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Original Article

Anat Cell Biol 2023; 56(4): 538-551

Published online December 31, 2023

https://doi.org/10.5115/acb.23.101

Copyright © Korean Association of ANATOMISTS.

A comparative study on the hepatoprotective effect of selenium-nanoparticles and dates flesh extract on carbon tetrachloride induced liver damage in albino rats

Ghada Nady Ouais1,2 , Doaa Mohamad Hassan3

1Department of Human Anatomy and Embryology, Faculty of Medicine, Cairo University, Cairo, 2Department of Human Anatomy and Embryology, Faculty of Medicine, Newgiza University, Giza, 3Department of Human Anatomy and Embryology, Faculty of Medicine, Beni-Suef University, Beni Suef, Egypt

Correspondence to:Doaa Mohamad Hassan
Department of Human Anatomy and Embryology, Faculty of Medicine, Beni-Suef University, Beni Suef 62511, Egypt
E-mail: doaa_eldary333@yahoo.com

Received: April 14, 2023; Revised: May 27, 2023; Accepted: June 30, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Exposure to environmental pollutants such as carbon tetrachloride (CCL4) causes liver damage. This study aimed to compare the ameliorative activity of the dates flesh extract (DFE) and selenium-nanoparticles (SeNPs) on CCL4-induced hepatotoxicity and if DFE could be a useful alternative supplement. Twenty-four male albino rats were enrolled and randomly divided into four equal groups (6 rats in each group): control group received only basal diet with no medications. Group II received CCL4 in a dose of 0.5 mg/kg intraperitoneal injection twice weekly for four weeks. Group III rats were pretreated with SeNPs in a dose of 2.5 mg/kg once a day orally three times/wk for four weeks alone then combined with the previously described dose of CCL4 for another four weeks. Group IV rats were pretreated with DFE in a dose of 8 ml of the aqueous extract/kg/d orally for four weeks alone then combined with the previously described dose of CCL4 for another four weeks. The liver damage was assessed by estimation of plasma concentration of albumin and enzymes activities of alanine aminotransferase and tissue genes expression. Liver oxidation levels were assessed by measuring the tissue concentration of the malondialdehyde, superoxide dismutase, and the total glutathione. Additionally, inflammatory mediators tumour necrosis factor--α and interleukin-6 were estimated. Detecting the liver’s cellular structural damage was done by histopathological and immunohistochemical examination. This study suggests that CCL4-induced liver damage in rats can be protected by administration whether the costly SeNPs or the economical DFE.

Keywords: Selenium-nanoparticles, Dates-flesh-extract, Hepatoprotection, Carbon tetrachloride

The liver plays an important role in the detoxification and the metabolic processes of many compounds whether endogenous or exogenous ones in the human body. Its exposure to toxic chemicals may overwhelm its antioxidant defense mechanism and cause hepatocellular damage because it is thought to be the target of many substances processing. The chemical molecule carbon tetrachloride (CCL4) is poisonous and predisposes to liver damage by producing the free radical trichloromethyl peroxyl radical, which results in an oxidative stress state [1]. Membrane lipids, enzymes, and DNA are all harmed in this hepatocellular injury [2-4].

Hepatic injury is a powerful activator of Kupffer cells and their cytokines production as well as the reactive oxygen species (ROS) such as malondialdehyde (MDA) and nitric oxide providing the pathological liver injury [1, 5]. Furthermore, the biochemical markers as alkaline phosphatase, alanine transaminase (ALT), bilirubin, aspartate transaminase, total cholesterol and total triglycerides were elevated in serum [6].

Since ROS plays an integral role in the development of liver disorders, antioxidants are thought to be protective against hepatic damage by increasing the activity of antioxidant enzymes [5, 7] and minimizing hepatic fibrosis [8].

Regretfully, the existing synthetic medications used to treat hepatic disorders may have harmful side effects, so that several studies intended to discover another substitute [9]. Based on studies, various oilseeds, fruits, vegetables, and medicinal plants have high antioxidant properties that may act against liver damage through their extracts, bioactive components, and oils [10].

Regarding folk medicine, the powder of date palm (Phoenix dactylifera L.) seeds is used by several producing nations to treat a variety of disorders including liver disorders, gastrointestinal disorders, diarrhea, throat diseases, cancer, toothaches, diabetes, and pulmonary disorders and infections [11]. It possesses anti-inflammatory, antioxidant, and hypolipidemic effects [12]. Date seeds show a substantial hepatoprotective effect in rats treated with CCL4, according to previous studies [13].

Date Flash or date seeds contain higher concentrations of polyphenols, including flavonoids, carotenoids, and other substances with antioxidant properties [14, 15]. The polyphenol profiles of the many date varieties are often not substantially different from one another. Moreover, dates contain high levels of condensed tannins, which give some varieties of dates an astringent flavor [16].

It has been shown that dates flash aqueous extract effectively scavenges dangerous superoxide and hydroxyl radicals. Additionally, it can lessen the effects of iron-induced lipid peroxidation in rat brain homogenate [17, 18]. Recent research has found that when dates were ingested during particular times, the mRNA expression of the antioxidant enzymes and genes superoxide dismutase (SOD2), catalase (CAT), nuclear factor erythroid-2-related factor 2, and glutathione peroxidase was upregulated [19, 20].

Selenium (Se) is considered as one of the most beneficial elements on the human health, such as performing as antioxidant and anticancer supplement it regulates the functions of selenoproteins which could reduce the tissue oxidation and balancing the redox [21]. Selenium nanoparticles (SeNPs) have a high bioavailability and proper biological activities, as its greater surface area, better absorption, minimal toxicity and increasing bioactivity so it is used widely in biomedicine [22].

The fact that Se has a hepatoprotective effect is usually attributed to its antioxidant ability to limit the tissue oxidation by decreasing the ROS. The cellular redox defense system depends on the production of ROS and enzymatic antioxidants levels as SOD, CAT, and glutathione (GSH) [23].

The purpose of our study is to compare between the costly SeNPs and available and economical dates flesh extract (DFE) as protective supplements on the CCL4-induced hepatic damage by raising the antioxidant ability and restraining the tissue oxidative damage.

Animals

The Institutional Animal Care and Use Committee of Beni-Suef University granted approval for the experiment, which followed all ethical guidelines for using animals in research (NO. 022-339). Twenty-four adult male albino Sprague-Dawley rats, weighing 200–240 g was kept in cages with free access to food and water at a temperature of (20°C±2°C), a normal 12-hour light/dark cycle, and unrestricted access to basic pellet food and water. Two weeks before the trial began; the animals were brought inside the lab so they could acclimatize.

Chemicals

CCL4 of 100% concentration were acquired from Merck (Darmstadt) obtained from Algomhoria Company. It was diluted in 100 ml of olive oil and administered intraperitoneal (IP) twice a week at a dose of 0.5 mg/kg body weight [24].

The SeNPs was obtained from Nanotech Egypt for the photo electronics communication center. It was in the size range of 3–5 nm and was prepared to agree to the processes reported previously by [25].

Plant material

Dates were obtained from the ISIS Organic Company. The flesh was separated manually from the pits and flooded in cold distilled water weight to volume about 1:3 and kept for 48 hours at a 4°C with continuous stirring. The extract was filtered then the aqueous supernatant was used. Aqueous extract was selected because most of the antioxidants and the active components in dates are extracted in water. Daily in the morning, a dose of 8 ml/kg of the DFE was given orally by gastric tube for all the experimental periods [26].

Experimental design

The animals were divided into four equal groups at random as follows: Group I: control group (n=6); no medicine was given to the rats in this group. Group II: CCL4 group (n=6); IP injection of CCL4 dissolved in olive oil at a dose of 0.5 ml/kg twice weekly for four weeks [27]. Group III: SeNPs+CCL4 group (n=6); the rats were pretreated with SeNPs orally in a dose 2.5 mg/kg once a day three times a week for eight weeks [28]. In addition to the SeNPs treatment, rats received IP injections of CCL4 dissolved in olive oil twice a week for the following four weeks at the end of the fourth week. Group IV: DFE+CCL4 group (n=6); the rats were pretreated with DFE orally in a dose 8 ml of the aqueous extract/kg/day for eight weeks [9]. In addition to DFE treatment, rats received IP injections of CCL4 dissolved in olive oil at a dose of 0.5 ml/kg twice weekly for the following four weeks at the end of the fourth week.

At the end of each group treatment, the rats were anesthetized with IP injection of a mixture of ketamine 2% and xylazine 2% in a dose of ketamine 87 mg/kg and xylazine 13 mg/kg and blood samples were taken from the retro-orbital vein. The collected blood samples were centrifugation at 3,000 r/min for 15 minutes at 4°C to prepare the blood serum and used to estimate the serum liver parameters, antioxidant, oxidative stress markers and inflammatory markers. After that, the animals were euthanized by CO2 narcosis, and their livers were immediately dissected. In ice-cold sodium, potassium, and phosphate buffer, 30 mg of liver tissues from each group were homogenized (0.01 M, pH 7.4). To isolate the total RNA, the homogenates were processed. The rest of the liver tissues were fixed overnight in 10% neutral buffered formalin at 4°C for farther histological study.

Biochemical analysis

Evaluation of hepatic injury was performed by determinations of ALT and albumin in blood plasma at the end of the experiment. The provider of the ALT kit was BioMed (CAT no. GPT11240). Albumin kit was provided by BioMed (CAT no. ALB100200). Utilizing the colorimetric endpoint method, ALT activity and serum albumin levels were evaluated [29].

In addition, the quantitative activity of the antioxidant enzymes MDA, SOD, and total GSH in the liver tissue were measured [30-32].

Moreover, the serum levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) (catalog No. CSB-E11987r), interleukin-6 (IL-6) (catalog No. CSB-E04640r) were estimated by using ELISA Sunlong Biotech kits supplied by IG Technology company, Egypt, regarding the manufacturer’s recommendations.

Real-time polymerase chain reaction

A Beckman dual spectrophotometer (Beckman Coulter) was used to measure the quantity and quality of total RNA after it had been extracted using the RNA easy Mini Kit from Qiagen in Valencia, California. Reverse transcription of the isolated RNA was carried out using a high-capacity cDNA Reverse Transcriptase kit from Applied Biosystem in Foster City, California. After that, the cDNA was amplified in a 48-well plate using the Step One instrument (Applied Biosystem) and PCR Master Kit Syber Green I (Fermentas) as follows: 10 minutes at 95°C for enzyme activation, then 40 cycles of 15 seconds at 95°C, 20 seconds at 55°C, and 30 seconds at 72°C for the amplification step. By using the DDCt approach, each target gene’s expression was normalized by comparison to the mean critical threshold values of the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase. Each target gene-specific primer was used in an amount of approximately 1 lM. Below is a list of the primer sequences for all examined genes: hepatocellular growth factor (HGF); forward 50-CCTATTTCCCGTTGTGAAG-30 and reverse 50-ACTAACCATCCA CCCTACTG-30 (gene bank accession number NC 005103.4); transforming growth factor-β1 (TGF-β1); forward 50-TGCGCCTGCAGATTCAAG-30 as well as reverse 50-AGGTAACGCCAGGAATTGTTGCTA-30 (gene bank accession number NM021578.2). Matrix metalloproteinase (MMP-2); forward 50-CAAGTGGGACAAG AATCAGA-30 and reverse 50-GAGAAAAGCGTAGTGGAGTTAC-30 (gene bank accession number NC_005118.4).

Histological study

The previously preserved liver specimens were embedded in paraffin blocks, and then sections made from those blocks were unstained. Haematoxylin and eosin (H&E) and Masson’s trichrome stains were used to identify the histopathological alterations. Antibodies against α-smooth muscle actin (α-SMA) that are marketed from (Abcam) were used for immunohistochemical (IHC) staining, followed by goat anti-mouse antibody conjugated to horseradish peroxidase (Dako) and colored with diaminobenzidine (DAB) tetrahydrochloride (Dako) [33].

The sections were initially blocked with 1.5% normal goat serum in poly(butylene succinate) (PBS) before being incubated for 45 minutes at room temperature with 6 ml of prediluted primary mouse monoclonal anti α-SMA antibody (ab5694), which was kept at 4°C, to immunostain them for α-SMA. The slices were then treated with a second stage biotinylated antibody (biotin-conjugated goat anti-rabbit IgG, 1:200, and one hour, at room temperature). The results of the reaction were then seen by soaking the slice in chromogen DAB after washing it in PBS. The sections were then mounted, dried, and counterstained with Mayer’s haematoxylin. A human tonsil sample served as the positive tissue control. Cells that were positive for α-SMA exhibited a brown cytoplasmic reaction. While ignoring the step of 1ry Ab, the negative control was managed the same way.

Morphometric study

At the Faculty of Medicine, Beni-Suef University, the analysis of Masson’s trichrome and IHC stained sections was performed at higher magnification (40 objective, scale bar 50 m). This study used 10 non-overlapping readings from each animal in different groups, which were calculated by two observers who were blind to the experimental group.

Statistical analysis

Using the SPSS version 16.0 statistical program (SPSS Inc.), the biochemical and morphometric data were statistically analyzed. Information presented as mean±SE. Analysis of variance with one way (ANOVA) was used to evaluate comparisons between the groups. For statistical significance, a difference has to be P<0.05.

Biochemical assays

The biochemical analyses of ALT and albumin in blood samples are presented in (Fig. 1). CCL4 treated group showed significantly (P≤0.05) increase in ALT and significantly decrease in the albumin levels compared to control group representing hepatic injury. Also, it was significantly (P≤0.05) altered in SeNPs and DFE treated groups compared to CCL4 group to levels similar to control group indicating their hepatoprotection ability against CCL4 hepatotoxicity. The presence of MDA, SOD, and GSH in liver tissues is shown in (Fig. 2). The tissue MDA level increased significantly (P≤0.05) in the CCL4 group compared to the control group, indicating lipid peroxidation and oxidative injury. Additionally, compared to the CCL4 group, it was (P≤0.05) significantly reduced in the SeNPs and DFE groups to levels comparable to the control group. Also, there was a significant (P≤0.05) decrease in the tissue antioxidants SOD and GSH in the CCL4 group compared to the control group. Furthermore, they were both moderately (P≤0.05) and significantly (P≤0.05) elevated in the SeNPs and DFE groups compared to CCL4 group levels similar to control, confirming the antioxidant abilities of both SeNPs and DFE.

Figure 1. The mean levels of liver functions: (A) ALT [U/L) and (B) albumin [g/dl) were assessed in all studied groups. *Significant difference versus control group, #significant difference versus CCL4 group (P-value<0.05). ALT, alanine aminotransferase; CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

Figure 2. Enzymatic activity of (A) SOD (U/ml), (B) GSH-px (mU/ml), and (C) MDA (nmol/ml) in liver tissue of all studied groups. *Significant difference versus control group, #significant difference versus CCL4 group (P-value<0.05). SOD, superoxide dismutase; GSH- px, glutathione peroxidase; MDA, malondialdehyde; CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

Concerning inflammatory markers (Fig. 3); TNF-α and IL-6 were significantly elevated in CCL4 group compared to control group. These levels were significantly reduced compared to CCL4 group in both SeNP and DEF treated groups (P<0.05). While, no statistically significant difference between the both treated groups (P>0.05).

Figure 3. Mean of serum levels of inflammatory markers (A) TNF-α (pg/mg) and (B) IL-6 (pg/mg). *Significant difference versus control group, #significant difference versus CCL4 group (P-value<0.05). TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

The quantitative reverse transcriptase-polymerase chain reaction analysis of the expression of the HGF, TGF-β1, and MMP-2 genes in liver tissue are reported in (Fig. 4). The level of TGF-β1 gene expression was considerably (P≤0.05) upregulated in the CCL4 group compared to the control group, this gene is a key regulator in all liver disorders. Additionally, its expression was downregulated in the SeNPs and DFE groups compared to the CCL4 group. However, no statistically significant difference between both treated groups (P>0.05). When compared to the control group, MMP-2, which is linked to matrix breakdown, was significantly (P≤0.05) downregulated in the CCL4 group. However, when compared to the CCL4 group, its expression was significantly (P≤0.05) upregulated in the SeNPs and DFE groups. However, no statistically significant difference between both treated groups (P>0.05). Both the SeNPs and DFE groups showed statistically significant upregulation in the levels of HGF gene expression as compared to the CCL4 group, suggesting increased cellular proliferation. No significant difference between the treated groups each other (P>0.05).

Figure 4. Mean of genes expression of (A) TGF-β1, (B) HGF, and (C) MMP-2 in liver tissue of all studied groups. *Significant difference versus control group, #significant difference versus CCL4 group (P-value<0.05). TGF-β1, transforming growth factor-β1; HGF, hepatocellular growth factor; MMP-2, matrix metalloproteinase; CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

Histological results

Histopathological examination of the liver H&E-stained sections: The histological pictures of all liver specimens of control groups represented practically the same findings as well defined central hepatic vein and the polygonal hepatocytes with vesicular nuclei and acidophilic cytoplasm. Thin blood sinusoids radiate between the hepatocytes cords (Fig. 5A, B). The CCL4 group demonstrated disarrangement of most of the hepatocytes and possessed cellular degeneration in the form of cytoplasmic vacuolations and cytoplasmic rarefaction. Other hepatocytes displayed pyknotic and vesicular nuclei. Additionally, regions with dilated, congested central veins and periportal cellular infiltration were demonstrated (Fig. 5C, D). The SeNPs group, the architecture of the hepatocytes in the liver tissue had improved. The hepatocytes relatively regained their normal arrangement in the form of hepatic cords. Although, small dark pyknotic nuclei surrounding mild congested central vein were also noticed (Fig. 5E, F). The DFE group obviously showed suppression of the inflammatory cellular response with relatively improvement in the hepatocyte’s degeneration as they appeared like those in the control group. Although, few hepatocytes were presented with slight vacuolated cytoplasm (Fig. 5G, H).

Figure 5. H&E-stained liver sections; (A, B) Control group, the hepatic central vein (CV) with polygonal hepatocytes (H) shows acidophilic cytoplasm and vesicular nucleus. Thin wall blood sinusoids (S) radiating between hepatocytes cords with their lining endothelium was seen. (C) CCL4 group, the hepatocytes show disorganization of hepatic cords. The portal regions show cellular degeneration (arrows), some hepatocytes showed pyknotic nuclei (arrow heads) while others have vesicular nuclei (H). Some hepatocytes show rarefication of their cytoplasm (curved arrows) while others have cytoplasmic vacuolations (V) and hemorrhage (stars). (D) Another section in the same group shows marked dilated congested vein in the periportal and pericentral areas (star). Also, cellular degeneration with inflammatory infiltrate (IF) in the portal region (arrows) was seen. (E, F) SeNPs group, the hepatocytes show more or less normal architecture (arrowheads) with normal hepatic cords. Note small dark pyknotic nuclei (arrows) and mild congestion of the CV. (G, H) DFE group, effectively cessation of inflammatory response with the improvement of hepatocytic degeneration and regain of the normal architecture excluding some areas (arrows). Some hepatocytes show cytoplasmic vacuolations (V). CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles.

Masson’s trichrome stained sections: the control group normal amount and distribution of collagen fibers around the portal area (Fig. 6A). CCL4 group, there is marked irregular collagen fibers deposition around the blood vessels in the portal area (Fig. 6B, C). While, SeNPs group showed obviously minimal collagen fibers deposition restricted to the portal area (Fig. 6D, E). DFE group showed decreased collagen fibers deposition (Fig. 6F, G). The area percentage of collagen fibers deposition around the portal area by morphometric analysis showed a significant increase (P<0.001) in CCL4 alone as compared to the other groups and a significant decrease SeNPs and DFE treated groups as compared to the only treated CCL4 group. Additionally, there is no significant deference in the results of both SeNPs and DFE treated groups (Fig. 6H).

Figure 6. Masson’s trichrome-stained liver sections; (A) Control group, show collagen fibers normally distributed (arrows) around the portal tract. (B, C) CCL4 group, shows a marked increase in collagen fibers (arrows) deposition around the blood vessels in the portal area. (D, E) SeNPs group, shows minimal collagen fibers deposition (long arrows) around the blood vessels in the portal area and between cells (short arrows). (F, G) DFE group, shows decreased collagen fibers deposition (arrows) around the blood vessels in the portal area. (H) Statistical analysis of the area percentage of collagen fibers deposition shows *significant difference against the control group. #Represents a significant difference against the CCL4 group. CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

Immunohistochemical stained sections with α-SMA of control group revealed a mild immune-reacted α-SMA positive cells, in between the hepatic lobules and around the central veins (Fig. 7A, B). Additionally, in the CCL4 group, numerous intensely immune-reacted α-SMA positive cells were detected between the hepatocytes in the connective tissue septa between the hepatic lobules (Fig. 7C, D), as the hepatocytes showed positive dense brown cytoplasmic reaction. These α-SMA positive cells were apparently decreased in the SeNPs group (Fig. 7E, F) and were moderately decreased in the DFE group (Fig. 7G, H).The percentage of α-SMA positive reactions by morphometric analysis showed a significant increase (P<0.001) in CCL4 alone comparable with the other groups and a significant decrease SeNPs and DFE treated groups as compared to the only treated CCL4 group. Additionally, no significant deference in the results of both SeNPs and DFE treated groups (Fig. 7I).

Figure 7. Immunohistochemical staining with α-SMA of (A, B) control group, most of the hepatocytes show negative reaction except few with mild cytoplasmic immune reaction. (C, D) CCL4 group, most hepatocytes have an intense immune reaction in hepatocytes and in-between hepatic lobules. (E, F) SeNPs group, shows a minimal immune reaction in vascular walls (arrows) and very mild reaction in between hepatic lobules. (G, H) DFE group, show mild to a moderate immune reaction in between hepatic lobules and among the hepatocytes (arrows). (I) Statistical analysis of the percentage of α-SMA positive reactions shows *significant difference against the control group. #Represents a significant difference against the CCL4 group. α-SMA, α-smooth muscle actin; CCL4, carbon tetrachloride; SeNPs, selenium-nanoparticles; DFE, dates flesh extract.

CCL4 is known as a hepatotoxicant component, and transformed into several metabolites that have a role in cirrhosis, hepatocellular carcinoma, and other liver diseases. Ebaid et al. [34] reported that accompanying its free radical’s production, the hepatocytes of animal models were damaged inducing hepatic injuries. Moreover, it altered lipid profiles and had a strong impact on how much ALT and aspartate aminotransferase were released into the plasma.

Various therapy strategies have been proposed in both clinical and experimental trials to reduce or stop the hepatotoxicity caused by CCL4 [35]. Selenium has a wide range of biological activity and a high bioavailability, making it highly predictable in biomedicine. Also, it has been claimed that its nano form possesses antioxidant and anti-inflammatory properties [36].

Abdelaziz and Ali [13] reported that date seeds show hepatoprotective properties in rats treated with CCL4 on top of its high content in caffeic acid, p-coumaric acid, quercetin, and rutin, as well as their antioxidant properties. The prior authors reported that dates could improve the alterations in the liver tissue vacuolization, and fibroblast proliferation and the liver function enzymes affected by CCL4. Parallel to our findings Bouhlali et al. [37] proved that dates seed extracts have powerful liver protective impact.

The goal of the current study was to compare the effects of SeNPs with highly nutritive and economical DFE by assessing the histopathological, IHC, and biochemical alterations linked to hepatotoxicity caused by CCL4 in an adult albino rat model.

In the current study, comparing the CCL4 treated group to the healthy control group, there was increased in the blood ALT and decreased in albumin levels, both of which were statistically significant and indicating hepatocellular damage. These outcomes were agreeing with Dong et al. [38], Shrestha et al. [39] and Salem et al. [40]. The previous authors suggested that the raised level of ALT was due to an insufficiency of the cell membrane selective permeability, following hepatotoxicity resulted in cellular leakage of hepatic enzymes into the bloodstream. Also, in the current study by consumption of SeNPs or DFE showed a significant decrease in the ALT and increase in albumin blood levels indicating their hepatoprotective properties.

In line with our study, Pettie et al. [41] demonstrated that Se provided protective effect against hepatotoxicity through maintaining the structural integrity of the hepatocellular membrane in rats. The authors used the biochemical markers; ALT and albumin to assess hepatic injury. Moreover, Abdeen et al. [42] described that date palm fruit caused significant improvement of hepato-renal function in gentamicin intoxicated animals by changing ALT and albumin serum levels close to normal. On the other hand, Habib and Ibrahim [43] reported that date seed supplementation did not alter serum ALT or albumin.

In the current study, the group treated with CCL4 had significantly lower levels of GSH and SOD and higher levels of MDA than control group, which were both indicators of the enhanced oxidative stress status. This may indicate that the CCL4-induced hepatotoxicity involves an oxidative effect and disturbed the balance between the antioxidant capability and harmful products in the tissues. In the current study, the SeNPs group showed a statistically increased in the tissue antioxidants GSH and SOD while in DFE group there was a moderate increase, these results supported by Essa et al. [44] and Xiao et al. [45]. Contradictory to our results Siddiqui et al. [46] reported that the date pulp extract delighted the production of ROS in human hepatocellular carcinoma cells, inducing oxidative stress thus promoting apoptosis. Also, the results of the current study clearly showed that SeNPs and DFE groups revealed lower levels of hepatocellular lipid peroxidation and decreasing MDA level. These findings were in agreement with Habib and Ibrahim [43] who reported that, date seeds significantly reduced liver and serum MDA and lipid peroxidation. These results were in line with Xiao et al. [45] who assessed the Se antioxidant activities.

Regarding liver injury, the hepatic Kupffer cells produce the pro-inflammatory cytokines causing activation and stimulation of hepatic stellate cells (HSCs) and enhancing extra cellular fibrogenesis [47]. HSCs can be altered to myofibroblasts like cells which express α-SMA and produce inflammatory cytokines like TGF-β1, IL-6, and TNF-α that produce excessive fibrosis in the extracellular matrix and liver inflammation [48].

The current study has shown that the serum level of the proinflammatory mediators TNF-α and IL-6 is substantially reduced in the SeNPs and DFE treated groups compared to the CCL4 group, demonstrating an impressive anti-inflammatory impact. According to Kang et al. [49], toxic substances cause inflammation by activating macrophages and triggering the production of cytokines that promote inflammation. Our results match with that reported by Gangemi et al. [50] and Ali et al. [51], who reach the conclusion that the pathogenesis of inflammation involves the involvement of oxidative stress in the activation of redox-sensitive transcription factors that regulate the expression of pro-inflammatory mediators and antioxidants.

Additionally, the current work demonstrated statistically significant increase in the expression of a pro-fibrogenic factor; TGF-β1 gene in the CCL4 group compared to the control group. Likewise, consumption of DFE and SeNPs reduced its expression. Also, by Comparing between the group treated by SeNPs and DFE on one side and CCL4 group on the other side, there were induction of the gene expression of the MMP-2 and HGF. In line with our findings, by using dates aqueous extracts Khan et al. [52] reported the extract could restore liver damage and return gene expression and enzyme activity of antioxidant enzymes, liver enzymes, and cytokines to normal levels. Harmonious with previous studies by Malyar et al. [53] who suggested that Se supplementation could decrease α-SMA and TGF-β1 mRNA expression level, indicating that the liver fibrosis caused by heat stress could be improved by Se.

Tissue histopathological evaluations are crucial instruments that are regarded as a cornerstone method for assessing the impact of drugs or chemicals in research on experimental animals for human disease. In the current study, histological analysis using H&E stain showed that the CCL4 treated group had dilated congested central veins, altered architecture, and inflammatory cell infiltrate. In the liver portal area, Masson’s trichrome staining revealed extensive collagen deposition, a sign of fibrosis in the CCL4 treated group. According to numerous reports, CCL4 causes free radicals to form and activates macrophages, which produces inflammatory and profibrogenic mediators [54]. The production of free radicals is the major process causing membrane lipid peroxidation, apoptosis, and necrosis [55].

In this experiment, co-treatment with SeNPs reduced or eliminated the histopathological changes brought on by CCL4 induced hepatotoxicity. Their antioxidant function, which is mediated by free radical scavenging, may be the source of this protective effect. In a study of acetaminophen-induced hepatotoxicity, Rashid et al. [56] demonstrated that Se co-treatment eliminated the histopathological consequences. Moreover, Yan et al. [57] found that SeNP has protective potentials as a powerful antioxidant in restoring the integrity of liver mitochondria to maintaining the normal function of cells, resisting lowered ROS production, and protecting against CCL4 induced hepatotoxicity. The mitochondrial membrane may be stable, preserving the structure and activities of the mitochondria [58].

Our findings regarding DFE co-treatment were consistent with those made by Roshankhah et al. [59], who suggested that date seeds extract could lessen the effects of vacuolization, congestion, and fibroblast proliferation on liver histopathology in rats with mercury-induced hepatotoxicity.

In the current study, compared to the control group, the area percentage of α-SMA positive cells was significantly higher in the CCL4 group, and it returned to be lower than the control in the SeNPs and DFE groups. Myofibroblasts-like cells can be identified by the detection of α-SMA [60]. According to previous information, hepatic fibrosis typically begins with hepatocyte injury, which stimulates Kupffer cells and secretes cytokines and growth factors. This caused fibroblast-like cells to multiply and release a lot of connective tissue components [61]. Another element that promotes the growth of fibroblasts and a component of connective tissue is the oxidative stress caused by CCL4 [62]. In this study, the substantial antifibrogenic impact of SeNPs and DFE after liver damage may be shown by the healing mechanism. In concurrence with these findings, Hamid et al. [63] reported that CCL4 increases hepatic immune-expression of α-SMA but there was a moderate expression in the Se-enriched yeast groups. Also, Gad et al. [64] observed a mild brown immuno-expression of α-SMA in the portal area by using methanolic date flesh extract against cisplatin-induced hepatic injury.

In agreement with our findings, Al-Qarawi et al. [65] found that date seed extract can restore the liver normal functional status and defend against CCL4 induced liver damage in rats. This is corroborated by Mansouri et al. [66], who found that dates’ aqueous extracts exhibit potent antioxidant activity. The current study used SeNPs and dates to reduce tissue damage and restore the normal oxidative stress balance, potentially via causing oxidative stability because of their antioxidant and anti-inflammatory properties.

Our results are consistent with the commonly accepted view that Se is an essential mineral for humans and that it is suggested as a dietary supplement because of its multiple health benefits inflammatory macrophages function as a physiological defense against toxins and injury. The result is considerable liver fibrosis and inflammation, according to histopathological examinations of the immune cells’ aggressive behavior. The inflammatory infiltration of immune cells into the hepatic tissue was shown to be greatly decreased by SeNPs [34].

Recent studies have shown that several natural compounds contain anti-inflammatory and antioxidant properties as well as damage repair mechanisms that protect against cytotoxicity. The presence of numerous phenolic chemicals in dates, including p-coumaric, ferulic, sinapic acids, flavonoids, and procyanidins, is thought to be the cause of DFE antioxidant action [58].

According to our findings, numerous research has demonstrated that dates have anti-inflammatory, immune-stimulating, anti-hyperglycemic, anti-cholesterol, antioxidant, and reno-protective characteristics [67, 68]. In a recently published a clinical control study that significantly demonstrated an improvement in sperm count, motility, and morphology in both fertile and infertile men [19]. The prior author reported that date consumption resulted in upregulation of mRNA expression of antioxidant enzymes and genes.

The current results point to a possible potential of SeNPs to treat in vivo CCL4 induced liver damage. It was found that SeNPs significantly reduced immune cells’ inflammatory infiltration of the hepatic tissue. Even further study is needed to understand the underlying pathways. The high concentrations of antioxidant molecules in dates may help to explain the protective properties, at least in part (polyphenols and flavonoids). It would also be a financially viable alternative to SeNPs. However, more study is required to fully comprehend the beneficial elements and mechanisms underlying this protective impact of data.

Conceptualization: GNO. Data acquisition: GNO. Data analysis or interpretation: DMH. Drafting of the manuscript: DMH. Critical revision of the manuscript: GNO, DMH. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

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