Hepatic and immune modulatory effectiveness of lactoferrin loaded Selenium nanoparticles on bleomycin induced hepatic injury

This study aimed to estimate the hepatic and immune ameliorating potential of extracted bovine lactoferrin (LF), Selenium nanoparticles (SeNPs) or their combination (LF/SeNPs) against bleomycin (BLM) induced hepatic injury. Fifty adult male rats (160–200 g) were equally divided into five groups: (1) the saline control group, (2) BLM-injected (15 mg/kg twice a week, ip), and (3–5) groups treated orally with LF (200 mg/kg/day), SeNPs (0.0486 mg/kg/day) or LF/SeNPs combination (200.0486 mg/kg/day) for 6 weeks post BLM-intoxication. Blood and liver samples were subjected to biochemical, histopathological, and immunohistochemical analyses. The results revealed that BLM caused a significant increase in hepatic lipid peroxidation and nitric oxide, as well as serum markers of liver functions (AST, ALT and GGT activities), and levels of GM-CSF, CD4, TNF-α, IL-1β, TGF-β1, fibronectin, triglycerides, cholesterol and LDL-C. Additionally, hepatic glutathione, Na+/K+-ATPase, and glutathione peroxidase, as well as serum HDL-C, total protein and albumin levels were significantly reduced. Moreover, BLM injection resulted in marked histopathological alterations and severe expression of caspase 3. Post-treatment of BLM-intoxicated rats with LF, SeNPs or LF/SeNPs combination obviously improved the BLM-induced hepatic damages; this was achieved from the marked modulations in the mentioned parameters, besides improving the histopathological hepatic architecture. It is worth mentioning that LF/SeNPs exerted the greatest potency. In conclusion, the obtained results demonstrated that LF, SeNPs and LF/SeNPs succeeded in attenuating the BLM-induced hepatic dysfunction. Therefore, these supplements might be used to protect against drug-associated side effects.


Lactoferrin-Selenium nanoparticles (LF/SeNPs) combination
A combination of LF/SeNPs was prepared by dissolving LF (1 g) and SeNPs (1 mg) in 10 ml deionized water, then the solution was cool-stirred (4 °C) for 60 min, then followed by cool-centrifugation at 4500 rpm for 10 min at 4 °C, then the precipitate was washed with phosphate buffer saline and recentrifuged 17 .The un-ligated metal ions, in the precipitated LF/SeNPs combination, were then removed thorough washing by 0.1 M sodium bicarbonate though dialysis.Then, the dialyzed LF/SeNPs were freeze-dried to produce a solid powder before being stored at -80 °C for further applications.Regarding the capacity of lactoferrin to bound Se, Higuchi et al. 17 reported that each 100 g lactoferrin can bound 24.3 mg of Selenium; therefore, the calculated dose that used in this study was 200.0486 mg of LF/SeNPs combination per kg; this dose is equivalent to a dose of 200 mg LF/kg 18 or equivalent to dose of 0.0486 mg SeNPs/kg 19 .

Experimental design
Adult male Wistar albino rats weighing 160-200 g were obtained from Animal Colony, National Research Centre, Giza, Egypt; the animals were housed in plastic cages and fed with standard laboratory diet and water ad libitum.All animals received human care in accordance with the institutional standards for the handling and use of experimental animals, where the study proposal was approved by the ethical committee of faculty of science, Al-Azhar university, Assuit (approval No AZHAR 14/2023).All methods were carried out in accordance with ARRIVE guidelines.
After acclimation for a week, all animals were randomly divided into five groups (10 rats each) as follows: the first group was injected intraperitoneally with saline and served as control; the second group was injected intraperitoneally, twice a week, with 35 U/kg of BLM dissolved in sterile saline for six weeks 20 ; the third and fourth groups were injected intraperitoneally with BLM for 6 weeks, then orally administrated with either LF (200 mg/kg/day) 18 or SeNPs (0.0486 mg/kg/day) 19 for other 6 weeks; the fifth group was intoxicated with BLM for 6 weeks, then treated with LF/SeNPs formulation (200.0486mg/kg/day that included 200 mg LF and 0.0486 mg SeNPs) for other 6 weeks.

Blood and tissue sampling
At the end of the treatments period, rats were fasted overnight and following anesthesia by isoflurane inhalation, blood samples were withdrawn from the retro-orbital plexus using heparinized-sterile glass capillaries.The coagulated blood samples were cool centrifuged at 3000 rpm for 15 min and the sera were separated and stored at − 80 °C till biochemical analysis.After blood collection, the animals were killed by sudden decapitation, and

Immunohistochemistry (IHC) analysis
Five µm thickness sections of the processed liver tissues from each rat for all groups were immune-stained for 90 min with anti-Caspase-3 primary antibody.Following the application of the secondary antibody via the immunoperoxidase method, the sections were stained using the Universal Staining Kit, "DAB."(ScyTekLaboratories, Inc. Logan, UT, USA) at room temperature for half an hour 24 .

Quantitative evaluation of IHC staining
Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues.The semi-quantitative IHC involves using ImageJ Fiji software (Johannes Schindelin, Albert Cardona, Mark Longair, Benjamin Schmid, and others, https:// imagej.net/ Fiji/ Downl oads), version 1.2 (no specific plugin was used) to conduct deconvolution and downstream analysis.The area percent for positive caspase-3 immunoreaction was measured at magnification X 400 for all groups 25 .

Statistical analysis
Comparisons between means were carried out using one-way analysis of variance (ANOVA) followed by post hock (Tukey) multiple comparisons test at p ≥ 0.05.This was carried out using statistical analysis system (SAS) program software; copyright (c) 1998 by SAS Institute Inc., Cary, NC, USA.

Ethics declarations
All animals received human care in accordance with the institutional standards for the handling and use of experimental animals, where the study proposal was approved by the ethical committee of faculty of science, Al-Azhar university, Assuit (approval No AZHAR 14/2023).All methods were carried out in accordance with ARRIVE guidelines.

Extraction and characterization of lactoferrin
The extraction, isolation, and purification process of lactoferrin revealed that bovine milk contains 3.92 mg/ml.The HPLC characterization of the pure-isolated lactoferrin is illustrated in Fig. 1 www.nature.com/scientificreports/

Biochemical results
In the present study, BLM-intoxication resulted in a significant elevation in the activities of serum ALT, AST, ALP and GGT coupled with a significant decrease in levels of albumin and total protein as compared to the control group.In a favorable manner, post-treatment of BLM-intoxicated animals with either lactoferrin or SeNPs showed a significant improvement in the mentioned parameters although their values were still away from those of the control group.Moreover, animals intoxicated with BLM and having received the LF/SeNPs recorded values comparable to those of the control (Table 1).
Post-BLM-intoxication, the animals showed significant increases in serum cholesterol, triglycerides, and LDL-c levels, matched with a marked drop in HDL-c level in compared to the control group.Treatment of BLMintoxicated animals with either LF, SeNPs or LF/SeNPs formulation significantly reversed the levels of the above parameters of lipid profile toward the corresponding values of the normal controls; Attention showed he drown here, the combined LF/SeNPs was more effective than the single treatment of LF or SeNPs (Table 2).
The present study revealed that BLM-intoxication induced a meaningful elevation in the hepatic MDA and NO levels, associated with a remarkable reduction in the values of hepatic content of GSH and activities of GPx and ATPase when compared with the control group.On the other hand, post-treatment with LF, SeNPs, or LF/ SeNPs formulation succeeded to markedly decrease hepatic MDA and NO levels; moreover, hepatic GSH content  www.nature.com/scientificreports/as well as GPx and Na + /K + -ATPase activities showed a significant increase in compared to the corresponding results of the untreated BLM-intoxicated-group.These supplements, either singly or combined, exhibited an antioxidant-ameliorating potency; this exhibition was more pronounced in the group treated with LF/SeNPs formulation (Table 3).The obtained data revealed that animals intoxicated with BLM showed significant increases in the serum level of apoptotic markers (GM-CSF and CD4), pro-inflammatory cytokines (TNF-α and IL-1β), and the pro-fibrotic markers (TGF-β1 and fibronectin) in compared to their corresponding values of control group.Favorably, treatment of BLM-intoxicated animals with LF, SeNPs, or LF/SeNPs formulation showed a significant amelioration in the values of all above-mentioned markers.It is worth noting that treatment with LF/SeNPs formulation exerted the most remarkable improvement (Fig. 2).

Immunohistochemistry analysis
The immunohistochemistry analysis revealed the occurrence of a very weak expression of caspase-3 in liver tissue of the control rats (Fig. 3A).The BLM-intoxication group showed severe expression of caspase-3 (Fig. 3B).The other groups revealed a reduced caspase-3 immunoreactivity in compared to the untreated BLM-intoxicated group (Fig. 3C-E), with approximately normal caspase-3 expression in LF/SeNPs formulation treated-group (Fig. 3E).There was a significant difference between BLM-intoxicated group and the groups treated with LF, SeNPs or LF/SeNPs formulation regarding caspase-3 IHC immunoreactivity with substantial reduced caspase-3 positivity in the treatment groups (Fig. 4).

Histopathological examination
The histopathological examination of the control group liver tissues showed normal structure of hepatic lobules with branching, radiating, and anastomosing hepatic cords with the central vein.The hepatic sinusoids in between the hepatic cords were lined with squamous endothelial cells with flat nuclei.The hepatocytes were polygonal in shape with central rounded vesicular nuclei and acidophilic cytoplasm with few binucleated hepatocytes (Fig. 5A).BLM-treated group revealed marked histopathological alterations.Most of the hepatocytes displayed marked necrosis with remarkable pyknotic nuclei (Fig. 5B).BLM /LF and BLM /Selenium NPs-treated groups showed normal hepatic structures with some congested hepatic sinusoids and focal necrosis in BLM / LF group (Fig. 5C, D).The BLM /LF/Selenium NPs-treated group showed improved histological structure of hepatic lobules and rearranged radiating hepatic ducts (Fig. 5E).

Discussion
The liver has a vital role in metabolism and detoxification of xenobiotics, metabolites, and drugs 26 .Bleomycin, an anticancer drug, is inactivated by bleomycin hydrolase in hepatic cells.With the deficiency of BLM hydrolase in liver, BLM exerts its toxic damage through the induction of oxidative stress and DNA damage 27 .BLM can damage DNA through the formation of DNA/Fe2+/BLM complex which leads to the generation of ROS, including superoxide and hydroxyl radicals that damage DNA, lipids, and proteins 28 .Oxidative stress occurs when the capability of the anti-oxidative defense system in eliminating the free radicals and ROS decreases; oxidative damage occurs because of increased lipid peroxidation and diminution of the antioxidant effects of thiol groups in organs 29 .
In this study, hepatocytes damage is evidenced by the marked increments in the values of serum ALT, AST, ALP and GGT and the decrement in levels of albumin and total protein in BLM-treated group.Similar observations were recorded by Kandhare et al. 28 .These markers are considered as indicators that confirm the presence of liver dysfunction.In the case of hepatic membrane damage, liver enzymes are released into the blood and are measured in blood serum as markers of liver injury 30 .Additionally, lipid profile was disturbed also by BLM treatment.Lipids concentration is correlated with the metabolic functions which are affected by liver integrity.Lipids like cholesterol and triglycerides were found to increase in various hepatic diseases.Also, Seki et al. 31 reported that lipid content in liver cells is affected by the integrated activities of cellular enzymes that catalyze lipids metabolism.
In the present study, oxidative injury in BLM-treated rats appeared from the marked reduction in GSH and GPx values.GSH is an antioxidant tripeptide, it improves protection against oxidative agents-induced cell damage 32 .GPx which is a Seleno-proteins enzyme; it functions as protector and detoxifier from oxidative injury via free radicals scavenging 33 .Oxidative damage was asserted also by increased MDA and NO content, and histological changes in liver tissue.MDA is the end aldehyde product of peroxidation of the polyunsaturated Table 3. Oxidative stress markers and Na + /K + -ATPase activity of the studied groups.Data are presented as mean ± standard error.* is significant versus the control group, while # is significant versus BLM group at p ≤ 0.05.LF (lactoferrin); BLM (bleomycin); SeNPs (Selenium nanoparticles).
Vol:.( 1234567890 www.nature.com/scientificreports/fatty acids of cell membrane 34 .Our results are consistent with other studies, which demonstrated the involvement of lipid peroxidation and oxidative stress in BLM -induced liver injury 2 .An increase in NO levels has been reported also because of BLM-induced increases in inducible nitric oxide synthase (iNOS) at gen and protein levels 35 .The overproduction of NO by iNOS plays a vital role in inducing various inflammatory diseases including liver fibrosis 36 as NO reacts with superoxide radical and forms highly reactive peroxynitrites which lead to tissue injury 37 .Fig. 4. Intensity quantitative measurement for positive caspase-3 immune reactions.Data are presented as mean ± standard error; subjected to one-way ANOVA followed by post hoc (Duncan) test at p ≤ 0.05; * is significant versus the control group, while # is significant versus BLM group; LF (lactoferrin); BLM (bleomycin); SeNPs (Selenium nanoparticles).
Inflammatory mediators have been reported to be involved in BLM toxicity 38 .Elevated levels of GM-CSF and CD4 were observed in serum of bleomycin group.GM-CSF is a monomeric glycoprotein, functions as a cytokine, secreted by activated T cells, natural killer cells, and mast cells.GM-CSF stimulates stem cells to produce dendritic cells and macrophages 39 and stimulates also macrophages and monocytes to secrete pro-inflammatory cytokines 40 .CD4, a monomeric glycoprotein, is produced also by blood T lymphocytes; it has been suggested that CD4 participates in the signal transduction during T cell activation 41 .It has been reported that the CD4 T lymphocyte can induce some degree of fibrotic response; in this concern, Piguet 42 reported that the depletion of CD4 T lymphocytes by treatment with the anti CD4 mAb restricted the bleomycin-induced elevation of TNF mRNA level and fibrosis.
Serum levels of TNF-α, IL-1β and TGF-β1 in the current study exhibited a marked increase in BLM -treated animals.The release of these cytokines appears to be the main mediators of hepatic fibrosis 43 , Fibronectin also increased by BLM treatment.BLM has been reported to increase the release of the pro-inflammatory cytokines (TNF-α and IL-1β) via elevating ROS through the formation of the DNA/Fe2+/BLM complex 38 .These sinister events cause increased expression of TGF-β1, and activation of hepatic stellate cells that ultimately leads to the synthesis of scar-forming collagen, and consequently the development of liver fibrosis 28 .TGF-β1 is a pro-fibrotic www.nature.com/scientificreports/cytokine that can directly stimulate the differentiation of fibroblasts into myofibroblasts 44 , through stimulation of overexpression of fibronectin and collagen which are the main proteins of extra cellular matrix involved in the fibrotic process 45 .Although there is an elevation in the level of fibronectin in the BLM group in our study, this increment did not enough to develop liver fibrosis.This may be due to the short duration of BLM treatment.These results are asserted by the results of Vásquez-Garzón et al. 2 who reported that the fibrotic process in the liver takes place similarly to that has been occurred in the lung, but to a lesser extent due to the presence of hepatic bleomycin hydrolase.The inflammatory effects have been reported to increase expression of the pro-apoptotic markers 46 .The immunohistochemistry analysis revealed the occurrence of severe expression of the apoptotic marker, caspase-3, in liver cells of BLM-treated group.This marker is markedly stimulated in the cells that are exposed to cytotoxic agents and it plays a vital role in tumorigenesis after cell injury 47 .
Na + /K + -ATPase is an enzyme found in the plasma membrane in all animals 48 .It controls the active transport of potassium and sodium across the cell membrane; it also controls cell volume, muscle, and nerve signals, and boosts the transport of amino acids and sugars across the cell membrane.Na + /K + -ATPase is highly lipid-dependent; therefore, peroxidation of membrane lipids affects this enzyme; Bleomycin hepatotoxicity therefore leads to membrane lipid peroxidation that causes disturbance in membranes lipid structure, resulting in inhibiting Na + /K + -ATPase activity that was observed in the current study.Li et al. 49 found a significant negative correlation between the Na + /K + -ATPase and profibrotic molecules in alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF).The findings of these authors revealed that the downregulation of the Na + / K + ATPase β1 subunit enhances the expression of profibrotic protein in patients with IPF and a mouse model of pulmonary fibrosis.
The oxidative damage, caused by BLM, is considered the main mechanism that leads to the subsequent hepatic injury; therefore, the supplementation of natural antioxidants has been proposed to counteract the harmful effects of BLM and prevent liver injury.Administration of lactoferrin, SeNPs or their combination to BLM -treated animals was greatly effective in decreasing lipid peroxidation and improving antioxidant capability in the liver of BLM-treated group.This emerged from the marked decrease in hepatic MDA and NO contents and the significant rise in GSH and GPx values.This improvement was more pronounced in the animals received the combined treatment of lactoferrin and SeNPs, indicating the synergistic effect between lactoferrin and SeNPs.Mostly, the benefit role of both substances might be attributed to the antioxidant effect and their free radical scavenging property.These results were in line with that reported for lactoferrin which was characterized in the current study by the HPLC analysis and confirmed that of earlier study 11 .The obtained results were in line also with that reported for SeNPs 50 .Consequently, the possible antioxidant effect of SeNPs may be attributed mainly to its participation in the Seleno-proteins structure such as selenocysteine which is the active center of GPx enzyme 51 .This enzyme can help in reducing lipid peroxidation, intracellular hydrogen peroxide 52 and ROS generated by all the cells 53 .On the other hand, lactoferrin can act as an iron scavenger by direct iron-chelating property or by modification of the key iron-binding proteins 10 .
In addition, lactoferrin, SeNPs and their combination could also significantly counteract the values of serum ALT, AST, ALP, GGT, albumin and total protein, suggesting their membrane stabilizing effect that based on their antioxidant activity, which may be responsible for saving the hepatocytes against the oxidative damage.This in turn improves the liver integrity and function and consequently improves Na + /K + -ATPase activity, protein synthesis and lipid metabolism.This improvement was more pronounced in the group that taken lactoferrin plus SeNPs.
In consistence with our results, Wang et al. 50observed the protective effect of SeNPs against nickel-induced developmental hepatic damage in rats.SeNPs have been found to protect the liver from cadmium-induced toxicity by ameliorating markers of liver function and restoring the activity of antioxidant enzymes.Amin et al. 4 demonstrated the ameliorative effect of SeNPs against oxidative stress mediating liver injuries induced by acetaminophen toxicity through modulating the oxidative stress and decreasing DNA fragmentation.In another study conducted on rats, SeNPs have been found to attenuate the deleterious effects of hypothyroidism on renal and hepatic functions by normalizing the values of AST, ALT, ALP, creatinine, and BUN 54 .Additionally, Rizk et al. 55 confirmed the hypolipidemic activity of SeNPs as they noted that adding SeNPs to laying hens diet resulted in lowering cholesterol, triglycerides, and LDL-C, and raised HDL-C compared to the control group.These authors attributed this effect to the lipolysis that increased with Se intake.Moreover, Li et al. 56 indicated that lactoferrin treatment prevented ethanol-induced liver injury and counteracted the values of AST and ALT in mice by modification of hepatic alcohol metabolism and modulating gut microbiota.Furthermore, ingestion of lactoferrin can help in preventing disturbance of hepatic lipid metabolism, which is associated with nonalcoholic steatohepatitis 57 , feeding with high-fat diet and ethanol ingestion 56 .
The obtained data revealed that lactoferrin, SeNPs and their combination succeeded to decrease the levels of inflammatory mediators (GM-CSF, CD4, TNF-α, IL-1β and TGF-β1) to reach near the normal levels and reduced the fibrotic marker, fibronectin, in rats treated with BLM, suggesting the anti-inflammatory activity of these supplements.Moreover, the combined treatment was more effective than the treatment with lactoferrin or SeNPs alone.SeNPs are known to have anti-inflammatory and antioxidant effects 58 .In addition, it was reported that Se deficiency induces inflammation through the activation of NF-κB 59 which plays critical role in regulating gene expression of iNOS and inflammatory cytokines such as TNF-α, IL-6, and IL-8 60 .Indeed, as indicated previously, administration of SeNPs to nickel-exposed animals showed a significant amelioration of apoptosis and inhibition of cytochrome c, caspase-3 and caspase-9 through activation of PI3K/AKT signaling pathway 50 .SeNPs have been found also to improve cardiac fibrosis through the modulation of oxidative stress status in hypothyroid rats 61 .Additionally, Aoyam et al. 11 found that lactoferrin treatment reduced the expression the inflammatory cytokine (TNF-a, IL-6, and IL-1b) and fibrotic related molecules (TGF-β, Timp2, and Col1a1) mRNAs in liver via NF-κB inactivation.Thus, the protection, afforded by either lactoferrin and/or SeNPs against

Table 1 .
Liver function tests of the studied groups.Data are presented as mean ± standard error.* is significant versus the control group, while # is significant versus BLM group at p ≤ 0.05.LF (lactoferrin); BLM (bleomycin); SeNPs (Selenium nanoparticles).