Phenolic composition and neuroprotective effects of the ethyl-acetate fraction from Inonotus sanghuang against H2O2-induced apoptotic cell death of primary cortical neuronal cells

Abstract

The study aimed to characterize phenolic compounds of the Inonotus sanghuang's ethyl-acetate fraction (EAF) and assess the neuroprotective effect of EAF using the H₂O₂-treated primary cortical neuronal cells (PCNC) model. Using HPLC-ECD, 5 phenolics were identified and quantified from EAF. H₂O₂-treated PCNC experiments in vitro showed that pretreatment with EAF increased the GSH-PX and SOD activities and reduced the NO, MDA, and Aβ contents. Furthermore, EAF suppressed the production of IL-1β, IFN-γ, IL-6, and TNF-α in H₂O₂-treated PCNC. Other mechanisms found that EAF reduced Bax, caspase 9, and caspase 3 expressions at the mRNA and protein levels while increasing Bcl-2 expression at the mRNA and protein levels. These results showed that EAF could serve as potential agents for anti-NDD.

Supplementary information: The online version contains supplementary material available at 10.1007/s10068-022-01107-x.

Keywords: Anti-apoptosis; Anti-inflammatory; Antioxidation; Inonotus sanghuang; Neuroprotective effect.

Fig. 1

EAF impacts the cell viability and morphology in primary cortical neuronal cells treated with or without H₂O₂. (A) Effect of EAF on cell morphology of H₂O₂-damaged primary cortical neuronal cells. (B) Primary cortical neuronal cells were treated with H₂O₂ (300–1200 μM) for 6–24 h and (C) the EAF (1.6–200 μg/mL) for 8 h. (D) Cells were precultured with the EAF (1.6–40 μg/mL) for 8 h and then administrated with H₂O₂ (900 μM) for another 6 h. Results are presented as mean ± SD. These experiments were repeated four times. Values with different letters have a statistical difference (p ≤ 0.05), tested using Tukey’s HSD Test

Fig. 2

Effects of EAF on GSH-PX and SOD activities, MDA, NO, Aβ, and pro-inflammatory cytokines levels in H2O2-damaged primary cortical neuronal cells. (A) GSH-PX activity; (B) SOD activity; (C) MAD level; (D) NO level; (E) Aβ level; (F) IFN-γ level; (G) IL-1β level, (H) IL-6 level; (D) TNF-α level (mean ± SD, n = 4). Values with different letters have a statistical difference (p ≤ 0.05), tested using Tukey’s HSD Test

Fig. 3

Inhibitory influence of EAF on H₂O₂-induced primary cortical neuronal cell apoptosis by flow cytometry. Values with different letters have a statistical difference (p ≤ 0.05), tested using Tukey’s HSD Test

Fig. 4

Impact of EAF on the expression of caspases3, caspase 9, Bax, and Bcl-2 in H₂O₂-damaged primary cortical neuronal cells. (A, B, C, D, and E) represents an analysis of mRNA expression of caspase 3, caspase 9, Bcl-2, Bax, and Bcl-2/Bax (mean ± SD, n = 3). F Represents western blot analysis of caspase 3, caspase 9, Bcl-2, Bax, and GAPDH is shown as the loading control. (G, H, I, J, and K) represents a quantitative analysis of the expression of caspase 3, caspase 9, Bcl-2, Bax, and Bcl-2/Bax. Cells were no treated (Control) or only treated with H₂O₂, EAF (40 μg/mL + H₂O₂), EAF (8 μg/mL + H₂O₂), EAF (1.6 μg/mL + H₂O₂) and HA (2 μg/mL + H₂O₂) (mean ± SD, n = 3). Values with different letters have a statistical difference (p ≤ 0.05), tested using Tukey’s HSD Test