Forsythoside A Mitigates Alzheimer's-like Pathology by Inhibiting Ferroptosis-mediated Neuroinflammation via Nrf2/GPX4 Axis Activation

Abstract

Ferroptosis and neuroinflammation play crucial roles in Alzheimer's disease (AD) pathophysiology. Forsythoside A (FA), the main constituent of Forsythia suspensa (Thunb.) Vahl., possesses anti-inflammatory, antibacterial, antioxidant, and neuroprotective properties. The present study aimed to investigate the potential role of FA in AD neuropathology using male APP/PS1 double transgenic AD mice, Aβ_1-42-exposed N2a cells, erastin-stimulated HT22 cells, and LPS-induced BV2 cells. FA treatment significantly improved mitochondrial function and inhibited lipid peroxidation in Aβ_1-42-exposed N2a cells. In LPS-stimulated BV2 cells, FA treatment decreased the formation of the pro-inflammatory factors IL-6, IL-1β, and NO. In male APP/PS1 mice, FA treatment ameliorated memory and cognitive impairments and suppressed Aβ deposition and p-tau levels in the brain. Analyses using proteomics, immunohistochemistry, ELISA, and western blot revealed that FA treatment significantly augmented dopaminergic signaling, inhibited iron deposition and lipid peroxidation, prevented the activation of IKK/IκB/NF-κB signaling, reduced the secretion of pro-inflammatory factors, and promoted the production of anti-inflammatory factors in the brain. FA treatment exerted anti-ferroptosis and anti-neuroinflammatory effects in erastin-stimulated HT22 cells, and the Nrf2/GPX4 axis played a key role in these effects. Collectively, these results demonstrate the protective effects of FA and highlight its therapeutic potential as a drug component for AD treatment.

Keywords: Alzheimer's disease; Nrf2/GPX4 axis; ferroptosis; forsythoside A; neuroinflammation; neuroprotection.

Figure 1

FA treatment protects N2a cells and BV2 cells against Aβ_1-42 and LPS toxicity. (A) Chemical structure of FA (CAS: 79916-77-1). (B) FA treatment enhanced cell viability in Aβ_1-42-exposed N2a cells without influencing cell viability alone (n = 8). (C) FA treatment downregulated MDA levels in Aβ_1-42-exposed N2a cells (n = 8). (D) FA treatment prevented the dissipation of MMP in Aβ_1-42-exposed N2a cells (n = 3). Scale bar: 100 µm. Red and green fluorescence signals represent J-aggregates and monomers, respectively. FA treatment decreased the concentration of (E) NO, (F) IL-1β, and (G) IL-6 in LPS-exposed BV2 cells (n = 8). The data are presented as mean ± S.E.M. ^##p < 0.01, and ^###p < 0.001 vs. CTRL N2a cells for (B) and (C); ^###p < 0.001 vs. CTRL BV2 cells for (E), (F), and (G); *p < 0.05, and ***p < 0.001 vs. Aβ_1-42-stimulated N2a cells for (B) and (C); ***p < 0.001 vs. LPS-exposed BV2 cells for (E), (F), and (G).

Figure 2

FA treatment ameliorates AD symptoms in APP/PS1 mice. (A) Schematic diagram of animal experiments. (B) FA treatment shortened foraging time of APP/PS1 mice in the Y-maze test (n = 12). (C) FA treatment shortened the escape latency of APP/PS1 mice in the MWM navigation test (n = 12). (D) FA treatment increased the time of APP/PS1 mice spent in the effective area in the MWM probe trials (no platform) (n = 12). The blue and green circles indicate the position of the original platform and effective area, respectively. (E) FA treatment decreased Aβ_1-42 (red arrow) deposition in the hippocampus of APP/PS1 mice (n = 3). (F) FA treatment suppressed the levels of phosphorylated tau protein (green arrow) in the hippocampus of APP/PS1 mice (n = 3). Scale bars: 400 µm for 4× magnification and 100 µm for 20× magnification. The data are presented as mean ± S.E.M. ^###p < 0.001 vs. WT mice; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. APP/PS1 mice.

Figure 3

Proteomics and bioinformatics analysis of the hippocampus in APP/PS1 mice. (A) Heat map of 15 proteins. Red and blue indicate high-abundance and low-abundance proteins, respectively. (B) GO enrichment analysis of differentially expressed proteins among WT, APP/PS1, and FA-treated APP/PS1 mice. Molecular function, cellular component, and biological process are marked in blue, green, and pink, respectively. (C) Pie chart of GO molecular function classification. (D) Pie chart of GO cellular component classification. (E) Pie chart of GO biological process classification. (F) KEGG enrichment analysis of differentially expressed proteins among WT, APP/PS1, and FA-treated APP/PS1 mice. Rows represent individual biological processes and the number of related altered proteins. (G) Protein-protein interaction network analysis generated using STRING software.

Figure 4

FA treatment regulates the dopaminergic system and ferroptosis in APP/PS1 mice. FA treatment increased the expression of (A) cAMP (red arrows), (B) p-AKT (green arrows), and (C) GPX4 (blue arrows) in the cortex in APP/PS1 mice (n = 3). Scale bar: 400 μm for 4× magnification and 100 μm for 20× magnification. (D) FA treatment enhanced the expression levels of DRD1, GNAL, Adcy5, cAMP, and p-PKA in the brains of APP/PS1 mice (n = 3). (E) FA treatment upregulated the phosphorylation of CREB, TrkB, PI3K, and AKT, and BDNF expression in the brains of APP/PS1 mice (n = 3). (F) FA treatment suppressed the expression of p-Fyn and upregulated the expression of p-GSK-3β, GPX4, Nrf2, and downstream proteins in the brains of APP/PS1 mice (n = 3). (G) FA treatment suppressed the expression of TFRC and DMT1 and upregulated the expression of FTH, and FTL in the brains of APP/PS1 mice (n = 3).

Figure 5

FA treatment alleviates neuroinflammation in APP/PS1 mice. FA treatment decreased the expression levels of Iba1 (red arrows) in the (A) hippocampus and (B) cerebral cortex in APP/PS1 mice (n = 3). FA treatment decreased the expression levels of GFAP (green arrows) in the (C) hippocampus and (D) cerebral cortex in APP/PS1 mice (n = 3). Scale bar: 400 µm for 4× magnification and 100 µm for 20× magnification. FA treatment upregulated the expression of (E) TGF-β and suppressed the expression levels of (F) MCP-1, (G) IL-1β, (H) IL-6, and (I) TNF-α in the brains of APP/PS1 mice (n = 8) detected with ELISA. (J) FA treatment downregulated the expression of Iba1, GFAP, ALOX5, CD33, and iNOS in the brains of APP/PS1 mice (n = 3). (K) FA treatment ameliorated the levels of ILs and TNF-α in the brains of APP/PS1 mice (n = 3). (L) FA treatment suppressed the phosphorylation of IKK, IκB, and NF-κB in the brains of APP/PS1 mice (n = 3). The data are presented as mean ± S.E.M. ^#p < 0.05, ^##p < 0.01 vs. WT mice; **p < 0.01 vs. APP/PS1 mice.

Figure 6

FA alleviates ferroptosis-related inflammation in erastin-exposed HT22 cells. FA treatment (A) improved cell viability, (B) decreased MDA levels, and (C) increased GSH levels in erastin-exposed HT22 cells (n = 8). (D) FA treatment suppressed lipid peroxidation in erastin-exposed HT22 cells (n = 3) as determined using BODIPY 581/591 C11. Red and green fluorescence signals represent non-oxidized and oxidized states, respectively. Scale bar: 100 µm. (E) Ultrastructure of HT22 cells in each group was analyzed using TEM (n = 3). Green, pink, and red arrows indicate disrupted mitochondrial cristae, increased mitochondrial electron density, and rough endoplasmic reticulum expansion. Scale bar: 5 µm for 1000× magnification and 2 µm for 2500× magnification. (F) FA treatment upregulated the expression of p-PI3K, p-AKT, p-GSK-3β, Nrf2, and NQO1, and downregulated the expression of p-Fyn in erastin-exposed HT22 cells (n = 3). (G) FA treatment upregulated the expression of GPX4, xCT, FTH, FTL, and FPN, and suppressed the expression of TFRC and DMT1 in erastin-exposed HT22 cells (n = 3). (H) FA treatment downregulated the expression of IL-1β, IL-6, and TNF-α; suppressed the phosphorylation of IKK, IκB, and NF-κB; and upregulated the expression of IL-4 in erastin-exposed HT22 cells (n = 3). The data are presented as mean ± S.E.M. ^###p < 0.001 vs. CTRL HT22 cells; ***p < 0.001 vs. erastin-exposed HT22 cells.

Figure 7

GPX4 and Nrf2 are involved in the anti-ferroptosis and anti-neuroinflammatory effects of FA treatment. (A) FA treatment-induced decreases in MDA levels were blocked in GPX4 siRNA-transfected erastin-exposed HT22 cells (n = 8). (B) FA treatment-induced increases in GPX4, xCT and FPN levels; reduction in IL-6 and IL-1β levels; and suppression of NF-κB signaling were abolished in GPX4 siRNA-transfected erastin-exposed HT22 cells (n = 3). (C) FA treatment-induced decreases in MDA levels were blocked in Nrf2 siRNA-transfected erastin-exposed HT22 cells (n = 8). (D) FA treatment-induced upregulation of Nrf2, GPX4, xCT, and FPN was abolished and NF-κB signaling was activated in Nrf2 siRNA-transfected erastin-exposed HT22 cells (n = 3). The data are presented as mean ± S.E.M. ^###p < 0.001 vs. CTRL HT22 cells transfected with NC siRNA; **p < 0.01, ***p < 0.001 vs. erastin-exposed HT22 cells transfected with NC siRNA; ^&&&p < 0.001 vs. FA-treated HT22 cells transfected with NC siRNA.