Neuroprotective effect of sea urchins ( Diadema savignyi ) extract in an animal model of aluminum neurotoxicity

Human exposure to heavy metals causes major health consequences. Aluminum (Al) toxicity primarily affects the brain and has been implicated in neurodegenerative disorders. Natural products offer a cheap and safe source of protective agents against heavy metal toxicity. This study investigates the neuroprotective role of the shell extract (SH) from the Sea urchin (Diadema savignyi) collected from the Red Sea in an Aluminum-induced neurotoxicity model. Aluminum (Al, 250 µg/ml) caused cell death in a dose-dependent manner on neuroblastoma (SH-SY5Y) cells. The shell extract (50, 100, and 200 µg/ml) improved cell viability of the neuroblastoma cells. In vivo , toxicity assessment showed that Aluminum administration increased the levels of blood Urea, creatinine, and liver enzymes. Treatment with Shell extract reversed the levels back to normal. Oxidative stress assessment in vitro and in vivo showed that Al caused an increase in Nitric Oxide (NO) concentration and a significant reduction in Catalase and Glutathione (GSH) activity. Treatment with Shell extract (SH) improved these changes. Microscopic examination of the cerebral cortex showed that Aluminum-treated animals had significant neuronal damage, as evidenced by the degenerated neurons and increased apoptosis marker Caspase 3. There was also an increase in glial activation seen by an increase in the expression of Glial fibrillary Acidic Protein (GFAP). Treatment with Shell extract (100 and 200 µg/kg) showed neuroprotective effects all over cortical layers with minimal neuronal degenerative changes. The current work proves the potential antitoxic and neuroprotective properties of Sea Urchins ( Diadema savignyi ) extract as a cheap and safe therapeutic alternative against heavy metal toxicity.


Introduction
Heavy metals, especially Aluminum (Al), are used in various household, medical, and industrial applications, e.g., Antacids, dialysis solutions, and water treatment [1].The nervous system is particularly sensitive to Aluminum exposure as it leads to endoplasmic reticulum (ER) stress and the production of reactive oxidant species (ROS), resulting in DNA damage and neuronal death [1][2][3].Al exposure has also been linked to learning disabilities, memory problems, and neural tube defects, as evidenced by high levels of Aluminum in the hair of newborn babies [4].
To this day, the evidence linking Aluminum and neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease, is controversial [5][6][7].However, long-term exposure to Al in various animal studies caused Al accumulation in degenerated neurons within different parts of the brain, especially the Hippocampus [8,9].Organic and organic Al complexes caused the accumulation of neurofibrillary aggregates in the hippocampus and cortex.In the case of Al-maltolate-treated rabbits, paired helical filaments (PHF) were observed in the axons of hippocampal neurons [10].This makes Al-induced neurotoxicity a good candidate for experimental studies of neurotoxicity and experimental AD models [10].
The marine environment with its biodiversity shows a rich source of bioactive substances.Mediterranean jellyfish [11], sea Cucumber, and Starfish cerebrosides [12] and Sponges [13] may potentially provide a wide variety of therapeutic compounds.Sea Urchins' extracts (class Echinoidea, Subphylum Eleutherozea, Phylum Echinodermata), exhibit abundant pharmacological effects due to the variety of bioactive compounds derived from their shells, spines, gonads, or intestinal extracts.
Previous studies show that Sea urchins have antioxidant [14], anticancer, antibiotic, antiviral, antiprotozoal and antifungal compounds [15].Previously, Sea Urchins extract were shown to contain a good number of phenolic compounds, such as Bisabolol oxide, Oleic acid, Hexadecanoic acid, Eugenol and Levomenthol [16,17].These extracts were shown to have a high safety profile in vitro and in vivo as well as significant neuroprotective properties due to the presence of carotenoids such as astaxanthins [16][17][18].
Therefore, the current study focuses on Sea urchins (Diadema savignyi) shell extracts as a natural neuroprotective agent against Aluminum induced toxicity both in vivo and in vitro.

Animals
All procedures were performed in compliance with the National Institute of Health (NIH) guidelines for the Care and Use of Laboratory Animals.Male Sprague-Dawley (SD) rats (weighing 175-200 gm) were allowed free access to water and food.At the end of the experiment, animals were euthanized by an overdose of Ketamine/ Xylazine (75 mg/kg).The rats were dissected, and samples were collected.

Sea urchins (Diadema savignyi) shell extract preparation
Sea urchins were collected from the red sea and the extraction and characterization were performed as previously described by Khalil et al. 2022 [16].Briefly, the urchins' internal organs were removed, and the empty shells dried at 40 °C.The dried shells were crushed then ground into a fine powder followed by suspension in 100% ethanol (1:1/ w: v).The extract was centrifuged at 8000 RPM for 15 min at 4 °C.The supernatant was concentrated in a rotary evaporator.Extract samples were analyzed by GCMS to determine their chemical composition [16].The extracts were stored at -20 °C until the time of use.

Cytotoxicity assessment
Cells were treated as described in the experimental design section above.Cytotoxicity was assessed using neuroblastoma cells (SH-SY5Y).After treatment for 24 h cells were collected and centrifuged at 12,000 RPM for 4 min at 4 °C.The supernatant was discarded, and the pellet was resuspended in RPMI media.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye was added to the cells and incubated for 4 h.The dye was removed and DMSO was added [21].
The optical density (OD) of color was measured, using a SPECTROstar ® Nano plate reader at 570 nm.The following formula was used to calculate the cell viability:

Oxidative stress assessment
Preparation of neuroblastoma cell lysate Neuroblastoma cells (SH-SY5Y) treated with 250 ug/ml Al + 100 ug/ ml and 200 ug/ml SH extract were collected and centrifuged for 10 min at 4,000 RPM at 4 °C.Supernatants were discarded and the pellets were re-suspended in Phosphate Buffered Saline (PBS) and homogenized using a sonicator (T 25 digital ULTRA-TURRAX ® , IKA ® -Werke GmbH, Germany) for 1 min.The cells were centrifuged again for 15 min to remove cell debris.The supernatant was then used to perform the oxidative stress assays [22].
Brain tissue oxidative stress assessment At the end of the experiment, the brains were harvested, and washed with PBS solution pH 7.4.The brain tissues were homogenized in a 5-10 ml cold buffer using the sonicator (T 25 digital ULTRA-TURRAX ® , IKA ® -Werke GmbH, Germany), then centrifuged for 4,000 RPM for 15 min at 4 °C [23].
The cell and brain tissue homogenates were used for oxidative stress assays NO, CAT and GSH as mentioned below.
Nitric Oxide (NO) assay Nitric Oxide (NO) was assessed using Griess reagent which converts NO 2 into a deep purple azo compound that could be estimated at 540 nm.

Glutathione (GSH) reduced
The method is based on the reduction of Ellman's reagent, 5, 5 dithiobis-(2-nitrobenzoic acid) (DTNB) with glutathione to produce a yellow compound.The reduced chromogen absorbance is directly proportional to GSH concentration in the sample.The absorbance was measured at 405 nm.
Catalase The Catalase activity in cell lysates brain tissues homogenate was measured by the addition of peroxidase (H 2 O 2 ) which reacts with 4-aminophenazone (AAP) and 3,5-dichloro -2-hydroxybenzene sulfonic acid (DHBS), forming a chromophore that is inversely proportionate to the amount of catalase in the sample [23].

In vivo biochemical analysis
After 4 weeks animals were euthanized as previously described and blood samples were collected for renal (Urea, Creatinine) and liver (Alanine Amino Transferase (ALT) and Aspartate Aminotransferase (AST)) functional assessment.

Histological examination
After euthanasia the brains were dissected, washed in PBS and fixed in 4% paraformaldehyde for 24 h.Tissues were dehydrated in alcohol, cleared in terpineol for 24 h and infiltrated in Paraplast at 60 °C for 2 h.Finally, the tissues were embedded in paraffin wax then sectioned using Leica Rotary Microtome TM (Model: 1512).5-6 µm thick sections were mounted on clean glass slides.Sections were deparaffinized and stained with Mayer's Hematoxylin and counterstained with 1% Eosin (H&E).Slides were examined using an Olympus light microscope.

Quantitative analysis
Six non-overlapping fields were randomly scanned per tissue section of each sample to determine the relative area of immunohistochemical expression levels of GFAP and Cleaved Caspase3 in the cerebral cortex of the different groups.Morphological measurements and analyzed data were obtained using Leica Application module for tissue sections analysis attached to Full HD microscopic imaging system (Leica Microsystems GmbH, Germany) [24].

Statistical analysis
Data were analyzed using GraphPad Prism v.7 Software Inc. (San Diego, CA, USA).Data were expressed as the mean ± standard deviation (SD), and P-values < 0.05 were considered statistically significant.One-way ANOVA test was followed by Dunnett's test for multiple comparisons.

In vitro cytotoxicity assessment
Cytotoxicity assessment showed that Aluminum induced reduction in neuroblastoma (SH-SY5Y) cell viability ranging from 65% with Al 250 µg/ml, decreasing to about 25% with Al 2000 µg/ml.The shell extract caused a significant increase in cell viability compared to the control confirming its neuroprotective effect.Treatment of cells with Al (250 µg/ml) followed by the extract restored the cell viability to 100% (Fig. 1).

Oxidative stress assessment
Aluminum caused a significant increase in the Nitric oxide (NO) concentration in both neuroblastoma cells and brain tissue compared to the control.Treatment of the cells with Shell extract (100 and 200 µg/ml) caused a reduction in Nitric oxide (NO) concentration back to the control levels (Fig. 2).Aluminum caused a significant reduction in GSH and Catalase levels in both the cells and brain tissue.Shell extract (100 and 200 µg/ml) increased the concentration level of GSH and Catalase back to control levels (Fig. 2).

Biochemical assessment
Aluminum treated animals showed an increase in blood Urea and Creatinine concentrations (Fig. 3).Treatment with Shell extract reversed this increase back to the control levels.
Liver enzymes (ALT and AST, Fig. 3) almost doubled in the Aluminum group compared to the control.Treatment with the shell extract reversed this increase back to the control levels (Fig. 3).

Histological investigation
Examination of cerebral cortex sections (Fig. 4), showed that Aluminum administration did cause severe histological alterations in the form of marked neuronal degenerative changes in the outer cortical layers with many congested cerebral blood vessels and high reactive astroglial cells infiltrate, compared to the control which showed normal organized morphological features of cortical layers (Fig. 4), with intact intercellular matrix and normal glial cells.Treatment with Shell extracts (100 ug and 200 ug) showed neuroprotective effects all over cortical layers with minimal neuronal degenerative changes in outer cortical layers, and intact intercellular matrix and mild reactive glial cells infiltrates.

Discussion
Heavy metals exposure especially Aluminum (Al) remains a major problem especially in low and middle income countries where Al is used in cooking utensils and in various industrial applications [1,4].The consumption of Aluminum in its many forms is associated with neurological symptoms.Therefore, a cheap treatment alternative is necessary to prevent longterm damage to the nervous system, especially in poor economies where access to high quality healthcare is limited.
Natural products propose safe and cheap solutions to fight Al-induced toxicity.Many studies provide evidence that supports the role of natural plant extracts' vital role in counteracting the damage induced by the Al.For instance, Pomegranate peel extract, Ginkgo Biloba, Bacopa monniera, Pistacia lentiscus and Fenugreek seeds reversed Al-induced toxicity in various studies [2,[25][26][27][28].
Heavy metals such as Cisplatin and Aluminum induce oxidative stress which often leads to disturbances in antioxidant defense enzymes such as superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase, and catalase (CAT).This oxidative stress induces lipid peroxidation, and DNA damage ultimately causing neuronal cell death in the brain [17,29,30].
The current study confirms the safety and neuroprotective activity of the shell extract in vitro [35,36].The extracts also improved oxidative stress parameters in vitro and in vivo.This is consistent with previous reports [5,17,37].Sea Urchin's extracts were previously reported to reverse oxidative stress induced by Cisplatin in brain tissue [17].Aluminum caused significant systemic toxicity in vivo, the extract improved the renal and liver functions which is consistent with previous reports [17,38,39].Previous reports showed an improvement in the histological examination of the liver and kidney tissues showed normal architecture without signs of degeneration [17,40].These results show the great potential of using such extracts either orally or by parenteral administration.It is worth noting that the safety of the sea Urchin extracts in vivo was previously confirmed as evidenced by liver and kidney function testing and histological assessment of liver and kidney tissues [16].
Histological examination of the cerebral cortex shows the neuronal damage caused by Aluminum administration.The apoptotic marker Caspase-3 was significantly increased with aluminum administration.Caspase 3 is involved in the cleavage of amyloid-beta 4A precursor protein linked in Alzheimer disease [41].This elevation in Caspase 3 was reversed with Shell extract treatment, which shows the neuroprotective properties of the extract and its potential in reducing the long-term effects of Al toxicity.The nervous tissue damage was also evident by the glial cell infiltration of the cerebral cortex as seen by the increased expression of the Glial Fibrillary Acidic Protein (GFAP) which is a protein normally found in astrocytes, and radial glia [42].Previous reports also confirm the neuroprotective effects of the Urchin extract following Cisplatin induced neurotoxicity [17].Previous reports showed that exposure to heavy metals caused neuronal apoptosis and reduced Bcl-2 protein levels possibly due to proteolytic cleavage by caspase3/7 or by transcriptional downregulation, and enhanced Bax causing a decreased Bcl-2/Bax ratio, which is key regulator of apoptosis [43,44].

Conclusion
The current work proves the potential for the use of natural products such as Sea Urchins (Diadema savignyi) as a natural remedy against heavy metal toxicity.Chronic exposure to heavy metals such as Aluminum cause permanent damage to the nervous system.Currently there are limited therapeutic approaches to combat the effects of such toxicity.Natural extracts offer a cheap, and safe alternative which is available worldwide.

(Fig. 1
Fig. 1 In vitro cytotoxicity assessment of aluminum and shell extract.a Aluminum (Al) caused cell death in a dose dependent manner compared to the control group.b Shell extract (SH) showed increased cell viability by 50% compared to control.c Shell extract maintained the cell viability of the neuroblastoma cells treated with Al (250 ug/ml) + Shell extract (50, 100, and 200 µg/ml) as compared to control.(n = 4, * p-value < 0.05; **** p-value < 0.0001)

Fig. 3 Fig. 4
Fig. 3 Biochemical analysis of renal and liver functions.Aluminum administration caused an increase in blood Urea and creatinine (A, B) and liver AST and ALT enzymes (D, E).Treatment with Shell extract reversed the levels back to normal.There was no significant difference between the 100 and 200 µg/ml.(n = 4, **** p-value < 0.0001)