidneys injury by promoting lipid peroxidation, cellular damage, and impairment of antioxidant defense systems³⁷. The liver is a vital organ involved in key metabolic and physiological functions, including bile secretion, energy production, vitamin storage, and the metabolism of carbohydrates, proteins, and fats. After absorption in the intestine, blood which is rich in nutrients and xenobiotics reaches the liver through the portal vein, exposing it to various toxic agents. This makes the liver particularly prone to toxic damage. The kidneys, which filter blood and eliminate waste, also play a crucial role in detoxification and are equally vulnerable to toxic insults³⁸. This study is, therefore, designed to assess whether a sustained dietary supplementation with Silybum marianum seeds and leaves extracts over one month can effectively attenuate the toxic effects induced by nickel exposure. In this investigation, the hematological, biochemical, and oxidative stress parameters were examined and compared across the experimental groups to evaluate the protective effects of Silybum marianum seed and leaf extracts against nickel chloride-induced toxicity in rats. The study aimed to determine how each extract from Silybum marianum parts modulates the toxic impact of nickel on blood, liver, and kidneys functions, as well as their ability to alleviate oxidative damage.

Effects of Silybum marianum seeds and leaves on body weight gain and relative organ weights in nickel chloride-treated rats

In this study, nickel chloride treatment resulted in a marked reduction in body weight gain accompanied by a significant increase in relative liver weight. The reduction in weight gain observed in NiCl₂-treated rats is consistent with previous reports demonstrating that nickel exposure adversely affects growth, partly through decreased food intake and disturbances in protein and lipid metabolism³⁹. Such reduction of weight gain has been described with different nickel compounds, including nickel chloride and nickel nanoparticles, and is considered a hallmark of systemic nickel toxicity^40,41. The hepatic hypertrophy observed in our study is also in agreement with earlier experimental findings. Sidhu et al.⁴² reported a significant increase in liver weight in female Sprague–Dawley rats exposed to NiSO₄ in drinking water, while Dumala et al.⁴³ showed that repeated oral administration of NiO nanoparticles induced a dose-dependent increase in both liver and kidney weights. Similarly, Das et al.¹¹ attributed nickel-induced hepatic hypertrophy to oxidative stress and the bioaccumulation of the metal in hepatic tissue. Taken together, these convergent findings confirm that the liver is a primary target organ of nickel toxicity.

Interestingly, in our experiment, no significant variations in relative kidney weight were observed between groups. This may be due to the relatively short duration of exposure, which might not have been sufficient to trigger measurable renal hypertrophy.

Importantly, one month of dietary supplementation with Silybum marianum seed extract prior to NiCl₂ exposure completely prevented the increase in relative liver weight, restoring values to those of the control group. In contrast, the leaf extract exerted only partial protection.

Effects of Silybum marianum seeds and leaves on nickel chloride-induced hematological alterations

Hematological parameters related to the protective effects of Silybum marianum against heavy metal-induced toxicity remain insufficiently explored in depth in the literature. In our investigation, exposure to nickel chloride at a dose of 4 mg/kg for 10 days caused significant changes in blood-related hematological markers. Notably, during the 10 days of the exposure, red blood cells (RBC) have decreased as well as the hemoglobin (HGB) which is related to a severe anemia caused by nickel chloride. Additionally, the mean corpuscular volume (MCV) has increased along with the mean corpuscular hemoglobin (MCH), suggesting the development of macrocytic anemia due to nickel exposure. The decrease in hematological parameters may be linked to impaired iron absorption and reduced hemoglobin synthesis which, in turn, causes anemia. Nickel can alter hemoglobin structure and lower its oxygen affinity, weakening its binding capacity and increasing erythrocyte fragility and permeability. These effects may lead to cell swelling, deformation, and damage. The drop in hemoglobin levels could also result from structural alterations of heme and the inhibition of enzymes involved in hemoglobin synthesis^44,45. Our findings differ from those reported by Bouhalit et al.⁴⁶, who observed that nickel exposure led to microcytic anemia, as indicated by reductions in hemoglobin levels and red blood cell count. Indeed, this may vary depending on the dose and duration of exposure. One month of dietary supplementation with Silybum marianum extracts, particularly seeds extract, has been shown to offer protective effects against hematological alterations caused by NiCl_2, as evidenced by improved hematological parameters compared to Ni-exposure group. Red blood cells (RBC), hemoglobin (HGB), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were not significantly altered, suggesting the maintenance of red blood cell morphology and hemoglobin content. This indicates that one month of Silybum marianum seeds extract supplementation provided a significant protection of hematological parameters against NiCl₂. On the other hand, leaves extract showed a partial protection on hemoglobin (HGB), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). This indicates that the leaf extract was insufficient to fully prevent anemia or counteract the hematological disturbances caused by nickel.

Effects of Silybum marianum seeds and leaves on nickel chloride-induced biochemical alterations

Nickel induced significant changes in serum biochemical markers. Notably, a dose of 4 mg/kg during 10 days showed a significant increase of urea level in Ni-exposure group compared to the control, which indicates an alteration of kidneys. Urea are metabolic waste products primarily eliminated by the kidneys. When kidney function is impaired, their excretion becomes inefficient, leading to elevated plasma levels. Thus, the observed increase in this biomarker in the NiCl₂-treated group may be attributed to the nephrotoxic effects of NiCl₂ on renal cells⁴⁷. However, creatinine level did not show significant changes between the control group and the Ni-exposed group, which may be due to the dose and duration of exposure being insufficient to cause alterations in creatinine levels. Our result is consistent with those from El Brouzi et al.⁴⁸ who found that creatinine levels remained unchanged during both acute and chronic exposure to NiCl₂ at a dose of 0.25 mg/kg. In contrast in their investigation, urea levels did not significantly change during acute exposure but exhibited a significant elevation following chronic exposure. Additionally, compared to the control group, the nickel group exhibited a marked increase in liver and kidney enzyme levels. Specifically, AST levels increased by approximately 1.6-fold, while ALT levels showed a twofold increase. These elevations indicate significant hepatic and kidney injury induced by nickel chloride. One month of dietary supplementation with Silybum marianum extracts prior to NiCl₂ exposure did not cause any significant changes in ALT, AST and urea compared to the control group. This implies that giving the extracts before exposure conditions the body to activate protective responses, reducing potential damage. However, when comparing the parts of Silybum marianum administered at the same dose of 100 mg/kg, the leaf extract did not provide the same level of protection as the seed extract. This is evidenced by the lack of significant differences between the control and Ni-exposed groups treated with the leaf extract, indicating that the leaves did not confer complete protection. Ni-exposure resulted in a significant increase in triglyceride levels compared to those in the control group, while HDL levels were significantly lower, indicating the presence of dyslipidemia. The elevation in serum triglycerides could be due to diminished lipoprotein lipase activity in the blood vessels, which normally facilitates the breakdown of triglycerides⁴⁹. However, cholesterol level was not affected. One month of dietary supplementation with Silybum marianum seeds prior to NiCl₂ exposure did not show changes in triglycerides and HDL levels, which suggests that Silybum marianum seeds provided a protective effect against nickel-induced lipid alterations, while leaf extract did not show a complete protection in these biomarkers.

Effect of Silybum marianum seeds and leaves on nickel chloride induced oxidative stress

Oxidative stress plays an important role in causing and worsening damage by creating an imbalance between harmful free radicals and protective antioxidants. Nickel can induce the formation of reactive oxygen species (ROS), including superoxide anions (O₂^•-), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH), and singlet oxygen (¹O₂). At physiological levels, ROS are naturally generated within cells and contribute to important functions. However, when ROS production exceeds the cell’s antioxidant capacity, oxidative stress occurs, which can damage cellular membranes, lipids, proteins, lipoproteins, and DNA⁵⁰. As demonstrated in this present investigation, 4 mg/kg during 10 days resulted in a significant decrease in oxidative stress parameters compared to the control group. Notably, there is a significant increase in MDA in the liver and kidneys, which is a compound formed during the final stages of lipid peroxidation and recognized for its mutagenic and cytotoxic properties⁵¹. Indeed, Nickel can promote the bioaccumulation of iron, which can participate in the Fenton reaction, leading to increased generation of reactive species particularly hydroxyl radicals that drive membrane lipid peroxidation and exacerbate oxidative stress⁵². A significant reduction in superoxide dismutase (SOD) activity, the enzyme responsible for converting the superoxide anion radical (•O₂⁻) into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂), was observed⁵³. There is a significant decrease in catalase (CAT) in liver while the kidney showed no significant change. Catalase is a key antioxidant enzyme that rapidly converts millions of H₂O₂ molecules into water and oxygen each second, protecting cells from ROS-induced oxidative stress⁵⁴. There is a significant decrease in reduced glutathione (GSH), which is involved in antioxidant defense by reducing oxidative stress and maintaining redox balance, detoxification of xenobiotics and other exogenous compounds along with a reduction in glutathione S-transferase (GST), which is the enzyme that utilizes GSH to detoxify harmful substances⁵⁵. Nickel exposure resulted in a marked alteration of oxidative stress biomarkers in both the liver and kidneys, reflecting its significant impact on the body’s redox balance. This suggests that a dose of 4 mg/kg over 10 days is sufficient to induce kidney alterations as well. However, the liver was more severely affected.

The administration of Silybum marianum seeds extract for one month prior to nickel exposure seems to maintain antioxidant defenses, as evidenced by the absence of a significant difference compared to the control group. This is explained by the fact that administrating the seeds extract prepares the body’s defense systems by enhancing antioxidant mechanisms, reducing oxidative stress, and protecting liver and kidney tissues. However, administration of leaf extract did not confer significant protection. This could be due to the dose of 100 mg/kg administered and the duration as well being too low to achieve a protection against 10 days on nickel exposure. Moreover, phytochemicals differ among the plant. The types and amounts of compounds vary between different parts of the plant. Its composition is also influenced by the time of harvest. In this study, the leaf extract was collected at the mid flowering stage. Silybum marianum seeds extract contains silymarin, the mixture of flavonolignans that has been shown to provide protection against various toxins by enhancing antioxidant defenses and supporting liver function¹³. A study has been carried out on the leaf and seeds extract of Silybum marianum against diethylnitrosamine/phenobarbital-induced nephrotoxicity; a dose of 50 mg/kg for 14 weeks has been shown to protect kidneys⁵⁶. In this investigation, it may be advisable to increase the dose of the leaf extract or to extend the duration of administration, as both factors could enhance its protective potential and allow a more pronounced biological response to be observed.

Histopathological studies

The biochemical alterations observed in this study (elevated transaminases and urea, together with disruption of the metabolic profile) reflect functional impairment of the liver and kidneys following NiCl₂ exposure. These changes are consistent with the histopathological findings, including hepatic cord disorganization, hydropic degeneration, sinusoidal congestion, focal necrosis, and inflammatory infiltration in the liver, as well as diffuse glomerular and tubular damage in the kidney. Such results are in line with previous studies reporting that nickel exposure induces massive oxidative stress, lipid peroxidation, and activation of apoptotic pathways in these organs^46,57. Oxidative stress appears to be the central mechanism underlying these observations. The rise in MDA, along with the depletion of antioxidant enzymes such as SOD, CAT, GST, and reduced glutathione (GSH), indicates a radical overload that surpasses cellular defense capacity. This leads to oxidation of membrane proteins, impairment of metabolic enzymes, and leakage of ALT and AST into the bloodstream. In the kidney, elevated urea reflects membrane disruption and tubular dysfunction, secondary to lipid peroxidation^58–61. The histological lesions can be explained by these molecular disturbances: hydropic degeneration results from ionic and osmotic imbalance, necrosis from irreversible damage to membranes and mitochondria, inflammatory infiltration from NF-κB activation and release of pro-inflammatory cytokines. Furthermore, intracellular accumulation of nickel promotes Fenton-type reactions, leading to overproduction of reactive oxygen species (ROS) and subsequent damage to lipids, proteins, and DNA, thereby triggering apoptosis and progressive fibrosis⁶². Administration of Silybum marianum seed extract showed nearly complete protection against these alterations. This finding agrees with the literature, where silymarin exerts strong antioxidant and anti-inflammatory effects, stabilizes membranes, and stimulates hepatocyte regeneration^63–65.

At the mechanistic level, the protective effects of Silybum marianum extract can be explained by the modulation of interconnected signaling pathways governing redox homeostasis, inflammation, and cell survival^66,67. Although no direct molecular assays were performed, the strong recovery of antioxidant enzymes and the improvement of tissue architecture strongly suggest activation of key cytoprotective networks.

A central mechanism involves the Nrf2/Keap1/ARE signaling pathway, recognized as the master regulator of cellular antioxidant defense. Under oxidative stress, electrophilic modification of Keap1 cysteine residues disrupts the Keap1-Nrf2 complex, allowing Nrf2 stabilization and nuclear translocation. Once in the nucleus, Nrf2 binds to antioxidant response elements (ARE) and induces transcription of genes encoding phase II detoxifying enzymes and antioxidant proteins, including SOD, CAT, GST, HO-1, and enzymes involved in glutathione synthesis (GCL and GSH-related systems)^68,69. The restoration of these enzymatic activities in milk thistle extract-treated groups strongly suggests activation of this pathway, which is consistent with the known ability of silymarin constituents to act as Nrf2 inducers⁶⁶.

In addition, NF-κB signaling represents a major pro-inflammatory pathway activated under oxidative stress conditions. Normally, ROS-mediated activation of upstream kinases (such as IKK) leads to degradation of IκB and subsequent nuclear translocation of NF-κB, resulting in transcription of pro-inflammatory mediators (TNF-α, IL-1β, IL-6). The reduced inflammatory infiltration observed in liver and kidney tissues suggests that milk thistle extract interferes with this cascade, possibly through ROS scavenging and indirect inhibition of NF-κB activation, thereby attenuating cytokine-driven tissue injury^70,71.

Furthermore, mitochondrial integrity appears to be preserved by Silybum marianum extract through regulation of intrinsic apoptotic signaling. Oxidative stress typically induces mitochondrial membrane permeabilization, cytochrome c release, and activation of caspase-dependent apoptosis. In this context, silymarin and its major active compound, silibinin, have been shown to attenuate apoptotic signaling by modulating the balance between pro- and anti-apoptotic proteins, notably through decreasing the Bax/Bcl-2 ratio and inhibiting caspase-3 activation, thereby limiting mitochondrial dysfunction and preventing progression toward cell death and fibrosis^72,73. Silybum marianum bioactive compounds have also been reported to stabilize mitochondrial membranes, reduce Bax translocation, and enhance anti-apoptotic Bcl-2 expression, ultimately preventing activation of the caspase cascade^67,69.

In addition, emerging evidence suggests the involvement of AMPK and SIRT1 as upstream metabolic regulators modulated by flavonolignans. AMPK activation promotes cellular energy balance and enhances antioxidant defenses, while SIRT1 deacetylates key transcription factors including Nrf2 and NF-κB, thereby simultaneously enhancing antioxidant gene expression and suppressing inflammatory signaling. The AMPK/SIRT1/Nrf2 axis, therefore, represents a potential integrative pathway linking energy metabolism to redox regulation and inflammation control; however, its implication in Silybum marianum extract effects remains to be further experimentally validated^74–76.

Collectively, these findings support a multi-target mechanism of action involving activation of the Nrf2 antioxidant pathway, inhibition of NF-κB-mediated inflammation, and suppression of mitochondrial-dependent apoptosis, which together contribute to the observed protection against nickel-induced hepatic and renal toxicity ^67,68.

By contrast, the leaf extract exhibited only partial protection, likely due to lower concentrations of silymarin-type flavonolignans, reduced bioavailability, and weaker capacity to activate Nrf2 and related signaling pathways, resulting in limited modulation of downstream antioxidant and anti-apoptotic responses^66,69.

Conclusion

The results indicate that supplementation with Silybum marianum seeds extract prior to nickel exposure for one month conditioned the body to mount protective responses, potentially through the upregulation of antioxidant systems and increased resistance to oxidative and toxic stress of nickel chloride. This may be beneficial for humans exposed to heavy metals through multiple pathways, such as contaminated food, water, soil, or occupational settings. Unlike the seeds, the leaf extract has not been shown to offer significant protection in all parameters. This lack of protection by the leaf extract may be due to an insufficient dose or duration of administration or even the low bioavailability of its compounds, as the phytochemical composition can vary among different parts of the same plant. Further research is warranted to extend the duration of pretreatment and/or increase the dose. Further studies should also assess the protective effects of Silybum marianum extracts on pulmonary function in humans exposed to nickel through inhalation in industrial settings.

Author contributions

O. I, M.J: Investigation, conceptualization, methodology, original draft writing. M. L, O. M, A. N, M. Y.E, H. N: Formal analysis, reviewing, and editing. O. N, A.A. S, A. D, J.M.B, R. N, S. C: Resources, data validation, data curation, funding acquisition. Y. Taboz, A. Habsaoui: Conceptualization, Supervision, Writing – review & editing.

Funding

This was funded by King Saud University through the Ongoing Research Funding program (ORF-2026-1057), King Saud University, Riyadh, Saudi Arabia.

Data availability

All data analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

The experimental procedures were approved by the Animal Ethics Committee (Local Institutional Research Committee). All animals were treated humanely in compliance with the principles outlined in the ARRIVE Guidelines.