Fig 1.
Chemical structure of geniposide.
Fig 2.
Immunocytochemsitry analysis of neurons labelling nuclei with NeuN.
Neuronal nuclei were labelled with specific neuronal marker-NeuN (red), while all cells’ nuclei were stained in blue.
Fig 3.
Geniposide prevents the cytotoxicity of oligomeric Aβ1–42 in neurons.
A, Effects of geniposide on the cell viability in neurons. Cells were treated with the indicated doses of geniposide for 24 h, and the cell viability was determined with MTT. B, Effects of oligomeric Aβ1–42 on the cell viability in the primary cultured cortical neurons. After cells were treated with the indicated doses of Aβ1–42 for 24 h, the cell viability was determined with MTT. C, Geniposide increases the cell viability of neurons induced by Aβ1–42. Cells were pre-incubated with geniposide at 2.5–10 μM for 24h, and then exposed to oligomeric Aβ1–42 at 5.0 μM for 24 h in the presence and absence of geniposide. The cell viability was measured by MTT. NS: non significance. N = 6 per group of cells. Studies were repeated four times and data were expressed as means ± SEM of percentage of vehicle-treated cells.
Fig 4.
The effect of geniposide on Aβ1-42-induced apoptosis.
A, Photomicrographs of TUNEL and DAPI fluorescence staining. Neurons were cultured in the presence of 5 μM oligomeric Aβ1–42 and in the presence and absence of geniposide (2.5 μM, 5 μM, 10 μM) for 24 h. TUNEL-positive cells were stained in green while all cells’ nuclei were stained in blue. B, Quantitative analysis the ratio of TUNEL-positive cells to total neuron number. The ratio of TUNEL-labelled neurons was significantly increased in neurons treated with oligomeric Aβ1–42 for 24 hours while the geniposide treatment dramatically reduced the Aβ-induced TUNEL-positive cells in a dose-dependent manner. NS: non significance. N = 6 per group of cells. Studies were repeated thrice and data were expressed as mean ± SEM of percentage of vehicle-treated cells.
Fig 5.
Effects of geniposide on the ROS and MDA levels in primary cultured cortical neurons.
The levels of DCF fluorescence intensity (an indicator of ROS generation (A)) and MDA production (B) were significantly elevated in Aβ1-42-treated neurons compared to vehicle-treated neurons, and partially restored in the geniposide-treated neurons in a dose-dependent manner. NS: non significance. N = 6 per group of cells. Studies were repeated four times and data were expressed as mean ± SEM of percentage of vehicle-treated cells.
Fig 6.
The effects of geniposide on Aβ1-42-induced mitochondrial abnormalities.
A, Mitochondrial membrane potential. Cells were labeled with JC-1 dye and analyzed by laser-scanning confocal microscope per the manufacturer’s protocol. In the live non-apoptotic cells, the mitochondria appearred following aggregation of JC-1 reagent. In the apoptotic cells, the dye remains in its monomeric form and appears green. Scalebar = 250 μm. B, Quantitative analysis of the Δψm among groups. The ratio of red to green fluorescence in A was used as the indicator for mitochondrial membrane potential. C, The ATP level was determined by fluorescence microplate reader. D, CcO activity was tested by spectrophotometer. NS: non significance. N = 6 per group of cells. Studies were repeated four times and data were expressed as mean ± SEM of percentage of vehicle-treated cells.
Fig 7.
The effects of geniposide on Aβ1-42-induced release of mitochondrial cytochrome c and caspase-3/9 activity.
Neurons were cultured in the presence of 5 μM oligomeric Aβ1–42 and geniposide (2.5 μM, 5 μM, 10 μM) for 24 h, harvested and the levels of specific proteins were assessed by western blot analysis (A). Levels of cytochrome c in the cytosolic fraction (B) or mitochondrial fractions (C), Geniposide attenuated mitochondrial cytochrome c releasing to cytosolic fraction in neurons induced by oligomeric Aβ1–42 at 5 μM. Caspase-3/9 activity was assessed by microplate fluorometer. The increased activity of caspase-3/9 was attenuated (d, e) by the present of geniposide in a dose-independent manner. NS: non significance. N = 6 per group of cells. Studies were repeated four times and data were expressed as mean ± SEM of percentage of vehicle-treated cells.
Fig 8.
The effect of geniposide on Aβ1-42-induced Bax and Bcl-2 proteins abnormally expression level.
Neurons were cultured in the presence of 5 μM oligomeric Aβ1–42 and geniposide (2.5 μM, 5 μM, 10 μM) for 24 h, harvested and the levels of specific proteins were assessed by immunoblot analysis (A). Pretreatment of primary cortical neurons with geniposide (2.5–10 μM) could upregulate Bcl-2 expression (B), downregulate Bax expression (C) and decrease the Bax/Bcl-2 ratio (D) induced by Aβ1–42. NS: non significance. N = 6 per group of cells. Studies were repeated four times and data were expressed as mean ± SEM of percentage of vehicle-treated cells.