Research Article
Rhinacanthin C Alleviates Amyloid-β Fibrils’ Toxicity on Neurons and Attenuates Neuroinflammation Triggered by LPS, Amyloid-β, and Interferon-γ in Glial Cells
Figure 1
Protective effect of RC on Aβ25–35-induced cytotoxicity. (a) Toxicity of RC on primary rat hippocampal neurons was determined using the MTT assay after 24 h of incubation with RC at the indicated concentrations. Data are expressed as mean ± SEM (). Statistical analysis was performed by Student’s t-test, and no significant difference was noted. (b) A representative image of Aβ25–35 after incubation at 37°C for 7 days. Aβ25–35 formed fibrillar morphology with ribbon-like structure. Scale bar, 200 nm. (c) Rat hippocampal neurons were treated with 0.1% DMSO or 25 μM Aβ25–35 fibrils for 24 h and observed by phase-contrast microscopy. Note that cells treated with Aβ25–35 caused cell shrinkage (arrows) and neurite breakdown (arrowheads). (d) Cells with the above treatments were fixed and visualized by immunofluorescence microscope using anti-β-III tubulin antibody (green channel) costained with antitubulinΔCsp6 antibody that specifically recognized caspase-6-cleaved tubulin (red channel). Nuclei were revealed by staining with DAPI. Merged images show the superimposition of both green and red signals, with overlapping areas appearing yellow. Aβ25–35 treatment induced caspase-mediated cleavage of tubulin, indicative of apoptosis. (e) Rat hippocampal neurons were treated with 0.1% DMSO or 25 μM Aβ25–35 for 24 h. The supernatant (S) and pellet (P) fractions were prepared from these cultures and analyzed by immunoblotting using the indicated antibodies. Caspase-6-cleaved tubulin (tubulinΔCsp6) and caspase-3-cleaved fragment (cleaved Csp3) indicate apoptosis. (f) RC reverses Aβ-induced cytotoxicity. Rat hippocampal neurons were pretreated with RC (0.25 and 0.5 μM) for 1 h, followed by 24-hour incubation with 25 μM Aβ25–35. Cell viability was measured by the MTT assay. Data are represented as mean ± SEM (). Statistical analysis was performed using one-way ANOVA with post hoc Dunnett’s test. ### versus control; and versus Aβ25–35.
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