Organic silicon protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide effects

Background Hydrogen peroxide (H2O2) is a toxic agent that induces oxidative stress and cell death. Silicon (Si) is a biological element involved in limiting aluminium (Al) absorption with possible preventive effects in Alzheimer’s disease. However, Si has not yet been associated with other neuroprotective mechanisms. Methods The present experiments evaluated in the SH-SY5Y human neuroblastoma cell line the possible role of different Si G5 (50-1000 ng/mL) concentrations in preventing cellular death induced by H2O2 (400 μM, 24 hours). Results Our findings showed that H2O2 promoted cell death in the human SH-SY5Y cell cultures and this could be prevented by Si treatment. The loss in cell viability mediated by H2O2 was due to an apoptotic and necrotic process. Apoptotic death was incurred by regulating caspase-8 activity in the extrinsic pathway. The apoptotic and necrotic cell death induced by H2O2 was almost totally reversed by Si (50-500 ng/mL), indicating that it down-regulates both processes in H2O2 treated cells. Conclusions According to our data, Si is able to increase SH-SY5Y cell survival throughout partially blocking cellular damage related to oxidative stress through a mechanism that would affect H2O2/ROS elimination.


Background
Silicon (Si) is a biologically important element that is soluble in water as silicic acid, Si(OH) 4 . It has been reported that Si is a decisive factor in limiting absorption of dietary Aluminium (Al) [1]. Si administration reduced Al accumulation in several tissues, including brain areas of rats orally exposed to Al [2,3]. In the form of silicon dioxide (SiO 2 ), Si is the most abundant element on the Earth's crust together with oxygen. Nevertheless, its biological role has not yet been well studied [4]. Si has been considered an "essential" element since chicks receiving Si-deprived diet showed poor growth [5], and because it is also necessary to assure normal growth impairment [6]. Apart from its physiological role in bone and cartilage formation, Si has been associated with cardiovascular protection, immune system enhancement and protective effects in the Alzheimer's disease [7,8]. Despite its numerous properties, its biological function remains unclear. Its blood concentration is similar to other elements like zinc [9] and in urine the concentration is like that of calcium, but contrary to other elements and although it is quite ubiquitous [10], it does not associate with plasma proteins [11] and does not have an identified binding site [12]. Finally, Si does not seem to take part in any biochemical reactions/interactions because it does not have bioorganicinorganic chemistry [13]. Ortho-silicic acid, or monomeric Si(OH) 4 , is water soluble and stable in highly diluted aqueous solutions. It plays a crucial role in delivering Si to the living organisms' cells. Thus, it represents the major source of Si for both humans and animals. The recommended daily Si intake (RDI) has not yet been set [14,15]. Some studies suggest that serum Si levels decrease in pregnancy [16] and with aging, particularly in women [17]; thus, supplementation with Si in available forms, such as organic Si, could prevent degenerative processes. As already commented, Si reduces Al bioavailability, thereby possibly limiting Al neurotoxicity. Consumption of moderately large amounts of beer and ortho-silicic acid, in both humans and mice, reduced Al uptake from the digestive tract and prevented the accumulation of Al in the brain [1,18]. Silicic acid has also been found to induce down-regulation of endogenous antioxidant enzymes associated with Al administration and to normalize tumour necrosis factor alpha (TNF-α) mRNA expression [19]. Davenward et al. [20] showed that Si-rich mineral waters can be used as a non-invasive method to reduce the body burden of Al in both Alzheimer's patients and a control group by facilitating the removal of Al via the urine without any other concomitant effect. Si has also been shown to induce clinically relevant cognitive improvements in a pilot study with 3 of 15 patients with Alzheimer's disease [20][21][22]. This suggests a possible use for ortho-silicic acid as long-term non-invasive therapy for Al reduction in Alzheimer's disease patients.
To the best of our knowledge, the effect of organic Si on oxidative stress induced by H 2 O 2 pre-treatment of SH-SY5Y cells has not been evaluated. The present paper hypothesized that Si plays a neuroprotective role against the H 2 O 2 oxidative aggression by removing ROS. It has been analysed whether organic Si G5 could induces protective effects against H 2 O 2 neurotoxicity in human neuroblastoma SH-SY5Y cell line. Cellular viability on the MTT assay, caspase activation (caspase-3,-8,-9) and LDH release were all quantified to evaluate the effects of Si and H 2 O 2. In the present study H 2 O 2 was chosen as toxic agent because: a) it is the most stable of the longlived reactive oxygen species (ROS) [23]; b) it passes through cellular membranes [24]; and c) free radicals are involved in the pathogenesis of many degenerative diseases such as Alzheimer's, Parkinson's and Multiple sclerosis [25].

Cell culture and treatment
Human SH-SY5Y neuroblastoma cells were grown in DMEM supplemented with 10% foetal bovine serum (FBS) and 100 IU/ml penicillin/streptomycin, maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. The culture medium was removed every three days and sub-cultures were used once they reached 80-90% confluence. 24 h later, cells were treated in DMEM containing 1% FBS in the presence/absence of organic silicon (50 ng/mL -20,000 ng/mL), H 2 O 2 400 μM or both, together and added at the same time. H 2 O 2 was freshly prepared from a 30% stock solution prior to each experiment. Control cells without Si treatment were included in all experiments. Treatment was maintained for 24 h except in the experiment of ROS measurement in which cells were treated for only 2 h.

Protein estimation
The protein concentrations of cell extracts were determined by the Bradford [26] method using bovine serum albumin as standard.

MTT assay
This assay is based on the ability of living metabolically active cells to reduce the MTT, a soluble formazan salt, given a purple colour. Therefore, the conversion only occurs in living cells. The MTT assay provides a sensitive measurement of the metabolic status of cells and is used to evaluate cell viability. SH-SY5Y were seeded into 96well culture plates (3×10 4 cells/well) and allowed to attach. After the above treatment MTT, solution (2 mg/mL) was added to each well and incubated in the dark for 1 hour at 37°C. Supernatants were removed, blue formazan crystals were dissolved in DMSO and absorbance at 595 nm was measured with a microplate reader (LT-4000, Labtech International Ltd, United Kingdom). All MTT assays were performed in quadruplicate. The results are expressed as the percentage of MTT reduction relative to control cells.

LDH release measurement
Intracellular enzyme lactate dehydrogenase is released into the cell culture medium when cell membrane damage occurs. LDH release is a good marker to detect necrosis. Enzyme activity was determined by spectrophotometric assay according to López et al. [27]. Briefly, NADH oxidation produces a decrease in the absorbance at 340 nm, and this was measured to determined LDH activity by the following formula: SH-SY5Y cells (3×10 5 cells/well in 24-well plates) were treated and incubated 24 h at 37°C. Culture medium from all samples was collected. In addition, the cells were homogenized with 0.1 M KH 2 PO 4 /K 2 HPO 4 (pH 7.4), containing 0.1% Triton X-100. Cells homogenates were centrifuged at 13,000 g for 10 min. LDH activity was measured both in cell supernatant as well as in the culture medium. Activity of the LDH release is expressed as percentage with respect to the total LDH content (LDH in the supernatant + LDH inside the cells) according to the following formula:

Evaluation of the TNF-α
Release of TNF-α was measured in culture medium after 24 h of treatment using enzyme-linked immunoabsorbent assay (ELISA) (Human TNF-α ELISA Kit, 950.090.096, Diaclone, France) according to the manufacturer's manual. The increase in colour intensity was evaluated at 450 nm using a microplate reader (LT-4000, Labtech International Ltd, Acorn House, East Sussex, United Kingdom).

Statistical analysis
Data are shown as mean ± SEM from either two or four independent experiments using different cultures, each experiment performed in triplicate with different cell batches (total 6-12 measurement/condition). Statistical analyses were made with a One-way ANOVA followed by multiple comparisons when the P value was significant. The Dunn's test was chosen because there were at least six different values per group. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed using Sigma Plot 11.0 software.

Results
Action of Si in cell viability Cells exposed to 400 μM H 2 O 2 during 24 h showed a significant of decreased (40%) cellular viability. This decline was, in part, reversed by Si G5 at concentrations ranging from 200 to 1000 ng/mL (Figure 1). The lack of a 40% cellular viability induced by H 2 O 2 in the MTT assay could reflect two events: 1) cellular death or 2) loss of metabolic ability. In order to check these options, LDH release, which measures necrotic death and caspase-3 increase (sensors of apoptosis), were quantified under our experimental conditions.

LDH assay
Results from Figure 2 show that adding 400 μM of H 2 O 2 , to the cells culture for 24 h, induced significant (p < 0.001) increase of LDH release. This release was partially reversed by Si G5 in a dose dependent manner at concentrations ranging from 50 to 500 ng/mL but never returned to basal values. The highest Si concentration tested (750 ng/mL) had no effect on necrotic death mediated by H 2 O 2 in this human neuroblastoma cell line.
Since necrotic death can be a consequence of lipid peroxidation and ROS production, both markers were also evaluated.   Si action on death mediated by caspase-3

Action of Si on ROS formation
Once it was found that H 2 O 2 could induce cellular death through necrosis, the possibility that H 2 O 2 could also induce cell death through apoptosis was analysed. Figure 5 shows H 2 O 2 was a potent apoptotic inductor in the SH-SY5Y cell line. Interestingly, Si G5 at concentrations ranging from 250 to 750 ng/mL almost totally reversed the caspase-3 activity that had been increased by H 2 O 2 .
Since caspase-3 can be activated through intrinsic as well as extrinsic pathways, caspase-9 activity (activated by mitochondrial dysfunction), as well as caspase-8 (activated through death receptors) were tested in the present study.

Action of H 2 O 2 on caspase-9 activity
Caspase-9 is activated by mitochondrial dysfunction through the release of cytochrome-c which binds to cytosolic protein (Apaf-1) and procaspase-9. In the presence of ATP, a complex that converts inactive procaspase-9 to active caspase-9 is formed. No caspase-9 activation could be observed in the H 2 O 2 -exposed SH-SY5Y cell culture (data not shown). Figure 6 shows that exposure to 400 μM of H 2 O 2 during 24 h further induced caspase-8 activation in the SH-SY5Y culture. Si G5, at concentrations between 100 to 750 ng/mL, completely reversed this H 2 O 2 -mediated effect, and reduced caspase-8 to lower than basal levels.

Effects on TNF-α
Since caspase-8 activation is mediated though death receptor activation, TNF-α levels were measured by ELISA in H 2 O 2 treated cells. There was not measurable TNF-α release mediated by H 2 O 2 (data not shown).

Discussion
H 2 O 2 is a classic ROS that is normally produced in cells, including neurons [29,30] and thus, it has been considered an appropriate molecule to study the possible protector effect of Si under cell death conditions in the human neuroblastoma SH-SY5Y cell line.
Our data clearly demonstrate that H 2 O 2 induced a great cell viability lost (by about 40%), in agreement with Chetsawang et al. [31] who found SH-SY5Y cells loss viability mediated by H 2 O 2 . Those authors suggest that Ras protein could be involved in this cell survival decrease.  However, Si levels ranging 200 to 1000 ng/mL increased cell survival, confirming our hypothesis that Si can alleviate H 2 O 2 neurotoxicity. At higher Si concentration (2000 ng/mL) did not protect cellular viability and this could be because Si may be toxic at high concentrations. Toxicity from high Si concentrations has been suggested by several researchers [32,33]. Neuronal cell death by apoptosis induced by oxidative stress has already been reported [34] but acute oxidative stress also often induces necrotic outcomes [35].
Oxidative stress, produced by excess ROS, is considered to be the major contributor to cell death in several CNS pathologies [36] such as Parkinson's disease [37] and Alzheimer's disease [38] or cerebral ischemia reperfusion after stroke [39,40]. Since ROS may induce apoptosis or necrosis depending on the level of intracellular ROS and ATP levels [41], the mechanism by which H 2 O 2 induces cellular death and the possible protector effects of Si against both types of cell death requires study. The results showed that the loss of cell viability  induced by H 2 O 2 in the SH-SY5Y was the result of both necrosis and apoptosis (Figure 7). With regard to the necrosis, H 2 O 2 increased LDL release 4.5 times. Si, doses of 50 to 500 ng/mL, reversed the H 2 O 2 induced LDH release on a great extent. However, the highest concentration of Si (750 ng/mL) employed did not exert any significant effect on cell necrosis death although it did reduce the caspase-3 activation. The protective effect of Si against H 2 O 2 toxicity might be a consequence of its capacity to remove ROS because Si at doses ranging from 50 to 500 ng/mL completely prevented the ROS increase in H 2 O 2 treated cells.
Moreover, regarding apoptotic death, H 2 O 2 produced caspase-3 activation in the treated SH-SY5Y cells. This activation was not mediated via the mitochondria pathway but through the death receptors because caspase-8 but not caspase-9 was activated by H 2 O 2 . Effects of H 2 O 2 as inductor of apoptosis or necrosis have been described in different cell types [42][43][44][45]. Generally, ROSmediated apoptosis is linked to the intrinsic pathway through Bax activation, which aggregates into mitochondria and leads to cytochrome-c release. Consequently, this event activates caspase-9 which finally leads to caspase-3 activation. However, in our case we found no caspase-9 activation. On the other hand, apoptosis mechanisms acting through the extrinsic pathway have also been described in HeLa cells treated with different H 2 O 2 concentrations for 8 h. Since apoptosis, via the extrinsic pathway, is mediated through death receptors, the possible involvement of TNF-α in cell death was investigated in our study. The results suggested that H 2 O 2 was not able to increase TNF-α release under our experimental conditions. Nonetheless, this result does not mean that activation of caspase-8 cannot be mediated by death receptors other than TNF-α, such as Fas, TRAS, or DR4,5, which would probably also be involved. In this way, Medan et al. [46] reported that H 2 O 2 and hydroxyl radical (OH • ) were key mediators of apoptosis in macrophages, increasing FasL; and Wang et al. [47], using an elegant approximation to study the ROS species responsible for the implication of FasL in apoptosis induction, demonstrated that H 2 O 2 and O 2 •were the ROS species that were most involved in this process.
As a whole, our findings suggest that H 2 O 2 induction of cellular death may be explained according to the scheme in Figure 7. H 2 O 2 , by an unknown mechanism, perhaps induction of a death ligand, that would activate the death receptor responsible to activate caspase -8 (inductor enzyme) which activates caspase -3 (effector enzyme). This last enzyme would damages DNA and protein and as a consequence, produces apoptosis. On the other hand, H 2 O 2 induces lipid peroxidation, which conduces to LDH release and necrosis death. In the presence of Si, treated cells seem to remove H 2 O 2 (ROS), preventing the neurotoxicity induced by H 2 O 2 . Taken together these neuroprotective effects from Si could suggest   As a whole, these mechanisms suggest that Si G5 could be a neuroprotective agent that would help prevent cell death under vulnerability conditions associated to strong ROS production.

Competing interest
There are no conflicts of interests. The authors declare that they have no competing financial interest. All authors only work in basic research and official Scientific Institutions.
Authors' contributions AG and MPG carried out the experiments, participated in data analysis and drafted the manuscript. JJM, performed the statistical analysis and helped to review the manuscript. FJSM and JB participated in its design and coordination. MISR and SB helped to review the manuscript. All authors read and approved the final manuscript.