Multifunctional Anti-Alzheimer's Disease Effects of Natural Xanthone Derivatives: A Primary Structure-Activity Evaluation

Abstract

Background: A series of α-Mangostin (α-M) derivatives were designed and synthesized. α-M and four analogues were evaluated for their multifunctional anti-Alzheimer's disease (anti-AD) effects on fibrillogenesis, microglial uptake, microglial degradation, and anti-neurotoxicity of Aβ, as well as LPS-induced neuroinflammation. The differences in bioactivities were analyzed to understand the structure-activity relationship for further modifications. Purpose: This study aims to investigate the anti-AD effects of α-M and elucidate its structure-activity relationship by comparing difference between α-M and several analogues. Methods: Aβ fibrillogenesis was detected by Thioflavin T fluorometric assay. The levels of Aβ_1-42 and inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Neuron viability was examined by the CCK-8 assay. The morphology of ZO-1 of bEnd.3 cultured in BV-2-conditioned medium was evaluated by immunofluorescence staining. Results: Aβ fibrillogenesis was significantly inhibited by co-incubation with α-M, Zcbd-2 or Zcbd-3. α-M, Zcbd-2, Zcbd-3, and Zcbd-4 decreased the levels of Aβ_1-42 and inflammatory cytokines, and promoted Aβ uptake, degradation and anti-inflammation effects inflammation in microglia. α-M and Zcbd-3 protected neuron viability from Aβ-induced neurotoxicity, and preserved tight junction integrity of bEnd.3 against LPS-induced neuroinflammation. Conclusion: Zcbd-3 acted as α-M almost in all effects. The structure-activity analysis indicated that the 3-methyl-2-butenyl group at C-8 is essential for the bioactivity of α-M, while modifying the double hydroxylation at the C-2 position may improve the multifunctional anti-AD effects.

Keywords: Alzheimer’s disease; amyloid beta; multifunctional; neuroinflammation; structure-activity; α-mangostin.

FIGURE 1

The chemical structure formulas of α-Mangostin (α-M), Zcbd-2, Zcbd-3, Zcbd-4 and Zcbd-5. Molecular formula: C₂₄H₂₆O₆ (α-M), C₂₄H₂₈O₈ (Zcbd-2), C₂₄H₃₀O₁₀ (Zcbd-3), C₂₄H₃₀O₈ (Zcbd-4), and C₂₁H₂₂O₉ (Zcbd-5). Molecular weight: 410.47 (α-M), 444.48 (Zcbd-2), 478.49 (Zcbd-3), 446.50 (Zcbd-4), and 418.40 (Zcbd-5).

FIGURE 2

α-M and its analogues inhibited Aβ fibrillogenesis (A) Inhibitory effects of α-M and its analogues effect on Aβ fibril formation 24 h kinetic detection. 10 μM of ThT was incubated with Aβ_1-40 or the mixture of Aβ_1-40 and α-M or its analogues at 37°C. The newly formed fibrils were measured by a 24 h continuous ThT fluorescence at 30 min intervals (B) Disaggregation of preformed Aβ fibrils by α-M and its analogues. Aβ_1-40 was firstly incubated at 37°C for 48 h. Then α-M or its analogues were added and incubated for another 8 h. ThT was subsequently added into the mixture with a final concentration of 10 μM. The fluorescence was monitored with excitation at 446 nm and emission at 490 nm (C) and (D) Dose-effect relationship in the disaggregation of preformed Aβ fibrils by Zcbd-2 and Zcbd-3. Zcbd-2 or Zcbd-3 (100 nM, 1 μM, and 10 μM) was added to preformed Aβ fibrils and incubated for additional 2 days. Then ThT fluorescence was detected. All data represent mean ± SEM from 4 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the control group (CTRL).

FIGURE 3

α-M and its analogues enhanced Aβ uptake and degradation in microglia BV-2 cells (A) The enhancement of Aβ uptake in microglia by α-M or its analogues. Microglia BV-2 cells were pre-treated with α-M (250 nM) or its analogues (100 nM) for 24 h. Then Aβ (1 μg/ml) was added into the medium for co-incubation. After 4 h incubation at 37°C, the medium was collected to extract the supernatant by centrifugation. The amount of Aβ in the supernate was detected (B) The acceleration of Aβ degradation in microglia by α-M or its analogues. BV-2 were pre-treated with α-M (250 nM) or its analogues (100 nM) for 24 h. Then Aβ was added into the medium for co-incubation. After 4 h incubation at 37°C, both the medium and the cell suspension were collected. Moreover, the amount of Aβ in the medium and cell lysate was detected, respectively. The degradation of Aβ was evaluated by measuring the difference between the initial total Aβ and the rest Aβ both in the medium and lysis of BV-2. All data represent mean ± SEM from 3 independent experiments. ∗p < 0.05 compared to the control group (CTRL).

FIGURE 4

α-M and its analogues suppressed LPS-induced production of pro-inflammatory cytokines in microglia BV-2 cells. BV-2 cells were pre-treated with α-M or its analogues for 2 h prior to stimulation with LPS (100 ng/ml) for another 24 h. Then the protein levels of (A) tumour necrosis factor-α (TNF-α) and (B) interleukin-6 (IL-6) were measured by ELISA in LPS-stimulated BV-2. All data represent mean ± SEM from at least 3 independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001 compared to the LPS-stimulated group (LPS); ####p < 0.0001 compared to the control group (CTRL).

FIGURE 5

α-M and its analogues rescued the Aβ_1-42 induced neurotoxicity in primary cultured cerebral cortical neurons from C57BL/6 mice. Primary cultured mouse cerebral cortical neurons were incubated with Aβ_1-42 oligomers (1 μM) and various concentrations of α-M or Zcbd-3 (0.5, 5, and 50 nM) for 24 h. Then the cell viability was measured by CCK-8 assay. All data represent mean ± SEM from 4 independent experiments. *p < 0.05, **p < 0.01 compared to the Aβ_1-42-stimulated group (Aβ_1-42); ####p < 0.0001 compared to the control group (CTRL).

FIGURE 6

α-M and Zcbd-3 ameliorated the alternation of tight junction (TJ) protein Zonula Occluden-1 (ZO-1) in LPS-stimulated bEnd.3 cells (A) α-M and Zcbd-3 preserved the expression and location of ZO-1 localization in LPS-treated bEnd.3 cells. Mouse brain endothelial bEnd.3 cells were pre-treated with α-M or its analogues for 2 h prior to stimulation with LPS (100 ng/ml) for another 24 h. Then the cells were fixed and stained. The morphology and fluorescence intensity of ZO-1 was detected by a confocal laser-scanning microscope (B) The bar graph showed the quantitation of the average fluorescence area fraction in bEnd.3 cells. All data represent mean ± SEM from 3 independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to the LPS-stimulated group (LPS); ####p < 0.0001 compared to the control group (CTRL). DAPI: blue, ZO-1: red, Scale bar: 50 μM.

FIGURE 7

Multifunctional anti-AD effects of α-M and its analogues. The radar plot of properties included eight aspects, which were Aβ fibrillogenesis, inhibition of TNF-α, inhibition of IL-6, Aβ disaggregation, Aβ uptake, Aβ degradation, neuroprotection and ZO-1 expression. The performance of the four analogues was compared with the control group (CTRL) and normalized to the effects of α-M. Polylines were used instead of the value “0” to represent analogues’ performance, which was eliminated during the experimental process.