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Evaluation of derived compounds from sponges against induced oxidative stress in cortical neurons
No full textIntroduction
Marine sponges are a huge source of different and active biochemical structures (Sagar et al., 2010;Abbas et al., 2011;Mollica et al., 2012). Many of these secondary metabolites are created with defensive purposes (Sagar et al., 2010;Mollica et al., 2012) and liberated into the water, so its action occurs at very low concentrations and therefore they are usually very potent and active compounds (Rane et al., 2014). The family of makaluvamines (MKs) is one of these active groups of secondary metabolites. They are iminoquinone alkaloids isolated from sponges of the genera Zyzzya (Zhang et al., 2012) that have been described as anticancer (Wang et al., 2009), antimalarial (Davis et al., 2012), topoisomerase II inhibitors (Shinkre et al., 2007) and antioxidant compounds (Utkina, 2013). Since previously antioxidant research was done in silica, in this work, we delve into the activity of this compounds family testing five MKs (H, J, F, L and G) in a cellular in vitro model of oxidative stress, also used for the study of neurodegenerative diseases (Facecchia et al., 2011). This model consists on the treatment of primary cortical neurons with hydrogen peroxide, an oxidant inductor, and the co-treatment with the tested compounds to evaluate the neuroprotection and the antioxidant capacity thereof. The fact is that oxidative stress is associated to mitochondrial dysfunction and a common feature in neurodegenerative diseases. Thus, increasing antioxidant defenses activity, in neurodegeneration cellular models, can elicit cellular protection and therefore diminish the risk of neurodegenerative diseases (Bonda et al., 2010).
Methods
Makaluvamines information: The Marine Biodiscovery Centre (Department of Chemistry, University of Aberdeen) supplied the library of pure compounds isolated from Zyzzya sources. Makaluvamines H, J, F, L and G, we will refer to them by its letter.
Primary cortical neurons: were obtained from embryonic day 15−18 mice fetuses as described elsewhere (Vale et al., 2010).
Cytotoxicity assay: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay was used to analyzed cell viability as previously described (Alonso et al., 2011a;b). Primary cortical neurons were grown in 96 well plates and exposed for 48h to different compound concentrations (0.01, 0.05, 0.1 and 1 µM) added to the culture medium. Saponin was used as cellular death control. After treatment, MTT was performed and absorbance was measured at 595 nm in a spectrophotometer plate reader.
Neuroprotection assays: Treatments were carried out in 96-well plate, as co-incubations of 200 µM H2O2 and the compounds H, L and G at 1 and 0.1 µM and J and F at 0.1 and 0.05 µM for 12h on 4-5 div neurons.
Mitochondrial function: mitochondrial function wastudied by MTT test following the method previously described.
Mitochondrial membrane potential (Ψm): Ψm was determined by the tetramethylrhodamine methyl ester (TMRM) assay (White et al., 2011). TMRM incubation was with 1 µM for 30 min and cells were solubilized with 50% DMSO/water. Fluorescence was measured at 535 nm excitation, 590 nm emission.
Reactive oxygen species (ROS) generation: ROS production was analyzed by a fluorescence test using 7′,2′-dichlorofluorescein diacetate (DCFH-DA), as previously described (Kim et al., 2002). Neurons were loaded with 20 µM DCF-DA for 30 min at 37 °C. Cells were washed and kept at room temperature for 30 min to allow a complete de-esterification. DCF fluorescence was measured at 475 nm and emission at 525 nm.
Glutathione (GSH) levels measurement: GSH levels were evaluated with a thiol tracker dye. Cells were loaded with 10 µM ThiolTracker™ Violet dye for 1 h at 37 °C. After incubation, fluorescence was read at 404 nm excitation and 526 nm emission.
Catalase (CAT) activity determination: CAT activity was measured with Amplex® Red Catalase Assay Kit, following the commercial protocol.
Statistical analysis: Statistical analysis was performed by Student's t-test. P values < 0.05 were considered statistically significant. Results, expressed in percentage of no-treated neurons control, are presented as means ± SEM of three or more experiments. Assays were carried out at least by triplicate.
Results and discussion
Neuronal viability is well related with mitochondrial activity, so MKs cytotoxicity was studied by MTT assay. Cortical neurons were exposed for 48h to MKs H, L and G at 0.01, 0.05, 0.1 and 1 µM. No signs of cytotoxicity were observed, even at the highest concentration tested and we follow the investigations with 1 and 0.1 µM. However, MKs J and F were cytotoxic at the highest concentration and therefore, 0.1 and 0.05 µM were chosen for the experiments.
In order to study the MKs antioxidant activity, compounds were tested in a cellular oxidative stress in vitro model previously set up [25], consisting of cortical neurons co-incubated with H2O2 (200 µM) and the MKs at the selected concentrations for 12h. Then, we evaluated the mitochondrial function, Ψm, ROS and GSH levels and CAT activity.
Firstly, the possible MKs protection against mitochondrial dysfunction caused by oxidative stress was tested. Mitochondrial function was analyzed by MTT, also correlated with neurons survival measurements (Varming et al., 1996). MKs, at the two chosen concentrations, were co-incubated with H2O2 (200 µM) for 12h, and viability assays were performed. Results demonstrated that the viability of neurons treated with the oxidant decreased a 31.6 ± 2.0% (p < 0.001), and MKs H, J and L protected neurons at this level. MK L protected cells reaching a basal percentage of viability, with a 90.5 ± 8.0% (p = 0.013) at 0.1 µM.
Ψm analyses contribute to comprehend the MKs activity against H2O2 insults. TRMR test reveals a diminution of 33.6 ± 4.3% (p < 0.001) in Ψm when neurons were treated with the oxidant inductor for 12h. The same MKs treatments were performed, and the TRMR was executed. The Ψm decrease produced by the oxidant agent, was only reduced by MK J at 0.1 µM (84.5 ± 1.5%, p = 0.009).
Oxidative stress is an imbalance between ROS generation and antioxidants levels (Gandhi and Abramov, 2012). Thus, in oxidative stress conditions ROS levels are augmented compared to basal conditions. H2O2 treatments in neurons elevated ROS production in a 20.0 ± 2.5% (p 2O2 as previously described and ROS levels were measured. A reduction of ROS levels regarding the oxidant treatment was observed in MKs H, J, F and G treatments.
In physiological conditions, low concentrations of H2O2 are transformed to water and molecular oxygen by GSH–peroxidase, with GSH as a proton donor. But when H2O2 amounts are high, they are instead eliminated by CAT. GSH is one of the antioxidant mitochondrial systems of protection against oxidative damage (Bains and Shaw, 1997). So to conclude the antioxidant research, MKs effects over GSH and CAT were evaluated. GSH is the main intracellular thiol in cells (Zampagni et al., 2012) and a thiol tracker was used to evaluate it. 12h H2O2 incubation produces a GSH level reduction of 25.8 ± 3.1% (p 2O2, as detailed above, and only MK J increased its levels to a 92.5 ± 9.4% (p = 0.048), achieving GSH basal amounts. Moreover the oxidation treatment decreases CAT activity in neurons in a 24.4 ± 5.5% (p < 0.01) however, the co-incubation with MKs increased CAT activity. MKs J, L and G treatments produced a significant elevation with a complete reestablishment of the activity.
Neurons consume an elevated percentage of total body oxygen and consequently they are one of the most vulnerable cell populations to oxidative stress, which plays an important role in neurodegenerative pathology . After MKs evaluation in neurons under oxidative stress condition, we conclude that all of them afford some protection against oxidation, which is consistent with the already published about MKs H, L and G (Utkina, 2013). Once again compound H was the less active in our cellular model and MKs L and G denoted some antioxidant protection. Above all the MKs tested, the no-previously tested MK J at 0.1 µM highlights with a complete neuroprotection, reducing oxidation consequences, such as mitochondrial dysfunction and ROS generation, and increasing antioxidant defenses by maintaining GSH basal levels and CAT activity. All these antioxidant effects might be explained for an activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE) pathway, the main sensor and modulator of oxidative stress, that trigger the transcription of genes like superoxide dismutase 1, CAT, sulforedoxin, thioredoxin, peroxiredoxin and proteins responsible for the synthesis and metabolism of GSH. It has been reported that Nrf2-ARE pathway activation ameliorates the animal symptoms in research models for neurodegenerative diseases (Gan and Johnson, 2013) and numerous scientists of this area are focusing their experiments on the modulation of enzymatic regulatory components, that protect against oxidative stress, to emulate their restorative effects and consequently slow down the illness progression (Andersen, 2004).
The results presented in this work elucidate that makaluvamine J is a potent molecule for neuroprotection against oxidative stress. Nevertheless, the precise mechanism by which MK J activates the antioxidant cell defenses is still unknown. For that reason, further studies about the MK J activity over the Nrf2-ARE pathway and its possible implications in neurodegenerative disorders will be required
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