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Mitochondrial Protection and Anti-inflammatory Effects Induced by Emodin in the Human Neuroblastoma SH-SY5Y Cells Exposed to Hydrogen Peroxide: Involvement of the AMPK/Nrf2 Signaling Pathway

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Abstract

Emodin (EM; 1,3,8-trihydroxy-6-methylanthracene-9,10-dione; C15H10O5) is an anthraquinone and exerts cytoprotective effects, as observed in both in vitro and in vivo experimental models. Mitochondrial dysfunction induced by reactive species plays a central role in the onset and progression of different human diseases. Thus, we have tested here whether a pretreatment (for 4 h) with EM (at 40 µM) would be able to promote mitochondrial protection in the human neuroblastoma SH-SY5Y cells exposed to the pro-oxidant agent hydrogen peroxide (H2O2). We found that the pretreatment with EM suppressed the effects of H2O2 on the activity of the mitochondrial complexes I and V, as well as on the production of adenosine triphosphate (ATP) and on the mitochondrial membrane potential (MMP). EM also prevented the H2O2-induced collapse in the tricarboxylic acid cycle (TCA) function. An anti-inflammatory role for EM was also observed in this experimental model, since this anthraquinone decreased the secretion of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) by the H2O2-challenged cells. Inhibition of the adenosine monophosphate-activated protein kinase (AMPK) or silencing of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) abolished the protection induced by EM in the H2O2-treated cells. Therefore, EM prevented the H2O2-induced mitochondrial dysfunction and pro-inflammatory state in the SH-SY5Y cells by an AMPK/Nrf2-dependent manner.

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Acknowledgements

MRO receives a “Bolsa de Produtividade em Pesquisa 2—PQ2” fellow from the Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) (Protocol Number 301273/2018-9). This work received financial support from CNPq (Protocol Numbers 400216/2016-7 and 460903/2014-4).

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Correspondence to Marcos Roberto de Oliveira.

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11064_2020_3181_MOESM1_ESM.pdf

Electronic supplementary material 1 (PDF 21 kb). Figure S1. EM prevents the H2O2- induced decline in the viability of SH-SY5Y cells. The SH-SY5Y cells were treated with EM at 1 - 40 µM for 4 h prior to the challenge with H2O2 at 300 µM for additional 24 h. The data are presented as the mean ± S.E.M. of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 vs control cells, # p < 0.05 vs H2O2-challenged cells

11064_2020_3181_MOESM2_ESM.pdf

Electronic supplementary material 2 (PDF 89 kb). Figure S2. EM prevents the H2O2-induced alterations in the levels of cytochrome c in the cytosol (A) and in the mitochondria (B), and the upregulation in the activity of the pro-apoptotic enzymes caspase-9 (C) and caspase-3 (D). The SH-SY5Y cells were treated with EM at 40 µM for 4 h prior to the challenge with H2O2 at 300 µM for additional 24 h. The data are presented as the mean ± S.E.M. of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 vs control cells, # p < 0.05 vs H2O2-challenged cells

11064_2020_3181_MOESM3_ESM.pdf

Electronic supplementary material 3 (PDF 86 kb). Figure S3. EM prevents the H2O2-induced effects on the levels of cleaved PARP (A) and DNA fragmentation (B). The SH-SY5Y cells were treated with EM at 40 µM for 4 h prior to the challenge with H2O2 at 300 µM for additional 24 h. The data are presented as the mean ± S.E.M. of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 vs control cells, # p < 0.05 vs H2O2-challenged cells

11064_2020_3181_MOESM4_ESM.pdf

Electronic supplementary material 4 (PDF 88 kb). Figure S4. EM prevents the H2O2-induced cellular lipid peroxidation (A), protein carbonylation (B), and DNA oxidation (C). The SH-SY5Y cells were treated with EM at 40 µM for 4 h prior to the challenge with H2O2 at 300 µM for additional 24 h. The data are presented as the mean ± S.E.M. of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 vs control cells, # p < 0.05 vs H2O2-challenged cells

11064_2020_3181_MOESM5_ESM.pdf

Electronic supplementary material 5 (PDF 7 kb). Figure S5. Compound C inhibits the effect of EM on the activity of Nrf2. The SH-SY5Y cells were treated with Compound C at 20 µM for 1 before the administration of EM at 40 µM for 4 h. The data are presented as the mean ± S.E.M. of three or five independent experiments each done in triplicate. One-way ANOVA followed by the post hoc Tukey’s test, * p < 0.05 vs control cells, # p < 0.05 vs EM-treated cells

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de Oliveira, M.R., de Souza, I.C.C. & Brasil, F.B. Mitochondrial Protection and Anti-inflammatory Effects Induced by Emodin in the Human Neuroblastoma SH-SY5Y Cells Exposed to Hydrogen Peroxide: Involvement of the AMPK/Nrf2 Signaling Pathway. Neurochem Res 46, 482–493 (2021). https://doi.org/10.1007/s11064-020-03181-1

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