Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide - PubMed (original) (raw)

Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

Hua Liu et al. Cell Commun Signal. 2018.

Abstract

Background: Four-octyl itaconate (OI), the itaconate's cell-permeable derivative, can activate Nrf2 signaling via alkylation of Keap1 at its cysteine residues. The current study tested the potential neuroprotective function of OI in hydrogen peroxide (H2O2)-treated neuronal cells.

Methods: SH-SY5Y neuronal cells and epigenetically de-repressed (by TSA treatment) primary murine neurons were treated with OI and/or H2O2. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Neuronal cell death was tested by the LDH and trypan blue staining assays. Apoptosis was tested by TUNEL and Annexin V assays.

Results: In SH-SY5Y neuronal cells and primary murine neurons, OI activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, as well as expression of Nrf2-regulated genes (HO1, NQO1 and GCLC) and ninjurin2 (Ninj2). Functional studies showed that OI attenuated H2O2-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage as well as neuronal cell death and apoptosis. shRNA-mediated knockdown, or CRISPR/Cas9-induced knockout of Nrf2 almost abolished OI-induced neuroprotection against H2O2. Keap1 is the primary target of OI. Keap1 knockout by CRISPR/Cas9 method mimicked and abolished OI-induced actions in SH-SY5Y cells. Introduction of a Cys151S mutant Keap1 in SH-SY5Y cells reversed OI-induced Nrf2 activation and anti-H2O2 neuroprotection.

Conclusions: OI activates Keap1-Nrf2 signaling to protect SH-SY5Y cells and epigenetically de-repressed primary neurons from H2O2 in vitro.

Keywords: Four-octyl itaconate; Keap1-Nrf2 signaling; Neuronal cells; Oxidative stress.

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Conflict of interest statement

This study was approved by the Ethics Committee of Jiangsu University.

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1

Fig. 1

Four-octyl itaconate activates Nrf2 signaling in neuronal cells. SH-SY5Y cells (a-d) or the primary murine neurons (e-h) were treated with applied concentration of 4-octyl itaconate (OI) for indicated time, mRNA expression of Nrf2-regulated genes and Ninj2 were tested by qPCR assay (a and e); Expression of listed proteins in total cellular lysates (b and f) and nuclear lysates (c and g) were tested by the Western blotting assays. Keap1-Nrf2 association was detected by co-immunoprecipitation assays (d and h). Expression of listed proteins were quantified and normalized to the loading control (b, c, f and g). Keap1-bound Nrf2 was quantified as well (d and h). Lamin-B1 was tested as a marker of nuclear protein (c and g). “Ctrl” stands for untreated control cells (Same for all Figures). Bars stand for mean ± standard deviation (S.D). * P < 0.05 vs. “Ctrl” cells

Fig. 2

Fig. 2

Four-octyl itaconate attenuates H2O2-induced neuronal cell death and apoptosis. SH-SY5Y cells (a-h) or the primary murine neurons (i-k) were pretreated for 30 min with applied concentration of 4-octyl itaconate (OI), followed by stimulation of H2O2 (300 μM) for indicated time, cell viability, cell death and apoptosis were tested by the listed assays mentioned in the text (a-k). Expression of listed proteins were quantified and normalized to the loading control (d and k). Bars stand for mean ± standard deviation (S.D., n = 5). * P < 0.05 vs. “Ctrl” cells. # P < 0.05 vs. H2O2 treatment only

Fig. 3

Fig. 3

OI inhibits H2O2-induced oxidative stress in neuronal cells. SH-SY5Y cells (a-d) or the primary murine neurons (e-f) were pretreated for 30 min with 4-octyl itaconate (OI, 25 μM), followed by stimulation of H2O2 (300 μM) for indicated time, relative DCF fluorescent intensity (a and e) and relative superoxide level (b and f) were tested; The lipid peroxidation (c) and DNA damage (d) were tested by TBAR activity assay and p-H2AX FACS assay, respectively. Bars stand for mean ± standard deviation (S.D., n = 5). “Vehicle” stands for PBS control (Same for all Figures). * P < 0.05 vs. “Ctrl” cells. # P < 0.05 vs. H2O2 treatment only

Fig. 4

Fig. 4

Nrf2 activation mediates 4-octyl itaconate-induced neuronal cell protection against H2O2. SH-SY5Y cells (a-e) or the primary murine neurons (i-k), with the applied Nrf2 shRNA or the scramble control shRNA (“shC”), were either untreated or treated with 4-octyl itaconate (OI), mRNA and protein expression of listed genes were shown (a-c, and i); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H2O2 (300 μM) for indicated time, cell viability (CCK-8 OD, d), cell death (LDH release, j) and apoptosis (TUNEL ratio increase, e, and k) were tested. Stable SH-SY5Y cells, with the CRISPR/Cas9-Nrf2 KO construct (“Nrf2-KO”) or the CRISPR/Cas9 control construct (“Cas9-c”), were treated with 4-octyl itaconate (OI), listed proteins were shown (f); Cells were pretreated for 30 min with OI (25 μM), followed by stimulation of H2O2 (300 μM) for indicated time, cell viability (g) and apoptosis (h) were tested. Expression of listed proteins were quantified and normalized to the loading control (c, f and i). “shNrf2 (m)” stands for murine Nrf2 shRNA (I-K). Bars stand for mean ± standard deviation (S.D., n = 5). # P < 0.05 vs. “shC” cells (a, b, d and e). # P < 0.05 (g, h, j and k)

Fig. 5

Fig. 5

Keap1 knockout or Cys151S mutation abolishes 4-octyl itaconate-induced neuroprotection against H2O2. Stable SH-SY5Y cells with the lentiviral CRISPR/Cas9 Keap1 vector (“Keap1-KO”) or the CRISPR/Cas9 control vector (“Cas9-c”) were treated with/without 4-octyl itaconate (“+OI”, 25 μM, for 12 h), mRNA and protein expression of listed genes were shown (a and b). Cells were pretreated with/without OI (“+OI”, 25 μM), followed by stimulation of H2O2 (300 μM) for indicated time, cell viability (c) and apoptosis (d and e) were tested by the listed assays. Stable SH-SY5Y cells with the Cys151S mutant Keap1 [“Keap1 (c151s)”] or the empty vector (“Vector”) were treated with 4-octyl itaconate (“OI”, 25 μM) for 12 h, expression of listed proteins were shown (f). Cells were pretreated with/without OI (“+OI”, 25 μM), followed by stimulation of H2O2 (300 μM) for indicated time, cell viability (g), and apoptosis (h) were tested. Expression of listed proteins were quantified and normalized to the loading control (a and f). Bars stand for mean ± standard deviation (S.D., n = 5). * P < 0.05 vs. “Cas9-c” cells (b). * P < 0.05 vs. “Ctrl” cells (c-e). # P < 0.05 vs. H2O2 treatment of “Cas9-c” cells (c-e). # P < 0.05 (g and h)

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