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Molecular Biology ReportsAn International Journal on Molecularand Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-018-4451-x
Royal jelly mitigates cadmium-inducedneuronal damage in mouse cortex
Rafa S. Almeer, Rami B. Kassab,Gadah I. AlBasher, Saud Alarifi, SaadAlkahtani, Daoud Ali & Ahmed E. AbdelMoneim
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Molecular Biology Reports https://doi.org/10.1007/s11033-018-4451-x
ORIGINAL ARTICLE
Royal jelly mitigates cadmium-induced neuronal damage in mouse cortex
Rafa S. Almeer1 · Rami B. Kassab2 · Gadah I. AlBasher1 · Saud Alarifi1 · Saad Alkahtani1 · Daoud Ali1 · Ahmed E. Abdel Moneim2
Received: 22 August 2018 / Accepted: 18 October 2018 © Springer Nature B.V. 2018
AbstractThis study aimed to evaluate the potential neuroprotective effect of royal jelly (RJ) against Cd-induced neuronal damage. Twenty-eight adult mice were placed equally into four groups. The control group received intraperitoneal (IP) injections of normal saline; the cadmium chloride (CdCl2) group was IP-injected 6.5 mg/kg (mg per kg of bodyweight) CdCl2; the RJ group was gavaged 85 mg/kg RJ; and the RJ + CdCl2 group was orally administered 85 mg/kg RJ 2 h before receiving IP-injections of 6.5 mg/kg CdCl2. All groups were treated for seven consecutive days and the mice were decapitated 24 h after the final dose. Cd accumulation was recorded in the cortical homogenates, accompanied by elevated levels of lipid peroxi-dation, nitric oxide, tumor necrosis factor-α, interleukin-1β, and the pro-apoptotic mRNA Bax and caspase-3. Meanwhile, significantly decreased levels of detoxifying antioxidant enzymes including GSH-Px, GSH-R, SOD, and CAT, anti-apoptotic mRNA Bcl-2, and monoamines such as norepinephrine, dopamine, and serotonin were also observed, along with reduced gene expression of Nrf2-dependent antioxidants. Interestingly, in mice pretreated with RJ, the assessed parameters remained near normal levels. Our data provide evidence that RJ treatment has the potential to protect cortical neurons in Cd-intoxicated mice via its antioxidant, anti-inflammatory, anti-apoptotic, and neuromodulatory activity.
Keywords Royal jelly · Cadmium · Neurotoxicity · Mice
Introduction
Cadmium (Cd) is a heavy metal that occurs widely in nature; it has been characterized as a highly dangerous environmen-tal pollutant known to cause severe adverse health effects in living organisms [1]. Cd exposure results mainly through inhalation and ingestion from various sources, including cigarette smoke, water, contaminated food, and industrial pollution [2]. Given that Cd is nondegradable and has a long biological half-life, it accumulates and produces toxicity in
Rafa S. Almeera, Rami B. Kassabb, and Ahmed E. Abdel Moneimb have contributed equally to this work.
Gadah I. AlBashera, Saud Alarifia, Saad Alkahtania, and Daoud Alia have also contributed equally to this work.
Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1103 3-018-4451-x) contains supplementary material, which is available to authorized users.
* Rafa S. Almeer [email protected]
Rami B. Kassab [email protected]
Gadah I. AlBasher [email protected]
Saud Alarifi [email protected]
Saad Alkahtani [email protected]
Daoud Ali [email protected]
Ahmed E. Abdel Moneim [email protected]
1 Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia
2 Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt
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organs such as the liver, kidney, brain, testes, and lung [3, 4]. Numerous neurological disturbances have been observed following Cd exposure, including Alzheimer’s disease, Par-kinson’s symptoms, schizophrenia, depression, hyperactiv-ity disorder, anxiety, learning disabilities, mood disorders, and attention deficit disorder [5, 6]. The mechanisms of Cd-induced neurotoxicity remain unclear. However, sev-eral reports have suggested that Cd may mediate its toxicity through the production of reactive oxygen species (ROS), disturbing differentiation and inducing neurochemical altera-tions, inflammation, and apoptosis. Moreover, Cd is reported to be positively associated with DNA oxidation [7, 8] and to disturb the integrity and permeability of the blood brain bar-rier, accumulating in the frontal cortex which in turn affects cognitive abilities [9, 10].
Owing to the fact that Cd is closely associated with oxida-tive stress, the use of antioxidants as a therapeutic strategy may mitigate its toxicity, rather than chelation therapy which causes several side effects [11].
Royal jelly (RJ) is a milky-white liquid secreted by the hypopharyngeal glands of nurse bees to feed young larvae and the queen [12]. The composition of RJ includes water (60–70%), carbohydrates (11–23%), proteins (9–18%), lipids (4–8%) and other low-level substances such as vitamins and mineral salts [13–15]. However, RJ ingredients vary depending on seasonal variation, race and genetic variation of bees, and regional conditions [12]. RJ exhibits a mul-titude of pharmacological activities, including antitumor, anti-inflammatory, neuroprotective, antioxidant, and hypo-glycemic [16–20].
Recently, our group demonstrated the hepatoprotective effects of RJ on CdCl2-induced hepatotoxicity [10]. How-ever, little is known about the neuroprotective role of RJ. Therefore, the present study evaluated the potential protec-tive activity of royal jelly against cadmium-induced neuronal damage by assessing the oxidants, antioxidants, pro-inflam-matory cytokines, apoptotic protein markers, cholinergic neurons, and monoamine content in the frontal cortex of male mice with and without the administration of RJ.
Materials and methods
Chemicals
Cadmium (II) chloride anhydrous was sourced from Sigma-Aldrich (St. Louis, MO, USA). Lyophilized royal jelly was purchased from PHARCO Pharmaceuticals (Alexandria, Egypt) and it contains 6% 10-hydroxy-2-decenoic acid equivalent to 250 mg crude royal jelly. PCR primers and TRIzol reagent were sourced from Invitrogen (Carlsbad, CA, USA). Thermo Scientific First Strand cDNA Synthesis Kit was obtained from Thermo Fisher Scientific Inc. (Waltham,
MA, USA). All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.
Experimental animals
Experiments were carried out on 28 adult male Swiss mice, weighing 22–27 g, obtained from VACSERA (Cairo, Egypt). The animals were maintained at 22–25 °C and exposed to a 12-h photoperiod. They had free access to a standard diet and water ad libitum. All protocols and animal handling procedures were approved by the Committee on Research Ethics for Laboratory Animal Care at the Department of Zoology, Faculty of Science, Helwan University (approval no, HU2017/Z/03). After 1 week of acclimatization, animals were randomly allocated into four groups (n = 7 mice/group). The control group received normal saline intraperitoneally; the CdCl2 group was IP-injected with 6.5 mg/kg bodyweight (bwt) CdCl2; the RJ group was gavaged with 85 mg/kg RJ; and the RJ + CdCl2 group was orally administered with 85 mg/kg RJ 2 h before IP-injection with 6.5 mg/kg CdCl2. All groups were treated for seven consecutive days, and the mice were decapitated 24 h after the final dose. Brain corti-ces were rapidly dissected, thoroughly washed with isotonic saline, then weighed. For the biochemical investigations, brain tissue was homogenized in ice-cold 10 mM phosphate buffer (pH 7.4) to produce a 10% (w/v) homogenate. In addi-tion, each cortical tissue sample was homogenized in 75% aqueous HPLC grade methanol (10% w/v). The homogenate was spun at 4000 rpm for 10 min for monoamine analysis. Total protein concentration of each cortical homogenate was estimated for all assays using the method described by Lowry et al. [21].
Cd concentration in brain tissue
Graphite furnace atomic absorption spectrophotometry (Per-kin-Elmer 3100) was used to estimate cadmium concentra-tion in brain tissue. Briefly, cortical tissue was dried in an oven at 100 °C for 8 h. Thereafter, 2 M hydrochloric acid and 2 M nitric acid added to digest the dried samples for 5 h at 150 °C. Samples were then diluted with deionized water to 50 mL and analyzed at 283.3 nm.
Biochemical analyses
Antioxidant parameters
Cortical glutathione (GSH) content was measured using the method reported by Ellman et al. [22]. Glutathione peroxi-dase (GSH-Px) and glutathione reductase (GSH-R) activities were determined utilizing procedures described by Paglia and Valentine [23] and De Vega et al. [24], respectively.
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Superoxide dismutase (SOD) activity was estimated using the protocol described by Nishikimi et al. [25] and catalase (CAT) activity was assayed according to Aebi [26].
Oxidant parameters
Thiobarbituric acid reactive substances (TBARSs) were estimated in the frontal cortex as an indicator of lipid per-oxidation (LPO) levels according to the protocol described by Ohkawa et al. [27]. Nitric oxide (NO) level was estimated with Griess reagent [28].
Inflammatory markers assays
Levels of tumor necrosis factor-α (TNF-α; Cat. No. EZMTNFA, MilliporeSigma, Burlington, MA, USA) and interleukin-1β (IL-1β; Cat. No. EM2IL1B, ThermoFisher Scientific) in the cortical homogenate were evaluated according to the manufacturers’ instructions.
Quantitative reverse transcription‑polymerase chain reaction analysis
Extraction of total RNA from cortical tissue was performed using standard TRIzol procedures (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed to obtain cDNA. Primer sequences used to investigate genes in the current study are listed in Table 1. Power SYBR® Green Master Mix was used for real-time PCR analysis which was performed in triplicate. Real-time PCR cycling condition was 10 min at 95 °C followed by 40 cycles involving denaturation at 94 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 20 s. The findings were estimated and reported with regard to the changes in the studied gene expression between
treated and control animals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene; its expression remained unchanged throughout the experiment.
Histological examination
To identify the cortical deformations and alterations using light microscopy, frontal cortices were dissected and trans-ferred into 10% buffered formalin for fixation. After 24 h, specimens were embedded in paraffin wax and stained with hematoxylin and eosin (H&E) after sectioning at a thickness of 5 µm. Images were captured at an original magnification of × 400 (Nikon Eclipse E200-LED, Tokyo, Japan).
The severity of the brain damage was graded using the numerical method of Shackelford et al. [29], with five grades of severity of lesions specified as follows: 1 = mini-mal (< 1%); 2 = slight (1–25%); 3 = moderate (26–50%); 4 = moderate/severe (51–75%); and 5 = severe (76–100%).
Immunohistochemistry analysis
To study the expression of apoptotic proteins in the frontal cortex, the prepared sections were mounted and depar-affinized. The slides were then washed with boiled water to unmask the antigen sites. Thereafter, sections were treated with 0.1% hydrogen peroxide in absolute metha-nol for 15 min to remove endogenous peroxidase activity. Then cortical sections were incubated overnight at 4 °C with rabbit polyclonal caspase-3 antibody. Sections were washed with phosphate-buffered saline (PBS) to remove the unbound primary antibodies and then incubated with goat-derived secondary anti-rabbit antibody. Thereaf-ter, sections were incubated at 30 °C for 30 min with streptavidin–peroxidase complexes. Diaminobenzidine
Table 1 Primer sequences of genes analyzed in real time PCR
The abbreviations of the genes; GAPDH glyceraldehyde-3-phosphate dehydrogenase; SOD2 superox-ide dismutase 2 mitochondrial (MnSOD); CAT catalase; GSH-Px1 glutathione peroxidase 1; GSH-R glu-tathione reductase; Nrf2 nuclear factor erythroid 2-related factor 2; iNOS inducible nitric oxide synthase; IL-1β interleukin 1 beta; TNF-α tumor necrosis factor; Bcl-2 B-cell lymphoma 2; Bax Bcl-2-like protein 4
Name Accession number Forward primer (5′–3′) Reverse primer (5′–3′)
GAPDH NM_001289726.1 TCA CCA CCA TGG AGA AGG C GCT AAG CAG TTG GTG GTG CASOD2 NM_013671.3 GCC CAA ACC TAT CGT GTC CA AGG GAA CCC TAA ATG CTG CCCAT NM_009804.2 CCG ACC AGG GCA TCA AAA GAG GCC ATA ATC CGG ATC TTC GSH-Px1 NM_001329527.1 CAG CCG GAA AGA AAG CGA TG TTG CCA TTC TGG TGT CCG AAGSH-R NM_010344.4 TGG CAC TTG CGT GAA TGT TG CGA ATG TTG CAT AGC CGT GGNrf2 NM_010902.4 CCT CTG TCA CCA GCT CAA GG TTC TGG GCG GCG ACT TTA TTiNOS NM_001313922.1 CGA AAC GCT TCA CTT CCA A TGA GCC TAT ATT GCT GTG GCT IL-1β NM_008361.4 TGC CAC CTT TTG ACA GTG ATG TTC TTG TGA CCC TGA GCG ACTNF-α NM_013693.3 AGA GGC ACT CCC CCA AAA GA CGA TCA CCC CGA AGT TCA GTBcl-2 NM_009741.5 GAC AGA AGA TCA TGC CGT CC GGT ACC AAT GGC ACT TCA AGBax NM_007527.3 CTG AGC TGA CCT TGG AGC GAC TCC AGC CAC AAA GAT GCaspase 3 NM_001284409.1 GAG CTT GGA ACG GTA CGC TA CCG TAC CAG AGC GAG ATG AC
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(DAB)-hydrogen peroxide was used to enhance peroxidase activity. Sections were visualized using × 400 magnifica-tion (Nikon Eclipse E200-LED, Tokyo, Japan).
Statistical analysis
Data were expressed as mean ± SD (standard deviation). One-way ANOVA followed by Tukey’s post hoc test using a statistical software package (SPSS version 17.0) were carried out for the statistical analysis. p values < 0.05 were considered statistically significant.
Results
A significant elevation (p < 0.05; F = 302.76) in Cd concen-tration was recorded in cortical tissue following CdCl2 injec-tion (6.5 mg/kg bwt) for seven consecutive days as compared to control mice. Administration of RJ (85 mg/kg bwt) was able to mitigate this increase significantly in Cd-exposed mice (Fig. 1).
The Cd accumulation was found to be associated with oxidative stress as evidenced by a significant increase in LPO and NO levels and a decrease in GSH content (p < 0.05; F = 55.87, 47.75 and 19.24 respectively) in cortical tissue with respect to the control values. The levels of the cor-tical LPO, NO and GSH were unchanged statistically in RJ treated mice as compared with the control mice. Con-versely, the mice pretreated with RJ along with CdCl2 sig-nificantly abrogated these conditions when compared to the CdCl2-intoxicated group, reflecting the antioxidant activity of RJ (Fig. 2).
Our findings also showed a disturbance in the endoge-nous enzymatic system. The activity of GSH-Px, GSH-R, SOD, and CAT was significantly reduced (p < 0.05; F = 8.60, 15.89, 30.44 and 12.96 respectively) in Cd-treated mice, in comparison with the untreated group. Meanwhile, the activ-ity of GSH-Px, GSH-R, SOD, and CAT was like normal control values in RJ treated group. Pre-treatment with RJ in the Cd-treated mice reversed these changes in the cortical homogenate as compared to Cd-intoxicated mice devoid of RJ. These results were confirmed by assessing the mRNA expression of GSH-Px1, GSH-R, SOD2, and CAT using qRT-PCR. Our results showed downregulation of these genes in the cortical tissue of Cd-intoxicated animals. Interestingly, the treated mice with RJ showed a significant upregulation in the expression of GSH-R, SOD, and CAT but not GSH-Px1 when compared with the control group. Meanwhile, the pre-administration with RJ significantly
Fig. 1 The effect of royal jelly (RJ) on cadmium accumulation in mouse cortical neurons after daily treatment with cadmium chloride (CdCl2) for 7 days. Data are expressed as mean ± SD (n = 7); results with different letters are significantly different at p < 0.05 using Tukey post hoc test
Fig. 2 Effects of royal jelly (RJ) on LPO, NO, and GSH in corti-cal homogenates of mice treated with 6.5 mg/kg cadmium chloride (CdCl2) daily for 7 days. Data are expressed as mean ± SD (n = 7);
LPO, lipid peroxidation; NO, nitric oxide; GSH, glutathione; results with different letters are significantly different at p < 0.05 using Tukey post hoc test
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upregulated the mRNA expression of the antioxidant genes tested in the cortical tissue with respect to Cd-injected mice (Fig. 3).
Cd was able also to decrease the gene expression of nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) sig-nificantly (p < 0.05; F = 30.91), which plays a protective role against oxidative damage in the cell. While the level of Nrf2 remained unchanged in RJ treated mice. RJ-pretreated mice exposed to Cd upregulated the transcriptional expression of the tested genes. Moreover, RT-qPCR results showed that, Cd injection caused upregulation of iNOS protein (p < 0.05; F = 36.20), which is responsible for the production of NO. In addition, the mRNA expression of iNOS in RJ treated mice was unchanged significantly. Whereas, RJ pretreated mice exhibited a downregulation of iNOS expression in the corti-cal tissue when compared against Cd-exposed mice (Fig. 4). These results demonstrate that RJ mediates its antioxidant defense capacity by activating Nrf2 and gene transcription of cytosolic antioxidant enzymes, providing a possible mecha-nism for its neuroprotective action in Cd-induced oxidative status in mouse cerebral cortex.
To study the potential anti-inflammatory properties of RJ, pro-inflammatory cytokines were measured using ELISA kits in the analysis of cortical tissue. The Cd-exposed group experienced a significant increase (p < 0.05; F = 46.83 and 27.06 respectively) in TNF-α and IL-1β levels as compared to the untreated mice. Animals pretreated with RJ one hour prior to Cd-injection exhibited a marked decrease in the lev-els of these inflammatory mediators when compared to mice treated with Cd devoid of RJ. Consistent with these results, RT-qPCR results showed upregulation of the gene expres-sion of TNF-α and IL-1β (F = 57.38 and 31.02 respectively) in Cd-treated mice as compared to control levels. In addi-tion, mice treated with RJ alone showed a non-significant change in the levels and the expression of TNF-α and IL-1β as compared to the control group. Meanwhile, RJ pretreat-ment was able to downregulate their expression in cortical tissue (Fig. 5).
Histological screening of cerebral cortices of control and RJ-treated mice showed normal cortical architecture (Fig. 6a, b). In contrast, degenerated nerve cells with deeply stained nuclei, several apoptotic neurons, inflammatory cell infiltration, and intracellular and extracellular vacuoles were recorded in Cd-exposed mice (Fig. 6c). Interestingly, royal jelly administration was able to abolish most of the cor-tical damage produced by cadmium (Supplementary data: Table S1).
To further elucidate the possible anti-apoptotic action of RJ against cadmium toxicity, the immunoreactivity of pro-apoptotic protein caspase-3 was assessed in cortical tissue. Mice injected with Cd exhibited a marked increase in the expression of caspase-3 in comparison to the control values. Conversely, pre-administration with RJ greatly alleviated
greatly these conditions in the tested apoptotic markers as compared to Cd-intoxicated mice (Fig. 7). In addition, RT-qPCR findings revealed a significant increase in mRNA expression levels of Bax and caspase-3 (p < 0.05; F = 51.14 and 23.40, respectively) and a decrease in the expression of Bcl-2 (p < 0.05; F = 10.26) in the cortical tissue of Cd-treated mice (Fig. 8). While, the levels of these apoptotic markers were non-significantly changed in RJ treated mice. However, RJ pretreatment was able to significantly reverse the recorded alterations in pro-apoptotic and anti-apoptotic proteins produced by cadmium.
Our study also showed alterations in the levels of bio-genic amines in the cortical homogenates of the experimen-tal mice (Fig. 9). We observed a significant decrease in the content of NE, DA, and 5-HT (p < 0.05; F = 53.89, 22.60 and 20.36, respectively) in CdCl2-intoxicated mice. The tested monoamines concentration was unchanged significantly in RJ treated mice. Moreover, the pre-administration with RJ significantly restored the levels of these monoamines to near normal, i.e., control values.
Discussion
Cd is a toxic heavy metal, occurring widely in nature that causes deleterious effects on animals and humans. Several reports highlight the link between Cd exposure and distur-bances in brain redox status, cell signaling pathways, neuro-inflammation, neurochemistry, and neurodegeneration.
Cd is known to accelerate the development of severe neurological complications such as autism, schizophrenia, depression, Parkinson’s disease, and Alzheimer’s disease [5, 8, 30]. Cd has been observed to alter the permeability properties of the blood brain barrier, accumulating mainly in the cerebral cortex which has been classified as a target for Cd-intoxication [9, 31]. This may explain the marked elevation of cortical Cd concentration in the present study.
The accumulation of Cd in cortical tissue has been found to be closely associated with oxidant/antioxidant imbalance in our experiment as evidenced by increased levels of LPO and NO and the decrease in GSH content. The production of reactive oxygen species (ROS) is one of the most accepted potential mechanisms involved in Cd-induced oxidative damage in brain tissue due to its high oxygen consumption. Lipid peroxidation is a series of reactions that take place in lipid compartments of the plasma membrane as a result of ROS production following Cd exposure [32]. The increased LPO level in serum and brain homogenate has been used as an important marker for the development of several neuronal diseases, such as Huntington’s disease, Parkinson’s disease, Down syndrome, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease [33]. The excessive generation of reactive nitrogen species (RNS), including NO, produces
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severe neuronal injury through interaction with superoxide anions, producing peroxynitrite anion (ONOO−), which is more active than its precursor in causing cytotoxicity [34]. According to our findings, the increase in NO production is due to the over-expression of iNOS observed in corti-cal tissue. In this study, pretreatment with RJ decreased the previously elevated levels of LPO and NO to be near the control values and ameliorated Cd-induced oxidative damage in cortical tissue. Mohamed et al. [17] recorded a decrease in LPO levels in brain tissue after treatment with RJ in tartrazine-intoxicated rats, reflecting its neuroprotec-tive effect. The suppression of LPO by RJ could be due to the peptide content of RJ, which has the ability to quench hydroxyl radicals [35].
Under normal physiological conditions, the enzymatic and non-enzymatic antioxidant system (GSH, GSH-Px, GSH-R, SOD, and CAT) antagonizes oxidants (LPO and NO) to ameliorate oxidative damage. However, we found in the present study that this defense system is markedly depleted as a result of Cd-intoxication. Inhibition of the endogenous antioxidant system may be due to several mechanisms, including the ability of cadmium to bind with sulfhydryl groups which in turn inactivate the glutathione pool enzymes [36], the consumption of glutathione dur-ing free radical removal [37], elevated lipid peroxidation levels [33], over-production of superoxide anions [38], up-regulation of stress gene expression which could inhibit the activity of antioxidant enzymes [39], and downregulation of the expression of the antioxidant genes confirmed in our experiments. Interestingly, the mice pre-administered with RJ reversed the oxidative damage induced by Cd exposure by increasing glutathione content and enhancing GSH-Px, GSH-R, SOD, and CAT activities, demonstrating its potent antioxidant capacity in neuronal cells. Previous reports dem-onstrated that RJ maintained the brain antioxidant system by decreasing LPO levels and increasing the glutathione pool in different brain regions in a stressed rat model [40]. In addition, the administration of RJ in traumatized rabbits exhibited a marked decrease in LPO and NO levels and an increase in GSH content and CAT activity [41]. Further-more, RJ restored the alterations in the antioxidant system following cadmium-induced testicular dysfunction [42].
Furthermore, 10-hydroxy-2-decenoic acid, the major lipid constituents in RJ, has been recorded to have estrogen like effect [43]. Moreover, estrogens, notably estradiol is well known to provide neuroprotective effect through scavenging free radicals, chelating redox-active metal ions, inhibiting lipid peroxidation and increasing the antioxidant enzymes in the brain tissue [44]. Mladenović et al. [45] reported that estradiol reversed Cd-induced changes in the oxidative status via quenching ROS, suppressing lipid peroxidation level, increasing GSH content and enhancing the activities of SOD and CAT in blood of rats.
Transcription factor Nrf2 was found to protect nervous system tissue by enhancing the genetic expression of cellular detoxifying and antioxidant enzymes, suppressing inflam-matory responses following brain injury, and maintaining Ca2+ homeostasis [46, 47]. The decrease in Nrf2 expression in cortical tissue after Cd-intoxication may also explain the downregulation of mRNA expression of GSH-Px1, GSH-R, SOD2, and CAT in the present study. Saleh et al. [48] demonstrated that rabbits exposed to Cd showed Nrf2 down-regulation due to the overexpression of Keap1 mRNA which enhance the degradation of Nrf2 through ubiquitin protea-some pathway in brain homogenates. In the current experi-ment, the mice pretreated with RJ showed activation of Nrf2 expression in cortical tissue that was associated with genetic upregulation of the tested antioxidant enzymes.
Neuroinflammation has been closely associated with oxidative damage following exposure to heavy metals. Treatment with CdCl2 elicited an inflammatory response made evident by the excessive release of pro-inflamma-tory cytokines (TNF-α and IL-1β) in cortical cells. Sev-eral reports attributed the elevation of these inflammatory mediators after Cd-intoxication to the over-production of ROS which plays a crucial role in the progression of neuro-degenerative diseases [49, 50]. The cortical levels of TNF-α and IL-1β decreased in RJ pretreated mice. You et al. [51] studied the potential anti-inflammatory activity of RJ in microglia cells. The authors found that RJ downregulated mRNA expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α in microglia cells through the suppression of the NF-κB, JNK, and p38 signaling pathways.
In the current study, Cd enhanced cortical apoptosis as evidenced by the elevation of pro-apoptotic mRNA expres-sion (Bax and caspase-3), while Bcl-2, which protects the cell from pro-apoptotic proteins, was downregulated. Our findings are in agreement with previous reports [9, 52, 53]. Cd is well known to disturb calcium ion channels and mitochondrial membrane potential leading to the release of cytochrome C, which in turn activates an apoptotic cascade [9]. The elevated levels of LPO and NO have been impli-cated as a possible mechanism in the induction of neuronal apoptosis [34, 54]. The activation of the JNK, ERK1/2, and mTOR signaling pathways are also involved in Cd-induced
Fig. 3 Effects of royal jelly (RJ) on the activity of endogenous anti-oxidant enzymes and their gene expression in mouse cerebral cortices treated with cadmium chloride (CdCl2) for 7 days. Results of antioxi-dant activities are expressed as mean ± SD for 7 mice, whereas results of mRNA expression (mean ± SD of triplicate assays) were normal-ized to GAPDH and expressed as fold change (log2 scale), relative to mRNA levels in controls; SOD2 superoxide dismutase 2 mitochon-drial (MnSOD); CAT catalase; GSH-Px1 glutathione peroxidase 1; GSH-R glutathione reductase; GAPDH glyceraldehyde 3-phosphate dehydrogenase; results with different letters are significantly different at p < 0.05 using Tukey post hoc test
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neuronal apoptosis [55]. In our experiment, RJ suppressed Cd-induced cortical apoptosis through the up-regulation of the anti-apoptotic mRNA Bcl-2. Mohamed et al. [17] showed that RJ has an anti-apoptotic effect in rat brain tis-sue treated with the neurotoxin tartrazine.
Our findings reveal that Cd causes alterations in the corti-cal histological structure, as evidenced by the appearance of degenerated neurons with deeply stained nuclei, inflamma-tory cell infiltration, and intracellular and extracellular vacu-oles. It has been proposed that the presence of dark neurons reflects the development of an apoptotic cascade [56]. Vacu-olation could be due to the elevated lipid peroxidation, which
affects both cell membranes and organelle membranes. This effect enables the excessive flow of sodium into the cell, increasing water content and finally cellular swelling [57].
Neurotransmitters play an essential role during neurogen-esis; their role in the development of neurodegenerative dis-orders cannot be ignored. Therefore, chemicals that interfere with neurotransmitters may produce neurodevelopmental alterations [58]. Our experiment showed that Cd is able to decrease cortical NE, DA, and 5-HT contents. Monoaminer-gic disturbance has been recorded in animal models follow-ing Cd-exposure [8, 32]. The depletion of monoamines after Cd-intoxication might be due to ROS production, which
Fig. 4 Effects of royal jelly (RJ) on gene expression of nuclear fac-tor (erythroid-derived 2)-like-2 factor (Nrf2) and inducible nitric syn-thase (iNOS) in mouse cortical neurons treated with cadmium chlo-ride (CdCl2) for 7 days. Results (mean ± SD of triplicate assays) were
normalized to GAPDH and expressed as fold change (log2 scale), relative to mRNA levels in controls; results with different letters are significantly different at p < 0.05 using Tukey post hoc test
Fig. 5 Effects of royal jelly (RJ) on the levels and gene expression of TNF-α and IL-1β in mouse brain cortical neurons treated with cadmium chloride (CdCl2) for 7 days. Results of inflammatory markers are expressed as mean ± SD of 7 mice, whereas results of mRNA expression (mean ± SD of tripli-cate assays) were normalized to GAPDH and expressed as fold change (log2 scale), relative to mRNA levels in controls; TNF-α tumor necrosis factor-α; IL-1β interleukin 1β; results with different letters are significantly different at p < 0.05 using Tukey post hoc test
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inactivates enzymes involved in monoamine biosynthesis, enhancing their degradation and blocking their turnover [59, 60]. This decrease in the levels of NE, DA, and 5-HT in the cortex was found to affect cognitive, motor, and behavioral functions [61, 62]. Interestingly, monoaminergic produc-tion in the cortical cells was revived in RJ-treated mice, demonstrating the neuroprotective activity of RJ against the disturbances that occurred following Cd treatment. Similar results were also obtained by Mohamed et al. [17]. RJ was found to contain active ingredients such cAMP-N1 oxide and 10-hydroxy-trans-2-decenoic acid; these compounds act in neuronal differentiation and stimulate the generation of different brain cells [63].
Conclusion
In summary, oral supplementation with RJ has a beneficial role in protecting against Cd-induced cortical damage in mouse brain, due primarily to its antioxidant properties, demonstrated by its capacity to suppress LPO and NO levels, sustain detoxifying antioxidant enzymes includ-ing GSH-Px1, GSH-R, SOD2, and CAT, and regulate Nrf2-dependent antioxidant gene expression. In addition, we found that RJ has anti-inflammatory capabilities, ren-dered by reversing elevated TNF-α and IL-1β levels and downregulating their gene expression. Moreover, RJ was able to prevent neuronal loss via its anti-apoptotic effect, by upregulating Bcl-2 and downregulating Bax and cas-pase-3. Its neuroprotective effect was also extended to enhance monoaminergic transmission. These results may suggest mechanisms by which RJ might protect cortical neurons against Cd-induced neurotoxicity.
Fig. 6 Photomicrographs of cortical tissue. Cortical tissues from the control and royal jelly-treated groups (a and b, respectively) showing normal cortical structure; from the cadmium chloride (CdCl2)-treated mice (c) showing intracellular and extracellular vacuoles (black colored right pointed arrow), inflammatory cell infiltration (filled star), and degenerated neurons with deeply stained nuclei indicat-
ing apoptosis (green colored right pointed arrow). Pretreatment with royal jelly (d) markedly attenuated all cortical damage caused by cadmium. However, a few degenerative neurons in the cortical tissue are still found. Hematoxylin and eosin (H&E), scale bar = 100 µm. (Color figure online)
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Fig. 7 Photomicrographs of changes in expression level of caspase-3 in cortical tissue following treatment with royal jelly (RJ) and cadmium chloride (CdCl2); scale bar = 100 µm
Fig. 8 Effects of royal jelly (RJ) on gene expression of Bcl-2, Bax, and caspase-3 in cerebral cortices of CdCl2-treated mice. Data (mean ± SD of triplicate assays) were normalized to those of GAPDH
and expressed as fold change (log2 scale), relative to mRNA levels in controls; results with different letters are significantly different at p < 0.05 using Tukey post hoc test
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Acknowledgements The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the research Group Project No. RGP-180.
Funding This research was supported by the Deanship of Scientific Research at King Saud University. Research Project No. RGP-180.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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