Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

Figure 2

Protective effects of LBP on TMT-induced apoptosis in N2a cells. N2a cells were treated with different concentrations of TMT (1.875, 3.75, and 7.5 μM) or cotreated with LBP (300 μg/mL) for 24 h. DNA condensation and fragmentation in N2a cells were observed using Hoechst33258 staining under an inverted fluorescent microscope, scale bar: 25 μm (a). Effects of TMT treatment on expression of Bcl-2 and Bax as determined through Western blot analysis. Total protein extracts were prepared after incubating the cells with 1.875~7.5 μM TMT for 24 h (b). Effects of LBP on the expression of Bcl-2 and Bax induced by TMT, as determined by Western blot analysis. Total protein extracts were prepared after incubating the cells with 300 μg/mL LBP and 3.75 μM TMT for 24 h (c). All Western blot analysis results were expressed as fold changes of optical density, with GAPDH as the internal control. The mean protein expression of control is designated as 1 in the graph. After incubation with 1.875~7.5 μM TMT for 24 h, cells were collected and stained with PE-Annexin-V and 7-AAD for 5 min. Samples were analyzed by flow cytometry. The proportions of living and dead cells were determined by flow cytometric analysis of PE-Annexin-V and 7-AAD-labeled cells. Live cells are unlabeled with PE and 7-AAD (Q3), whereas PE labeling (Q4) represents the population of early apoptosis. Cells showing PE and 7-AAD double labeling (Q2) represent those that have already died due to apoptosis. Ten thousand cells were analyzed in each sample. The percentages of the total apoptosis analyzed were determined (d). The effects of TMT alone (e) or TMT with LBP intervention (f) on caspase-3 activity were assayed by ELISA method. Each bar represents mean ± SD (). , compared with the control group; , compared with the TMT-treated group.

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