Sulforaphane reduces reactive astrocyte-mediated neuron apoptosis in vitro by inhibiting the MAPK/NF-κB signaling pathway in Aβ42 oligomer-activated astrocytes

doi:10.12122/j.issn.1673-4254.2026.01.21

Abstract

Abstract:

Objective To explore the effects of sulforaphane (SFN) on Aβ42-activated U87 astrocyte-mediated apoptosis of SH-SY5Y neurons in vitro. Methods U87 cells treated with different concentrations of Aβ42, SFN or both were examined for changes in cell activity, IL-6 and TNF-α mRNA expression, release of IL-6 and TNF-α proteins, and expressions of p-p38, p-p65 and GFAP using CCK-8 assay, RT-qPCR, ELISA and Western blotting. SH-SY5Y neurons were co-cultured with U87 astrocytes treated with Aβ42 alone or in combination with SFN or SB203580 for 24 h, and the changes in Bax protein expression levels and viability of SH-SY5Y cells were examined. The effects of Aβ42, SFN, and their combination were also observed in astrocytes isolated from mouse brain tissues, and the indirect effects of astrocyte treatmentt on viability of the co-cultured primary neurons were assessed. Results The viability of U87 astrocytes increased significantly following treatment with 1.25 μmol/L Aβ42 but decreased after Aβ42 treatment above 5 μmol/L. SFN treatments for 24 h below 5 μmol/L did not significantly affect U87 cell viability. Aβ42 treatment significantly increased protein expressions of p-p38, p-p65 and GFAP, mRNA expression levels of IL-6 and TNF-α, and IL-6 and TNF-α levels in culture supernatant of U87 cells. SH-SY5Y cells co-cultured with Aβ42-treated U87 cells showed significantly increased protein expressions of Bax, and exhibited lowered viability following co-culture with 5 μmol/L Aβ42-treated U87 cells. The isolated mouse astrocytes showed lowered viability following Aβ42 treatment above 10 μmol/L, but SFN treatment below 5 μmol/L for 24 did not obviously affect the cell viability. The primary neurons co-cultured with Aβ42-treated mouse astrocytes showed significantly lower cell viability than those co-cultured with the astrocytes treated with Aβ+SFN or Aβ+SB203580. Conclusion SFN attenuates astrocyte-mediated neuron apoptosis by inhibiting the MAPK/NF-κB signaling pathway in Aβ42 oligomer-activated astrocytes

Key words: sulforaphane, Alzheimer's disease, astrocyte, β-amyloid, MAPK, nuclear factor-κB, neuroinflammation, apoptosis

Shufen ZHANG, Tianrong HUANG, Canhong YANG, Jiayi CHEN, Tianming LÜ, Jiafa ZHANG. Sulforaphane reduces reactive astrocyte-mediated neuron apoptosis in vitro by inhibiting the MAPK/NF-κB signaling pathway in Aβ42 oligomer-activated astrocytes[J]. Journal of Southern Medical University, 2026, 46(1): 191-199.

Figures/Tables 7

Fig.1 CCK-8 assay for assessing viability of U87 cells treated with different concentration of Aβ42, sulforaphane (SFN) and both. A: Viability of U87 cells treated with 0, 1.25, 2.5, 5, 10, and 20 μmol/L Aβ42 for 48 h (*P˂0.05, ***P˂0.001, ****P˂0.0001 vs 0 μmol/L group). B: Viability of U87 cells treated with 0, 5, 10, 20, 40, and 80 μmol/L SFN for 24 h (*P˂0.05, ****P˂0.0001 vs 0 μmol/L group). C: Viability of U87 cells treated with vehicle, Aβ42 (5 μmol/L), and Aβ42 (5 μmol/L)+SFN (5 μmol/L) (*P˂0.05, **P˂0.01).

Fig.2 Effects of Aβ42 (5 μmol/L), SFN (5 μmol/L) and SB203580 (20 μmol/L) on protein expressions of p-p38, p-p65 and GFAP, mRNA expressions of TNF‑α and IL-6, and TNF‑α and IL-6 levels in culture supernatant of U87 cells. A-D: Protein expression levels of p-p38, p-p65 and GFAP in U87 cells treated with vehicle, Aβ42, Aβ42+SFN, and Aβ42+ SB203580 detected by Western blotting (*P˂0.05, **P˂0.01, ***P˂0.001, ****P˂0.0001). E, F: Expression levels of TNF-α and IL-6 mRNA in treated U87 cells detected by RT-qPCR (*P˂0.05, **P˂0.01). G-H: TNF-α and IL-6 levels in culture supernatant of the treated U87 cells (***P˂0.001, ****P˂0.0001).

Fig.3 Indirect effects of Aβ42, SFN and SB203580 treatment of U87 cells on co-cultured SH-SY5Y neurons. A: Transwell co-culture scheme of U87 and SH-SY5Y Cells. B: Protein expression of Bax in SH-SY5Y cells co-cultured with U87 cells treated with vehicle, Aβ42, Aβ42+SFN, and Aβ42+SB203580. C: Quantification of Bax expression levels. *P˂0.05, **P˂0.01.

Tab.1 Primers sequences for qRT-PCR

Primer	Sequuence (5' to 3')
GAPDH-F	CTA GGC CAC AGA ATT GAA AGA TCT
GAPDH-R	GTA GGT GGA AAT TCT AGC ATC ATC C
TNF-α-F	CTG TGA AGG GAA TGG GTG TT
TNF-α-R	CAG GGA AGA ATC TGG AAA GGT C
IL-6-F	GAG AGC ATT GGA AGT TGG GG
IL-6-R	CTT CCA GCC AGT TGC CTT CT

Fig.4 Purity identification of astrocytes and primary neurons isolated from mouse brain tissues. A-C: Immunofluorescence staining for assessing purity of the astrocytes labeled with GFAP (green). DAPI (blue) was used to label the cell nuclei (Scale bar=50 μm). D-F: Immunofluorescence staining for assessing purity of the primary neurons labeled with MAP-2 (green). DAPI (blue) was used to label the cell nuclei (Scale bar=20 μm).

Fig.5 CCK-8 assay for assessing the effect of different concentrations of Aβ42, SFN and their combination on viability of isolated mouse astrocytes. A: Viability of mouse astrocytes treated with 0, 1.25, 2.5, 5, 10, and 20 μmol/L Aβ42 for 48 h (*P˂0.05, ***P˂0.001 vs control group). B: Viability of mouse astrocytes treated with 0, 5, 10, 20, 40, and 80 μmol/L SFN for 24 h (****P˂0.0001 vs control group). C: Viability of mouse astrocytes treated with vehicle, Aβ42 (10 μmol/L), and Aβ42 (10 μmol/L) + SFN (5 μmol/L) (*P˂0.05, **P˂0.01).

Fig.6 Indirect effects of Aβ42, SFN and SB203580 treatment of isolated mouse astrocytes on the primary neurons. A: Transwell co-culture scheme for mouse astrocytes and primary neurons. B: Viability of primary neurons co-cultured with mouse astrocytes treated with vehicle, Aβ42 (10 μmol/L), Aβ42 (10 μmol/L)+SFN (5 μmol/L), and Aβ42 (10 μmol/L)+SB203580 (20 μmol/L). *P˂0.05.

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