Cysteine persulfides and polysulfides produced by exchange reactions with H2S protect SH-SY5Y cells from methylglyoxal-induced toxicity through Nrf2 activation

Abstract

Many physiological functions of hydrogen sulfide (H₂S) have been reported in mammalian cells over the last 20 years. These physiological effects have been ascertained through in vitro treatment of cells with Na₂S or NaHS, both of which are precursors of H₂S. Since H₂S exists as HS^- in a neutral solution, a disulfide compound such as cystine could react with HS^- in culture medium as well as in the cell. This study demonstrated that after the addition of Na₂S solution into culture medium, HS^- was transiently generated and disappeared immediately through the reaction between HS^- and cystine to form cysteine persulfides and polysulfides in the culture medium (bound sulfur mixture: BS-Mix). Furthermore, we found that the addition of Na₂S solution resulted in an increase of intracellular cysteine persulfide levels in SH-SY5Y cells. This alteration in intracellular persulfide was also observed in cystine-free medium. Considering this reaction of HS^- as a precursor of BS-Mix, we highlighted the cytoprotective effect of Na₂S on human neuroblastoma SH-SY5Y cells against methylglyoxal (MG)-induced toxicity. BS-Mix produced with Na₂S in cystine-containing medium provided SH-SY5Y cells significant protective effect against MG-induced toxicity. However, the protective effect was attenuated in cystine-free medium. Moreover, we observed that Na₂S or BS-Mix activated the Keap1/Nrf2 system and increased glutathione (GSH) levels in the cell. In addition, the activation of Nrf2 is significantly attenuated in cystine-free medium. These results suggested that Na₂S protects SH-SY5Y cells from MG cytotoxicity through the activation of Nrf2, mediated by cysteine persulfides and polysulfides that were generated by Na₂S addition.

Keywords: Bound sulfur species; Hydrogen sulfide; Methylglyoxal; Nrf2; Persulfide; Polysulfide.

Graphical abstract

Fig. 1

H₂S and cysteine concentrations in culture medium. DMEM/F12 medium containing 10% FBS was treated with or without 200 μM Na₂S for the indicated time. (A) Protocol for the addition of Na₂S in the medium. (B and C) H₂S and cysteine in the medium were derivatized with mBB and (B) H₂S and (C) cysteine concentrations measured using HPLC-FL. Data are representative of at least three independent experiments. Values indicate means±S.D.

Fig. 2

Measurement of BSS levels in culture medium. DMEM/F12 medium containing 10% FBS was treated with or without 200 μM Na₂S for the indicated time. (A) Protocol for the addition of Na₂S in the medium. Medium was derivatized with (B) SSP4 to measure BSS levels, using a microplate reader or (C-G) mBB for (C) Cys-SSS-Cys, (D) Cys-SSH, (E) Cys-SSSH, (F) Cys-SSSSH or (G) cystine using LC-MS/MS. Data are representative of at least three independent experiments. Values indicate means±S.D. *P<0.05, **P<0.01 and ***P<0.001 significantly different from the Ctr (PBS) level.

Fig. 3

Measurement of Cys-SS⁻ levels in df-SH-SY5Y cells. (A and B) Cells were treated with or without 200 μM Na₂S for (A) 30 min or (B) 2 h in DMEM/F12 medium containing 10% FBS with or without cystine. (C) Protocol of the preparation and treatment of a medium containing BS-Mix (prepared by 200 μM Na₂S). (D and E) Cells were treated with or without BS-Mix for (D) 30 min or (E) 2 h in DMEM/F12 medium containing 10% FBS and cystine. Cell lysates were derivatized with mBB to measure Cys-SS⁻ levels using LC-MS/MS. Values indicate means±S.D. *P<0.05, **P<0.01 and ***P<0.001 significantly different from the Ctr (PBS) level. N.S.; not significant. All data are representative of at least three independent experiments.

Fig. 4

Protective effects of Na₂S against MG-induced cytotoxicity in df-SH-SY5Y cells. (A) Cell viability was measured by a LDH assay. Cells were treated with the indicated concentrations of MG for 24 h. ***P<0.001 compared to the value obtained following treatment with 0 μM MG. (B) Cells were pretreated with or without the indicated concentrations of Na₂S for 2 h in DMEM/F12 containing 10% FBS, and culture medium was exchanged to remove Na₂S; cells were then treated with 900 µM MG for 24 h in DMEM/F12 containing 2% FBS. ***P<0.001 significantly different from the control (Ctr) level. ###P<0.001 significantly different from 0 μM Na₂S (C) Cells were pretreated with 200 μM Na₂S or vehicle (PBS) for 2 h in DMEM/F12 containing 10% FBS, and culture medium exchanged to remove Na₂S. Cells were then treated with 900 µM MG for 24 h in DMEM/F12 containing 2% FBS with or without cystine. Cell viability was assessed by the LDH assay. **P<0.01 significantly different from the control (Ctr) level. Values indicate means±S.D. All data are representative of at least three independent experiments.

Fig. 5

Protective effects of BS-Mix without free H₂S against MG-induced cytotoxicity in df-SH-SY5Y cells. (A) Protocol of the preparation and treatment of a medium containing BS-Mix before the treatment of MG in df-SH-SY5Y cells. Cells were pretreated with or without the indicated concentrations of BS-Mix or 50 μM Na₂S₃ for 2 h, and the culture medium exchanged to remove BS-Mix. Cells were then treated with 900 µM MG for 24 h in DMEM/F12 containing 2% FBS. Values indicate means±S.D. ***P<0.001 significantly different from the Ctr level. ###P<0.001 significantly different from 0 μM Na₂S. All data are representative of at least three independent experiments.

Fig. 6

Accumulation of Nrf2 in the nuclear fraction in df-SH-SY5Y cells. (A) Cells were treated with or without 200 μM Na₂S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na₂S) for 2 h, and Nrf2 protein levels in nuclear fractions were analyzed by western blotting. Lamin B1 was used as nuclear loading control (bottom panel). (B) Cells were treated with or without 200 μM Na₂S in DMEM/F12 containing 10% FBS medium or BS-Mix (prepared by 200 μM Na₂S) for 10 h, and NQO1 protein levels in cytosol fractions were analyzed by western blotting. β-actin was used as cytosol loading control (bottom panel). (C) Cells were treated with 0, 100 and 200 μM Na₂S or BS-Mix (prepared by 200 μM Na₂S) for 2 h, and GSH levels were measured using HPLC-FL with ABD-F derivatized samples. Values indicate means±S.D. *P<0.05 and ***P<0.001. All data are representative of at least three independent experiments. p-Nrf2, phosphorylated Nrf2.

Fig. 7

Accumulation of Nrf2 in the nuclear fraction from df-SH-SY5Y cells. (A–C) Cells were treated with 200 μM Na₂S in DMEM/F12 medium with 10% FBS and cystine or without FBS and cystine; Nrf2 protein levels in nuclear fractions were analyzed by western blotting (A and B) and GSH levels were measured by HPLC-FL with ABD-F derivatized samples (C). Lamin B1 was used as the nuclear loading control (bottom panel of A). (B) The densities of p-Nrf2 bands were measured and the ratio to LaminB1 calculated and expressed as fold-change of the band levels relative to control (Ctr) samples. Values indicate means±S.D. *P<0.05, **P<0.01 and ***P<0.001. All data are representative of at least three independent experiments. Cys-free, cystine-free; p-Nrf2, phosphorylated Nrf2.

Fig. 8

Influence of MG on endogenous GSH and Cys-SS⁻ levels in df-SH-SY5Y cells. (A and B) Cells were pretreated with or without 200 μM Na₂S for 2 h in DMEM/F12 containing 10% FBS, and culture medium exchanged to remove Na₂S. Cells were then treated with 900 µM MG for 1 h in DMEM/F12 containing 2% FBS. Cell lysates were derivatized with ABD-F to measure GSH by HPLC-FL (A) or mBB to measure Cys-SS⁻ (B) levels by LC-MS/MS. Values indicate means±S.D. **P<0.01 and ***P<0.001. All data are representative of at least three independent experiments.

Fig. 9

Proposed mechanism for the activation of Nrf2 mediated by BSS. (A) HS⁻ does not react with Keap1 cysteine residues and Nrf2 remains inactive. (B) BSS (hydrogen polysulfides, cysteine persulfides and polysulfides) react with Keap1 cysteine residues and induce the activation of Nrf2.

Fig. 10

The exchange reactions between HS⁻ and cystine to form persulfides and polysulfides in the cultured medium and differentiated-SH-SY5Y cells.