Protective Role of Chichorium intybus Extract Against Renal Toxicity Induced by Magnetite Silver Nanoparticles in Male Rats

Corresponding author: Doha M. Beltagy Biochemistry Division, Chemistry Department, Faculty of Science, Damanhour University, Egypt E-mail: dohabel4@yahoo.com Abstract: Magnetite silver nanoparticles (Fe3O4/AgNPs) symbolize a new candidate in several industrial and biological products for their activities, simple preparation and low synthesis costs. Many studies recommended the usage of Fe3O4/AgNPs to overcome lacking biocompatibility of AgNPs inside the body. However, Fe3O4/AgNPs also has toxic effects on kidney tissues. In this study, AgNPs was synthesized on the surface of magnetic nanoparticles (Fe3O4) as a core-shell hetero-structure and used to induce renal injury. The microstructure, morphology and magnetic properties of Fe3O4/AgNPs were characterized using different analysis as X-ray diffraction, scanning electron microscopy and vibrating sample magnetometry. This study aimed to evaluate the protective effects of chicory (Chichorium intybus) fruit extract against kidney injury induced by Fe3O4/AgNPs administration in rats. The study was carried out on 40 male adult Sprague Dawley rats (12 weeks old, 140-145 g). Rats were randomly classified into four groups. Group-1 included control rats; group-2 contained the administered chicory extract rats; group-3 was the rats injected withFe3O4/AgNPs; and group-4-in which rats administered with Fe3O4/AgNPs then treated by chicory extract. The obtained results showed that kidney function tests as urea and creatinine increased by about 1.5 folds in rats injected with Fe3O4/AgNPs (G3). Malondialdehyde (MDA) concentrations in kidney tissue duplicated and serum potassium levels increased by about 1.2 folds, while serum sodium and calcium ions slightly decreased in G3. On the other hand, reduced glutathione declined in G3 to nearly one-half normal values in G1. Superoxide dismutase and catalase enzymes decreased to about 0.7 folds in G3. Chicory extract administration in G4 improved all these parameters to nearly their normal values. In conclusion, the obtained results in the current study indicated the protective effects of chicory extract against kidney injury and oxidative stress induced by Fe3O4/AgNPs.


Introduction
With the immense spread of nanoparticles (NPs, ranged from 1-100 nm) in industry and purchaser markets, the increased accumulation of nanomaterials in the environment and human exposure to NPs grew to be fact (Yousef et al., 2019). Silver nanoparticles (AgNPs) can be considered the most largely related nanomaterial in the medicinal and biochemical fields, which leads to take care about security restricts and the danger related to dissociate producing active Ag + into the human body. Assessments of AgNP toxicity are restricted and limited to only in vitro studies of its cytotoxicity and genotoxicity properties against different cell lines and tissue cultures (Hassanen et al., 2019;Altwaijry et al., 2021). All in vivo studies indicated that AgNPs transferred through blood stream once after absorption regardless the route of application. Inside the body, AgNPs can easily accumulate in several organelles and give rise to many pathological change such as hepatic dysfunction, renal injury and brain damage (Barbir et al., 2019). In order to try to overcome this limitation and improve its properties, many previous studies tried to use Magnetite silver nanoparticles (Fe3O4/AgNPs) which symbolized a promising candidate for their amazing biocidal activity, simple preparation and low production costs. However, it also still has toxic effects on kidney tissues (Hosu et al., 2019;Ivashchenko et al., 2015).
Acute Kidney Injury (AKI) can lead to nephron injuries which in turn, can lead to death. The major sign of AKI is the great decrease in Glomerular Filtration Rate (GFR) due to renal blood flow reduction. Inflammation is an important reason of AKI leading to extensive stage of injury, with losing sensitivity to the vasodilator therapy (Basile et al., 2012). The usage of traditional and alternative medicine has been recently increased. The antioxidant impacts of different plant extracts play important roles in several diseases treatment (Tousson et al., 2018;Mutar et al., 2020). The ancient Egyptians planted Cichorium intybus Linn to be used as a medicinal plant. It was used because of its many advisable outcomes as hepatoprotective, neuroprotective, hypolipidemic, antioxidant and anti-inflammatory activities (Keshk et al., 2019). Chemical analysis of the fresh chicory revealed that typically it composed of mainly inulin and sucrose (68 and 14%, respectively), other components as protein, cellulose and ash present small amounts. On the other hand, dried chicory composed approximately of 98% inulin (Hassan and Yousef, 2010).
The current study purposed to synthesize and characterize magnetite (Fe3O4)core-(Ag)shell nanoparticles and examine its toxic effect on kidney in rats. The study also aimed to evaluate the protective effects of chicory (Chichorium intybus) fruit extract against kidney injury induced by Fe3O4/AgNPs administration in rats.

Materials and Methods
All required chemicals as ferric chloride (FeCl3·6H2O), ferrous sulphate (FeSO4·6H2O), ammonia, silver nitrate and glucose were obtained with high purity degree from Sigma-Aldrich

Synthesis of Magnetite (Fe3O4)core-Agshell Nanoparticles
Magnetite nanoparticles (Fe3O4) had been prepared using the coprecipitation method as reported previously (Abdo et al., 2019). Fe3O4 nanoparticles were each coated using the noble metal silver (Ag). To coat the magnetite particles (Fe3O4) with silver, 0.5 g of glucose was added to Fe3O4 and silver nitrate with a molar ratio of 1: 0.5. Then, mixture was sonicated for about 15 min and incubated in a hot water bath with a slow stirring for 1 h. As the particles are gradually coated by silver, the black Fe3O4 particles converted gradually into a brownish color. After a while, the brown mass was precipitated at the bottom of the flask. After cooling to room temperature, it was centrifuged and the brownish precipitate was washed several times with water and then dried at 60°C.

Characterization of the Synthesized Fe3O4/AgNPs
The synthesized particles were characterized by Scanning Electron Microscopy (SEM), X-Ray Diffraction pattern (XRD) and Vibrating Sample Magnetometer (VSM).
The morphology of Fe3O4-Ag nanoparticles were analyzed using Scanning Electron Microscopy (JEOL SEM, JSM-636OLA, Japan) at an accelerated voltage 20 kV.
XRD measurements were recorded on (Shimadzu LabX XRD-6100 X-ray diffractometer, Japan). That was operated at a voltage of 40 kV and a current of 30 mA with an excitation source of CuKα radiation (λ= 1.541 Å), in the range of scanning angle 30 to 80 o at a scan rate of 5 o /min with the step width 0.02 o . For XRD measurements, Fe3O4-Ag nanoparticles data obtained as powder samples.
Magnetic characteristics were measured by VSM (Lake Shore-7410 vibrating sample magnetometer, USA), magnetic field up to 30000 Oe. Parameters like specific saturation Magnetization (Ms), coercivity (Hc) and retentivity (remanence Mr) were evaluated.
The average size of Fe3O4-Ag NPs calculated by applying Debye-Scherrer equation (Malapati et al., 2018), which gives a relationship between peak broadening in XRD and particle size that is demonstrated by following equation: D = kλ/(β·cosθ), where D is the particle size of the crystal, k is Scherrer constant (0.9), λ is the X-ray wavelength (0.154 nm), β is the width of the XRD peak at half-height (FWHM) and θ is the Bragg diffraction angle for calculating d-spacing from Bragg`s law as; d = n/2sin. The lattice constant (a) of the cubic crystal was computed using the d-spacing values and the respective (hkl) parameters as; a = d*√(h 2 +k 2 +l 2 ).

Extraction of Chicory
The fruits of chicory (Chichorium intybus Linn, edible herb growing wildly in Egypt among many other plants, blooming in late spring till mid-fall) were collected from the Agricultural Research Center, Giza, Egypt.
The extraction assay followed the hydro-alcoholic extract protocol as previously described (Saggu et al., 2014). Exactly 100 gram of Chicory fruits were immersed in one liter of methanol for 8 h or overnight at room temperature, after that it filtered to obtain the methanolic crude extract. One liter of methanol was added to the sample residue and heated to reach boiling and refluxed in a water bath for at least 2 h. Filtration process was repeated and the filtrate was added to the previously obtained methanolic crude extract. By a similar method, all the previous steps were repeated but by using distilled water (instead of methanol). The watery filtrate was collected to the methanolic extract to achieve the final hydro-alcoholic crude extract. This hydro-alcoholic crude extract was evaporated and dried in reduced pressure conditions at 60°C and then it stored in dark bottles and frozen till using.

In Vivo Study
Adult Sprague Dawley rats (40 male, 12 weeks old, 140-145 g) were obtained from the Egyptian Organization for Biological Products and Vaccines in Cairo and fed on the standard rodent diet (Egyptian Company of Oils and Soap Kafr-El Zayat Egypt) and water available ad libitum. The study was carried out according to the ethical protocol of Institutional Animal Care and Use Committee of Faculty of Science, Tanta University, code No. IACUC-SCI-TU-00125.

Animal Treatments
The rats were acclimatized for only one week in the animal house and then they were randomly divided into four groups (10 rats for each): Group 1 (control): Rats did not receive any treatment and served as control.

Blood and Tissue Homogenate Preparation
After consummation of the experiment strategy, rats were fasted for 10 h and were euthanized with intraperitoneal infusion of sodium pentobarbital and exposed to a total necropsy. Blood samples of each rat were gathered from sub-par vena cava and collected in non-heparinized tubes and left for 20-30 min. Then, it was centrifuged at 5000 rpm for 10 min to isolate the sera which were stored as aliquots at -80°C until required.
Kidneys were carefully removed from rats, then washed with ice-cold saline three times then chilled on ice, divided into four sections, one part were fixed in 10% neutral buffered formalin that used for histological and immunohistological investigations . The rest of organs kept in aluminum foil and stored at -80°C until tissue homogenization at required.
Kidney tissues were prepared for determination of antioxidant parameters. To evacuate any red blood cells and clots from tissues, they were washed with a mixed solution of Phosphate Buffered Saline (PBS) pH 7.4 and 0.16 mg/mL heparin. Then, the tissues were homogenized in 5-10 mL cold buffer (around 50 mM potassium phosphate pH 7.5, 1 mM EDTA) per gram tissue utilizing tissue homogenizer. At that point the samples were centrifuged at 5000 rpm for 15 min at 4°C. The supernatant were evacuated for examine and put away on ice.

Kidney Functions Biomarkers and Electrolytes
Urea levels were measured according to technique depending on oxidation of two moles of NADH into NAD+ for each mole of urea hydrolyzed using urease (EC 3.5.1.5) and glutamate dehydrogenase (EC 1.4.1.2) (Sampson et al., 1980). Creatinine concentration was detected by kinetic colorimetric assay depending on formation of a yellow-orange complex by creatinine in alkaline solution. The rate of dye formation was proportional to creatinine concentration in the specimen (Toora, 2002). All the biochemical analysis of kidney function was detected using commercial kit (Diamond, Egypt) according to Roche/Hitachi Cobas c systems. The levels of serum electrolytes (Potassium, sodium and calcium ions) using commercial kits (Sensa core electrolyte, India).

Oxidative Stress Biomarkers
Determination of reduced Glutathione (GSH) was colorimetrically determined using the kit of BioDiagnostic, Egypt (CAT. No GR 2511) relying on conversion of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) into 5′-Thionitrobenzoate (TNB) and the yellow colored precipitate is detected at 412 nm (Beutler, 1963). The lipid peroxide (Malondialdehyde, MDA) was detected depending on its reaction with Thiobarbituric Acid (TBA) at 95°C for 30 min in acidic medium to give pink product which can be evaluated at 534 nm (Ohkawa et al., 1979). Super Oxides Dismutase enzyme (SOD) (EC 1.15.1.1) assessment was done using the commercial kit (Biodiagnostic kit, Egypt, CAT. No # SD2521) depending on the ability of enzyme to inhibit the reduction of nitro-blue terazosin dye by phenazine methosulfate. At 25°C the absorbance determined at 560 nm for both control and sample (Nishikimi, 1975). Catalase enzyme (EC 1.11.1.6) was estimated by colorimetric method following the instruction of Biodiagnostic kit, Egypt (CAT. No # SD2517). The measuring of the end product (quinoneimine dye) was determined at 510 nm. The density colored product quinoneimine dye is due to the interaction between the catalase and standard amount of H2O2 (Saggu et al., 2014).

Histopathological and Immunohistochemical Detection of p53 and TNFα Expression
Fixed kidney in 10% neutral buffered formalin from different groups were stained using routine haematoxylin and eosin counterstain methods (Tousson, 2016).

Statistical Analysis
Al the obtained data were statistically analyzed using the Statistical Package for the Social Sciences (SPSS software version 16). Data were expressed as mean ± Standard Error of Mean (SEM). One-way ANOVA (Analysis of Variances) followed by Dennett test were performed to evaluate the significance of differences between groups. The criterion of the statistical significance was set at p<0.05.

Characterization of Fe3O4/Ag Core-Shell Nanoparticles (SEM Analysis, XRD Analysis and Magnetic Analysis)
The scanning electron micrograph (Fig. 1) shows more compact and better separated particles and the aggregates with size of 33.66 nm. Clearly, the SEM data serve as an important piece of evidence for the formation of Fe3O4/Ag core-shell nanoparticles.
The phase identification and crystalline structures of the nanoparticles was characterized by X-ray powder diffraction (Table 1 and Fig 2).
Magnetic properties of Fe3O4-Ag composite were studied with a magnetometer with vibrating sample (a maximum applied field of 30 kOe) at ambient temperature (Table 2).

Kidney Functions Biomarkers and Electrolytes
Rats injected with Fe3O4/AgNPs (G3) Fe3O4/AgNPs-group) displayed significant elevation level of serum urea, creatinine, potassium ions and calcium ions. In contrast, serum sodium and calcium ions levels significantly reduced in G3 if compared to control (G1) and G2 (chicory administered group). Improvement in electrolytes and renal functions were observed after the treatment with Chicory after Fe3O4/AgNPs (G4) if compared with G3 (Table 3).

Oxidative Stress Biomarkers
Oxidative stress parameter showed significant decrease in GSH concentrations, SOD and Catalase enzymes activities in G3 if compared to G1 and G2. However, MDA concentrations significantly increased in G3 compared with G1 and G2. By chicory treatment, there were ameliorations in all oxidative parameter (GSH, SOD, Catalase and MDA) in comparison with G3 (Table 4).

Kidney Histopathology
Kidney sections in the control and chicory groups showed normal histological structures of the glomeruli and tubules in the cortical and medullary portions ( Fig. 4A and 4B). Kidney section in G3 showed severe atrophy of tubular cells and glomeruli, necrotic tubular cells and mild inflammatory cellular infiltration (Fig 4C). On the other hand, G4 exhibited a good degree of improvement in the glomeruli and renal tubules (Fig 4D).

Immunohistochemical Changes of Apoptotic p53 Protein and TNFα Expressions in Kidney
Detection of p53 and TNFα expressions in kidney sections of G3 showed strong positive reaction for p53 and TNFα expression as compared to the expression in G1 and G2 groups which showed faint positive expression for the both (Fig. 5A-2C and 6A-6C, respectively).
On the other hand; mild positive expression for p53 and TNFαwere detected in kidney sections of treated rats withFe3O4/AgNPs and Chicory extract ( Fig. 5D and 6D, respectively).

Discussion
XRD peaks characteristic for the Fe3O4 phase are observed in our previously prepared magnetite nanoparticles without adding silver metal (Abdo et al., 2019). Additional peaks, at angular positions 32.21 o (111), 38.09 o (200), 46.22 o (211) and 57.45 o (300), are attributed to metallic silver. This is a proof that silver salt was reduced to metal and successfully introduced into the iron oxide substrate.
The average crystal size of Fe3O4-Ag NPs was about 37.85 nm after the Fe3O4 nanoparticles were coated with silver. Furthermore, the most intensive peak located at 2θ = 32.21° corresponding to the diffractions of nanoparticles crystallized in the FCC structure with basal (111) lattice plane. The results were somewhat in good agreement with the data obtained from the SEM image (Fig. 1). Thus, both XRD and SEM provide strong evidence that the Fe3O4-Ag composite magnetic nanoparticles have a core-shell structure.
Magnetic saturation (Ms) of Fe3O4-Ag nanoparticles has shown some changes in field-dependent magnetization after the incorporation of a silver shell as compared with our previously prepared magnetite nanoparticles (Abdo et al., 2019). There was a decrease in the saturation Magnetization (Ms) from 57.4 to 51.1 emu/g, decrease in the remnant Magnetization (Mr) from 0.39 to 0.14 emu/g and a decrease in coercivity (Hc) from 4.21 to 1.53 Oe. Additional coverage with silver weakens magnetization intensity further, which diminishes Magnetic remanence value (Mr) for the Fe3O4-Ag nanostructures.
The obtained results in this study indicated that Fe3O4/AgNPs administration in rats (G3) causes renal destruction and elevation of both urea and creatinine concentrations by about 1.5 folds compared to control Group (G1). Elevation was due to severe atrophy in tubular cells and glomeruli, necrotic tubular cells and mild inflammatory cellular infiltration as confirmed by histological result. These results were in line with previous studies (Vasanth and Kurian, 2017).
The important functions of kidney are the precision of the body fluids, electrolytes and acid-base balance. In Chronic Kidney Disease (CKD) prognosticate outcome multiple disturbance including hyperkalemia, metabolic acidosis and hyperphosphatemia (Dhondup and Qian, 2017). Assessment of electrolytes showed that the concentrations of K + significantly increased in G3 (rats injected with Fe3O4/AgNPs) by about 1.2 folds if compared to G1 (control). This increase was due to kidney injury. On the other hand, Na + and Ca +2 concentrations significantly decreased in G3 in comparison with G1 by 0.9 and 0.8 folds, respectively. Hyperkalemia and hyponatremia are common features of chronic renal failure. This can be explained by low and passive back diffusion through damaged tubular cells. Decrease in Glomerular Filtration Rate (GFR), tubular flow and reabsorption dysfunction, also tissue injury and acute K + load are occurred (Singh et al., 2017;Kovesdy, 2014). CKD caused marked disruption in bone and mineral metabolism which in turn resulted in decreased serum calcium (Gallant and Spiegel, 2017).
The study of oxidative biomarker showed that induction of Fe3O4/AgNPs in G3 caused redundant damage in kidney by increasing the concentration of MDA by about 1.86 fold compared to its normal concentrations in G1. However, GSH concentrations SOD and catalase enzyme activities decreased by about 0.71-0.74 folds in comparison with their normal values in G1. It has been demonstrated by a number of in vivo studies that AgNPs induce cytotoxicity and DNA damage in the kidney. The mechanism of cytotoxicity related to oxidation of AgNPs which causes H2O2 releasing. Reactive Oxygen Species (ROS) generation in the kidney have a significant role in the AKI genesis. ROS related to renal injury including superoxide ions, hydrogen peroxide and hydroxyl radicals cause decreasing in antioxidant enzyme status in both blood and tissue (Barbir et al., 2019;Shrivastava et al., 2016).
TNFα is a proinflammatory cytokine. It can be considered as antitumorigenic agent that is mainly synthesized by immune cells as macrophages and lymphocytes. Additionally, several studies indicated that TNFα originated inside the kidney in podocytes, mesangial cells and epithelial cells from proximal tubules, thick ascending limbs and collecting ducts. TNFα concentration increases in different injuries as renal failure, glomerulonephritis, diabetic nephropathy and interstitial tubular nephritis. Our result showed high expression of TNFα in the both glomeruli and tubules. The high expression of TNFα was demonstrated in G3 which administrated by Fe3O4/AgNPs compared with normal rats in G1. That was because that oxidative stress caused by Fe3O4/AgNPs enhances TNFα production. This stimuli cause migration of dendritic cell, macrophage and lymphocyte to the kidney. This migration enhanced expression of endothelial adhesion molecules and chemokines by the kidney in response to elevations in renal derived TNFα (Ramseyer and Garvin, 2013;Sonkar and Singh, 2009).
The result also showed high expression of p53 in kidney tissues in rats of G3 compared to G1. This elevation confirmed that Fe3O4/AgNPs causes DNA destruction, which can lead to activation of p53-dependent signalling pathway. Activation of p53 intercede cell cycle arrest and activated the apoptotic pathway and lead to cellular failure such as oxidative stress, hypoxia, heat shock and activation of oncogenes (Kovács et al., 2016). Treatment of rats by chicory extract in G4 showed excellent regression to nearly normal values in all studied parameters as urea, creatinine, electrolytes and oxidative stress markers. Even histological studies and immunohistochemical staining (p53 and TNFα) showed good recovery from the toxic effect of Fe3O4/AgNPs administration after chicory extract treatment. It can be discussed in consideration with the fact that chicory extract contains different chemical compound such as inulin, coumarins, flavonoids, sesquiterpene lactones (lactenin and lactucopicrin), tannins, alkaloids, vitamins, minerals and volatile oils. These chemical compounds especially flavonoid, tannins and coumarins have several antioxidant, antitumor, anti-inflammatory activities (Nwafor et al., 2017).

Conclusion
This study assessed the protective effect of hydro-alcoholic extract of Chicory fruits (Cichorium intybus) administration against renal toxic effects produced due to chronic exposure of Fe3O4/AgNPs. Fe3O4/AgNPs were synthesized as core-shell nanoparticles by a co-precipitation method. Fe3O4/AgNPs were characterized by XRD, SEM and VSM. SEM showed core-shell nanoparticles with size of 33.66 nm, which somewhat in a good agreement with the data obtained from XRD. Magnetic measurement revealed the superparamagnetic nature of the synthesized Fe3O4/AgNPs. The obtained results indicated that chicory extract can enhance renal activity and recover kidney injury and oxidative stress induced by Fe3O4/AgNPs.

Acknowledgement
This study is supported by the Biochemistry division, Faculty of Science, Damanhur University where all the biochemical markers were assessed. The animal experiment was carried out in the animal house of Faculty of Science, Tanta University.

Author's Contribution
Doha M. Beltagy: The experiment idea with performing the biochemical analysis with its interpretation and the manuscript writing and revision. Ehab Tousson: Histological and immunochemical analysis.
Nabiha I Abdo: Synthesis of nanoparticles and its analysis.
Batoul M. Izzularab: biochemical and statistical analysis with its interpretation with writing the draft of the manuscript.

Ethics
All the authors declared that this article is original with no any ethical conflict of interest.