Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor

Abstract

Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

Figure 1

NGF induces the expression of neurofilaments in cultured PC12 cells. Cultured PC12 cells were treated with NGF (0.3 to 50 ng/mL) for 72 hours. The cell lysates were collected to determine the expressions of NF68 (M _r ~ 68 kDa), NF160 (M _r ~ 160 kDa), and NF200 (M _r ~ 200 kDa). GADPH (M _r ~ 38 kDa) served as a loading control (upper panel). Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in Mean ± SEM, n = 4, each with triplicate samples. Representative images were shown. **P < 0.01 compared to the control.

Figure 2

Isorhamnetin induces the neurofilament expression in cultured PC12 cells but not the neurite outgrowth. (a) The chemical structure of isorhamnetin is illustrated. (b) Cultured PC12 cells were treated with isorhamnetin (1 to10 μM) for 72 hours. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. The lower panel shows the quantitation from the blots by a densitometer. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4, each with triplicate samples. (c) Cultures were treated with isorhamnetin (3 or 10 μM) and NGF (50 ng/mL), as indicated, for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 μm. Representative images were shown. (d) Cultured PC12 cell was treated as in (c). The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, mean ± SEM, n = 4. **P < 0.01 compared to the control.

Figure 3

NGF induces the neurite outgrowth in a dose-dependent manner. Cultured PC12 cultures were treated with NGF (0.3 to 50 ng/mL) for 72 hours. Cells were fixed with ice-cold 4% paraformaldehyde. The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Method section. Values are expressed as % of total cells in 100 counted cells, Mean ± SEM, n = 4. **P < 0.01 compared to the control.

Figure 4

Isorhamnetin potentiates the NGF-induced neurofilament expression. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μM), and NGF (0.5 ng/mL) + isorhamnetin (10 μM) for 72 hours. NGF at 50 ng/mL was applied as a control. The cell lysates were collected to determine the expressions of NF68, NF160, and NF200 (upper panel). GADPH served as a loading control. Quantification plot was shown in lower panel. Values are expressed as the fold of change (× Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P < 0.01 compared to the control.

Figure 5

Isorhamnetin potentiates the NGF-induced neurite outgrowth. Cultured PC12 cells were treated with NGF (0.5 ng/mL), isorhamnetin (10 μM), and NGF (0.5 ng/mL) + isorhamnetin (10 μM) for 72 hours, as in Figure 4. (a) Cells were fixed with ice-cold 4% paraformaldehyde and the extension of neurites was revealed. Bar = 10 μm. (b) The % of differentiated cell (upper panel) and length of neurite (lower panel) were counted as described in the Method section. Values are expressed as % of cells in 100 counted cells, mean ± SEM, n = 4. **P < 0.01 compared to the control.

Figure 6

The potentiating effect of isorhamnetin is not mediated by NGF-induced signaling cascade. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (5 ng/mL), isorhamnetin (Iso; 10 μM), and NGF (5 ng/mL) + isorhamnetin (Iso; 10 μM) for different time. Total TrkA and phosphorylated TrkA (a) total Akt and phosphorylated Akt (b) total Erk1/2 and phosphorylated Erk1/2 (c) were revealed by using specific antibodies. (d) Quantification plot of the phosphorylation level in treatment of 5 min was shown. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. ** where P < 0.01 compared to the control.

Figure 7

The potentiating effect of isorhamnetin on NGF-induced response could not be blocked by U0126. Cultured PC12 cells, serum starvation for 5 hours, were treated with NGF (0.5 ng/mL), isorhamnetin (Iso; 10 μM), and NGF (0.5 ng/mL) + isorhamnetin (Iso; 10 μM) for 72 hours with or without the pretreatment of U0126 (20 μM) for 3 hours. NGF at 50 ng/mL served as a positive control. (a) The cell lysates were collected to determine the expressions of NF68, NF160, and NF200. GADPH served as a loading control. (b) Quantification plot was shown in lower panel. Values are expressed as the fold of change (×Basal) against the control (no treatment; set as 1), and in mean ± SEM, n = 4. Representative images were shown. **where P < 0.01 compared to the control.