Abstract
Mitochondria are a primary source and a target of reactive oxygen species (ROS). Increased mitochondrial production of ROS is associated with bioenergetics decline, cell death, and inflammation. Here we investigated whether a pretreatment (for 24 h) with sesamol (SES; at 12.5—50 µM) would be efficient in preventing the mitochondrial collapse induced by hydrogen peroxide (H2O2, at 300 µM) in the human neuroblastoma SH-SY5Y cell line. We have found that a pretreatment with SES at 25 µM decreased the effects of H2O2 on lipid peroxidation, protein carbonylation, and protein nitration in membranes obtained from the mitochondria isolated from the SH-SY5Y cells. In this regard, SES pretreatment decreased the production of superoxide anion radical (O2−•) by the mitochondria of H2O2-treated cells. SES also prevented the mitochondrial dysfunction induced by H2O2, as assessed by analyzing the activity of the complexes I and V. The H2O2-induced reduction in the production of adenosine triphosphate (ATP) was also prevented by SES. The levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as the activity of the transcription factor nuclear factor-κB (NF-κB) were downregulated by the SES pretreatment in the H2O2-challenged cells. Silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor abolished the protection induced by SES regarding mitochondrial function and inflammation. Thus, SES depends on Nrf2 to promote mitochondrial protection in cells facing redox impairment.
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Acknowledgements
This work was supported by the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq; Edital Universal 2016 protocol number 400216/2016-7). MRO receives a “Bolsista Produtividade PQ2” fellow (protocol number 301273/2018-9). SMSN received a fellow from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Funding
This study was funded by Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) [grant number 301273/2018-9 (Bolsa de Produtividade em Pesquisa 2-PQ2) and grant number 400216/2016-7].
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Conceptualization: Marcos Roberto de Oliveira; Methodology: Sônia Mendes da Silva Navarro, Fhelipe Jolner Souza de Almeida, Matheus Dargesso Luckachaki, Marcos Roberto de Oliveira; Formal analysis and investigation: Sônia Mendes da Silva Navarro, Fhelipe Jolner Souza de Almeida, Matheus Dargesso Luckachaki, Marcos Roberto de Oliveira; Writing—original draft preparation: Marcos Roberto de Oliveira; Writing—review and editing: Sônia Mendes da Silva Navarro, Fhelipe Jolner Souza de Almeida, Matheus Dargesso Luckachaki, Marcos Roberto de Oliveira; Funding acquisition: Marcos Roberto de Oliveira; Resources: Marcos Roberto de Oliveira; Supervision: Marcos Roberto de Oliveira.
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Supplementary file1 Figure S1. Quality control for the extraction of mitochondria from the SH-SY5Y cells. The activity of the lactate dehydrogenase (LDH), which is located in the cytosol of mammalian cells, was checked aiming to evaluate whether the mitochondrial and nuclear samples were contaminated by cytosolic components. Figure S2. Quality control for the silencing of Nrf2. The cells were treated with SES at 25 µM for 24 h and the activity of the transcription factor Nrf2 was accessed by using a commercial assay kit, as described in the Material and Methods section. Data are shown as the mean ± S.D. of five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 different from control cells transfected with negative control (NC) siRNA; # p < 0.05 different from SES-treated cells transfected with NC siRNA. Figure S3. SES prevented the effects of H2O2 on the viability of the SH-SY5Y cells. SES was tested at 12.5 - 50 μM for 24 h before the exposure to H2O2 at 300 μM for further 24 h. Data are shown as the mean ± S.D. of five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey's test, * p < 0.05 different from the control group; # p < 0.05 different from H2O2-treated group. Figure S4. The mitochondria-related anti-apoptotic effects induced by SES pretreatment in H2O2-challenged SH-SY5Y cells. The levels of Bcl-2 (A), Bax (B), and cytosolic (C) and mitochondrial cytochrome c (D), as well as the activity of the caspases-9 (E) and -3 (F), were modulated by SES in the cells exposed to H2O2. SES at 25 μM was administrated to the cells for 24 h before the challenge with H2O2 at 300 μM for further 24 h. Data are shown as the mean ± S.D. of five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey's test, * p < 0.05 different from the control group; # p < 0.05 different from H2O2-treated group. Figure S5. SES decreased the oxidation of DPPH. SES at 25 and 50 µM inhibited the oxidation of DPPH. Data are shown as the mean ± S.D. of five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey's test, * p < 0.05 different from the control group. Figure S6. Nrf2 silencing abrogated the reduction elicited by SES on the mitochondrial production of O2-• in H2O2-challenged cells. Data are shown as the mean ± S.D. of five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, a p < 0.05 different from the control; b p < 0.05 different from H2O2-challenged cells transfected with negative control (NC) siRNA; * p < 0.05 different from H2O2-challenged cells transfected with NC siRNA; # p < 0.05 different from SES + H2O2-treated cells transfected with NC siRNA (PDF 243 KB)
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da Silva Navarro, S.M., de Almeida, F.J.S., Luckachaki, M.D. et al. Sesamol prevents mitochondrial impairment and pro-inflammatory alterations in the human neuroblastoma SH-SY5Y cells: role for Nrf2. Metab Brain Dis 37, 607–617 (2022). https://doi.org/10.1007/s11011-021-00875-5
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DOI: https://doi.org/10.1007/s11011-021-00875-5