Neuroprotective effects of quercetin on the cerebellum of zinc oxide nanoparticles (ZnoNps)-exposed rats

ABSTRACT

Engineered nanomaterials induce hazardous effects at the cellular and molecular levels. We investigated different mechanisms underlying the neurotoxic potential of zinc oxide nanoparticles (ZnONPs) on cerebellar tissue and clarified the ameliorative role of Quercetin supplementation. Forty adult male albino rats were divided into control group (I), ZnONPs-exposed group (II), and ZnONPs and Quercetin group (III). Oxidative stress biomarkers (MDA & TOS), antioxidant biomarkers (SOD, GSH, GR, and TAC), serum interleukins (IL-1β, IL-6, IL-8), and tumor necrosis factor alpha (TNF-α) were measured. Serum micro-RNA (miRNA): miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-3p expression levels were quantified by real-time quantitative polymerase-chain reaction (RT-QPCR). Cerebellar tissue sections were stained with Hematoxylin & Eosin and Silver stains and examined microscopically. Expression levels of Calbindin D28k, GFAP, and BAX proteins in cerebellar tissue were detected by immunohistochemistry. Quercetin supplementation lowered oxidative stress biomarkers levels and ameliorated the antioxidant parameters that were decreased by ZnONPs. No significant differences in GR activity were detected between the study groups. ZnONPs significantly increased serum IL-1β, IL-6, IL-8, and TNF-α which were improved with Quercetin. Serum miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-p expression levels showed significant increase in ZnONPs group, while no significant difference was observed between Quercetin-treated group and control group. ZnONPs markedly impaired cerebellar tissue structure with decreased levels of calbindin D28k, increased BAX and GFAP expression. Quercetin supplementation ameliorated cerebellar tissue apoptosis, gliosis and improved calbindin levels. In conclusion: Quercetin supplementation ameliorated cerebellar neurotoxicity induced by ZnONPs at cellular and molecular basis by different studied mechanisms.Abbreviations: NPs: Nanoparticles, ROS: reactive oxygen species, ZnONPs: Zinc oxide nanoparticles, AgNPs: silver nanoparticles, BBB: blood–brain barrier, ncRNAs: Non-coding RNAs, miRNA: Micro RNA, DMSO: Dimethyl sulfoxide, LPO: lipid peroxidation, MDA: malondialdehyde, TBA: thiobarbituric acid, TOS: total oxidative status, ELISA: enzyme-linked immunosorbent assay, H₂O₂: hydrogen peroxide, SOD: superoxide dismutase, GR: glutathione reductase, TAC: total antioxidant capacity, IL-1: interleukin-1, TNF: tumor necrosis factor alpha, cDNA: complementary DNA, RT-QPCR: Real-time quantitative polymerase-chain reaction, ABC: Avidin biotin complex technique, DAB: 3′, 3-diaminobenzidine, SPSS: Statistical Package for Social Sciences, ANOVA: One way analysis of variance, Tukey’s HSD: Tukey’s Honestly Significant Difference, GFAP: glial fiberillar acitic protein, iNOS: Inducible nitric oxide synthase, NO: nitric oxide, HO-1: heme oxygenase-1, Nrf2: nuclear factor erythroid 2-related factor 2, NF-B: nuclear factor-B, SCI: spinal cord injury, CB: Calbindin

KEYWORDS:

• Quercetin
• zinc oxide nanoparticles
• miRNA
• cerebellum
• neurotoxicity

Introduction

Nanotechnology has prompted scientific research advancements. Nanoparticles (NPs) are being considered nowadays as the materials of the future because of their unique properties and numerous applications.^1,2 Therefore, they are fast rising in the industrial, medicinal, and military domains. Natural or produced materials, present in aggregate or dispersed condition, with a diameter of less than 100 nm, are defined as NPs by the European Union. They’re exciting new materials with potential applications in medicine, imaging, cosmetics, and food preservation additives.³ They have various characteristics, including a high surface area/mass ratio, small size, and surface reactive chemical groups, all of which aid in endocytosis and contact with intracellular structures. Despite their beneficial effects, several studies reported their ability to generate reactive oxygen species (ROS), as they interact with intracellular biomolecules such proteins, enzymes, and DNA, and potentially affect their function.^4,5

Zinc oxide nanoparticles (ZnONPs) are a type of nanoparticles that are widely present in several products such as building materials, wall paints, cosmetics, toothpaste, and sunscreens. In addition, they’re widely employed in the rubber industry, electronics, and food additives.^6,7 The microbial selectivity, stability, and low cost of ZnONPs make them a possible alternative to silver nanoparticles (AgNPs) or antimicrobial peptides. Human exposure to ZnONPs occurs via several routes. They can reach the brain through oral intake or along the olfactory nerve. It has recently been evidenced that NPs can cross the blood–brain barrier (BBB) and induce oxidative stress, cytotoxicity, and inflammatory reactions.^8–12 Their toxic effects may produce alterations in synaptic plasticity leading to changes in spatial learning and memory abilities.^13,14 The size, shape, and surface material of ZnONPs affect their cytotoxicity.^15,16

We all can be frequently exposed to the hazardous effects of NPs therefore, searching for natural substances having the ability to counteract these drawbacks is essential. Quercetin (3,3′,4′,5,7- pentahydroxy-flavanone) is a flavonoid found in seeds, vegetables, fruits, coffee, tea, and bracken fern. It acts as an antioxidant with anticancer, anti-inflammatory, and antiviral properties.¹⁷ In addition, previous studies also reported their antihypertensive, antibacterial, and antiprotozoal properties.^18,19 Its potential to protect neurodegenerative,²⁰ cardiovascular,²¹ and neoplastic disorders²² has also gotten a lot of interest.

Studying in-depth the molecular basis underlying the effect of each of ZnONPs or Quercetin on the brain tissue is very essential to explain how they affected it. Non-coding RNAs (ncRNAs) were formerly assumed to be “junk” DNA but recently, they were found to have critical regulatory functions in several vital cellular pathways. They also impact cell physiological and pathological states. Micro RNA (miRNA) with 18–25 nucleotides, is a type of short noncoding RNA (ncRNA) with a length fewer than 200 nucleotides that has recently been implicated in gene regulation.²³ According to research, micro-RNA (miRNA) regulates gene expression via several ways as translational suppression, mRNA degradation, and binding to the 5′ UTR and other coding areas.²⁴ Other investigations, on the other hand, have shown that miRNA can stimulate transcription and translation.^25,26

As a result, more research is required to fully comprehend and validate the functional connection. Considering the foregoing, we explored the potential harmful effects of ZnONPs on the molecular and histological structure of the rat cerebellum, as well as the many pathways driving Quercetin’s potential preventive role.

Chemicals

All chemicals were purchased from Sigma-Aldrich, 3050 Spruce Street, Saint Louis, MO 63103, USA

ZnONPs: In the form of dispersion of less than 100 nm particle size (≤40 nm average particle size), 20 wt.% in H2O. Product number 721077, CAS number 1314–13-2.²⁷

Quercetin: In the form of solid (CAS-No.: 117–39-5, ≥95% HPLC) that was dissolved in DMSO solution (Dimethyl sulfoxide, CAS-No.:67–68-5) to be administered orally.

Characterization of ZnONPs

The size and shape of ZnONPs were examined by transmission electron microscopy (TEM, JEOL JEM 1010; Jeol Ltd., Tokyo, Japan) at the Electron Microscope Unit, Faculty of Agriculture, Mansoura University, Mansoura, Egypt. Aqueous dispersion of nanoparticles was dropped onto a carbon coated copper grid, which was then dried and studied (Figure 1).

Figure 1. Electron micrograph of ZnONPS nanoparticles less than 100 nm.

Experimental animals

Forty adult Wistar male albino rats (5–7 months) weighing (200–250 gm.) were purchased from the Breeding Animal House at Faculty of Medicine, Zagazig University, Zagazig, Egypt. They were kept for acclimatization in stainless-steel cages at room temperature under standard laboratory settings. They were fed standard laboratory food and water ad libitum during the experiment. The experimental protocols were carried out according to the Institutional Animal Care and Use Committee’s guidelines, approved and accepted by the Faculty of Medicine, Zagazig University; Zagazig, Egypt.

Experimental design

The rats were divided into three groups:

Group I (control group): Included 20 rats that were divided into two equal subgroups:

Subgroup Ia (negative control): Ten rats received no treatment.

Subgroup Ib: Ten rats were daily administrated 50 mg/kg body weight orally of Quercetin dissolved in DMSO for 1 month.²⁸

Group II (ZnONPs-treated group): Ten rats were intraperitonially injected with 400 mg/kg b. wt. of ZnONPs, every other day for 1 month.²⁹

Group III (ZnONPs and Quercetin group): Ten rats were injected with ZnONPs at a similar dose and duration as group II and simultaneously administrated a daily dose of Quercetin dissolved in DMSO (50 mg/kg body weight), orally of for 1 month.

Blood collection and tissue preparation

Rats were anaesthetized intraperitoneally with 60 mg/kg body weight of pentobarbital. Blood was collected from the ophthalmic plexus then it was centrifuged at 2000 rpm for 20 minutes to obtain serum samples. The supernatant sera were then kept at −20°C. Biochemical assays were performed on serum samples that had been stored. Each animal’s cerebellar tissue was separated into two parts: one for histological evaluation and the other for biochemical and molecular analysis. 5–10 ml cold buffer (50 mM potassium phosphate, pH 7.4, 1 mM EDTA; Catalog Number G8789) per gramme tissue homogenized the latter. The homogenate was centrifuged at 4,000 rpm for 15 minutes at 4°C, and the supernatant was frozen at −80°C for analysis of oxidative stress markers.

Oxidative biomarkers assay

The lipid peroxidation (LPO) marker malondialdehyde (MDA) was evaluated calorimetrically in cerebellar homogenate using a (Biodiagnostic, Egypt; Catalog Number: MD 25 29) kit. Ohkawa et al.³⁰ developed a method that involves reacting thiobarbituric acid (TBA) with MDA in an acidic medium at 95°C for 30 minutes, yielding a TBA reactive pink product. This product’s absorbance can be measured at 534 nm. In addition, reactive oxygen species (ROS) levels were measured by ELISA kit (Catalog Number: MBS2802061), according to the manufacturer’s instructions (MyBioSource, USA).

Measurement of antioxidant biomarkers

The cerebellar homogenate’s total oxidative status (TOS) was estimated calorimetrically with a (Rel Assay Diagnostics kit, Turkey). The results were expressed in mol H₂O₂ equivalents per liter after the test was calibrated with hydrogen peroxide (H₂O₂). The Nishikimi et al. method was used to evaluate the activity of superoxide dismutase (SOD) in U/g cerebellar tissue.³¹ The level of reduced glutathione (GSH) was tested using Beutler’s technique.³² In addition, the activity of glutathione reductase (GR) was determined, as described by Erden & Bor.³³ Serum total antioxidant capacity (TAC) was measured according to Koracevic et al.³⁴ Bio diagnostic, Cairo, Egypt, developed all the above kits (Catalog Number: SD 25 21, Catalog Number: GR 25 11, Catalog Number: GR 25 23, Catalog Number: TA 25 13, respectively).

Measurement of serum cytokines concentrations

The serum levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) were determined using (ELISA) kits (MyBioSource, Inc.; USA) according to the manufacturer’s protocol (Catalog Number: MBS825017, Catalog Number: MBS269892, Catalog Number: MBS9141543, Catalog Number: MBS175904, respectively).

miRNA isolation and reverse transcription

Total miRNA was isolated from serum using (miRcut Tiangen biotech, China, Catalog Number: 4992860) kit according to the manufacturer’s instructions. The miRNA was then reverse-transcribed, and polyadenylated to complementary DNA (cDNA) by using (miRCURY LNA RT Kit, Qiagen GmbH, Germany, Catalog Number:339340) kit. The mixture was centrifuged and incubated at 37°C for 60 minutes. After mixing with template RNA, miScript Reverse Transcriptase mix, 5Χ miScript buffer, and RNase-free water in a final volume of 20 μl., samples were finally incubated at 95°C for 5 min and put on ice for inactivation of the reverse transcriptase enzyme.

Expression levels of miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-3p in serum were assessed

Real-time quantitative polymerase-chain reaction (RT-QPCR) with an applied biosystem real-time instrument was used to determine the expression levels miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-3p in serum. Primers were designed and purchased using miRCURY LNA miRNA PCR Assay (Qiagen GmbH, Germany) for amplification of hsa-miR-21-5p (MIMAT0000076), hsa-miR-122-5p (MIMAT0000421), hsa-miR-125b-5p (MIMAT0000423) and has-miR-155-3p (MIMAT0016993). We performed PCR in a 20 µ1 final volume including 2 µl 10X miScript universal reverse primer, 10 µl 2X Quanti-Tect SYBR-Green Polymerase Chain Reaction master mix, and 2 µl template cDNA after dissolving template cDNA and reagents. 2 μl 10 pmol forward specific stem-loop primer, and 4 μl RNase-free water. RT-PCR conditions: initial activation at 95°C for 15 minutes (one cycle), denaturation at 94°C for 40 cycles, annealing at 55°C for 30 seconds, and extension at 70°C for 30 seconds. In quantification investigations of miRNA utilizing the miScript PCR System,³⁵ we employed SNORD68 as a control for normalization of RT-PCR data. We compared the expression of miR-96-5p and miR-183-5p to that of the SNORD68 gene, and all data was standardized to that of the SNORD68 gene.³⁶ In every case, SNORD-68 was found to be highly expressed and stable.

Histopathological study

Hematoxylin and Eosin staining

Sagittal cerebellar specimens were fixed in 10% buffered formalin, dehydrated, embedded in paraffin wax, and cut into 5 µm-thick paraffin sections for light microscopic examination.³⁷

Silver staining

Deparaffinized sections were hydrated then, the slides were immersed in a 1.0% silver nitrate solution in a plastic Coplin jar before being rinsed with distilled water. In a flask, 1% warm silver nitrate was poured, and 28% ammonium hydroxide was added drop by drop with constant shaking until the first precipitate vanished and the solution became clear. 5% silver nitrate was then pipetted, with constant shaking, until the solution became slightly cloudy. The ammoniacal silver solution was poured into a plastic Coplin jar and the slides were placed in this solution and microwave at power level 3 (180 W) for 1 minute, then in 1% ammonium hydroxide solution for not more than 20 seconds. Developer was added to the ammoniacal silver solution for no more than 15 seconds, slides were immersed in a 1% ammonium hydroxide solution, rinsed three times with distilled water followed by 2% sodium thiosulfate solution for 30 seconds. The slides were rinsed three times in distilled water, 5 min each. Finally, they were dehydrated in graded alcohols, cleared in three or four changes of xylene, and mount with synthetic resin.³⁸

Immunohistochemical study

Avidin biotin complex technique (ABC Peroxidase Staining Kits, Code No. 32020, Thermo Scientific, Rockford, IL, USA) was utilized for immunohistochemical staining of the calcium binding protein; Calbindin, GFAP, and Bax protein. Paraffin-embedded specimens were deparaffinized in xylene and hydrated in distilled water. Antigen retrieval was done in the microwave for 15 minutes using citrate buffer. Bovine serum albumin was used for tissue block. Sections were then incubated overnight at 4°C with the following primary antibodies: Anti-CbD28k (rabbit polyclonal antibody; Cat. No. PA5-85669; dilution 1/500; Thermo Scientific, San Jose, CA, USA). Anti-GFAP (Cat. No. MS- 280- R7, Lab Vision Corporation, Fremont, USA) was diluted to1:100 in Lab Vision antibody diluent (Cat. TA- 125- UD). Finally, Bax mouse monoclonal antibody (CAT. MAS-14003, Lab Vision Corporation, Fremont, USA) was diluted to 1:200 in PBS (for 30 min at room temperature). Recognition was accomplished by secondary antibodies and labeled horseradish peroxidase followed by colorimetric detection by 3′, 3-diaminobenzidine (DAB). As a counterstain, Mayer’s Hematoxylin was utilized. As a substitute for the primary antibody, negative control slides were immersed in phosphate-buffered saline. The brown color indicated the antigen location under light microscopy.³⁹

Histomorphometric study and statistical analysis

The Pathology Department, Faculty of Dentistry, Cairo University, Cairo, Egypt, used a Leica Qwin 500 (Leica Imaging system, Ltd., Cambridge, England) image analyzer computer system to perform a morphometric study of the area percentage of calbindin-positive cells, GFAP, and BAX protein immunoexpression in the studied groups. Data analysis by Leica QWin 500 software and a digital camera connected to an optical microscope (Olympus, Tokyo, Japan). Ten non-overlapping fields were randomly selected from each rat in each group, and the means of the parameter measurements in each section were recorded for each animal. The Statistical Package for Social Sciences (SPSS) version 22.0 was used to analyze the data (IBM Corp., Armonk, NY, USA). One way analysis of variance (ANOVA) was used to express the data as means ± standard deviations, followed by Tukey’s Honestly Significant Difference (Tukey’s HSD) test as a post hoc test. The probability (P) values less than 0.05 were considered significant, while P values less than 0.001 were considered highly significant.

Results

Oxidative stress biomarkers concentrations (Table 1

• No statistically significant differences were detected between the control subgroups regarding levels of the studied parameters.

• Significant variations were detected in MDA and TOS in cerebellar homogenate between the three groups (P < .001 for each). Group II showed significantly higher levels of cerebellar MDA and TOS while Quercetin supplementation with ZnONPs (group III), significantly decreased their levels.

• On the other side, we detected significant differences of antioxidant parameters (SOD & GSH) in cerebellar homogenate, and TAC in serum between the three groups (P = .004, P < .001, P < .001, respectively). ZnONPs decreased the antioxidant parameters which were improved by Quercetin treatment.

• No significant differences were detected regarding GR activity between the study groups (P = .18).

Table 1. Oxidative stress parameters in the study groups (Mean ±SD).

	Group Ia(No.: 10 rats)	Group Ib(No.: 10 rats)	Group II(No.: 10 rats)	Group III(No.: 10 rats)	P-value
MDA (nmol/g tissue)	1.7 ± 0.09	1.6 ± 0.1	4.5 ± 0.3	2.3 ± 0.1	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
TOS umo H2O2 equivl/l	19.3 ± 1.0	20.2 ± 1.1	41.8 ± 2.2	18.7 ± 1.3	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
SOD (U/g)	29.9 ± 2.7	30.1 ± 2.6	21.5 ± 2.0	27.3 ± 2.8	0.004
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
GSH (mmol/g) tissue	9.2 ± 0.2	9.3 ± 0.4	4.1 ± 0.2	6.2 ± 0.8	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
GR (U/g)	4.2 ± 1.6	4.3 ± 1.9	3.0 ± 1.2	4.0 ± 1.8	0.18
Post-hoc	-	-	-	-
TAC(Mm/l)	0.78 ± 0.09	0.9 ± 0.06	0.01 ± 0.02	0.65 ± 0.06	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II

Serum inflammatory cytokines levels in different study groups (Table 2)

• No significant changes were detected in serum cytokines levels between both control subgroups.

• Significant differences in IL-1, IL-6, IL-8, and TNF-α levels were detected between the study groups (P < .001).

• Significantly higher serum concentrations of inflammatory cytokines levels in ZnONPs group (group II) compared to the control group.

• Quercetin supplementation with ZnONPs (group III), improved the inflammatory status through decreasing serum cytokines levels but still significantly higher than the control subgroups, suggesting potential anti-inflammatory effect.

Table 2. Serum inflammatory cytokines in the study groups (Mean ±SD).

	Group Ia(No.: 10 rats)	Group Ib(No.: 10 rats)	Group II(No.: 10 rats)	Group III(No.: 10 rats)	P-value
IL-1β (pg/mL)	1.2 ± 0.08	1.1 ± 0.1	5.5 ± 0.3	3.3 ± 0.5	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
IL-6 (pg/mL)	0.9 ± 0.3	0.9 ± 0.2	4.7 ± 1.4	2.9 ± 0.7	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
IL-8 (pg/mL)	1.5 ± 0.1	1.3 ± 0.2	6.1 ± 1.4	3.8 ± 1.2	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
TNF-α (pg/mL)	1.4 ± 0.8	1.1 ± 0.5	8.5 ± 1.4	6.2 ± 0.9	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II

Serum miRNAs expression in different study groups (Table 3)

• miR-21-5p expression value was significantly higher in group II compared to control subgroups and Quercetin treated group (III). However, we did not find significant changes between groups I and III.

• miR-122-5p and miR-155-p expression levels were significantly increased in ZnONPs exposed group as compared to control subgroups or Quercetin treatment group (III).

• ZnONPs group also showed significantly higher miR-125b-5p expression levels than the control subgroups while, Quercetin treatment (group III) showed no significant changes in comparison to control subgroups.

Table 3. Serum miR-21-5p, miR-122-5p, miR-125b-5p, and miR-155-p expression levels in the study groups (Mean ± SD).

	Group Ia(No.: 10 rats)	Group Ib(No.: 10 rats)	Group II(No.: 10 rats)	Group III(No.: 10 rats)	P-value
miR-21-5p	1.1 ± 0.2	1.0 ± 0.3	4.7 ± 1.5	2.2 ± 1.3	<0.001
Post-hoc	II	II	Ia, Ib, III	II
miR-122-5p	0.89 ± 0.1	0.7 ± 0.09	3.9 ± 1.4	2.0 ± 0.8	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II
miR-125b-5p	1.1 ± 0.2	1.0 ± 0.3	4.7 ± 1.5	2.9 ± 1.1	<0.001
Post-hoc	II, III	II, III	Ia, Ib	Ia, Ib
miR-155-p	0.93 ± 0.07	0.85 ± 0.09	4.6 ± 1.5	2.4 ± 0.8	<0.001
Post-hoc	II, III	II, III	Ia, Ib, III	Ia, Ib, II

Histological results

• H&E-stained cerebellar sections of the control group showed the three layers of cerebellar cortex; the molecular layer, Purkinje cell layer, and the granular layer. The molecular layer showed small, scattered stellate and basket cells. Purkinje cell layer was formed of pyriform-shaped Purkinje cells with vesicular nuclei, prominent nucleoli, and basophilic cytoplasm. The granular layer composed of closely packed small, rounded granule cells with darkly stained nuclei. Among these cells, cerebellar islands or glomeruli were found. ZnONPs exposure for one month, markedly distorted the cerebellar tissue structure as the molecular layer showed cells with small dark nuclei, and vacuolated neuropil. Purkinje cells were shrunken, irregular in shape and deeply stained. Peri-cellular halos were observed around Purkinje cells and granule cells. Quercetin supplementation with ZnONPs, ameliorated the cerebellar histological structure in the form of normal appearance of the cerebellar layers while some cells showed peri-cellular halo (Figure 2).

Figure 2. H&E-stained sections of rat cerebellar cortex showing: (a) the control groupshows cerebellar cortex formed of three layers; the molecular layer (ML) shows small, scattered stellate (sc), larger basket cells (bc) and blood vessels (bv). Purkinje cell layer (PL) is formed of pyriform-shaped Purkinje cells (p) with vesicular nuclei, prominent nucleoli, and basophilic cytoplasm. The granular layer (GL) shows tightly packed small, rounded granule cells (arrow) with deeply stained nuclei. Among these cells, cerebellar islands, or glomerulus (asterisks) are located. (b) In group II, the molecular layer (ML) shows cells with small dark nuclei, and vacuolated neuropil (v). Purkinje cells are shrunken, irregular shaped and deeply stained (arrow). Pericellular halos (curved arrows) are observed around Purkinje cells and granule cells. (c) Group III shows normal shaped molecular layer (ML) containing stellate cells (Sc), basket cells (bc) and blood vessels (bv). Some cells show pericellular halo (curved arrow). Purkinje cells (P) are pyriform in shape. They had vesicular nuclei with prominent nucleoli and basophilic cytoplasm. Granule cells (arrowhead) are tightly packed with darkly stained nuclei. Cerebellar islands or glomerulus (asterisks) are located among granule cells (H&E × 400; Scale bar = 30 µm).

• Silver impregnation of cerebellar sections showed many polymorphic neuronal cells in the molecular layer of control group in the form of outer stellate cells and inner basket cells. The Purkinje cells were large, pyriform shaped with vesicular nuclei. The granular layer contained plenty of granule cells arranged in rosettes or cords. ZnONPs exposure showed neuronal degeneration in the three layers in the form of marked decreased cellularity in molecular layer, darkly stained Purkinje cells with abnormal shapes, and vacuolated neuropil. The granular layer showed peri-neuronal spaces around dark stained granule cells. Quercetin supplementation with ZnONPs showed normal appearance of the three cerebellar layers (Figure 3).

Figure 3. Silver impregnation of cerebellar sections showing (a): the control group shows more polymorphic neuronal cells in the molecular layer (ML) in the form of outer stellate cells (Sc) and inner basket cells (Bc). The Purkinje cells (arrow) are large, pyriform shaped with vesicular nuclei. The granular layer (GL) contains plenty of granule cells arranged in rosettes or cords (circles). (b): Group II shows marked decreased cellularity in molecular layer (ML), Purkinje cells are darkly stained with abnormal shape (arrow), and vacuolated neuropil (asterisks). The granular layer (GL) shows peri-neuronal spaces (curved arrow) around dark stained granule cells. (c): Group III shows normal shaped molecular layer (ML) containing stellate cells (Sc) and basket cells (Bc). The Purkinje cells (arrow) are pyriform shaped with vesicular nuclei. The granular layer (GL) contains plenty of granule cells arranged in rosettes (circles) (Silver impregnation×400; Scale bar = 30 µm).

• Calbindin D28k immunostaining showed intense cytoplasmic immunoreaction in many Purkinje cells and their processes in control group and Quercetin-treated group (III) while decreased intensity of immunoreaction was observed in few irregular-shaped Purkinje cells of group II. (Figure 4).

Figure 4. Immunohistochemically stained cerebellar sections for Calbindin D28k showing (a) Intense cytoplasmic immunoreaction in many Purkinje cells (arrow) and their processes (red arrow) in control group. (b): Decreased intensity of immunoreactions in few Purkinje cells of group II (arrow). (c): Positive cytoplasmic immune reaction in Purkinje cells (arrow) and their processes (red arrow) of group III (Anti calbindin D28k ×400; Scale bar = 30 µm).

• Bax protein immunostaining revealed negative expression in the control group, high expression in the three layers of cerebellar cortex of group II, and weak expression in Purkinje cells of group III. (Figure 5).

Figure 5. Immunohistochemically stained cerebellar sections for Bax protein showing (a): Negative expression in control group (arrow). (b): Intense expression in Purkinje cells and basket cells (arrow) and some granule cells (red arrow) of group II, while (c): shows weak expression in Purkinje cells of group III (arrow). (Anti Bax×400; Scale bar = 30 µm).

• GFAP immunostaining showed positive expression in few astrocytes of control group and Quercetin-treated group (III) while intense expression was observed in cytoplasm and processes of astrocytes in all layers of group II. (Figure 6).

Figure 6. Immunohistochemically stained cerebellar sections for GFAP showing (a): positive expression in astrocytes of control group. (b): intense expression in cytoplasm and processes of astrocytes in all layers of group II. (c): positive expression in astrocytes of group III (arrows indicate immunoexpression for GFAP) (Anti GFAP ×400; Scale bar = 30 µm).

Morphometric and statistical results (Table 4)

• Highly significant (P < .00001) decrease in the main area % of Calbindin D28k was observed in ZnONPs-exposed rats compared to all groups while, it showed highly significant (P < .00001) increase in Quercetin-treated rats (group III) relative to group II rats.

• Highly significant (P < .00001) increase in the mean area % of BAX, and GFAP immunoexpression was observed in ZnONPs-exposed rats compared to all groups while Quercetin-treated rats (group III), showed highly significant (P < .00001) decrease in their levels in comparison to group II.

• Non-significant difference was noted between the control subgroups regarding the three measured proteins.

Table 4. Area % of Calbindin D28k, BAX, and GFAP immunoexpression levels in the study groups (Mean ±SD).

	Group Ia(No.: 10 rats)	Group Ib(No.: 10 rats)	Group II(No.: 10 rats)	Group III(No.: 10 rats)	P-value
Calbindin D28kPost-hoc	58.2 ± 7.3II, III	57.6 ± 6.8II, III	25.2 ± 4. 8Ia, Ib, III	44.4 ± 5.1Ia, Ib, II	< .00001
BAXPost-hoc	2.2 ± 0.6II, III	2.18 ± 0.7II, III	10.11 ± 0.9Ia, Ib, III	3.45 ± 0.7Ia, Ib, II	< .00001
GFAPPost-hoc	4.37 ± 1.1II, III	4.35 ± 1.1II, III	14.05 ± 1.5Ia, Ib, III	6.35 ± 1.04Ia, Ib, II	< .00001

Discussion

ZnONPs are one of the most utilized engineered nanomaterials so, exposure to their harmful effects is inevitable.⁴⁰ Neurotoxic potential of ZnONPs has not been explored in detail so; we aimed to investigate different cellular and molecular mechanisms underlying the neurotoxic effect of ZnONPs and the ameliorative effect of Quercetin.

The present research revealed that oxidative stress caused by ZnONPs can be reversed by Quercetin administered simultaneously with ZnONPs. It could be explained as ZnONPs interfere with cell zinc homeostasis, producing an excess of reactive oxygen species (ROS) that deplete intracellular antioxidants. ZnONPs also induce mitochondrial dysfunction leading to inflammation.^41,42 In addition, endothelial cells could internalize ZnONPs of 20–70 nm in diameter, causing inflammatory reaction due to the accumulation of the particles rather than ZnO-released Zn2 + .⁴³ Qiao et al.⁴⁴ reported that ZnONPs induced significant increases in the serum levels of IL-8, IL-1β, and TNF-α shedding the light on the proinflammatory effect of ZnONPs. IL-1β is a strong pro-inflammatory mediator and is crucial for inflammation induced by nanoparticles.⁴⁵ In contrast, Abdulmalek et al.⁴⁶ reported decreased IL-1β, and TNF-α expression values accompanied by increased GSH and TAC in ZnONPs exposed rats as compared to healthy rats, proposing anti-inflammatory and antioxidant effect of ZnONPs. Also, a previous study showed similar anti-inflammatory effects of ZnONPs on downregulating the expression of iNOS, IL-1β, and TNF-α in the cell.⁴⁷ This discrepancy in results could be attributed to changes in doses and duration of exposure to the nanoparticles.

Much prior research suggested that Quercetin may possess antioxidant and anti-inflammatory properties due to its free radical scavenging and metal-chelating capacities.^48–50 The anti-inflammatory/anti-allergic property of Quercetin was attributed to mast cell membranes stabilization so, preventing the release of histamine and other inflammatory mediators in the body. It was shown to reduce IL-6 and TNFα in lipopolysaccharide in murine macrophages.⁵¹ Quercetin also prevents glial cells from releasing proinflammatory cytokines. It protects against neuroinflammation by decreasing the formation of nitric oxide (NO) in microglial cells, which inhibits NF-B signaling.^52–55 Similarly, Quercetin has been shown to reduce manganese neurotoxicity via reducing neuroinflammation-mediated neurodegeneration, by regulating the heme oxygenase-1 (HO-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear NF-B pathways.⁵⁶

According to Wang et al.,⁵⁷ Quercetin administration has alleviated cerebral I/R-induced neurological impairments, brain infarction, disruption of the blood–brain barrier, oxidative stress, TNF- and IL-1 mRNA expression, as well as apoptotic caspase3. Additionally, their findings showed that Quercetin reduced the rise in protein tyrosine and serine/threonine phosphatase activity, as well as the decline in extracellular signal-regulated kinase and Akt phosphorylation, all of which may be crucial in the progression of brain injury following cerebral ischemia/reperfusion.

In the same context, we proved significant differences between the three groups of the present study regarding serum miR-21-5p, miR-122-5p, miR-125b-5p, and miR-155-3p expression levels (P < .001 for each). Recent research showed that miRNAs are important regulators of inflammation. miRNAs can influence a variety of biological processes by modulating the expression of several target genes.⁵⁸ miRNAs are one of the primary epigenetic mechanisms that have been linked to toxicity through altering gene expression post-transcriptionally, according to.⁵⁹ Differential expression of miRNAs has been observed in neurodegenerative illnesses such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia. Environmental neurotoxicants, such as metals, have been connected to brain miRNA expression and function.⁶⁰ These genes are involved in apoptosis, vesicle-mediated transport, oxidative stress, chemokine-mediated signaling, IL-1 signaling, inflammation, and autophagy according to Qiao et al.⁴⁴ Our findings could be explained as miR-155 has been implicated in NF-κB and other inflammation-based signaling pathways.^61,62 Furthermore, abnormal miR-155 production and function have been linked to inflammation and have been shown to influence immune cell functioning at several levels by targeting inflammation-related genes, such as TLRs.⁶³

Hu et al.⁶⁴ discovered six significant miRNAs in response to different NPs. They involve MiR-124, miR-144, miR-148, miR-155, miR-19a, and miR-223. These miRNAs have already been validated in mammalian and zebrafish miRNA profiling investigations, and their predicted targets have been discovered to interact with a variety of signaling pathways. The accumulation of NPs in the environment is unregulated, which is a rising source of worry. It worth noting that, miR-21 is well-known as oncogenic and an unfavorable prognostic factor,⁶⁵ and increased miR-21 expression was reported in mouse hippocampal neurons associated with neurotoxicity.⁶⁶ Many studies evaluated miR-21 expression, and Quercetin treatment downregulated its expression regardless the tested model was in vitro or in vivo according to Dostal & Modriansky.⁶⁷ In addition, Boesch-Saadatmandi et al.⁶⁸ assumed the mechanism of Quercetin-induced miR-155 regulation to direct and indirect modulation through NFκB. In agreement with our results, the study of Garelnabi and Mahini,⁶⁹ proved that Quercetin alone caused non-significant downregulation of miR-125b expression in the liver but when administered in combination with exercise, it attenuated the exercise induced upregulation of miR-125b.

The histopathological changes in the present study represented the consequence of the aforementioned molecular results. Both H&E and silver staining revealed the toxic effects of ZnONPs on the three cortical layers that were improved with Quercetin supplementation. Muller et al.⁷⁰ explained this damaging effect by release of ionic Zn²⁺ from ZnONPs due to intracellular dissolution. Vacuolated neuropils might result from degeneration and shrinkage of Purkinje cells with retraction of their processes leaving empty spaces according to Garman.⁷¹ These findings can be also attributed to apoptotic and oxidative stress pathways which are responsible for the accumulation of damaged molecules (proteins, nucleic acids, and lipids).⁷² Vacuolation, on the other hand, was described by Eluwa et al.⁷³ as a form of cellular defense mechanism against neurotoxins in the cerebellar cortex. In the same context, the fall in the number of granule cells with ZnONPs, could be linked to oxidative and metabolic stress. Moreover, Purkinje cells are synaptically dependent on granule cells; hence, the loss of Purkinje cells may result in granule cell death.⁷⁴

Quercetin supplementation in group III showed nearly normal appearance of cerebellar tissue apart from some peri-cellular halos. Accordingly, the antioxidant and anti-inflammatory effects of Quercetin that were proved in our study, were reflected on the histological structure of the rat cerebellar cortex where most of Purkinje cells retained their normal characters. These results were in line with Fereidounni and Dhawan⁷⁵ and Haq and Al Amro⁷⁶ who proved a remarkable protective effect of Quercetin on the overall morphology and viability of neuronal cells.

Neuronal degeneration may result from mitochondrial fragmentation. However, Quercetin can increase the expression of H2AX gene and mitochondrial transcription factor A to minimize the damages in the mitochondrial structure and activity, while also reducing axonal and neuronal damage.⁷⁷ Also, Quercetin can reduce the damage of myelin sheath of white matter after cerebral hypoperfusion by increasing the secretion of IL-10 by M2-like microglia.⁷⁸ Quercetin decreases the apoptosis of cortical neurons, improves degenerative lesions of the central nervous system, and has a neuroprotective effect in Alzheimer’s disease.⁷⁹ Additionally, Quercetin lessens the gray and white matter damage following spinal cord injury (SCI), which may suggest its potential use in the treatment of SCI.⁸⁰

Calbindin D28k (CB) is a calcium-binding protein that helps to maintain calcium balance. It inhibits various pro-apoptotic pathways so, prevents neuronal death.⁸¹ CB is an antioxidant that protects against oxidative stress and harmful substances.⁸² Its levels drop dramatically in degenerative neuronal disorders.⁸³ In our investigation, exposure to ZnONPs reduced the strength of CB immunoexpression in Purkinje cells. The buffering ability of neurons is reduced when CB is depleted, resulting in an increase in intracellular and intranuclear calcium.⁸⁴

From all mentioned above, we can deduce enhancement of cerebellar neuronal cells apoptosis which was evident by immunohistochemical detection of Bax protein as intense expression was found in Purkinje cells and basket cells of group II, while weak expression was observed in Purkinje cells of group III. The genes Bax and Bcl-2 control the intrinsic apoptotic pathway, and ZnONPs significantly raises Bax mRNA expression and protein levels, as well as the Bax/Bcl-2 ratio.^85,86

Every chemical, mechanical, or degenerative input to the brain causes astrocyte proliferation and hypertrophy, which lead to increased GFAP production and astrogliosis, according to Sofroniew and Vinters.⁸⁷ The molecular mechanism of astrocyte activation has been proposed as oxidative stress, which is a result of the increased need for neural protection.⁸⁸ So, antioxidants administration could play a crucial protective role against astrogliosis. This explains the significant increase in GFAP immunoexpression in ZnONPs-exposed rats of the present study and reversal after Quercetin supplementation. GFAP is an intermediate filament protein known to be specifically expressed in astrocytes; the glial cells that are responsible for repairing and scarring of the brain following injuries.⁸⁹ The increase in GFAP expression has been identified as a biomarker of neurotoxicity.⁹⁰ In the CNS, Quercetin reduced the activation of astrocytes and reduced neuroinflammation.⁹¹ The blood-brain barrier is crucial in relation to the toxicity of the nanoparticles in the brain tissue. This expanded membrane between the cerebral capillaries and surrounding endothelial cells contains tight junctions.^92,93 When nanoparticles enter the circulation, they may change the permeability of the membrane or cause a cascade of chemicals that damage tight junctions, causing direct or indirect toxicity in the brain tissues. NPs may also stimulate the vesicular transport, to get entry inside the microenvironment of CNS where they further disrupt several molecular pathways.^94,95

Conclusion

The current study’s findings supported the idea of integrating epigenetic testing during the toxicological evaluation of nanomaterials’ biological interactions. We have shown for the first time that frequently used NPs cause alterations in serum miRNA expression. In addition, these NPs induced changes in oxidative stress parameters and inflammatory cytokines levels which resulted in observable alteration in cerebellar structure with associated apoptosis, gliosis and diminished neuronal Calbindin level. To completely understand the association between observed changes in miRNA expression and NP effects at the cellular level, more research is required. In addition, we also clarified the different mechanisms underlying the neuroprotective effect of Quercetin against the oxidative stress and neuronal injury induced by ZnONPs administration.

Authorship contribution statement

Abdelrahman SA, El-Shal AS, Abdelrahman AA, Saleh EZH, and Mahmoud AA: designed the research and manuscript writing. Abdelrahman SA, Saleh EZH and Mahmoud AA: performed the practical part of histological examination with analysis of data. El-Shal AS and Abdelrahman AA: performed the practical part of biochemical examination and interpretation of data.

Consent for Publication

All authors have read and approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.