Deciphering the Potential Neuroprotective Effects of Luteolin against Aβ1-42-Induced Alzheimer's Disease

Abstract

The current study was undertaken to unveil the protective effects of Luteolin, a natural flavonoid, against amyloid-beta (Aβ₁-₄₂)-induced neuroinflammation, amyloidogenesis, and synaptic dysfunction in mice. For the development of an AD mouse model, amyloid-beta (Aβ₁-₄₂, 5 μL/5 min/mouse) oligomers were injected intracerebroventricularly (i.c.v.) into mice's brain by using a stereotaxic frame. After that, the mice were treated with Luteolin for two weeks at a dose of 80 mg/kg/day. To monitor the biochemical changes, we conducted western blotting and immunofluorescence analysis. According to our findings, the infusion of amyloid-beta activated c-Jun N-terminal kinases (p-JNK), p38 mitogen-activated protein kinases, glial fibrillary acidic protein (GFAP), and ionized calcium adaptor molecule 1 (Iba-1) in the cortex and hippocampus of the experimental mice; these changes were significantly inhibited in Aβ₁-₄₂ + Luteolin-treated mice. Likewise, we also checked the expression of inflammatory markers, such as p-nuclear factor-kB p65 (p-NF-kB p65 (Ser536), tissue necrosis factor (TNF-α), and Interleukin1-β (IL-1β), in Aβ₁-₄₂-injected mice brain, which was attenuated in Aβ₁-₄₂ + Luteolin-treated mice brains. Further, we investigated the expression of pro- and anti-apoptotic cell death markers such as Bax, Bcl-2, Caspase-3, and Cox-2, which was significantly reduced in Aβ₁-₄₂ + Lut-treated mice brains compared to the brains of the Aβ-injected group. The results also indicated that with the administration of Aβ₁-₄₂, the expression levels of β-site amyloid precursor protein cleaving enzyme (BACE-1) and amyloid-beta (Aβ₁-₄₂) were significantly enhanced, while they were reduced in Aβ₁-₄₂ + Luteolin-treated mice. We also checked the expression of synaptic markers such as PSD-95 and SNAP-25, which was significantly enhanced in Aβ₁-₄₂ + Lut-treated mice. To unveil the underlying factors responsible for the protective effects of Luteolin against AD, we used a specific JNK inhibitor, which suggested that Luteolin reduced Aβ-associated neuroinflammation and neurodegeneration via inhibition of JNK. Collectively, our results indicate that Luteolin could serve as a novel therapeutic agent against AD-like pathological changes in mice.

Keywords: Alzheimer’s disease; amyloid-beta; luteolin; neurodegeneration; neuroprotection.

Figure 1

Graphical Abstract and Study design. (a) Proposed hypothesis and (b) study design.

Figure 2

Luteolin reversed the activation of stress kinases and glial cells-mediated neuroinflammation, i.e., p-JNK/p-38/GFAP/Iba-1 in Aβ₁–₄₂-treated mice brains. (a) Western blots results of p-JNK/p-38/GFAP/Iba-1 in the frontal cortex and hippocampus of the experimental mice brains. β-Actin was used as a loading control. (b,c) Immunofluorescent co-localization of p-JNK/Iba-1 in the frontal cortex and DG region of the hippocampus in the experimental mice. Magnifications10×. Scale bar 50 μm. confocal image analysis was done using ImageJ software. Values are the means ± SEM from three independent experiments. For the statistical analysis, Graph Pad Prism 5 was used, and one-way ANOVA and posthoc multiple comparison tests were employed. * p < 0.05 represents control versus Aβ-treated mice, # p < 0.05 represents Aβ + Luteolin-treated mice.

Figure 3

Luteolin ameliorated the activation of p-NF-kB p65 (Ser536) and of inflammatory mediators in the brains of Aβ₁–₄₂-treated mice. (a) Western blot results of p-NF-kB p65 (Ser536), TNF-α, and IL-1β in the frontal cortex and hippocampus of experimental mice’s brains. β-Actin was used as a loading control. (b) Immunofluorescence results of p-NF-kB p65 in the frontal cortex and DG region of the hippocampus in experimental mice. Values are given as mean ± SEM from three independent experiments. Magnification 10×. Scale bar 50 μm. For the statistical analysis, Graph Pad Prism 5 software was used, and one-way ANOVA and posthoc multiple comparison tests were employed. * p < 0.05 represents control versus Aβ-treated mice, # p < 0.05 represents Aβ + Luteolin-treated mice.

Figure 4

Luteolin suppressed apoptotic cell death in the brains of Aβ₁–₄₂-treated mice. (a) Western blots results of the expression of Bax, Bcl2, caspase-3, and Cox-2 in the frontal cortex and hippocampus of the experimental mice. β-Actin was used as a loading control. (b) Immunofluorescence images of Caspase-3 in the frontal cortex and DG region of the hippocampus of the experimental mice. Values are given as means ± SEM for three independent experiments. Magnification 10×. Scale bar 50 μm. For the statistical analysis, Graph Pad Prism 5 software was used, and one-way ANOVA and posthoc multiple comparison tests were employed. * p < 0.05 represents control versus Aβ-treated mice, # p < 0.05 represents Aβ + Luteolin-treated mice.

Figure 5

Luteolin improved the synaptic dysfunction and reduced the accumulation of Aβ in the brains of Aβ₁–₄₂-treated mice. (a) Western blot results of Aβ, BACE-1, PSD-95, and SNAP-25 in the cortex and hippocampus of experimental mice. β-actin was used as a loading control. (b,c) Immunofluorescence images of Aβ and PSD-95 in the frontal cortex and DG region of the hippocampus of experimental mice. Values are given from three independent experiments. Magnification 10×. Scale bar 50 μm. For the statistical analysis, Graph Pad Prism 5 software was used, and one-way ANOVA and posthoc multiple comparison tests were employed. * p < 0.05 represents control versus Aβ-treated mice, # p < 0.05 represents Aβ + Luteolin-treated mice.

Figure 6

Luteolin conferred neuroprotection in a JNK-dependent manner. Western blot results of p-JNK, p-NF-kB, TNFα, Bax, and Bcl-2 in HT22 cells; β-actin was used as a loading control. For the statistical analysis, Graph Pad Prism 5 software was used, and one-way ANOVA and posthoc multiple comparison tests were employed. Significance = 0.05, δ = Significant difference between Aβ-treated cells and the control group, ε = significance difference between Aβ and Aβ + Luteolin groups, α = significance difference between Aβ and Aβ + SP600125, β = significance difference between Aβ and Aβ + Luteolin + SP600125.