Korean black ginseng extract alleviates Alzheimer's disease-related cognitive impairment by activating the Nrf2/HO-1 pathway and suppressing the p38 MAPK/NF-κB/STAT3 pathways and NLRP3 inflammasome via TLR2 and TLR4 modulation

Abstract

Background: Korean black ginseng, a specially processed ginseng through repeat steaming and drying, has various pharmacological effects. However, its role i n cognitive impairment remains unclear.

Purpose and methods: This study examined whether Korean black ginseng extract (BGE; 50 and 100 mg/kg, orally, 18 weeks) may mitigate cognitive impairment in a 5xFAD mouse model of Alzheimer's disease (AD).

Results: BGE significantly improved cognitive performance in 5xFAD mice, associated with reduced Aβ accumulation in the frontal cortex and hippocampus. BGE suppressed microglial and astrocytic activation, alongside the downregulation of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and enzymes (cyclooxygenase-2 and inducible nitric oxide synthase). These changes coincided with the inhibition of key inflammatory signaling pathways, such as p38 mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB)/p65, signal transducer and activator of transcription (STAT) 3, and NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, BGE reduced the generation of reactive oxygen species and enhanced the nuclear-E2-related factor 2 (Nrf2)-heme oxygenase 1 (HO-1) signaling pathway in the brains linked to the downregulation of toll-like receptors (TLR)-2 and TLR-4 in the brain.

Conclusion: Taken together, BGE could improve AD-related cognitive decline and neurodegeneration by simultaneously regulating anti-inflammatory pathways (p38 MAPK/NF-κB/STAT3 and NLRP3 inflammasome) and an antioxidant pathway (Nrf2/HO-1) via modulation of TLR2/4.

Keywords: Alzheimer's disease; Anti-inflammation; Anti-oxidation; Korean black ginseng; Toll-like receptor 2/4.

Graphical abstract

Fig. 1

Schematic representation of the experimental protocol and the impact of Korean black ginseng extract (BGE) on cognitive impairment. (A) Schematic overview of the experimental design and timeline. 5xFAD mice (24–44 weeks old; five females and four males) were administered BGE (50 and 100 mg/kg) orally once per day for 4 months. WT and 5xFAD mice received a vehicle (saline). Cognitive function was evaluated using the Y-maze, MWM, and PA task, in that sequence, with the intensity of stress increasing across the tests. Brain tissue was collected 7 days after the final behavioral assessment. (B) Body weight (n = 9 per group) was recorded twice a week throughout the entire experimental period. (C) Y-maze performance was quantified as the spontaneous alternation rate (%). (D–I) MWM assessments are presented as delayed latency to reach the platform (escape time) (D), representative swimming paths (E), swim speed (F), total distance swum (G), number of platform area crossings (H), and time spent in the target quadrant (I). (J) PA task performance is expressed as the latency to enter the dark chamber. The data are presented as mean behavioral scores ± SEM (one-way ANOVA with post hoc test for B and D; two-way ANOVA with post hoc test for C and E; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. WT mice; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. 5xFAD mice). Abbreviations; 5xFAD, 5xFAD transgenic mouse; MWM, Morris water maze; PA, Passive avoidance; SEM, Standard error of the mean; WT, Wild type.

Fig. 2

Effect of Korean black ginseng extract (BGE) in alleviating Aβ accumulation in the brains of 5xFAD mice. Seven days after completing the last behavioral tests, brains of female mice (n = 5 per group) were collected from the WT, 5xFAD, 5xFAD + BGE (50 and 100 mg/kg), and BGE (100 mg/kg) groups. (A–C) Cryo-sections (3 sections per brain), including the hippocampus (n = 5 per group), were subjected to immunohistochemical staining with anti-Aβ antiserum (A). Panels B and C present quantified graphs for the cortex and hippocampus regions shown in A. Aa highlights the quantified regions (cortex and hippocampus) corresponding to B and C. (D and E) The expression levels of Aβ protein and p-tau in forebrain lysates were analyzed via Western blotting using anti-Aβ and anti-p-tau antibodies, respectively, followed by quantification. Bars represent 100 μm; Data are presented as the mean ± SEM (one-way ANOVA with post hoc test; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. WT mice; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. 5xFAD mice). Abbreviations; 5xFAD, 5XFAD transgenic mice; Aβ, Beta-amyloid; SEM, Standard error of the mean; WT, Wild type.

Fig. 3

Effect of Korean black ginseng extract (BGE) on alleviating microglial and astrocyte activation in the brains of 5xFAD mice. Seven days after the final behavioral tests, brains were harvested from female mice (n = 5 per group) in the WT, 5xFAD, 5xFAD + BGE (50 and 100 mg/kg), and BGE (100 mg/kg) groups. (A–C and E–G) Cryo-sections (3 sections per brain), including the hippocampus (n = 5 per group), were processed for immunohistochemical staining using anti-Iba-1 (A) and anti-GFAP (E) antibodies. The images in the middle and bottom rows are magnified views of the square regions in the top-row images. Panels B, C, F, and G present quantified graphs of layer 4–6 of the cerebral cortex (B and F) and the stratum oriens of the hippocampus (CA1) (C and G) corresponding to A or E. (D and H) To assess the activation levels of microglia and astrocytes, forebrain lysates were analyzed by Western blotting with anti-Iba-1 and anti-GFAP antisera, followed by quantification (D and H). Abbreviations; 5xFAD, 5XFAD transgenic mice; GFAP, Glial fibrillary acidic protein; Iba-1, Ionized calcium-binding adaptor molecule 1; WT, Wild type.

Fig. 4

Effect of Korean black ginseng extract (BGE) on the expression of pro-inflammatory cytokines/enzymes and p38 MAPK/NF-κB/STAT3 signaling pathways and NLRP3 inflammasome activation in the brains of 5xFAD mice. (A–J) Seven days post-final behavioral tests, forebrain lysates of male mice (n = 4 per group) were procured from the WT, 5xFAD, 5xFAD + BGE (50 and 100 mg/kg), and BGE (100 mg/kg) groups. These lysates underwent Western blot analysis to assess the protein expression levels of key pro-inflammatory cytokines [IL-6 (A) and TNF-α (B)], enzymes [COX-2 (C) and iNOS (D)], signaling pathways [p-p38 MAPK (E), p-NF-κB p65 (F), and p-STAT3 (G)], and NLRP4 inflammasome pathway [NLRP3 (H), ASC (I), IL-1β (J), and cleaved caspase-1 (K)] followed by subsequent quantification. Data are presented as mean expressive value (the ratio of each value relative to β-actin for each sample) ± SEM (one-way ANOVA test with post hoc analysis; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. WT mice; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. 5xFAD mice). Abbreviations; 5xFAD, 5XFAD transgenic mice; ASC, Apoptosis-associated speck-like protein; COX-2, Cyclooxygenase-2; IL, Interleukin; iNOS, Inducible nitric oxide synthase; NF-κB p65, Nuclear factor kappa-light-chain-enhancer of activated B cells/p65; NLRP3, NOD-like receptor protein 3; p38 MAPK, p38 mitogen-activated protein kinase; SEM, Standard error of the mean; STAT3, Signal transducer and activator of transcription 3; TNF-α, Tumor necrosis factor-α; WT, Wild type.

Fig. 5

Effect of Korean black ginseng extract (BGE) on ROS generation and the Nrf2/HO-1 signaling pathway in the brains of 5xFAD mice. (A–N) Seven days after the final behavioral tests, forebrain lysates were collected from male mice (n = 4 per group) in the WT, 5xFAD, 5xFAD + BGE (50 and 100 mg/kg), and BGE (100 mg/kg) groups. Cryo-sections (3 sections per brain), including the hippocampus, were subjected to the MitoSOX assay to assess mitochondrial ROS levels (A) and to immunofluorescence staining for Nrf2/Aβ and HO-1/Aβ to investigate the Nrf2/HO-1 pathway (E and I). Expression levels were quantified in the frontal cortices (B–D), CA1 (F–H), and dentate gyri (J–L). Bar = 100 μm. Data are presented as mean ± SEM (one-way ANOVA with post hoc test; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. WT mice; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. 5xFAD mice). Abbreviations; 5xFAD, 5XFAD transgenic mouse; HO-1, Heme oxygenase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; SEM, Standard error of the mean; WT, Wild type.

Fig. 6

Effect of Korean black ginseng extract (BGE) on TLR2 and TLR4 in the brains of 5xFAD mice. (A and B) Seven days after the final behavioral assessments, forebrain lysates were collected from male mice (n = 4 per group) in the WT, 5xFAD, 5xFAD + BGE (50 and 100 mg/kg), and BGE (100 mg/kg) groups. The lysates were analyzed by Western blotting to evaluate the activation levels of TLR2 (A) and TLR4 (B), followed by quantification. Each experiment was performed in triplicate, and protein levels were normalized to total protein. Data are expressed as mean ± SEM (one-way ANOVA with post hoc test; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. WT mice; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. 5xFAD mice). Abbreviations; 5xFAD, 5xFAD transgenic mouse; SEM, Standard error of the mean; TLR, Toll-like receptor; WT, Wild type.